Towards the synthesis of alpha-cyclo peptide nucleic acid monomers
by
Manish Radheshyam Biyani (M. Pharm.)
Submitted for the degree of Doctor of Philosophy
A thesis submitted to Heriot-Watt University
School of Engineering and Physical Sciences
March, 2012
The copyright in this thesis is owned by the author. Any quotation from the thesis or use
of any of the information contained in it must acknowledge this thesis as the source of
the quotation or information.
I
Dedicated
to
my
Teachers
II
and
Family
Abstract
Abstract
Peptide nucleic acids (PNAs) are potentially useful as mimics of DNA for gene therapy
treatment of cancer and other genetic disorders. The original PNA molecule developed
by Nielsen et al. lacks selectivity towards nucleic acid targets (DNA vs RNA) and the
ability to incorporate new monomers for each alteration to be examined. Our design of
L-α-PNA
allows for the incorporation of different appropriate α-amino acid spacers into
the oligomers during peptide synthesis. Selectivity towards nucleic acid targets is
improved by freezing out conformation with incorporating cyclopentane ring in L-αPNA.
The aim of the research reported in this thesis was to develop viable synthetic routes to
α-cycloPNA monomers, constrained analogues of PNA monomers. Our initial objective
was to optimize an established synthetic pathway to a key cyclopentane α–amino acid
intermediate suitably functionalized at the 3-position to enable subsequent preparation
of pyrimidine and purine α-cycloPNA monomers. However, reproduction of the key
cyclization
step
in
our
prototypical
route
to
a
key
3-alkoxy-1-
aminocyclopentanecaboxylate precursor proved to be problematic and so alternative
synthetic pathways were explored.
All four diastereoisomers (203, 143, 204 and 144) of the required cyclopentane α–
amino acid intermediate were prepared via hydroboration of ethyl N-Boc-1aminocyclopent-3-ene-1-carboxylic acid in overall yields of 6.2% for the (1S/R, 3S/R)alcohols (204 and 144) diastereoisomeric mixture and 13% for the (1R/S, 3S/R)-alcohol
(203 and 143), also isolated as an inseparable diastereoisomeric mixture. Unfortunately,
all attempts to resolve these mixtures failed. With both the (1S/R, 3S/R)-alcohols and
(1R/S, 3S/R)-alcohols to hand, synthesis of a cytosine derivative of the α-cycloPNA
monomer was investigated. The mixed (1R/S, 3S/R)-alcohol diastereoisomers were first
converted into their respective camphorsulfonate derivatives (232 and 233) and treated
with Cbz-protected cytosine. Unexpectedly, the corresponding O2-alkylated cytosine αcycloPNA monomers (266 and 267) were afforded.
Asymmetric hydroboration of the chiral cyclopentene intermediate bearing D-menthyl
III
Abstract
ester using either (+)- or (-)-IpcBH2.TMEDA were studied. This also gave unresolvable
diastereoisomeric mixtures of (1R/S, 3S/R)-alcohols (299 and 300), in 82% yield, and
(1R/S, 3S/R)-alcohols (299 and 300), in 69% yield. Finally, treatment of the (1R/S,
3S/R)-brosylate mixture derived from the (1R/S, 3S/R)-alcohols of D-menthyl esters
with Cbz-protected cytosine again yielded the O2-alkylated cytosine α-cycloPNA
monomers.
IV
Acknowledgements
Acknowledgements
It gives me immense pleasure to express gratitude to my research advisor Dr. Nicola
Howarth for her advice, guidance, support and encouragement during every stage of my
PhD work. This has helped me to improve both my bench skills and my insight into
research. I am very grateful to the School of Engineering and Physical Sciences, and in
particular Professor Alan Welch, for waiving my tuition fees.
I would like to thank Dr Alan Boyd for recording my NMR spectra, Dr Georgina Rosair
for determining X-Ray structure, Dr Roderick Ferguson and Mr Gerry Smith for
recording mass spectra, and Mrs Christine Graham for providing CHN analysis and
optical rotation analysis.
I have appreciated the constant assistance provided by my colleagues in labs 1.15, 1.18
and 1.20. I am grateful to Dr. Geoffrey Livesey for proofreading my Introduction
chapter of thesis. I would like to thank Dr Koen Collart for proofreading my final
version of R & D discussion. This PhD would have been incomplete without the support
of my friend Barry Gale. He did several times proofreading of all chapters in the thesis.
I am thankful to Granules India Ltd for their support
Finally, I would like to thank my family and friends for their encouragement, especially
my father Radheshyam Biyani, my mother Urmila Biyani, my wife Vishakha Biyani &
my daughter Stuthi Biyani, when things were drab.
V
Abbreviations
Abbreviations
[α]
specific rotation
A
9-adenyl
ADDP
1,1’-(azodicarbonyl)dipiperidine
ATP
adenosine-5-triphosphate
A
6-(3-aminopropyl)-7-methyl-3H-pyrrolo[2,3-d]pyrimidin-2(7H)-one
BAU
2’-aminoethoxy-5-(3-aminoprop-1-ynyl)uridine]
BH3.THF
borane- tetrahydrofuran complex
Boc
tert-butyloxycarbonyl
PP
t
Bu
tert-butyl
9-BBN
9-borabicyclo[3.3.1]nonyl
BOP
benzotriazolyl-tris-(dimethylamino) phosphonium hexafluorophosphate
[bmim][BF4]
1-butyl-3-methylimidazolium tetrafluoroborate
Bs
4-bromobenzene sulfonyl
Bz
benzoyl
CBZ
benzyl carbamate
C
degrees Celcius
C
1-cytosinyl
CI
chemical ionisation (in mass spectrometry)
cm
centimetre
COSY
correlation spectroscopy
dA
deoxyadenosine
dG
deoxyguanosine
dC
deoxycytidine
dT
deoxythymidine
DBU
1,5-diazabicyclo[5.4.0]undec-7-ene
DCC
dicyclohexylcarbodiimide
DCM
dichloromethane
d.e.
diastereomeric excess
DEAD
diethyl azodicarboxylate
DIAD
diisopropyl azodicarboxylate
DMF
dimethylformamide
VI
Abbreviations
DMSO
dimethyl sulfoxide
DNA
deoxyribonucleic acid
ds-DNA
double stranded deoxyribonucleic acid
DPM
diphenylmethyl
e.e.
enantiomeric excess
EI
electron impact (in mass spectrometry)
Et
ethyl
G
9-guanyl
Δ
heat
HCl
hydrochloric acid
HPLC
high-performance liquid chromatography
HRMS
high-resolution mass spectrometry
hnRNP
heterogenous nuclear ribonucleoproteins
IR
infrared
IpcBH2
monoisopinocampheylborane
LDA
lithium diisopropylamide
LiHMDS
lithium hexamethyldisilazide
LRMS
low-resolution mass spectrometry
Me
methyl
Me
3-methyl-2 aminopyridine
MP
melting point
Ms
methanesulfonyl
MS
mass spectrometry
m/z
mass to charge ratio (in mass spectrometry)
NMR
nuclear magnetic resonance
NOE
nuclear Overhauser effect
NOESY
nuclear Overhauser effect spectroscopy
ODN
oligodeoxynucleotide
P
pyridone
PS
phosphorothioate
PNA
peptide nucleic acid
L-α-PNA
L-α-peptide nucleic
α-cycloPNA
α-cyclo-peptide nucleic acid
RNA
ribonucleic acid
P
acid
VII
Abbreviations
RISC
RNA-induced silencing complex
r.t..
room temperature
S
N-(4-(3-acetamidophenyl)thiazol-2-yl)acetamide
siRNA
small interfering RNA
SN1
unimolecular nucleophilic substitution
SN2
bimolecular nucleophilic substitution
snRNPs
small nuclear ribonucleoproteins
ss-DNA
single stranded deoxyribonucleic acid
T
1-thyminyl
TAR
trans-activating responsive sequence
TBDPS
tert-butyldiphenylsilyl
TFA
trifluoroacetic acid
THF
tetrahydrofuran
TFO
triplex forming oligonucleotide
TMEDA
N,N,N',N'-tetramethyl-ethane-1,2-diamine
Tm
melting temperature
VIII
Index
Abstract
Acknowledgments
Abbreviations
Content
1.
Introduction...........................................................................................on page 1
1.1.
Structure, conformation and configurations of nucleic acids...........on page 2
1.2.
The Antisense Strategy.........................................................................on page 7
1.3.
The Antigene Strategy.........................................................................on page 11
1.3.1
Minor Groove Binders.........................................................................on page 11
1.3.2
Triplex Forming Oligonucleotides......................................................on page 15
1.3.3.
Melting temperature (Tm)....................................................................on page 17
1.4.
Oligonucleotide Analogues..................................................................on page 18
1.4.1
Furanose-Containing Phosphate Backbones.....................................on page 19
1.4.2
Furanose-Containing Nonphosphate Backbones..............................on page 23
1.4.3
Sugar and Backbone Replacements....................................................on page 27
1.4.4.
Sugar Modifications..............................................................................on page 28
1.4.5.
Sugar, backbone and nucleobase replacements.................................on page 34
1.5.
Peptide nucleic acid..............................................................................on page 38
1.5.1. Binding affinity of PNA with complementary ss-DNA, RNA and
PNA.........................................................................................................on page 39
1.5.2. Strand invasion......................................................................................on page 39
1.5.3. Hydribization Property of PNA ...........................................................on page 43
1.5.4. Stability, solubility and cellular uptake of PNA..................................on page 45
1.5.5. Potential therapeutic applications of PNA..........................................on page 45
1.5.6.
Antisense PNAs.....................................................................................on page 45
1.5.7.
Antigene PNAs......................................................................................on page 47
1.5.8.
PNA backbone modification................................................................on page 48
1.5.9.
α-PNA.....................................................................................................on page 51
1.5.10. ConstrainedPNAs.................................................................................on page 53
1.6.
Conclusion.............................................................................................on page 60
2.
Results and Discussion..........................................................................on page 62
2.1.
Retrosynthesis of target compound......................................................on page 63
2.2.
Synthesis of key intermediate alcohol (143 and 144) via Curtius
rearrangement........................................................................................on page 64
IX
Index
2.3.
Synthesis of key intermediate alcohol (143 and 144) via Schiff’s base route
using N-(diphenylmethylene)glycine ethyl ester................................on page 68
2.4.
Synthetic routes to the key cyclopentane-3-ol via hybroboration of the
corresponding cyclopent-3-ene (108)..................................................on page 81
2.4.1.
Ring-closing metathesis (RCM)..........................................................on page 82
2.4.2.
Curtius Rearrangement......................................................................on page 83
2.4.3.
Cycloalkylation of a glycine equivalent with a dielectrophile.........on page 86
2.4.4.
Preparation of intermediate alkene (108)..........................................on page 87
2.4.5.
Synthesis of key intermediate cis-alcohol (203 and 143) and trans-alcohol
(204 and 144).......................................................................................on page 90
2.4.6.
Preparation of cis-alcohol (203 and 143) and trans-alcohol (204 and 144)
via hydroboration with borane-THF complex...................................on page 95
2.4.7.
Asymmetric Hydroboration................................................................on page 96
2.4.8.
Chiral resolution.................................................................................. on page 98
2.4.9.
Sulfonation (Introduction of a chiral auxiliary)..............................on page 102
2.4.10. Cis-alcohol to Trans-alcohol...............................................................on page 106
2.4.11. Synthesis of thyminyl α-cycloPNA monomer................................. on page 107
2.4.12. Synthesis of cytosine α-cycloPNA monomer.................................. on page 111
2.4.13. Ester Hydrolysis................................................................................. on page 115
2.5.
Modified hydroboration route for the synthesis of α-cycloPNA cytosine
monomer.............................................................................................on page 116
2.5.1
Hydroboration after introduction of chiral auxiliary on the ester group with
(-)-IpcBH2 and (+)-IpcBH2............................................................... on page 122
2.5.2.
Sulfonation.........................................................................................on page 124
2.5.3.
Synthesis of cytosine α-cycloPNA monomer...................................on page 126
2.5.4. Menthol Hydrolysis.............................................................................on page 130
2.6
Final Conclusions.................................................................................on page 132
2.7
Future Work.........................................................................................on page 135
3.
Experimental........................................................................................on page 138
4.
Appendices............................................................................................on page 180
5.
References.............................................................................................on page 232
X
Introduction
1. Introduction
The recognition of both DNA (deoxyribonucleic acid) and RNA (ribonucleic acid)
sequences by corresponding oligonucleotides is a central feature in biotechnology, and is
important for hybridization-based biological applications. Such recognition underlies
widely used experimental techniques and diagnostic protocols, and makes possible a
consideration of antisense or antigene based inhibition of gene expression as a practical
approach to therapeutics. Specific inhibition of a biological or disease causing process
depends on Watson-Crick base-pairing between the heterocyclic bases within the
antisense oligonucleotide and the target nucleic acid, along-with Hoogsteen base-pairing,
which allows the third strand to bind around the duplex due to hydrogen binding in the
target nucleic acid. Various cellular processes can be inhibited, the success of which may
depend on the location of the target nucleic acid to be hybridized.1
The recognition of cellular nucleic acids by synthetic oligonucleotides is a versatile
strategy for selective manipulation of gene expression, which may make a major impact
in the treatment of a variety of human diseases such as cancer, HIV, and sickle cell
anaemia.1 In recent years, there has been surging interest in the design and synthesis of
antigene oligonucleotides that bind to ds-DNA in a sequence specific manner and high
affinity, so as to inhibit transcription of particular genes and the mechanisms governing
their regulation.1 On a cautionary note, however, agents used in gene therapy may give
epigenetic effects by targeting the ds-DNA2 [eg. ds-DNA methylation and chromatin
(complex between ds-DNA and histone protein), histone deacetylation] and RNA (RNA
methylation)3,4,5,6, thus inducing a phenotypic change (by altering gene expression)
without a change in the nucleotide sequence of the genome. These concerns have
prompted Wellcome-Trust to reduce funding in gene therapy area.
The synthetic natural oligonucleotides (DNA or RNA) have been reported to exhibit both
antisense and antigene properties in in vitro studies. However, they are rapidly degraded
by nucleases in in vivo studies. This led to development of synthetic oligonucleotides
with minor changes in either phosphate or sugar or both backbones of the synthetic
natural oligonucleotides. Although it circumvents nuclease degradation, use of these
oligonucleotides introduces new problems that need to be overcome, such as synthetic
1
Introduction
challenges in large scale preparation of oligomers by solid phase synthesis and,
conjugation to other ligands such as intercalators, metal binders, amino acid, etc.10,11,12,13
This inspired the replacement of deoxyribose-phosphate backbone of DNA with a
structurally homomorphous uncharged polyamide backbone composed of N-(2aminoethyl)glycine units.7 This revolutionary change extended to development of peptide
nucleic acid (PNA), because of the well-established robustness and flexibility of SPPS
technology. PNA exhibits improved nuclease as well as protease stability and binds to
single-stranded DNA (ss-DNA), double-stranded DNA (ds-DNA) and RNA with high
affinity and sequence specificity. PNAs were found to be 1st oligonucleotide analogs to
hybridize with ds-DNA through a comparatively unique mechanism called strand
invasion.7 The above developments have led us to design one such analogue of PNA, i.e.,
α-cyclo peptide nucleic acids.12,13 The focus of this thesis is to synthesize four
diastereoisomers of α-cyclo peptide nucleic acids.
1.1. Structure, conformation and configurations of nucleic acids
O
NH
Thymine
N
HO
O
O
NH2
N
O
O
P
-
O
Cytosine
O
5'
O
Sugar
N
O
4'
3'
2'
1'
O
O
P
NH2
N
Adenine
N
ON
O
N
O
O
N
O
Phosphate
O
P
NH
Guanine
ON
O
N
NH2
O
O
O
O-
P
-
O
Figure 1
Primary structure of DNA
The structure of DNA was described by Watson and Crick in 1953.14 DNA is a
biopolymer with a primary structure composed of monomeric units called nucleotides
(Figure 1). Each nucleotide is constructed from three components: a 2'-deoxyribose
2
Introduction
sugar, a heterocyclic base (often referred to as a nucleobase) and a phosphate group.
There are four different nucleobases in DNA. Two are substituted purines (adenine and
guanine) and two are substituted pyrimidines (cytosine and thymine). The heterocyclic
bases are attached to the C1' position of the sugar ring through a -glycosyl linkage. The
nucleotides are joined together in DNA by phosphodiester linkages, which form
specifically between the 5'-phosphate group of one nucleotide and the 3'-hydroxyl on the
sugar of the next nucleotide. The terminus of the resulting polynucleotide chain bearing
the free 3'-hydroxyl group is called the 3'-end while the other terminus carrying the 5'phosphate is known as the 5'-end.
DNA comprises two polynucleotide chains (strands) which are coiled around each other
to form a double helix. The two strands are aligned in opposite directions (i.e. they line
up in an anti-parallel orientation) and are held together by hydrogen bonds that form
between specific pairs of nucleobases. Thus, thymine on one strand only pairs with
adenine on the other strand through the formation of two hydrogen bonds and cytosine
only pairs with guanine through the formation of three hydrogen bonds. These two base
pairs are known as Watson-Crick base pairs.
The hydrophobic nature of the nucleobases keeps them on the inside of the double helix;
meanwhile the hydrophilic nature of the phosphate groups keeps the backbone on the
outside. The DNA double helix is further stabilised by the fact that the base pairs are
stacked one on top of another, which allows for  stacking interactions between the
heterocyclic rings.15,16,17
Figure 2
Cylindrical ribbon presentations in different helical forms of DNA15
3
Introduction
DNA exists mainly in three possible conformations that include A-DNA, B-DNA, and ZDNA forms. A- and B-DNA are both right-handed helices, while Z-DNA is left-handed
helical structure with Watson and Crick base pairings. DNA varieties are differentiated
from each other mainly by three structural features: glycosidic bonds, major and minor
grooves, and sugar ring puckering as shown Table 1.
In the case of B-DNA, a complete turn happens every ten base pairs, while in A-DNA
the turn occurs every eleven base pairs. In the case of Z-DNA, the complementary
strands form a left-handed double helix with twelve base pairs per turn. Z-DNA
structural features consist of mainly C-G base pairings; although A-T base pairing can
exist within the Z-DNA helix, it destabilizes the Z-DNA helix.15 In Z-DNA, the
conformation at C base is anti and G base is syn resulting in zigzag backbone, which
renders phosphate groups to come close together. This in turn causes there to be more
electrostatic repulsion between the phosphate groups than in B-DNA. Consequently, ZDNA can be stabilized by high salt concentrations or by polyvalent cations.15
Sr No
Parameter
A-DNA
B-DNA
Z-DNA
Right
Right
Left
1
Helix handedness
2
Base pair / turn
11
10
12
3
X displacement from bp to helix axis
-4.1
0.8
3.0
Anti
Anti
Anti, syn
C3’-endo
C2’-endo
C2’-endo
(Å)
4
Glycosidic bond orientation
5
Sugar conformation
C3’-endo
6
7
Table 1
Major groove depth
13.5
8.5
Convex
width (Å)
2.7
11.7
Minor groove depth
2.8
7.5
9
width (Å)
11.0
5.7
4
Average structural parameter for the different helical forms of DNA15
4
Introduction
O
O
H2N
N
N
HN
HOCH2
N
N
N
HOCH2
O
H
OH
Syn
NH2
H
H
OH
H
N
O
H
H
H
NH
H
Anti
Figure 3
Example of syn and anti deoxyguanosine
Characteristic parameters and shape of each type of DNA are described in Table 1 and
Figure 3. The anti conformation is observed when C4 and the six-membered ring of the
purine are away from the sugar; in pyrimidines, the C2 carbonyl is away from the sugar. The
syn conformation is observed when C4 of purines or C2 of pyrimidines above the sugar. The
position of the glycosyl bond between the heterocyclic base and the deoxyribose sugar is anti
in A-DNA and B-DNA, but it alternates between syn (at G) and anti (at C) in Z-DNA
(Figure 2). The displacement of the base pair from the helical axis (dx) influences the pattern
of major and minor grooves, which affects the ultimate shape of B-DNA (dx = average 0.8
Å), A-DNA (dx = about - 4 Å), and Z-DNA (dx = about 3 - 4 Å) as shown Figure 3. The
bend in the plane of the sugar ring (pucker) tends to be C3’-endo in A-DNA, C2’-endo in BDNA, and in Z-DNA it is C2’- endo at C while C3’- endo at G.
5
3
Base
North (N) conformersA-form double strands
O
4
5
3
Base
O
1
1
4
2
2
3 - endo
Phase angle, P = 18o
Envelope
3 - endo, 2 -exo
Phase angle ,0o < P < 18o
Twist
5
Base
2
5
South (S) conformersB-form double strands
O
4
3
Base
2
O
4
1
1
3
2 - endo
Phase angle, P = 162o
Envelope
Figure 4
2 endo, 3 - exo
Phase angle,162o < P <180o
Twist
The conformation of ribose and deoxyribose found in nucleotides
5
Introduction
The pseudorotation phase angle, P, characterizes the sugar pucker in a five membered ring,
which are mainly found with a range near P = 18 (C3’-endo) and P = 162 (C2’-endo),
while when P = 0 ± 18 (N-conformer) and P = 180 ± 18 (S-conformer). A planar five–
membered sugar ring is sterically and energetically unfavourable. However, this strain can
be released by placing one atom out of the plane (Figure 4). The conformation with the 3carbon out of the plane defined by C4’-O4’-C1’-C2’ (ν0=0) and on the same side of the
plane as the nucleobase is refered to as the C3’-endo conformer. In contrast, the C3’-exo or
C2’-endo conformation has the 3’-carbon on the opposite side of the plane, while C2’-exo
or C3’-endo conformation has the 2’ carbon out of the plane and on the same side as the
base. The nucleotide in the A-DNA double helix forms the N-conformer, while the sugar
pucker determines the C3’-endo or C2’-exo conformation i.e. the ring is twisted
symmetrically. The B-DNA double helix forms the S-conformer.15
Figure 5
Major and minor groove binders9
The spatial relationship between twin helical strands creates a minor groove, which is the
side of the base pairs facing towards the sugar phosphate backbone, while major groove is
the side away.15 As the strands are not directly opposite each other, the grooves are
unequally sized. These voids are adjacent to the base pairs and may provide a binding site.
The narrowness of the minor groove in B-DNA means that the edges of the bases are more
accessible in the major groove. The wider major groove in B-DNA makes this form more
accessible to interaction with other molecules and proteins in comparison to narrow minor
groove.15 The depth in both the grooves in B-DNA are of similar length. Compared to BDNA, the A-DNA has shallow, wide minor groove and a narrower, deeper major groove.
In Z-DNA, the major groove is convex and minor groove is deep but narrow.15 For more
6
Introduction
quantitative comparison, please refer Table 1.
1.2. The Antisense Strategy
Zamecnik and Stephensen were the first to propose the use of synthetic antisense
oligonucleotides for therapeutic purposes.18,19 For an antisense oligonucleotide to be able
to inhibit translation, it must reach the interior of the cell unaltered. This means that it
needs to be able to traverse the cell membrane and be stable to the extra- and intracellular enzymes. Once within the cytoplasm, the antisense oligonucleotide must bind to
the target mRNA with sufficient affinity and high specificity. In addition, it must possess
an adequate half-life in order to elicit its action. Finally, the toxicity of this
oligonucleotide should be negligible to the cell.20
There are a number of ways in which an antisense oligonucleotide may exert its
pharmacological effect as shown in Figure 6. These include:
i)
Modulation by splicing
Splicing is a modification of m-RNA that occurs immediately after transcription. In this
process, introns are removed and exons are joined. Splicing is catalyzed by the
spliceosome, which is a large RNA-protein complex composed of five small nuclear
ribonucleoproteins (snRNPs). Two well-characterized categories of splicing regulators
mediate intronic and exonic regulatory-element recognition. The first is the SR protein
family, which bind exonic splicing enhancer sequences and drive formation of
complexes to enable both exon identification and selection of splice sites. The second is
the heterogeneous nuclear RNP (hnRNP) family, which is known to associate with premRNA.21
Ideally, the accuracy of spliceosome-catalyzed reactions is dictated by base-pairing
between pre-mRNA and snRNPs and is assisted by specific proteins. The force-driving
activation of the spliceosome is ATP. The “splicing” reactions are sequence-specific and
require the concerted action of several spliceosomes sequentially.21
Alternative splicing offers perhaps more exciting therapeutic possibilities. This uses an
antisense oligonucleotide to induce the production of an alternative protein, in effect to
7
Introduction
produce “agonist-like” activities. A number of in vitro and in vivo studies involving
either inhibition of splicing or activation of alternative splicing have been reported for a
variety of genes using antisense oligonucleotide analogues.21,22,23,25 One such
investigation employed alternative splicing to alter splicing of the dystrophin gene
product.24 Dystrophin is an essential protein for the normal function of skeletal and
cardiac muscle. Mutation of the dystrophin gene has been found to result in early
termination of the synthesis of the truncated dysfunctional protein, which causes disease
like muscular dystrophy in humans. Antisense oligonucleotides have been used to alter
splicing in dystrophic mice in vivo.25
Large ribosomal unit
protein
Translation
RNA-BD
RNA
Cat
mRNA
5'
3'
MOE
MOE
small ribosomal unit
ASO
Figure 6B Protein synthesis due to translation
FAST
Spacer
RNA-BD
RNA-BD
Cat
Catalyst domain
Cat
RNA
mRNA
5'
3'
MOE
MOE
ASO
SLOW
RNA
Figure 6C
Antisense effect due to activation of RNase enzyme
Figure 6A RNase site specifity response
RNA-BD =RNA binding domain, MOE = 2'-OMethoxyethyl modification, ASO - Antisense
Antisense oligonucleotides
oligonucleotides
Figure 6D Antisense effect due to translational arrest
Figure 6
Ways by which antisense oligonucleotide exerts pharmacological effects
ii) Translational arrest
Prevention of binding of the protein translational machinery to the target mRNA by an
hybridization of antisense oligonucleotide to gene sequences involves any one of the
three steps (i.e. initiation, elongation, and termination) of the translation process and has
the potential to interrupt protein synthesis as shown in Figure 6D. In order to successfully
8
Introduction
mediate translational arrest, the antisense oligonucleotide needs to bind with very high
affinity to the target mRNA by Watson-Crick base pairing, thus sterically blocking the
translation of a transcript into a protein. Optimum inhibition has been observed with
antisense oligonucleotides being targeted by binding at the 5-untranslated region
(5UTR) in mRNA that codes for protein through translation without involvement of an
RNase H substrate.23,27
iii)
RNase H
RNase H is an enzyme that has a very reliable and robust mechanism both in vitro and in
vivo. RNase H1 is involved in the degradation of the RNA strand of an RNA:DNA
heteroduplex. It has been noticed that some antisense oligonucleotides, when bound to
mRNA, can also trigger RNase H activity (Figure 6C). The activation of RNase H has
been found to depend upon the antisense oligonucleotide bearing a conformationally
flexible sugar such that the O-4’-endo sugar pucker is adopted upon its hybridization to
RNA; it is the helical geometry of the heteroduplex substrate at the catalytic site of
human RNase H1 which directs the selective recognition of the substrate by the enzyme
(Figure 6A).25 The resulting cleavage products are then subject to degradation by
exonucleases. As the antisense oligodeoxyribonucleotide essentially remains untouched
during this process, this approach is in effect catalytic as, in principle, a single oligomer
can result in the degradation of many strands of target mRNA. This compensates in part
for the regeneration of the target mRNA through transcription. Although this strategy has
been the focus of much attention, to date, only phosphodiester and phosphorothioate
oligomers have been proven to work in this manner by activating the RNase H
mechanism. Unfortunately, both oligomers are degraded by exonucleases over time,
which limits their ultimate utility. 9,28
iv) Small interfering RNA (siRNA)
Small interfering RNA (siRNA) is a class of 20-25 nucleotide-long double-stranded
RNA molecules that play a variety of roles in biology. Each strand has a 5'-phosphate
group and a 3'-hydroxyl (-OH) group. This structure is the result of processing by ‘dicer’,
an enzyme that converts either long ds-RNA or small hairpin RNA molecules into
siRNAs. The short interfering duplexes are incorporated into a protein complex called
9
Introduction
the RNA-induced silencing complex (RISC). The two strands of the siRNA duplex are
dissociated and the “sense” strand is discarded. siRNA-RISC functions as an enzymatic,
multiple turnover complex that recognizes and cleaves mRNA strands complementary to
the “antisense” or “guide” strand of the siRNA. The RISC catalytic site appears,
therefore, to be fixed relative to the 5-end of the siRNA. Cleavage is magnesium-ion
dependent and yields 3-hydroxyl and 5-phosphate groups on the resulting mRNA
degradation products.22,23
A number of non-specific effects are triggered by the siRNA. When a mammalian cell
encounters a double-stranded RNA such as a siRNA, it may mistake it as a viral byproduct and initiate an immune response. However, delivery strategies that target siRNA
to certain organs or cell types, but avoid the cell types responsible for immune
stimulation, may circumvent this problem.23
Recently, siRNA has been used to target the splicing factor SC35 in order to restore
correct splicing in human fibroblasts derived from a patient with pyruvate dehydrogenase
(PDH) complex deficiency, which causes mental retardation.29
v) Disruption of necessary RNA structure
RNA can adopt various three dimensional conformations as a result of intramolecular
hybridization. These structures play crucial roles in defining functions of the RNA
molecule e.g. additional stability for RNA, recognition motifs for a number of proteins,
nucleic acids and ribonucleoproteins that participate in the intermediary metabolism, and
activities of RNA species.23 All human immunodeficiency virus (HIV) mRNAs contain a
sequence known as TAR (trans-activating responsive sequence). In theory, molecules
that bind to the TAR RNA structure would inhibit trans-activation by perturbing the
native RNA secondary structure and lead to anti-HIV drug activity. This inspired Vickers
et al. to synthesize a series of phosphodiester and phosphorothioate antisense
oligonucleotides for this purpose.30 They observed that while shorter phosphodiester and
phosphorothioate oligonucleotides (ranging from 12-14 mers) did not bind to TAR RNA,
longer oligonucleotides (ranging from 18-28 mers) did and lead to disruption of RNA
function. Phosphorothioates were found to bind with 4 – 5 fold lower affinity in
comparison to the corresponding diesters.30
10
Introduction
1.3. The Antigene Strategy
Another viable target for the regulation of a specific gene is the gene itself. This
approach, known as the antigene strategy, has a number of potential benefits over the
antisense strategy including:
i) A cell generally contains only two copies of a gene whereas there are thousands of
copies of mRNA. Therefore, the smaller population of the target in the antigene approach
should enable the use of lower concentrations of the antigene oligonucleotide. This should
lead to an increase in efficacy and reduced side effects and other undesired effects.9
ii) Frequency of re-administration of the antigene oligonucleotide formulation should
be lower as compared to the antisense approach mainly due to prevention of repopulation of mRNA through transcription.9
iii) Historically, there have been two broad approaches described in the literature for the
development of ligands that bind to ds-DNA in a sequence selective manner so as to
inhibit transcription. These are: a) Minor Groove Binders and b) Triplex Forming
Oligonucleotides; they shall be discussed in more detail in sections 1.3.1. and 1.3.2.9 The
nucleotide bases in DNA have the potential to form hydrogen bonds with ligands bound
in either the major grooves or minor grooves (Figure 7) and it is this finding that has
been exploited in the development of these sequence specific, DNA-binding agents.
1.3.1. Minor Groove Binders
H2N
H2N
NH2
H2N
NH2
NH2
H2N
N
N
O
N
H
H
H
H
H
O
O
H
H2N
Figure 7
Neptropsin
N
O
N
H
N
H
N
O
H2N
NH2
NH2
Distamycin
monocationic lexitropsin
Examples of minor groove binding agents
11
N
O
N
N
N
N
O
H
O
N
N
H
N
N
N
H
N
H
H
O
N
N
O
N
N
O
H
N
H
N
N
O
H
O
H
N
NH2
Dicationic lexitropsin
Introduction
C19
H2N
NH2
CH2 CH
CO
OC NH
T
H
NH
T
CO
H
H
H
H
A
CO
HN
CO
H
A
N
P
A15
N3
O 4'
P
A14
N3
O 4'
H
H
T
N
NH
A
H
F
P
T16
O2
O 4'
N
T
NH
CO
P
T17
O2
O 4'
HN
A
N
H
P
G18
N3
O 4'
N
A
H
CO
O 4'
H
HN
N
H
P
FH
CH2
H2N
CH
P
NH2
P
O 4'
O 4'
N10
N
A4
O
O4'
N8
MeN
N3 A5
N6
O 4'
O 4'
O
P
P
P
P
T6
O2
MeN
N4
O
N
N1
N
T7
O2
G8
O 4'
O 4'
O 4'
C9
G12
Figure 8A
Figure 8
C3
C13
HN CO
T
G2
O 4'
O 4'
P
P
P
P
Figure 8B
a) 2:1 Distamycin-ds-DNA complex b) 1:1 Distamycin-ds-DNA complex
Minor groove binding agents are ligands that interact with the minor groove of double
stranded DNA (ds-DNA) and bind to residues lying in this groove. They have been
extensively explored in the antigene strategy.31 Classic examples of this class of DNAbinding compounds are the antiviral antitumor antibiotics, distamycin32 and netropsin33
(Figure 7). These are constructed from N-methylpyrrole amide (Py) subunits and they
selectively bind in the minor groove of AT rich DNA.34 An X-ray crystal structure of
netropsin bound to duplex DNA of sequence d(CGCGAATTCGCG) showed it to be
located at the AATT centre, and that it had displaced the sequence of ordered solvent
molecule in the “spine of hydration” from the minor groove. X-ray and 2D-NMR
performed on netropsin (1:1 drug:ds-DNA complex) or distamycin (1:1 or 2:1 drug:dsDNA [Figure 8]) nucleic acid complex have shown that specific bifurcated hydrogen
bonds form between the amide NH group of the drug, which points in towards the minor
groove and both N3of adenine and O2 of thymine located on opposite strands of two
adjacent base pairs.35,36,37 In addition to hydrogen bonding, close ligand-DNA van der
Waals contacts shape complementary and polyelectrolyte interactions (between the
polyanionic DNA and the cationic drug) further enhances the stability of these
complexes. These non-covalent interactions hold the pyrrole rings roughly in a parallel
orientation with the walls of the minor groove.31,34 The discovery by Wemmer et al. that
the natural product distamycin could form antiparallel 2:1 complex in the minor groove,
12
Introduction
and interpret the sequence of DNA from both strands of the duplex inspired Dervan et
al.’s design of the recognition code for their extended polypyrroles.
In an attempt to try to change the selective binding of such ligands Lown38 and Dickerson34
designed lexitropsins (Figure 7). It was proposed that replacement of one or more pyrrole
rings (Py) in netropsin by imidazole (Im) units should lead to preferential recognization of
GC-rich minor groove tracts.39,40 It was reasoned that the N3 imidazole nitrogen would be
able to form hydrogen bond to the 2-amino group of guanine. However, NMR studies of
the complex formed between a lexitropsin containing an imidazole at the carboxyl
terminus and the decamer d-[CGCAATTGCG] showed that it too bound specifically in the
minor groove of the AATT.31,41
C.G G.C A.T T.A
Py/Im
+
-
-
-
Im/Py
-
+
-
-
Py/Hp
-
-
+
-
Hp/Py
-
-
-
+
Hz/Py
-
-
-
+
Table 2
The code for minor groove recognition by polyamides. Note: + and - signs
denote favourable and unfavourable interactions respectively.
In the development of sequence-selective DNA-binding agents, it is essential to be able to
distinguish A.T base pairs from T.A, T.A base pairs from A.T, G.C base pairs from C.G and
C.G base pairs from G.C. Dervan and collaborators developed a set of base pair recognition
rules for DNA-polyamide interactions as shown in Table 2.31 Although a pair of stacked
methylpyrroles was not able to distinguish between A.T or T.A base pairs, the pairing of
pyrrole with hydroxypyrrole (Py/Hp) was able to distinguish between A.T or T.A base
pairs,.42,43 The preference for A.T over T.A was mainly due to the presence of hydroxyl
pyrrole, which caused steric destabilisation of the complex when bound next to adenine but
stabilisation, through hydrogen bonding, when located near thymine.44,45 Two paired
imidazoles (Im/Im) showed degeneracy in their recognition of either G.C or C.G base pairs.
However, a pyrrole paired with an imidazole (Py/Im) was able to distinguish C.G from G.C,
13
Introduction
while Im/Py selectively bound G.C.42 These findings gave access to the required four base
pair recognition code, which is essential if oligopyrroles are to become part of an effective
sequence selective antigene strategy.46 Dervan et al. have also reported another pairing,
involving 4-hydroxyl benzimidazole and pyrrole (Hz/Py), where Py was directly connected
to Hz without an intervening amide bond. The Hz/Py was equivalent to Hp/Py in
distinguishing T.A over A.T, G.C or C.G. 47,42
NH3
O
N
O
N
H
N
A
O
C
N
H
G
H
N
O
T
O
H
N
H
A
N
Py / Im
N
H
N
O
N
H
T
N
H
T
A
N
Py / Hp
O
Hp / Py
N
H
G
N
N
N
H
C
O
N
T
H
A
O
Im / Py
N
H
N
N
O
O
N
H
Figure 9
Pairing rules for oligopyrroles
Dervan and co-workers have also shown that the presence of a terminal acetyl group
alters the binding orientation of hairpin polyamides from the normal 5'-to-3' orientation
to 3'-to-5' and that this change reduces the affinity between polyamide and the DNA.31 A
possible reason for this kind of selectivity in orientation is the shift in stacking behaviour
of side-by-side dimeric polyamides which then has a direct consequence for sequence
recognition.31,42,48,49
The original oligopyrrole minor groove binding sites for the natural products, distamycin
and netropsin, are sequence selective for only 4-5 adjacent AT base pairs.34 When
Dervan et al. attempted to bind longer DNA targets, using extended analogues of
distamycin, a decrease in binding affinity was observed when more than four or five
14
Introduction
pyrrole-carboxyamide residues were linked together. This was attributed to the inherent
structural over-curvature of the longer oligopyrroles.51 This problem was overcome by
strategic replacement of specific pyrrole rings by more flexible β-alanine groups, which
allowed the compound to adapt its conformation for optimal binding.52 Thus, the eightring hairpin ImHpPyPy(R)H2N‫ץ‬-ImHpPyPy-β-Dp (where ‫( = ץ‬R)-2,4–diaminobutyric acid,
Dp = Dimethylaminopropylamide, β = β-alanine are linker) has been shown to target the
six base pair sequence 5-TGTACA-3 with high affinity and sequence specificity.42,48
For an effective antigene therapeutic in humans,50 it has been reported that statistically
ca. 15-17 base pairs need to be recognised, in order to achieve target specificity and
sequence selectvity. Dervan et al. have designed and synthesized ImPy-β-ImPy-β- ImPyβ-PyPy-β –Dp based on the polyamide ring pairing rules, the A.T specificity of β / β
pairs, and the “slipped” dimer motif. As speculated, this polyamide bound to the target
16-mer 5-ATAAGCAGCTGCTTTT-3 of 3-32P-end-labeled DNA as a co-operative
anti-parallel dimer. However, it also bound to mismatch sites on the restriction fragment
albeit with lower affinity. Thus, further developments are required to improve their
sequence specificity before these polyamides minor groove binders can be used as
antigene theraputics.
For a more comprehensive coverage of this field, see review by Dervan et al.42,48
1.3.2. Triplex Forming Oligonucleotides
The majority of the work undertaken in this area has focused on single-stranded
oligonucleotides which bind to double-stranded DNA in the major groove through
Hoogsteen hydrogen bonding.9,53 Hoogsteen hydrogen bonds are typically formed between
the nucleotide bases in the third oligonucleotide strand and purines in one of the WatsonCrick strands making up the DNA duplex. Triplexes exploiting (C+), T or G in the third
strand can form both either Hoogsteen (Figure 10b) or reverse Hoogsteen hydrogen bonds
(Figure 10b).50 In the latter case, the bases in the third strand are rotated by 180.
Triple helical DNA/RNA was first discovered by Felsenfeld, Davis and Rich in 1959.54
Despite the potential biological importance of this discovery, it was not until the late 1980s
that oligonucleotide-directed triple helix formation was reported as a means for gene
15
Introduction
sequence regulation, independently by Moser et al.,55 Doan et al.56 and Cooney et al.57 Each
of these triplexes were found to inhibit sequence specific DNA binding proteins.
Antiparallel motif
3'
5'
3'
Rd
N
O
N
N
Reverse Hoogsteen
Thymine
O
O
Cytosine
O
N
H
dR
H
H
H
O
N
N
H
N
O
N
5'
3'
5'
Pu
Pu
Py
H
N
N
Rd
N
N
N
H
N
N
dR
Watson-Crick
N
N
Rd
O
Thymine
Adenine
N
H
N
Guanine
H
dR
O
H
Cytosine
3rd Strand dsDNA
H
Rd
Parallel motif
N
5'
dR
O
N
Hoogsteen
Thymine
3'
5'
N
H
O
O
N
H
H
H
N
N
N
3'
Py
5'
3'
Pu
Py
H
H
H
Rd
Cytosine
N
N
H
O
O
H
N
N
N
N
Adenine
H
N
Rd
O
Thymine
N
N
dR
Watson-Crick
N
Guanine
H
N
N
N
H
H
O
dR
Cytosine
3rd Strand dsDNA
Figure 10A
Figure 10
Figure 10B
a) Parallel and anti-parallel motif triplexes and b) structure of Watson-
Crick and Hoogsteen hydrogen bonds
Based on binding orientation, triplexes can be divided into two types: parallel and antiparallel. In the parallel motif, the third strand is a homopyrimidine sequence, and it forms
Hoogsteen hydrogen bonds with the purine strand of the DNA duplex in a parallel
orientation (Figure 10a).58 In the anti-parallel-motif triplex, however, the third strand is a
homopurine sequence, which forms reverse-Hoogsteen hydrogen bonds with the
pyrimidine strand of the duplex in an anti-parallel orientation (Figure 10 b).59
Most research in the area of triple helix forming oligonucleotides has focussed on DNA
recognition via formation of T·AT and C+·GC triplets as they are isomorphous. 56 This
means that if their structural geometry were to be superimposed, they would have
identical C1-C1 sugar distances.50,56 If triplets are not isomorphous, then the resulting
triplexes are heteromorphous with backbone distortion occurring due to the variation in
16
Introduction
distance between the base and backbone.50 However, despite the difficulties in forming
heteromorphs, a few have been reported to date.56,60,61
One problem associated with the use of triple helix forming oligonucleotides for gene
regulation is that, in order for a cytosine in the third strand to form two hydrogen bonds
to a guanine in the DNA duplex, the cytosine must be protonated.50,56 This is a pHdependent process and the resulting positive charge produces an electrostatic repulsion
between adjacent cytosines that further reduces the potential target sequences open to
triplex formation.50,56 In an attempt to overcome this pH-dependent process and reduce
repulsion,
a
number
of
methylpseudocytidine62 and
pH-independent
unnatural
bases,
such
as
2'-O-
methyl-8-oxo-deoxyadenosine63 (MODA), have been
developed (Figure 11).
Another major drawback to the use of triple helix-forming oligonucleotides for the
regulation of gene expression is that triplex forming oligonucleotides generally target
oligopurine sequences only in the ds-DNA.56 Such a limitation arises owing to purines
having two vacant ‘Hoogsteen’ hydrogen bonding sites in the major groove of ds-DNA
whereas pyrimidines possess only one. Consequently, pyrimidines cannot be bound as
efficiently as purines.50
NHMe
NH2
N
H
N
NH
N
O
HO
O
O
HO
HO
2'-O-Methylpseudocytidine
1.3.3.
N
O
OMe
Figure 11
N
HO
Methyl-8-oxo-deoxyadenosine
Unnatural modified bases for the triplex strategy.
Melting temperature (Tm)
Melting temperature, Tm, of nucleic acid duplexes can be used to predict the stability and
energetics of their hybridization. Melting temperature is defined as the temperature at which
equal amounts of base-paired duplex and unpaired single strands are in equilibrium when
standard free energy is zero. It depends on both the length of the molecule and the specific
17
Introduction
nucleotide sequence of the nucleic acid strand.20,64
Thermal studies of nucleic acids typically use either spectroscopic methods, such as
monitoring UV absorption at 260 nm, or calorimetric melting point measurements. A
temperature increase causes a disruption of the hydrogen bonds between the base-pairs
and diminishes the distance between adjacently stacked nucleobases, leading to departure
from characteristic secondary and tertiary structures. Such phenomenon, observed by UV
absorption at 260 nm, is termed ‘hyperchromicity’ and the magnitude of hyperchromicity
is taken as a measure of how much secondary structure is present in nucleic acid. The
plot of the UV absorbance at 260 nm vs temperature is usually sigmoidal. A nonsigmoidal (e.g., linear) transition with low hyperchromicity is a consequence of no
duplex formation (non-complementation). Generally, the transition is broad and so the
exact Tm is better obtained from first derivative plot.20
The binding stoichiometry of nucleic acids is determinable from UV-mixing or UVtitration experiments. UV-mixing experiments are carried out by mixing the appropriate
oligomers in different molar ratios, with the total concentration remaining constant. The
UV-absorbance plotted as a function of the mole fraction for one component is known as
a ‘Job’s plot’. In such plots, the absorbance steadily decreases until all the strands present
are involved in complex formation, and then it rises as one strand is present in excess.
The stoichiometry of complex formation is derived from the minimum in such a plot.20
1.4. Oligonucleotide Analogues
Over the last few years, many modifications to the structure of natural oligonucleotides
have been investigated in an attempt to improve their cellular uptake, nuclease stability,
DNA binding affinity, and/or sequence selectivity. The modifications undertaken have
been aimed principally at i) the backbone, ii) the sugar and iii) the heterocyclic base of the
nucleic acid monomer [covered under triplex forming oligonucleotide (TFO), Section
1.3.2.]. Additionally, many diverse moieties have been conjugated at various positions
within the oligomer subunits, usually in an attempt to alter their pharmacokinetic
properties.
As this is an extensive area of research, only the most promising oligonucleotide
18
Introduction
modifications reported to date will be discussed in the following sections. For more
comprehensive coverage of this field, see a review by Crookes et al.22,23
1.4.1. Furanose-Containing Phosphate Backbones.
1.4.1.1. Phosphorothioate (PS)
Phosphorothioate (PS) containing oligonucleotides represent, by far, the most widely
studied and used antisense drugs. The first FDA approved antisense drug vitraveneTM
(ISIS-2922),63 which is used to treat cytomegalovirus (CMV), is a PS derivative.
Introduction of a sulphur atom into the phosphate backbone generates a chiral centre
yielding the diastereoisomers Sp and Rp, as shown in Figure 12.
O
O
Base
O
O
P
O
O
(Sp)
S
O
O
S
O
Base
O
P (Rp)
O
O
Base
OH
OH
Phosphothiorate (Sp) diastereoisomer
Figure 12
Base
Phosphothiorate (Rp) diastereoisomer
Sp and Rp diastereoisomers of phosphorothioate oligonucleotides.
The PS backbone (Figure 12) is the only modification that has been shown to retain the
RNase H activity of its natural oligodeoxyribonucleotide counterparts. Although PS
analogues show reduced duplex stability with RNA (ΔTm = -0.8 C per modification),63
they exhibit increased stability towards nucleases in comparison to their phosphodiester
analogues; their plasma half-lives are 30-60 min rather than 1-2 min.66 Unfortunately,
despite finding that the SP isomer is substantially more resistant to nuclease enzymes than
the corresponding RP-enantiomer, thus far it has proved difficult to achieve a stereocontrolled synthesis of PS oligomers using cost-competitive methods appropriate for the
manufacturing industry. Consequently, PS oligomers are typically prepared and marketed
as diastereomeric mixtures.67,68 When phosphorothioate oligonucleotide (PS-ODN) were
investigated as antigene agent, homothymine PS-ODN or homoadenine PS-ODN
containing more than 20 mer on duplex formation with corresponding ss-DNA gave
19
Introduction
larger decrease in thermal stabilities in comparison to unmodified ds-DNA
i.e.
poly(dA)-poly(dT) = 50 C; poly(dsA)-poly(dT) = 44 C; poly(dA)-poly(dsT) = 33 C;
and poly(dsA)-poly(dsT) = 26 C. These results suggest that phosphorothioates were
unsuccessful in antigene strategies, since they led to unstable anti-parallel triplexes.69
A limitation of PS oligonucleotides is that they can be toxic due to non-specific binding
to enzymes and other proteins.70 This can be detrimental due to immunological responses
and causes severe hypotension. These non-specific interactions complicate elucidation of
the exact mechanism of action of PS oligomers.70 A further problem of PS
oligonucleotides is that, although they are more stable to nuclease than their natural
counterparts, they are gradually cleaved over time.71 The resulting shorter strands, which
can be as short as five or six bases, have been found to retain their ability to bind to
mRNA and to induce RNase H activity. However, these short strands obviously lack
specificity for their targets.71
1.4.1.2. N3→P5 Phosphoramidates and Thiophosphoroamidates
O
O
Base
O
O
HN
HN
O
P
Base
S
O
P
O
O
O
O
Base
Base
OH
OH
N3'  P5' Phosphoramidate
Figure 13
O
N3'  P5' 'Thiophosphoramidate
Phosphoramidate analogues
In phosphoramidate oligodeoxynucleotides, the 3-oxygen of the natural deoxyribose
sugar has been replaced by a 3-amino group and the nucleosides are joined together via
phosphoramidate monoester linkages (Figure 13). These analogues were first reported by
Gryaznov et al. in 1994.72,73
As shown in Table 3, N3→P5' phosphoramidates ODN exhibit a preference for binding to
RNA over DNA. Similar to the 2’-hydroxyl group in RNA, 3-amino groups in N3→P5'
20
Introduction
phosphoramidates increased solvation due to the hydrogen bonding properties, which in
turn stabilizes a string of water molecules that runs across the minor groove. This has been
considered to explain an increased thermodynamic stability of N3→P5' phosphoramidates
bound with high affinity to complementary RNA oligomers.
No
Target
Tm (C)
1
TTTTTTTTTT
Poly(dA)
29.7
2
TTTTTTTTTT
Poly(rA)
27.0
3
tNPTtNPTtNPTtNPTtNPT
Poly(dA)
25.8
4
tNPTtNPTtNPTtNPTtNPT
Poly(rA)
33.7
5
tNPtNPtNPtNPtNPtNPtNPtNPtNPtNP
Poly(dA)
36.0
6
tNPtNPtNPtNPtNPtNPtNPtNPtNPtNP
Poly(rA)
51.5
Table 3
tNP indicates thymine monomer of N3→P5' phosphoramidates, Tm = melting
temperature of resulting duplexes or triplexes.
Although these oligomers do not activate RNase H, they have shown encouraging in-vivo
results implying that they have potential antisense therapeutic applications. For example,
a phosphoramidate modified antisense oligonucleotide has been specifically used to
down-regulate the expression of the c-myc gene.78,79 Moreover, in a leukaemia model, the
phosphoramidates were found to outperform PS oligodeoxynucleotides for steric
blocking of translation initiation.78,79
The stability of this modification has been found to have a few drawbacks. For example,
N3→P5' phosphoramidates have been reported to show some affinity for intra- and
extracellular proteins and they have decreased acid stability relative to their phosphodiester
counterparts.74,75,76 To address stability in acidic conditions, Pongracz et al. have
synthesized and investigated N3→P5 thiophosphoramidate.74,77 These analogues were
found to be more acid stable when incorporated into oligonucleotides, and the introduction
of sulphur did not appear to alter the RNA-binding properties of these compounds.
1.4.1.3. Cationic aminoalkyl phosphoramidate
Electrostatic interactions have been exploited in order to increase the binding affinities of
many oligonucleotide analogues with both DNA and RNA. One such strategy reported
21
Introduction
O
Base
O
O
O
NH
N
H
P
O
Base
O
O
O
P
O
O
O
Base
O
O
NH
N
H
P
O
Base
O
O
Figure 14
Cationic phosphoramidate d[(t+T-)xT]
Poly (dA)
d(C2A15C2)
Poly (rA)
Tm value at different pH
Oligomers
No
0.1 M 1.0M No
Salt
Salt
0.1M 1.0M
No
0.1M 1.0M
salt
dT15
22
40
58
21.5
40
53
21
39
52
d(t+T-)7T (1) fast
58
58
58
52
52
52
37
39
41
d(t+T-)7T (2) slow
20
21
18
19
19
26
18
19
20
d(t+T-)7T (3) mixed
40
40
44
38.5
39
40
27
27
29
Table 4 Binding affinity of cationic phosphoramidate ODN with target DNA or RNA, t+
= thymine monomer of cationic phosphoramidate, T- = thymine monomer of
phosphodiester, Tm = melting temperature of resulting complexes at different pH.
concerns homothymidine oligomers containing alternating anionic phosphodiester and
stereo-uniform cationic phosphoramidate backbones e.g. d[(t+T-)7T] (Figure 14).79,80 The
stereochemistry of the two separated monomers of cationic phosphoramidate were not
determined but were distinguished on the basis of their separation during purification on
silica gel during chromatography. Oligomer 1 d[(t+T-)7T] is termed the "fast" isomer
22
Introduction
because it elutes before oligomer 2 d[(t+T-)7T], which is termed the "slow" isomer, while,
oligomers 3 is the mixture of oligomer 1 and oligomer 2, and termed as "mixed" isomer.
The nucleic acid hydridization properties of oligomers 1, 2 and 3 were examined using
two ss-DNA targets [poly(dA) and d(C2A15C2)] and an RNA target [poly(rA)] as shown
in Table 4. The key finding of this study was the high affinity binding exhibited by
oligomer 1 to both ss-DNA targets at all ionic strengths. Of the three chimeras
investigated, oligomer 1 was found to form the most stable complex, with poly(rA).81
The results also showed that both oligomers 1 and 3 had a stronger preference for
binding to DNA targets than to RNA. The stability of complexes formed by oligomer 1Poly (dA) in comparison to oligomer 2-Poly (dA) clearly indicates hybridization is highly
dependent on chirality at the phosphoramidate centres (Sp or Rp).
1.4.2. Furanose-Containing Nonphosphate Backbones.
1.4.2.1. Amides
O
O
Base
O
O
NH
HN
O
Base
O
O
Base
OH
OH
trans - Amide
Figure 15
Base
O
cis - Amide
trans- and cis-amide oligonucleotide
Oligonucleotides, in which the anionic phosphodiester backbone has been replaced by a
neutral amide linkage, have been synthesized with the intention to increase binding affinity.82
It was speculated that removal of the electrostatic forces of repulsion, which exist
between phosphodiester groups in natural duplexes, should both enhance the affinity of
the resulting oligomers and increase their permeability through cell membranes.
De Mesmaeker et al.82 have reported the synthesis of both trans and cis amide thymine
dimers (Figure 15). The thermal stability for the duplex formed between TTTtt(CT)5
(where t is the amide modified monomer) and complementary RNA was found to be
23
Introduction
slightly more stable (Tm/mod = +0.4 C) for the oligomer bearing the trans linkage and
slightly less stable (Tm/mod = -0.8 C) for the one containing the cis compared to the
complexes formed with the unmodified counterpart.69 The affinity for both oligomers to
bind to complementary DNA was found to be somewhat lower (Tm/mod = -1.7 C) than
the corresponding unmodified oligonucleotide. It has been argued that restricted rotation
about the amide bond pre-organizes this backbone into a conformation that gives the
oligomer a more favourable A-like geometry, and consequently such oligomers favour
hybridization to RNA.83
In a separate study, a 15 mer of sequence (TTTttCTCTCTCTCT) (where t indicates
amide monomer) has been investigated. The two amide monomers were incorporated
into DNA ODN and were found to bind to complementary RNA with small rise in
thermal stabilities of the resulting heteroduplex in comparison to the unmodified RNADNA duplex (Δ Tm = +0.4 °C per modification).
1.4.2.2. DNG
5'
5'
O
O
O P O
O P O
O
O
Base
O
O
O
Base
NH
O P O
H2 N
HN
O
O
Base
O
O
O
O P O
O P O
O
O
3'
DNA
Figure 16
Base
3'
DNA/DNG
DNA/DNG chimeras
Bruice et al.84 have reported the development of oligonucleotide analogues containing
cationic guanidinium linkages (DNG) (Figure 16). It was envisaged that the positive
charges along the backbone of DNG may induce greater cell membrane permeability
through electrostatic interactions between the linker and the negatively charged
24
Introduction
phosphate groups on the cell surface. This characteristic is thought to be important if any
oligononucleotide analogue is to be used as a therapeutic; poor cellular uptake of
oligomers is correctly the major drawback of both antisense and antigene strategies.
A study of duplex formation of ODNs bearing DNG/DNA chimeric ODNs was carried
out. ODN 1-3 are 18-mers with either three (where t indicates thymine monomer of
DNG, placed at the 5 end, 3 end, and centre), two (at the 5 and 3 ends), or one (at the
centre) guanidium linkages.85
ODN 1
5-d(tTGTTAGTtTTCTTGtTT)-3
ODN 2
5-d(tTGTTAGTTTTCTTGtTT)-3
ODN 3
d(TTGTTAGTtTTCTTGTTT)-3
DNA 4
(5-d(TTGTTAGTTTTCTTGTTT)-3)
DNA 5
5-d(AAACAAGAAAACTAACAA)-3
DNA 6
(5-d(AAACAAGATAACTAACAA)-3
RNA 7
5-r(AAACAAGAAAACUAACAA)-3
RNA 8
(5-r(AAACAAGAUAACUAACAA)-3
Duplex
Duplex
Tm (C)
Tm (C)
No
10
100
No
10
100
Salt
mM
mM
Salt
mM
mM
DNA 4:DNA 5
34.8
48.6
58.5
DNA4:DNA 6
-
39.5
-
DNA 4:RNA 7
34.0
49.7
59.9
DNA4:RNA 8
-
45.5
-
ODN 1:DNA 5
34.8
46.6
53.5
ODN 1:DNA 6
-
35.5
-
ODN 2:DNA 5
36.8
48.6
57.5
ODN 2:DNA 6
-
38.5
-
ODN 3:DNA 5
34.8
47.6
56.5
ODN 3:DNA 6
-
37.6
-
ODN 1:RNA 7
30.1
41.8
50.9
ODN 1:RNA 8
-
30.5
-
ODN 2:RNA 7
34.2
45.9
57.1
ODN 2:RNA 8
-
35.5
-
ODN 3:RNA 7
31.2
43.9
54.0
ODN 3:RNA 8
-
34.6
-
Table 5
Duplex melting temperature of DNG/DNA chimera with complementary
DNA or RNA at various ionic concentrations compared with unmodified duplexes of
DNA:DNA and DNA:RNA, and mismatch
25
Introduction
The single guanidinium linkage in ODN 3 and the three guanidinium linkages in ODN 1
appeared to have no effect on hybridization properties with complementary DNA 5, in
the absence of salt. However, as the salt concentration increased, the stabilities of
duplexes ODN 1:DNA 5 and ODN 3:DNA 5 were found to decrease, when compared the
unmodified duplex DNA 4:DNA 5. The heteroduplexes of ODN 1:RNA 7 and ODN
3:RNA 7 were destabilized at all salt concentrations compared to the corresponding
duplex DNA 4:RNA 7.85
To study the sequence specificity of DNG containing oligomers, ODN 1, ODN 2 or
ODN 3 were hybridized to either DNA 6 or RNA 8 (Table 6), both of which contained
one base mismatch in the centre. The resulting duplexes between DNA 4:DNA 6 (Δ Tm +
9.1 C) and DNA 4:RNA 8 (Δ Tm + 4.2 C) exhibit decrease in Tm in comparison to fully
complementary DNA 4:DNA 5 and DNA 4:RNA 7 duplexes. Approximately the same
mismatch discrimination was observed for ODN 1:DNA 6, ODN 2:DNA 6 and ODN
3:DNA 6 complexes (Δ Tm -10 to -11 C) in comparison to ODN 1:DNA 5, ODN 2:DNA
5 and ODN 3:DNA 5 respectively. Similarly, ODN 1:RNA 8, ODN 2:RNA 8 and ODN
3:RNA 8 complexes showed a mismatch discrimination, which was more than double
that of DNA 4:RNA 8 i.e. Δ Tm + 9.3 to 11.3 C. Thus it was demonstrated that the
binding of DNG/DNA chimera with complementary DNA and RNA is sequence
specific.85
To investigate their stability to nucleolytic digestion DNG/DNA ODNs 1–3 were exposed to
exonuclease and the resulting mixture was periodically carried out on RP-HPLC. The control
DNA 4 was found to be completely hydrolysed after 1 h of incubation. However, DNG/DNA
chimeras 1 and 2 were found to be completely stable towards exonuclease 1 digestion, even
after 12 h of incubation. The DNG/DNA chimera 3, which contains only one guanidinium
linkage at the centre of the ODN, was found to be partially hydrolyzed after 1 h.85
There are numerous other oligonucleotide backbone modifications that have been
reported in the literature to date including silyl,86 sulphone86, sulphoxide86, urea86,
carbamate86 and sulphide phosphodiester86 surrogates.23 These have been not discussed
here due to space restrictions. For a more thorough coverage of this whole field and
information about these analogues see a review by Micklefield et al.86
26
Introduction
1.4.3.
Sugar and Backbone Replacements
1.4.3.1. Morpholino Phosphorodiamidate
Phosphorodiamidate morpholino oligonucleotides (PMO) are a class of analogues in which
both the ribose sugar and phosphodiester linkage of natural nucleic acids have been replaced
by a morpholine ring and phosphorodiamidate unit, respectively (Figure 17).88
Base
O
N
N
P
O
O
Base
O
N
Figure 17
O
Morpholino oligonucleotide
Correlation between stacking and solubility has been demonstrated by Kang et al.87
Phosphorodiamidate morpholino oligonucleotide (PMO) shows excellent solubility in
aqueous solution (> 100 mg/ml).89 It is postulated that aqueous solubility of PMOs is
presumably due to the flexibility of the phosphorodiamidate linkages, which presumably
hides the hydrophobic faces of the bases from the aqueous environment. They exhibit
good binding affinities with target RNAs (Tm = 81.3 C for a 20-mer) when compared to
the identical sequence of phosphorothioate DNA (PS-DNA)-RNA duplex (Tm = 68.5 C)
and DNA-RNA duplex (Tm = 77.3 C).89 Owing to their high solubility in water,
excellent base stacking and good binding affinities towards target RNAs they have been
found to be effective antisense oligonucleotides.89 However, PMOs do not activate
RNase H, and so they are primarily used for translation arrest or other steric blocking
mechanisms, such as alteration of splicing.90 Potential efficacy of a neutrally charged cmyc antisense approach for the prevention of restenosis has been investigated in phase 3
clinical trials to evaluate PMOs safety and efficacy of local delivery to reduce restenosis
after coronary stenting.90,91,92,93
27
Introduction
1.4.3.1. Sugar Modifications
1.4.4.1. Arabinonucleic acid and 2’-Fluoro Modifications
O
O
O
O-
P
O
Base
O
O-
P
Base
O
O F
O
O
P
O OH
O
H
O-
O
Base
O
O
O
Figure 18
P
H
OBase
O
F
H
O OH
O
H
2’-FANA and ANA oligonucleotide
Arabinonucleic acid (ANA) is the 2’-epimer of RNA. ANA oligomer with sequence 5(AGC UCC CAG GCU CAG AUC)-3 forms more stable duplexes with complementary
RNA (Tm = 44.0 C) than DNA (Tm = 26.0 C). ANA:RNA hybrids had a lower value of
Tm compared to the corresponding DNA:RNA (Tm = 72.3 C) and RNA:RNA (Tm = 84.6
C) duplexes. From CD spectra studies, it has been suggested that ANA:RNA hybrid shares
the same A-like helical conformation as DNA:RNA hybrid. Furthemore from molecular
modelling studies, it has been proposed that the inversion of the stereochemistry of the 2’OH group, ongoing from RNA to ANA,94 causes a conformational change in the ribose
sugar from C3’-endo (northern conformation) to O4’-endo pucker (eastern conformation).
This change in the sugar pucker is thought to be a key determinant in the activation of the
enzyme RNase H by ANA. This prompted the researchers to investigate their RNase H
activity. The lack of complete cleavage of ANA:RNA by RNASE H suggest the inability
of RNase H to bind optimally to the minor groove of ANA:RNA hybrids or the low
thermal stability of this particular complex led to discovery of newer analogue like 2’deoxy-2’-fluoro-D-arabinonucleic acid (2’F-ANA).
Damha et al. have synthesized 2’F-ANA, Figure 18 in which the 2’-OH group of ANA
was replaced by a 2’-fluoro group,98 which is more electronegative than hydroxyl group,
thus could offer more hydration in the grooves.98 The replacement by 2’-fluoro group
28
Introduction
also facilitated in reducing the steric hindrance offered by the 2’-OH group in ANA.95
Structural studies have shown that 2’F-ANA preorganizes itself more towards O4’-endo
pucker or eastern conformation, which lies halfway between C2’-endo (South B-DNA)
and C3’-endo (North A-RNA form), due to this ability 2’F-ANA preorganize itself with
both DNA and RNA to form stable duplex.96,97
The selectivity of 2’F-ANA for hybridization to complementary RNA over ss-DNA has
been found to depend on the percentage content of purine and pyrimidine in the target
sequence e.g. when the purine content was low 2’F-ANA preferred to bind RNA over ssDNA. Furthermore, as the purine content of the 2’F-ANA strand increased from 0 to
60% so the Tm values of the resulting 2’F-ANA:RNA (Δ Tm + 12 to 34 C) and 2’FANA:DNA (Δ Tm – 1 to +19 C) duplexes increased.98
In an attempt to improve the pharmacokinetic properties and metabolic stabilities of
siRNAs, Damha et al. have prepared and evaluated 2’F-ANA-containing siRNAs.99,100
These researchers observed that two 21-mer siFANA oligomers, both of which were
compiled of a fully modified sense strand, had half lives of between 5 and 6 h, in foetal
bovine and human plasma compared to a half-life of 15 min for the analogous unmodified
siRNA. Remarkably, one of these siFANA oligomers showed only minimal loss of activity
when compared with the analogous unmodified siRNA. In fact, the resulting siFANA
(>50% FANA) duplex was more active than the native siRNA duplex.99,100
1.4.4.2. 2’-O-Methoxyethyl (MOE)
The 2’-O-methoxyethyl (MOE, Figure 19) oligonucleotide analogue is currently
clinically the most advanced 2’-sugar modified derivative in this series and MOE
oligomers have entered clinical trials for multiple indications.23 MOE oligonucleotides
exhibit several unique structural features that help to explain their properties.101 The
MOE substituent at the 2’-position of the ribose induces a C3’-endo (Northern)
conformation of the sugar, while the methoxyethyl moiety promotes a gauche orientation
by hydration with water molecules between oxygen acceptors of MOE. This geometry is
thought to largely account for the increased affinity of MOE oligomers that forms duplex
with RNA that are 2 C more stable on average per modification than the corresponding
PS-DNA:RNA hybrids.101 In addition, this conformation enables water be trapped to
29
Introduction
form a shell of hydration, and this could play a role in the improved cellular uptake
exhibited by MOE modification, Furthermore, this specific hydration pattern that bridges
substituent and phosphate oxygen atoms in the minor groove of MOE modification may
explain its high nuclease resistance.101,102
O
O
O P
O P O-
S-
O
O
Base
Base
O
O
O
O
O
O
-
O P O
O P
O
Base
O
O
-
S
O
O
Base
O
O
O
O
O
A) 2'-O-Methoxyethyl (MOE)
Figure 19
O
O
B) 2'-O-Methoxyethyl (MOE)
2’-O-Methoxyethyl Modification with PO (A) and PS (B) backbones
1.4.4.3. LNA
Oligonucleotides bearing locked sugar conformations have been investigated utilising
bicyclic sugars. This area of study was initiated with the development of 2,4-LNA (Figure
20), independently by Wengel et al.103 and Imanshi et al.104 in 1998. The LNA sugar is a
bicyclic ribose derivative in which the ribose C4’ and O2’ positions are covalently joined
together through a bridging methylene group. This bridge confers the sugar with rigidity;
the 2’,4’-LNA sugar moiety exhibits a preference for the C3’-endo pucker (Northern
conformation) as shown in Figure 20. From a thermodynamic point of view, locking
nucleoside units in this way afforded two benefits: (1) a reduction in the inherent entropy
loss during nucleic acid hybridization; and (2) the oligomer was preorganised into a highaffinity conformation for binding.104 Following hybridization with complementary target
nucleic acid sequences, the resulting LNA:RNA duplexes were found to adopt A-type
conformations.105 Furthermore, upon hybridization to ss-DNA, LNA oligomers promoted
B-type DNA to also adopt a A-type duplex structure.105
30
Introduction
When 3 residues of LNA (Table 6, entry 2) were incorporated in RNA oligonucleotide
respectively, the resulting duplexes of sequence 3:RNA 3 (∆ Tm/mod = + 7 C) were more stable
those of the other sequences. This suggested the preference of LNA for RNA duplex over
DNA duplex, which could be explained on the basis of conformation studies as explained
above.106
O
Base
O
O
O P OO
Base
O
O
O
O P OO
Base
Base
O
O
O
O
O
O P OO
O
DNA / LNA Chimera
Figure 20
P
O
O-
LNA
2’-4’-Locked nucleic acid (2’-4’-LNA) and DNA:2’-4’-LNA chimera
DNA
RNA
Tm C
Tm C
5-d(GTGATATGC)
28
28
LNA:DNA chimera
5-d(G tL GA tL A tL GC)
44
50
3
LNA:RNA chimera
5-r(G tL GA tL A tL GC)
55
63
4
Fully modified LNA
5-d(gL tL gLaL tL aL tL gLcL)
64
74
Sr
Code
Sequence
1
Reference DNA
2
Table 6 Comparison of Tm resulting complexes of RNA or DNA with ODN sequence 1
to 4. Bold and L indicates LNA backbone.
It has been reported that the LNA-DNA chimeras containing eight DNA monomers can
induce RNase H activity, e.g. the 18-mer LNA:DNA chimera of sequence 5d(cAtGtCATGACGGttAGg) (where lowercase letters indicate LNA monomers and
uppercase indicate DNA subunits) was shown to induce cleavage of 90% of the target
31
Introduction
RNA by activation of RNase H.107 However, LNA antisense oligonucleotides have the
potential to improve antisense potency, their use in vivo poses a significant risk of
hepatotoxicity.107
1.4.4.3.1 α- L-LNA
O O
Base
O
O
O
O
O O
O
O
Base
-L-LNA (S-type)
-L-LNA (S-type)
O
Base
O
O
O
Base
O
O
-D-LNA (N-type)
-D-LNA (N-type)
O
O
Base
O
O
O
O
O
-L-Xylo-LNA (S-type)
O
O
Base
O
O
O
-D-LNA (N-type)
O
Base
O
O
O
Base
-D-Xylo-LNA (N-type)
-L-LNA (S-type)
α-L-LNA (αLtL), β-D-LNA (LNA) (tL), α-LNA (α-D-LNA or α-D-ribo),
Figure 21
and β-L-LNA (L-LNA), α-L-Xylo-LNA (αLxtL), β-D-Xylo-LNA (XtL), β-L-Xylo-LNA, αD-Xylo-LNA
Sr
Sequence
r(A)14
5-r(A6CA7)
ent-rA14
ent-5-r(A6CA7)
Tm C
1
5-dT14
18
no Tm
no Tm
no Tm
2
5-(tL)9T
71
61
52
51
2
5-( α LtL)9T
66
49
40
no Tm
3
5-(xtL)9T
57
47
39
36
4
5-( α LxtL)9T
no Tm
no Tm
no Tm
no Tm
Table 7 Binding studies of homothymine diastereoisomeric LNAs towards RNA (rA14),
singly mismatched RNA 5-r(A6CA7), enantiomeric RNA (ent-rA14), and singly
mismatched enantiomeric RNA ent-5-r(A6CA7), as shown by melting temperature Tm
values (C).
After the discovery of LNA, the Wengel group108 investigated the duplex stability of the
32
Introduction
four diastereoisomers isomers of LNA [i.e. LNA (tL) (β-D-ribo), (α-L-LNA) (αLtL) (α-Lribo), xylo-LNA (XtL) (β-D-xylo) and α-L-xylo-LNA (αLxtL) (α-L-xylo)] bound to
complementary with RNA (Table 11). These four diastereoisomers i.e. L-LNA (β-L-ribo),
(α-LNA) (α-D-ribo), L-xylo-LNA (β-L-xylo) and α-xylo-LNA (α-D-xylo) were also
evaluated, indirectly by hybridization studies of the first mentioned stereoisomers (TL,
αL L
t , XtL and
αL
xtL) towards enantiomeric RNA targets (ent-RNA also known as L-RNA
or mirror-image of RNA).108
The formation of very stable complexes with ent-RNA was detected for ODNs composed
of entirely tL (∆ Tm/mod = +5.8 C), αLtL (∆ Tm/mod = +4.4 C) or XtL monomers (∆ Tm/mod =
+4.3 C), while no complex was formed with
αL
xtL ODN. It has been speculated that the
unnatural configuration might have prevented a suitable positioning of the thymine base
for hybridization in case of
αL
xtL ODN. Similarly, very stable complexes were observed
with RNA, when ODNs composed entirely of tL (∆ Tm/mod = +5.9 C),
αL L
t (∆ Tm/mod =
+5.3 C) or XtL (∆ Tm/mod = +4.3 C) monomers were used; again no complex was formed
between RNA and
αL
xtL ODN and the probable reason for this is similar as for ent-RNA
and αLxTL ODN.108
1.4.4.4. ENA
O
Base
O
O
Base
O
O
O
O P O-
Figure 22
O
O P
O
O-
2’-O,4’-C-Ethylene nucleic acids (ENA)
Imanshi et al. have reported 2’-O,4’-C-ethylene bridged nucleic acids (ENA)109 (Figure
22), which have conformational similarity with of LNA nucleosides. ENA has sixmembered rings with ribo-type sugar substituted with nucleobases.109 Due to the 2’-O,
4’-C-ethylene bridge, ENAs are fixed in the N-conformation like LNA.109 The intention
of increasing the methylene bridge in LNA with one more carbon in the linkage in ENA
was that it could form more rigid six-membered ring than the flexible five-membered
33
Introduction
ring, which might lead into better hybridization properties.
The heteroduplex formed between ENA ODN 5-d(GCGxxxxxxGCT)-3 (where x =
thymine monomer of ENA) and complementary RNA was found to have a higher
thermal stability (Tm = 75 C) than the corresponding DNA:RNA duplex (Tm = 43 C ).
The thermal stability of this heteroduplex was comparable to that of the heteroduplex
formed from the 2’,4’-LNA ODN of the same sequence and complementary RNA (Tm =
77 C).110
The 3-exonuclease snake venom phosphodiesterase has been used to investigate the
stability of oligonucleotides containing ENA monomers.110 The stability of the
oligonucleotide 5-dT10xT-3 (where X= thymidine ENA monomer) was found to be
identical to that of the analogous oligonucleotide bearing a single Rp -PS nucleotide i.e.
both were degraded in 30 min. When the oligonucleotide (5-T10xx-3:x=2’-O, 4’-C-ethylene thymidine) containing two ENA residues was evaluated, 10% of the oligomer
remained after 90 min. This oligonucleotide was shown to be more stable than the
analogous oligonucleotide containing a Sp-PS dinucleotide unit, which had degraded
completely within 90 min.110,111,112,113 When similar analogue of LNA was examined,
ENA was found to be 55 times more stable than LNA.
ENA in comparison to LNA has equivalent affinity for RNA and higher nucleaseresistance. Thus, it shows encouraging properties as an antisense agent.
1.4.4. Sugar, backbone and nucleobase replacements.
A range of oligonucleotide analogues bearing modified nucleobases, together with sugar
or backbone replacements, have been employed to address issues in triple helix
formation in order to improve recognition of mixed purine/pyrimidine sequences, pH
dependence and affinity.114
Keith et al.114 have reported an interesting strategy to overcome base pair recognition in
mixed sequence duplex target that contained four pyrimidine inversions at physiological
pH. They merged a phosphoramidite monomer with an existing nucleobase i.e. BAU [2’aminoethoxy-5-(3-aminoprop-1-ynyl)uridine] recognizes AT base pairs from T.A rich
34
Introduction
regions with high affinity. C-Nucleoside monomer was merged with other nucleobases
like
Me
P (3-methyl-2 aminopyridine), which selectively binds GC base pairs from C.G
rich regions with high affinity at higher pH than cytosine, APP (6-(3-aminopropyl)-7methyl-3H-pyrrolo[2,3-d]pyrimidin-2(7H)-one), which binds CG base pairs from G.C
rich regions and S [N-(4-(3-acetamidophenyl)-thiazol-2-yl-acetamide)] which recognizes
TA base pairs from A.T, rich regions (Figure 23). Subsequently, these four novel
nucleobases were incorporated into the oligomer TFO-1. Binding studies revealed that
this oligomer formed a stable triplex with ds-DNA 1 (D-1) at physiological pH, while the
analogous unmodified oligomer TFO-2 did not (Table 12). The inability to form stable
duplex between ds-DNA:TFO-2 was due to lack of recognition of pyrimidine residues,
as C and T have only one H-bond donor or acceptor site available for binding in the
major groove; also the recognition of T of a TA base pair is hampered by steric clash of
the 5-methyl group. The other reason could be the formation of the C+.GC triplet
requires low pH conditions (<6.0), necessary for protonation of the third strand cytosine.
BAU. .AT
Me
A
PP.CG
P.GC
OR
OR
OR
HO
NH3
HO
O
H3N
N
N
O
N
R
N
H
H
H
N
R
N
N
O
H
N
N
R
N
H
H
H
O
N
Me
N
O
H
O
N
O
N
R
A
H
N
Me
H
N
S
H
H
N
H
N
N
Me
H
Me
N
Figure 23
O
O
N
O
H
N
N
H
N
O
O
H
H
H
HO
N
N
N
OR
O
CH3
N
O
NH3
HO
O
O
S.TA
N
N
N
R
N
N
O
N
H
N
N
N
N
N
R
R
H
H
H
N
R
D
C
B
Chemical structures of the four nucleobases, which may selectively
recognise AT, GC, CG, and TA base pairs
The sequence specificity of triplex formation of TFO-1 was scrutinized by 12 duplexes,
each of which differed from duplex 1 (D-1) by a single base pair opposite to one of the
modified nucleotide (D-2 to D-5) to assess selective recognition of BAU.AT,
Each duplex (D-2 to D-5) formed triplex (X.YZ), where X is BAU,
YZ is each base pair (AT, GC, CG or TA in D-2 to D-5).
35
Me
Me
P.GC,.
P, APP or s, while
O
Introduction
Code
Sequence
X
BAU
TFO-1 (5-Q-bmbpbsbTmTmpTsmTmbT)
TFO-2 (5-Q-TCTTTGTTCTCTTGCTCTT)
D-1
D-2
Me
P
A
PP
s
Tm at pH 7.5 (C)
X.AT 37.4
n.d
n.d
n.d
5-F-AGACATAAGAGCATGAGAA
X.TA n.d
37.4 n.d
n.d
3-TCTGTATTCTCGTACTCTT
X.GC n.d
n.d
37.4 n.d
5-F-AGACA1AAGAGCATGAGAA
X.CG n.d
n.d
37.4 37.8
3-TCTGT2TTCTCGTACTCTT
D-3
5-F-AGACAT3AGAGCATGAGAA
3-TCTGTA4TCTCGTACTCTT
D-4
5-F-AGACATAA5AGCATGAGAA
3-TCTGTATT6TCGTACTCTT
D-5
5-F-AGACATAAGAG7ATGAGAA
3-TCTGTATTCTC8TACTCTT
Table 8 Where b = BAU, m =
P, p = APP, F= labelled with fluorescein (F) at the 5
Me
end of the purine strand, Q = labelled with methyl red serinol at the 5 end TFO-1 strand,
D-2 to D-5 are identical to D-1, except that single base pair changes are introduced at
different positions, opposite one of the modified third strand bases; positions 1.2, 3.4, 5.6
and 7.8 correspond to each base pair (A.T, T.A, G.C and C.G) successively. n.d.
indicates that no melting transition was detected (Tm < 30 C).
Except s i.e. s.TA and s.AT all the nucleobase BAU,
recognition for BAU.AT,
Me
P, and APP showed selective
Me
P.GC, and APP.CG respectively at physiological pH by
fluorescence melting studies; Tm values at pH 7.5 were 37.4 C. It has been postulated
that alternative hydrogen bonding for protonated form of S might be responsible for lack
of TA base pair recognition. Thus Keith et al. have demonstrated high binding affinity to
mixed sequence duplex DNA targets at physiological pH with TFO contaning four
different modified nucleosides [BAU MeP APP and s].
Satoshi et al.58 have reported another approach to improve TFO recognition of
pyrimidine interruptions in the homopurine target strand of ds-DNA. These researchers
utilized the LNA (see section 1.4.3.4. LNA) backbone attached to either pyridine or
36
Introduction
pyrimidone nucleobases.115
N
O
N
N
2
O
O
2
m
P
P
N
4
4
O
2 N
H3
2 N
3
O
4H
T
T
N
N
4
N
4
O
2
O
P
H
N
2 N
3 H
4HT
N
O
H
O
N
O
H
N
Rd
O
Cytosine
Figure 24
H
N
N
H
N
H
N
N
4H
T
O
H
N
N
N
N
2 N
3
H
N
H
N
dR
Guanine
Pyrid-2-one
Rd
O
H
(P),
N
H
Cytosine
N
N
N
Guanine
N
H
N
dR
Rd
N
N
O
H
dR
N
H
Cytosine
Guanine
1,5-dimethyl-1,2-dihydropyrimidin-2-one
(4HT),
Thymine (T), 5-methyl-pyrid-2-one
Pyrid-2-one (P) and 1,5-dimethyl-1,2-dihydropyrimidin-2-one (4HT) (Figure 24) are
thymine analogues.
4H
T was designed to recognize cytosine of a CG Watson-Crick base
pair through formation of a hydrogen bond between either the N3 atom of 4HT and the 4amino group of C or the O2 atom of 4HT and the 4-amino group in C (Figure 24). On the
other hand, P was designed to recognize cytosine of a CG Watson-Crick base pair
specifically by the formation of a hydrogen bond between the O2 atom of P and the 4amino group of C (Figure 24). These nucleobase modified LNA monomers were
incorporated into TFOs. Subsequent binding studies revealed that TFO of sequence 5T5mcTXTmcTmcTmcT-3 (where X = P-LNA monomer and
m
c indicates 5-
methylcytosine) was found to form a stable triplexes with ds-DNAs of sequence (5GCTA5GAYAGAGAGATCG-3 (when Y = A, C, G or T). This TFO also exhibited a
strong preference for the CG base pair over GC, TA and AT base pairs (∆Tm = +10-19 C
at pH 7.5)
Comprehensive coverage of nucleobase modification, has been reviewed by Duca et
al.116
37
Introduction
1.5. Peptide nucleic acids (PNAs)
O
O
R
NH
NH
HN
N
N
N
N
H
OO
O
O
O
N
O
O
NH2
N
6 bond
N
N
O
O-
P
N
O
O
O
HN
N
N
O
N
O
HN
N
O
N
O
O
HN
O
O
NH2
-
N
N
O-
O
P
N
N
O
N
O
N
P
-
N
OH
N
O-
O
Adenine
N
O
O
O
Guanine
NH2
NH2
O
O
O
NH
OH
O
NH2
N
O
NH2
O
N
N
P
O
N
N
O
P
OH
O-
O
O
R
PNA
Figure 25
N
O
O
O P
N
Cytosine
N
O-
O
-
O
H2 N
OH
NH
O
O
NH
P
3 bond
O
O
N
O
N
P
NH2
N
O
O
O
O
O
N
6 bond
3 bond
OO
O
O
NH2
O
HN
Uracil
Thymine
DNA
RNA
Structures of PNA and segments of DNA and RNA
PNAs were first reported by Nielsen et al.7 at the University of Copenhagen in 1991 and
later were licensed to Applied Biosystems and ISIS pharmaceuticals Inc. PNA chemistry
was first commercialized in 1993. At the time, PNA represented a radical change in
oligonucleotide analogue design since most derivatives that had been investigated up
until then had structures closely related to or derived from DNA.
In PNA, only the natural nucleobases of DNA have been retained (Figure 29). The
normal deoxyribose-phosphate backbone has been replaced by a structurally
homomorphous backbone composed of N-(2-aminoethyl) glycine subunits.
In this way, the distances between the nucleobases along the PNA backbone (6 bond) and
from nucleobase to the PNA backbone (3 bond) are the same as those found in DNA
(Figure 29). PNAs were developed as neutral compounds because it was thought that this
would lead to a reduction in electrostatic repulsion upon their binding to DNA through
triplex-helix formation. PNAs are depicted like peptides, with the N-terminus at the first
38
Introduction
(left) position and the C-terminus at the right.
1.5.1. Binding affinity of PNA with complementary ss-DNA, RNA and PNA
PNA has been found to bind with high affinity and sequence selectivity to ss-DNA, RNA
and PNA oligomers.65 Unlike DNA, PNA can bind both in parallel and antiparallel
orientation, whereby the PNA C-terminus corresponds to the 3' end, and the N-terminus to
the 5' end of normal oligonucleotides. Antiparallel PNA:DNA hybrids [Tm = 69.5 C for a
15-mer (tgtacgtcacaacta)] are considerably more stable than the corresponding parallel
PNA:DNA hybrids (Tm = 56.1 C) and DNA:DNA hybrids (Tm = 53.3 C) complexes.
PNA can bind RNA to form PNA:RNA hybrid both in a parallel (Tm = 51.2 C) for a 15mer (tgtacgtcacaacta) as well as for a slightly more favoured anti-parallel (Tm = 72.3 °C for
above sequence orientation with complementary nucleic acid (Tm = 50.6 C for a 15-mer
DNA:RNA).65 Consequently, PNA:RNA duplexes are more stable than the corresponding
PNA:DNA duplexes. When PNA hybridizes to RNA, it adopts an A-like helix, whereas
when it hybridized to a complementary DNA strand, it adopts a conformation that is a
hybrid structure between the A and B forms of DNA.119 PNAs form very stable duplexes
with complementary PNA sequences. For example, the duplex formed by the PNA
decamer H-gtagatcact-L-Lys-NH2 and the complementary anti-parallel sequence forms
PNA:PNA hydrid (Tm = 67 C). The corresponding anti-parallel DNA:PNA hybrid (Tm =
51 C) and the DNA:DNA duplex at (Tm = 33.5 C) shows that anti-parallel strand
orientation is characteristically more stable.
1.5.2.
Strand invasion
When PNAs were originally designed it was envisaged they would bind to ds-DNA in an
analogous manner to other oligonucleotide analogues i.e. in the major groove through
Hoogsteen hydrogen bonding to form DNA2/PNA triplexes. However, PNAs were found
to hybridize with ds-DNA through a comparatively unique mechanism called strand
invasion. Strand invasion involves one PNA oligomer binding to the complementary
oligonucleotide strand of the ds-DNA target by (-) Watson-Crick base pairing. During
this process the non-complementary oligonucleotide strand is displaced by analogous
strand to form a P-loop.121 In the case of homopyrimidine PNAs, the PNA:DNA
39
Introduction
heteroduplex formed is further stabilised by binding of a second PNA oligomer through
(*) Hoogsteen hydrogen bonding (usually in a parallel orientation) to ultimately yield a
PNA*DNA:PNA triplex. For homopurine PNAs, strand invasion stops at the PNA:DNA
heteroduplex stage and as a result their strand invasion complexes are less stable.121 To
date, only a few examples of strand invasion have been reported in the literature for
mixed sequences PNAs, presumably because their strand invasion complexes are even
less stable.121 Since these initial findings, a variety of other strand invasion complexes
between PNA and ds-DNA have been discovered as shown in Figure 26. Since each of
these complexes involves distinct physico-chemical characteristics, it is essential to
categorize these strand invasion complexes.
A
Figure 26
B
C
E
D
A) Triplex invasion – as formed by homopyrimidine bis-PNA. B)
Duplex invasion – as formed by homopurine PNA. C) Double duplex invasion – as
formed by sets of pseudo-complementary PNAs. D) “Conventional” PNA.DNA-DNA
triplex – as formed by single cytosine-rich PNA binding to its DNA target in the DNA
major groove via Hoogsteen hydrogen bonds. E) Triplex / duplex invasion – as formed
upon binding of Tail-Clamp PNA to a DNA target. The last complex (E) is a hybrid of
those shown at A and B but is formed with less sequence constraints by comparison with
conventional triplex invasion complexes. The lower constraint is due to the shortening of
the homopyrimidine “bis” moiety to 5-6 bp.117
1.5.2.1.
Triplex invasion (Figure 30A)
As stated above, conventional triplex invasion involves formation of an internal
PNA*DNA-PNA triplex in which two homopyrimidine PNA strands are bound to one
40
Introduction
oligonucleotide strand in the ds-DNA target through Hoogsteen (*) and Watson-Crick (:)
hydrogen bonds (Figure 31). For example, homopyrimidine (H-t10-LysNH2) PNAs were
found to hybridise to ds-DNA (dA)98-(dT)98 through a triplex invasion.120
bis-PNA
Figure 27
DNA
PNA (DNA)2 Intermediate
PNA (DNA)2 triplex
Binding of bis-PNA
The bis-PNAs (Figure 27) were designed in order to reduce the entropy losses by only
having 1 molecule having to bind rather than 2. They were designed in such a way that a
parallel and anti-parallel orientated PNA strand were covalently bound together through
a linker like flexible polyethylene glycol linker (PEG) and 8-amino-3,6-dioxaoctanoic
acid (egl).123 The most efficient binding was observed with the the bis-PNA of sequence
H-tctct3-(egl)3-t3jtjt-LysNH2 (Tm = 66 C), which contained cytosines residues in the
anti-parallel (Watson-Crick) PNA strand and the pseudo-isocytosine (j), in the parallel
(Hoogsteen) PNA strand. This bis-PNA exhibited pH independent triplex invasion.124
“Tail-clamp” PNAs (TC-PNA) are composed of a short (5 or 6-mer) homopyrimidine
triplex forming domain and a (10-mer) mixed sequence duplex forming extension. The
combined triplex/duplex strand invasion strategy has been reported for the PNA of
sequence H-tttttj-egl-ctttttgtcgacctgc (where egl is 8-amino-3,6-dioxaoctanoic acid),
while only triplex strand invasion was observed for the PNA with the sequence H-tttttjegl-cttttt and this strand invasion complex was less stable (∆Tm = - 40 C).122
41
Introduction
1.5.2.2. Duplex Invasion
As alluded to earlier, the duplex invasion complex is formed between homo-purine PNA
and the complementary homo-pyrimidine oligonucleotide strand of the ds-DNA target
(Figure 26B).120 This complex is typically much less stable than triplex-invasion
complexes although these complexes can exhibit greater stability if the DNA target is
negatively supercoiled or if the PNA employed is conjugated to a cationic peptide.120
1.5.2.3.
H
N
N
Double Duplex Invasion
H
N
N
N
H
O
N
H
N
H
N
H
N
N
N
N
R
H
N
R
O
O
H
N
N
H
N
H
S
H
H
H
N
N
N
H
H
O
N
N
N
N
H
O
N
N
O
N
N
R
N
H
N
H
R
S
N
H
H
Figure 28
Binding of pseudo-complementary bases
It was realised that an ideal PNA antigene strategy would be to use duplex-forming
PNAs to target both the sense and an antisense strands of DNA at the same time. A
problem with this approach is that unfortunately PNAs bind more strongly to themselves
than to natural nucleic acids and so two complementary PNA strands would self
aggregate rather than bind to their respective DNA target strands.125 In order to prevent
self aggregation and increase their base pair recognition ability, Nielsen et al. have
explored the use of PNAs bearing pseudo-complementary bases.
125,126
The two pseudo-
complementary (pc-PNA) bases developed were 2,6-diaminopurine (D) and 2-thiouracil
(sU) (Figure 28). Hybridization of these bases with each other was restricted by a steric
clash between the 2-amino group of the 2,6-diaminopurine and the thio group of 2thiouracil. Thus, binding between the PNA strand and its pseudo-complement was
42
Introduction
destabilized while binding of the modified PNA oligomers with their complementary
oligonucleotide strands in the ds-DNA target was maintained. Inclusion of these bases in
PNAs targeted to mixed sequences in ds-DNA resulted in the formation of double duplex
invasion complexes (Figure 26C). The formation of these complexes is thought to occur
via the formation of a transient complex between ds-DNA and two pseudocomplementary PNA strands by Watson Crick base pair type recognition. The previous
process is triggered through small scale opening in the ds-DNA caused by dynamic base
pair breathing and which allows the two pseudo-complementary PNA strands to trap the
exposed nucleobases in the open state.125,127 Although double duplex invasion complexes
(Figure 26C) are stable, they were found to be less stable than the corresponding triplex
invasion complexes.
1.5.2.4. Triplex Formation
Under certain circumstances PNA may bind ds-DNA in the DNA major groove without
displacement of the non-complementary DNA strand (Figure 26D).128,129 This leads to
formation of PNA*DNA:DNA triplexes in which the single PNA strand is bound
through (*) Hoogsteen hydrogen bonds. The DNA’s (:) Watson-Crick hydrogen bonds
are preserved. Formation of PNA*DNA-DNA triplexes appear to be most favourable for
homopyrimidine PNA oligomers with a high cytosine content.128,130
1.5.3.
Hybridization Property of PNA
1.5.3.1. Ionic effects
DNA/DNA hybridization is dependent on ionic strength, i.e. solubility and hybridization
generally increases with an increase in ionic strength.133 However, melting curve analysis
of fully complementary mixed sequence PNA-DNA heteroduplexes of different oligomer
lengths (6- to 20-mers) show that these complexes are significantly more stable at low
salt concentrations (10-100 nM) than at a high ionic strengths 1000 nM. This
destabilization of PNA-DNA duplex at high ionic concentration is due to the presence of
the polyelectrolytic single DNA strand, which releases counterions upon duplex
formation with the uncharged PNA strand.64,65,133,
43
Introduction
1.5.3.2. Effects of mismatches
PNA Sequence
ss-DNA
Xa
Tm C
Xa
Tm C
H-p(tcacaacta)
3-d(AGTGXTGAT)
a.T
42
a.C
29
a.A
28
a.G
25
a.T
42
g.T
34
c.T
10
t.T
13
c.G
62
c.C
41
c.A
43
c.T
47
H-p(tcacxacta)
H-p(acgtcacaacta)
3-d(AGTGTTGAT)
3-d(TGCAGTXTTGAT)
Table 9 The effect of a single mismatch on PNA binding affinity, where Xa mismatched
monomer, Tm C melting temperature of PNA-DNA duplex
To study the effects of mismatches on the thermal stability (Tm) of PNA-DNA duplexes,
two of the 9-mer PNA:DNA duplexes were investigated, aX series and xT series differ only in
the chemical nature of the two strand i.e P9/D9aX and P9xT/D9. In the 9-mer PNA:DNA
duplex aX series, melting temperatures decrease considerably (on average, Tm = - 15 C).
The two purine-purine base pairs (a.A and a.G) have greater entropic cost than the a.C
mismatch which induces the least destabilization (Tm values a.C > a.A > a.G). These results are
unexpected, since the stabilization conferred by stacking of the inherently larger purine bases
might be expected to be greater than that of the pyrimidine cytosine, as observed for DNADNA as well as for PNA-PNA duplexes with Tm values. For the 9-mer PNA-DNA duplex xT
series, the g.T mismatch (where the mismatched nucleobase resided on the PNA strand) was
the least destabilizing, with only a modest (8 °C) decrease in Tm as shown in Table 9. In DNADNA systems, the g.T mismatch is also found to be extraordinarily stable and this is believed
to be due to the fact that this wobble base pair has two hydrogen bonds.133 In contrast, a
substantial decrease in  Tm was observed (≥ 29 C) for 9-mer PNA-DNA duplexes bearing
both the c.T and t.T mismatches, where the mismatched base was incorporated into the PNA
strand. The 12-mer PNA-DNA duplex containing a cT mismatch (where the mismatched
nucleobase was located on the ss-DNA strand) was also destabilised this time by -15 °C. The
analogous complexes with c.C and c.A mismatches were marginally more destabilized (Table
9). In summary, for this series of PNA oligomers, the trends observed for the destabilisation of
the resulting PNA:DNA duplexes on inclusion of a single base mismatch into either strand
44
Introduction
were similar to those reported for complementary DNA-DNA duplexes.64,65, 132,133,
1.5.4. Stability, solubility and cellular uptake of PNA
PNAs have been shown to be stable to both nuclease and protease enzymes.135,136
However, as their chain length increases, neutral PNAs tend to self-aggregate and they
are only partially soluble in aqueous media,137 with the exception of homo-adenine
PNAs.137 To overcome poor cellular uptake and to aid delivery to the cell, it is crucial for
PNAs to be soluble in water. Thus, PNAs synthesized as PNA/DNA chimeras, bear an
overall net negative charge, which improves water solubility.138 As a result of PNA selfaggregation, a great deal of work is currently being undertaken to find an effective
cellular PNA delivery system.139 A number of PNA-peptide conjugates have been
developed for this reason. For example, peptides such as penetratin and transportan have
been used to facilitate cellular entry by passive transportation through the lipid bilayer
membrane.140,141 Hydrophobic motifs (eg. adamantyl acetic acid, biotinylated etc) and
cationic liposomes, like Lys4-chol and Lys4-palm,122 have also been shown to aid
delivery.139
1.5.5 Potential therapeutic applications of PNA
As stated in the previous section, PNAs bind with high affinity to both target RNA and
DNA and are stable to both nuclease and protease enzymes. As a result much research is
now focussed on their potential applications in both antisense and antigene therapeutic
strategies142 as well as in the development of genetic diagnostics.143 In addition to acting
on human DNA and RNA, PNAs offer the possibility of acting on bacterial and viral
nucleic acids, thereby widening the scope for their use as potential therapeutic agents.
1.5.6. Antisense PNAs
PNAs have been shown to be effective modulators of antisense mechanisms through
either inhibition of translation or splicing of mRNA.144 Thus, antisense PNAs have been
used to successfully sterically block transformation of the translational apparatus.23
Unfortunately, however, unlike 2-FANA and PS-ODN, PNAs are unable to activate
RNase H.144 The antisense activity of the PNA has been demonstrated by employing the
rabbit reticulocyte lysate model system as shown in Table 10.144 The PNA 7 oligomer,
45
Introduction
which had been designed to bind near to the 5-end of the AUG region, was found to
efficiently block translation of CAT mRNA in a dose-dependent manner, presumably by
physically blocking assembly of the 80S ribosome initiation complex.144 The analogous
phosphodiester ODN 2 targeted to the same region did not inhibit translation, even after
addition of RNase H to the translation reaction. However, when PNA 3, PNA 4 or PNA 5
were studied, all of which had been designed to bind to a sequence in the coding region
of CAT mRNA, no inhibition of translation was observed. Compared with the (PNA 5)2RNA triplex, which results in thermally more stable Tm = 81 C than PNA 1-RNA
duplex Tm = 69 C suggests stability of the complex is not the only criteria that
determines the translation elongation blockage.144 This indicated that duplex-forming
PNAs were not able to arrest the elongating ribosome.144 Clamp PNA 8 was developed
so that it would partly form both a triplex at the 5-end and a duplex at the 3end upon
hybridization to the same coding region of CAT mRNA and thereby inhibit translation in
a sequence specific manner.144 Thus, they could be potent antisense agent, although their
specific antisense effects in intact cells remain to be seen.
No
Sequence
Target
Effect
PNA 1
H-(ttc ttc ttt t)-Lys-NH2
Coding region
+++
PNA 2
H-(ctc ttt ttt t)-Lys-NH2
AUG region
+++
PNA 3
H-(aca tct tgc g)-Lys-NH2
Coding region
-
PNA 4
H-(acg cca cat ctt gcg)-Lys-NH2
Coding region
-
PNA 5
H-(gta aca cgc cac atc ttg cg)-LysNH2
Coding region
-
PNA 7
H-(ttt agc ttc ctt agc)-Lys-NH2
AUG region
+++
PNA 8
H-(tct caa taa acc ctt t)-(eg1)3-ttt jjj-Lys-NH2
Coding region
++
ODN 1 5-(ttt ttt tct cca)-3
AUG region
-
ODN 2 5-(ttt agc ttc ctt agc)-3
AUG region
-
Table 10
List of ODNs targeting CAT mRNA and AUG region in rabbit
reticulocyte lysate and their effect. eg1 indicates 8-amino-3,6- dioxaoctanoic acid; j
indicates pseudoisocytosine.
An attempt has been made to increase the antisense activity of PNA by activating RNase
H. Therefore, a 12-mer PNA/DNA chimera of sequence (5'-aca tca TGG TCG-h), (where
46
Introduction
a, c, t indicates adenine, cytosine, and thymine PNA monomers and T, G, C indicates
thymine, guanine and cytosine DNA monomers), was synthesized and evaluated. The
chimera was found to bind to complementary RNA with high affinity in an anti-parallel
orientation. The stability of the resulting duplex was greater than that for the
corresponding anti-parallel unmodified DNA-RNA heteroduplex (Tm = 52.1 C cf. Tm =
44.5 C respectively). The chimera was not observed to bind to complementary RNA in a
parallel orientation.138 Unfortunately, the chimera also failed to activate the RNase H
enzyme in vero cells, although it too was capable of inhibiting the expression of a
specific gene by sterically blocking the translational apparatus.
1.5.7. Antigene PNAs
PNA triplex strand invasion complexes i.e. (PNA2:DNA) form with such high stability
that they have been found to inhibit the recognition of DNA binding proteins e.g.
transcription factor and RNA polymerase.145 Thus, strand invasion was found to occur
more efficiently when the PNA oligomer was designed to bind to the non-template strand
of ds-DNA because then it did not compete with the machinery involved in the formation
of the nascent RNA chain.146 Thus, the salt inhibition of PNA strand invasion caused by
the transcription buffer could be eluded by transcriptional activity of RNA polymerase to
further facilitate formation of (PNA)2/ds-DNA complexes.146
In contrast to the finding that PNAs are able to inhibit the process of transcription, PNAs
have also been shown to have the capacity to activate specific genes, when bound to a
repressor site.148 Thus, PNAs can be used to switch genes both “on” and “off”. In
addition, it has been shown that the displaced P-loop produced upon binding of PNA to
ds-DNA by strand invasion, can act as an artificial transcription promoter.146
Furthermore, merged P-loops are capable of binding to ss-DNA, forming PD-loops,
which can then act as artificial primosomes; they initiate RNA primers on the lagging
DNA strand during DNA replication.147 Such PD-loops have a number of potential uses
in topological DNA labelling of non-denaturing ds-DNA sequences.
47
Introduction
1.5.8. PNA backbone modification
The original PNA is considered, even now, as being a highly optimised system.
However, a modelling study has suggested that there may be room for improvement.119
Furthermore, by modifying the structure of PNA, it is possible to get an idea about the
nucleic acid binding requirements for the 2-aminoethylglycine-based backbone and the
methylene carbonyl linkage joining the nucleobase to the backbone (Figure 25). Thus,
various modifications of PNA have been undertaken to date mainly with the intention to
improve the properties of PNA, such as its hybridization ability and aqueous solubility.
Furthermore, research efforts have been undertaken to expand PNA recognition alphabet
to enable to target all gene sequences and therefore enhance their therapeutic utility,
which is currently typically limited to homopurine or homopyridine sequences. Finally,
extensive investigations have also been performed to improve the cellular uptake of
PNA, and overcome its ambiguity in orientational selectivity (i.e. parallel vs antiparallel) during binding.136,149,150,151,152,153
An improvement in aqueous solubility of PNAs has been achieved by: i) the introduction
of charges into the molecule (described earlier in Section 1.5.4.), and; ii) replacing the
methylene carbonyl sidechain to the nucleobase with a flexible ethylene linker (eth PNA,
Figure 29).154,155 Eth PNA modifications eliminates the conformational constraint of the
amide and increases aqueous solubility through protonation of the tertiary amine at
physiological pH alongwith enhanced binding affinity due to favourable electrostatic
interactions. Although the sequence specificity of the original PNA was preserved in eth
PNA, based on mismatch evidence, which showed a decrease in Tm for the oligomer of
sequence H-gtaga-x-cact-LysNH2 (where g, t, a and c are the original PNA monomers
and x denotes the eth PNA monomers) to less than 20 C for complexes containing t-C
and t-T mismatches, the binding affinity of this oligomers was also dramatically reduced.
The stability of the resulting duplex formed upon hybridization to complementary antiparallel ss-DNA was considerably lower than the corresponding original unmodified
PNA-DNA duplex (ΔTm = 24 C). This indicates that the change in linker from a tertiary
amide to tertiary amine is not well tolerated as the fall in stability at least partly could be
attributed to the higher flexibility of the linker to the nucleobases and a less efficient
water exclusion from the helix.
48
Introduction
Base
O
N
BocHN
OH
BocHN
O
O
O
N
Base
Base
Base
O
OH
N
BocHN
H
O
O
OH
apg-PNA
Eth-PNA
N
H
BocHN
O
O
HO
N
NHBoc
Figure 29
N
Chain extensions not permitted
H
BocHN
O
Retro-inverso PNA
modifications may be tolerated
OH
OH
O
pa-PNA
H
Base
O
N
R
N
Base
Base
O
O
N
R
-PNA
O
O O
O
required elements
L-Ornithine based PNA
PNA backbone modifications and structure activity relationship of an
acylclic original PNA backbone
It has been found that, in general, changes in the distances between a) the terminal
ethylene amino N-atom and the tertiary amide N-atom in the PNA backbone, b) tertiary
amide N-atom and the glycine carbonyl group in the PNA backbone, and c) the backbone
and the nucleobase, are not favoured (Figure 29).153,158 The effect of increasing the
number of bonds in PNA by one carbon unit β-PNA158 has been studied by preparing
apg-PNA158 and pa-PNA158 (Figure 29), where β-PNA bears an ethyl β-alanine
backbone, apg-PNA an amino propylglycine backbone and pa-PNA a propionyl linker
from the ethylglycine backbone to the nucleobase. When either a β-PNA, apg-PNA or
pa-PNA monomer was incorporated individually into the middle of the original PNA
decamer H-t4-(x)t5-Lys-NH2 (where x= β-PNA, apg-PNA or pa-PNA thymine monomer),
the Δ Tm values of the resulting complexes formed with complementary ss-DNA were
depressed by 13, 11 and 18 C, respectively, compared to the unmodified PNA-DNA
duplex.158 The oligomers extended in all backbone units did not form stable complexes
with complementary ss-DNA, and these results indicate the importance of maintaining
the correct distance between the bases in order to preserve base pairing and allowing a
regular backbone geometry, whereas a change in length between the backbone and the
nucleobases is more easily accommodated as the sequence specificity was maintained.
In order to understand the structural requirements for high affinity binding, Krotz et al.
have synthesized a retro-inverso159 PNA monomer composed from a N-(aminomethyl)-βalanine (Retro-inverso PNA) backbone (Figure 29). The number of bonds separating the
nucleobases along the backbone (6 bonds) and from the backbone to the nucleobase (3
49
Introduction
bonds) in retro-inverso159 PNA were identical to those found in the original PNA. When
a single thymine derivatized retro-inverso PNA monomer was introduced into the PNA
oligomer of sequence H-tgtacgt*cacaacta-NH2, (where t* indicates the retro-inverso
thymine PNA subunit), the thermal stabilities of the resulting complexes formed upon
hybridization to either complementary ss-DNA or RNA oligomers decreased relative to
those produced with the unmodified original PNA (ΔTm -8 C per modification).
Subsequent molecular modelling has shown that the carbonyl groups of the backbone
amides in a retro-inverso PNA-DNA heteroduplex point in towards the helix upon
hybridisation.159 A number of reasons have been offered as to why this should ultimately
undermine the thermal stability of the resulting complex. Firstly, it has been suggested
that the close proximity of the backbone carbonyl to the linker carbonyl could give rise to
a destabilising electrostatic interaction. It is also possible that this conformation offers
less access to water, which is important because solvation is thought to stabilise the
complexes. This finding highlights that simple bond counting rules do not ensure the
success of any potential DNA mimic.
Only the α-position of glycine in the original PNA has been found to tolerate substitution.
For example, replacement of the glycine residue of PNA with either D- or L-alanine (Figure
30) resulted in only a slight depression of (Δ Tm = 0.8 C) in the stability of their resulting
complexes with complementary anti-parallel DNA.155 Haima et al. have synthesized the
PNA oligomer of sequence H-(gtxagatxcactx)-Lys-NH2 (where tx, represents the thymine αsubstituted glycine PNA monomer) with the intention of improving the binding of the PNA
strand to the target DNA by inducing positively charged side chains. Introduction of Dlysine (Figure 30) into the above sequence resulted in formation of a complex with
complementary anti-parallel ss-DNA, which was slightly more stable than the analogous
unmodified complex (∆Tm = +1 C per modification). By contrast, L-lysine (Figure 30) had
the opposite effect, whereby its inclusion resulted in destabilization of the complex, (∆Tm =
-1 C per modification).153 When D-serine and L-serine were substituted for glycine into the
original PNA backbone for the above sequence, the Tm values for the resulting duplexes
were both slightly decreased (-0.6 and -1 C per modification respectively). These findings
suggest that, of the two isomers, D-amino acids appear to be better accommodated into the
backbone of PNA.
50
Introduction
NH
NH


"
O
O
O

Base
'
N
NH

O
Base
'
N
'
"
O
OH
Base
N
'
O
NH

O
NH2

Base
'
N
PNA with L-Serine backbone
NH2
PNA with L-Lysine backbone
PNA with D-Lysine backbone
'
"
O
NH

Original PNA
NH

O

Base
'
N
"
O
NH

O

'
OH
PNA with D-Serine backbone
Base
'
N
'
"
O

Base
'
N
O
"
O
'
PNA with D-Alanine backbone
PNA with L-Alanine backbone
Modifications at α” position of original PNA backbone
Figure 30
1.5.9. α-PNA
R1
R1
R1
HN
HN
Base
N
O
O
O
NH
HN
O
HN
Base
N
O
O
Base
HN
Base
O
NH
NH
O
O
O
HN
Base
N
O
HN
Base
O
NH
O
O
OR2
OR2
OR2
PNA
Base
HN
O
NH
O
Figure 31
NH
O
O
Repeat
Unit
Base
HN
Base
-PNA
-cycloPNA
Structural comparison of PNA, α-PNA and α-cycloPNA
Modification of PNA through incorporation of natural amino acids can offer several
51
Introduction
advantages over the original PNA such as a) an increase in cellular uptake; b)
introduction of chirality and; c) preferential target orientation (parallel or anti-parallel). It
is not surprising therefore that many such modifications have been widely reported in the
literature.153,160 However, investigations of this type on the original PNA developed by
Nielsen et al. are complicated by its structure; whenever the change is not at either the Nor C-terminal position, there is a necessity to synthesize new monomers for each
alteration to be examined.
In order to ease studies in this area, Howarth et al. have reported on the development of
L-α-PNA
oligomers (Figure 31).161 The nucleobase containing α-amino acid monomers
were synthesized from L-homoserine and these were interspaced in the L-α-PNA
oligomers with other α-amino acids e.g. glycine. This design allowed for the
incorporation of different appropriate α-amino acid spacers into the oligomers during
peptide synthesis and thereby overcame the requirement to prepare new monomers in
order to examine the effect of each modification on binding. Molecular modelling studies
suggested that L-α-PNA would bind to complementary single-stranded DNA.162,163
Unfortunately, subsequent binding studies using suitable single-stranded DNA targets
and a range of L-α-PNA oligomers proved unsuccessful,164 despite the fact that the
structural features considered important for hybridization had been conserved in the L-αPNA design, i.e. the base-to-base (6 bonds) and base-to-backbone (3 bonds) distances
had been maintained. However, an L-α-PNA-PNA chimera was found to bind to
complementary single-stranded DNA and the L-α-PNA portion was shown to participate
in hybridisation, rather than merely stabilising the PNA-DNA complex. This was proved
by the findings that the Tm of the resulting complex was destabilised upon introducing a
single base mis-match into the L-α-PNA binding region.164
Base
Base
O
O
O
N
BocHN
H
ethPNA
Figure 32
OH
BocHN
N
OH
R
Glycine modified PNA
Charged and chiral PNA modifications; R = α-amino acid
A possible reason for the observed lack of hybridisation of pure L-α-PNA oligomers to
52
Introduction
complementary nucleic acid targets has been suggested upon studying the structures of
DNA, PNA and L-α-PNA in more detail. It is apparent that, although these oligomers share
several geometric similarities, L-α-PNA bears a more flexible side chain. The ethyl linker in
L-α-PNA gives the side chain three degrees of freedom compared to two in the original PNA
and one in DNA. Upon binding, the “freezing out” of movement in the extra bonds found in
the ethyl linkage of L-α-PNA would have an associated entropic cost. That such effects may
be significant is supported by the research reported previously for PNA. As described earlier
in section 1.5.7., a derivative of PNA was prepared in which the side chain methylene
carbonyl linker of the original PNA had been replaced by ethylene (ethPNA, Figure 32).155
The ethPNA oligomer too had formed less stable complexes with target ss-DNA relative to
the analogous unmodified PNA oligomer. This destabilisation had been attributed to the
removal of the amide bond which, through its partial double bond nature, afforded an
element of rigidity in the linker.
It was this finding that inspired Howarth et al. to investigate constrained L-α-PNA
analogues. The work reported in this thesis is concerned with one such analogue,
designated α-cycloPNA.165,166 Here, the flexible ethyl linkage in L-α-PNA has been
replaced by a rigid cyclopentane ring with intent to “freeze out” a more ideal
conformation for hybridisation to nucleic acid targets. As α-cycloPNA monomers have
two chiral centres, four diastereoisomers of α-cycloPNA are possible75,150,165,166 (Figure
33). Thus, the ultimate aim of this research was to develop suitable synthetic routes to all
four diastereoisomers of α-cycloPNA in order that the conformation most ideal for
binding to nucleic acids could be identified.
Base
CO2Et
Base
NHBoc
Figure 33
CO2Et
Base
NHBoc
CO2Et
NHBoc
Base
CO2Et
NHBoc
Four diastereoisomers of the α-cycloPNA monomer
1.5.10. Constrained PNAs
The concept of freezing out the optimum conformation for binding is not new to the field of
medicinal chemistry. A number of examples of this concept exist in the literature, including
in the area of peptide nucleic acids.8,167,168 The conformational restriction and
53
Introduction
preorganization of the original PNA structure has allowed for the development of PNA
analogues which are now able to discriminate between DNA and RNA targets and between
parallel and anti-parallel sequences. Important cyclic PNA analogues that have been reported
recently in the literature will be discussed over the following sections.
1.5.10.1 Cyclohexyl PNA (ch-PNA)
Base
Base
H
O
HN
O
O
OH
OH
H
H
O
Base
Figure 34
NH H
H H
H N
NH N
H
HN
O
N
HO
O
O
Base
N
HO
O
Four diastereoisomers of cyclohexyl PNA (ch-PNA) monomer
The flexibility in the aminoethyl segment of the original PNA has been “frozen” by
Nielsen et al.169 through its incorporation into a cyclohexyl (ch) ring (Figure 34). The
trans-(1S, 2S)-ch-PNA monomer was subsequently introduced into the middle of an
original PNA-T9 oligomer (H-t4tSSt5-Lys-NH2) (where t= thymine monomer of original
PNA and tSS = trans-(1S, 2S)-ch-PNA monomer) and the resulting oligomer was
hybridized to complementary ss-DNA. This resulted in formation of a PNA2DNA triplex
with Tm value of 70.0 C, which was comparable to the thermal stability of the triplex
formed between ss-DNA and the unmodified original PNA (Tm = 71.5 C). When the
other enantiomer, the trans-(1R, 2R)-ch-PNA monomer, was incorporated in the
corresponding sequence H-t4tRRt5-Lys-NH2 (where tRR = trans-(1R, 2R)-ch-PNA
monomer), the resulting triplex with complementary ss-DNA had a Tm value of 52.5C.
This suggest that single unit of trans-(1S, 2S)-ch-PNA monomer in above PNA sequence
may be entropically favorable, owing to significant preorganization of the duplex, while
binding with DNA in comparison to trans-(1R, 2R)-ch-PNA monomer. When the same
oligomer H-t4tSSt5-Lys-NH2 was formed to complementary RNA instead, the
heteroduplex yielded was slightly less stable than formed with the corresponding original
unmodified PNA (∆Tm = -5.5 C). However, when the analogous oligomer bearing the
tRR monomer was examined, the resulting heteroduplex formed with the RNA target was
found to be dramatically destabilised compared to the complex formed with the original
54
Introduction
unmodified PNA oligomer (∆Tm = -25 C), which again emphasizes that the (S, S) form
exhibits higher duplex stability than the (R, R) form in case of RNA as well. In addition
to these oligomers, the fully modified mixed sequence oligomer (H-(gtagatcact)SS-LysNH2) has been synthesized from the appropriate (trans-(1S, 2S)-ch-PNA monmers.
Subsequently, this oligomer was shown to bind to complementary anti-parallel ss-DNA,
but that the resulting heteroduplex was less stable than that produced by the
corresponding original PNA, (∆Tm = - 14 C)
These findings clearly demonstrate that inclusion of either enantiomeric forms of the
cyclohexyl PNA monomers into oligomers leads to the formation of less stable
complexes with complementary ss-DNA or RNA in comparison to the unmodified
original PNA. However, of the two, the trans-(S, S) isomer causes less destabilisation
than trans-(R, R) isomer. Molecular modelling studies suggested that the reason for this
may be due to the fact that the trans-(S, S) isomer was more easily accommodated in a
right-handed hybrid duplex with ss-DNA than the (R, R) isomer. Furthermore, it was
suggested that cyclohexyl 1,2-substituents are in a diaxial disposition in case of (R, R)
isomer, corresponding to a dihedral angle of 180. Thus resulting in PNA:DNA or
PNA:RNA complexes, which are incompatible with the geometric requirements.173

B
O
B
O
O
N
N
N
H
O

N
N
H
H
Dihedral angle in PNA-DNA (RNA) complexes
B

O
N
O
O


N
H
B
B
  
N
N
N
H

O
O
B
O


N
H
  
N
O
N
H
ch-PNA
cp-PNA
Figure 35
H
H
N
H
PNA
O
H
HN
O
O

NH
B
Dihedral angle in PNA, cp-PNA, ch-PNA, PNA-DNA and PNA-RNA
Despite, the drawbacks of the research reported by Neilsen et al. concerning cyclohexyl
PNAs, their results indicate that chemical constraint of the conformational freedom in the
original PNA backbone may lead to improved hybridization potency. A study of the X-
55
Introduction
ray structural data of the PNA2-DNA triplex170 and the PNA-DNA duplex171 (β=65-70°)
and NMR data of the PNA-RNA duplex172 (β=140°), has also revealed the importance of
a β dihedral angle (Figure 35) and the role that it plays in conformationally
distinguishable DNA/RNA binding preference. As the cyclohexyl ring is rigid it forbids
structural readjustments to bind to DNA or RNA due to the large difference in β dihedral
angle of PNA-DNA or PNA-RNA duplexes.175 Based on these findings, Ganesh et al.
have hypothesized that, by synthesizing a PNA analogue with a dihedral angle, β of 6570°, it could be possible to differentiate between ss-DNA and RNA targets of the same
sequence. Therefore, these researchers have prepared both the cis-(1S, 2R) and cis-(1R,
2S)-cyclohexylthyminyl PNA monomers (Figure 35).175 The crystal structures confirmed
that the dihedral angle β was -63° for the (1S, 2R) isomer and 66° for the (1R, 2S)
isomer.150,174,175
These two cis enantiomers, (1S, 2R) and (1R, 2S), were then separately incorporated
within an original T10-mer PNA oligomer to give the oligomers H-tSRt3tSRt5-Lys-NH2 and
H-tRSt3tRSt5-Lys-NH2 (where t= thymine monomer of original PNA, tSR = thymine
monomer of cis-(1S, 2R)-ch-PNA). Subsequent binding studies revealed that both
oligomers formed complexes with complementary poly dA, although the resulting
PNA2DNA triplexes were destabilized relative to the complex formed with the
unmodified PNA (∆Tm = - 34.8 C for oligomer containing tSR monomers; -x.y C for
oligomer containing tRS monomers). In the case of poly rA complexes, a reverse trend
was observed i.e. the stability of the (1S, 2R)-PNA:RNA heteroduplex was slightly more
than that of the (1R, 2S)-PNA-RNA duplex (Tm = 64.4 C cf. 58.6 C respectively) and
the cyclohexyl-PNA2-DNA triplexes as compared to the unmodified PNA2-DNA
triplexes Tm = >80 C).174,175,176 These results suggest that (S, R)/(R, S)-ch--PNAs
destabilize the DNA duplex enormously but stabilize the RNA duplex. This exceptional
binding selectivity of PNA to RNA over the same DNA sequence is based on the
difference in torsion angle as explained above and shown in Figure 35.
(1R, 2S)- or (1S, 2R)-cyclohexyl monomers have been incorporated within an original PNA
mixed oligomer to give corresponding oligomers (gtRSagatRScactRS) or (gtRSagatRScactRS).
(gtRSagatRScactRS) (where g, a and c = guanine, adenine and cytosine monomer of original
PNA respectively, tRS = thymine monomer of cis-(1R, 2S)-ch-PNA).176 The resulting
duplexes of PNA sequence (gtRSagatRScactRS) with complementary target anti-parallel ss56
Introduction
DNA or anti-parallel RNA gave thermal stabilities ∆Tm = -20 C or ∆Tm = > + 30 C
respectively, compared to unmodified PNA:ss-DNA or unmodified PNA:RNA. Similarly,
for the PNA sequence of gtSRagatSRcactSR (where tSR = thymine monomer of cis-(1S, 2R)-chPNA), the resulting duplexes with complementary target anti-parallel ss-DNA or antiparallel RNA gave thermal stabilities (∆Tm = - 30 C or ∆Tm = + 3 C) respectively, in
comparison to unmodified PNA:ss-DNA or unmodified PNA:RNA.
Hence, ch-(1R, 2S) oligomers could selectively discriminate between ss-DNA and RNA
targets but their binding affinities were lower compared to oligomers bearing the other
isomer.169,174,175,176
1.5.10.2. Cyclopentyl PNA (cp-PNA)
H
H
N
(R)
OH
H
OH
H
(S)
(S)
N
O
H
H
Figure 36
O
O
Base
Base
N
(S)
H
OH
(R)
N
O
H
H
N
(S)
H
N
O
N
(R)
H
OH
(R)
H
N
O
O
O
Base
Base
Four diastereoisomers of cyclopentyl PNA (cp-PNA) monomer
To account for the lower binding affinity for triplex formation, as seen with ch-PNA
compared to the original PNA, it was speculated that the substituted cyclohexyl ring was
inherently too rigid; it was reasoned that it could be locked into either of the two chair
conformations and that it may resist further retuning during complex formation.169,174
Independently, Ganesh et al.177 have examined substitution of (S, R)- and (R, S)diaminocyclohexane with (S, R)- and (R, S)-diaminocyclopentane (Figure 36). In both
cases, it was proposed that these less rigid systems, in which the characteristic endo-exo
puckering dictates the pseudoaxial/pseudoequatorial dispositions of substituents, may
allow for better torsional adjustments to attain the necessary hybridization-competent
conformations.150
57
Introduction
The (S, R)-cp-PNA and (R, S)-cp-PNA monomers have been included in the centre of the
original homothymidine PNA to give oligomers H-ttttSRtttt-LysNH2 [(S, R)-cp-PNA
oligomer] and H-ttttRStttt-LysNH2 [(R, S)-cp-PNA oligomer] (where t = thymine
monomer of original PNA, tSR = thymine monomer of cis-(1S, 2R)-cyclopentyl PNA and
tRS = thymine monomer of cis-(1R, 2S)-cyclopentyl PNA). The (S, R)-cp-PNA oligomer
destabilized the hybrid with DNA (∆Tm = -23 °C), whereas (R, S)-cp-PNA oligomer
stabilized the complex (∆Tm = +17 °C), compared to the unmodified PNA:DNA
hybrid.These oligomers displayed their binding to complementary RNA with (S, R)-cpPNA oligomer > (R, S)-cp-PNA oligomer having Tm that differ by 15 C as compared to
binding with DNA d(CGCAAAAAAAACGC), where as (R, S)-cp-PNA oligomer > (S,
R)-cp-PNA oligomer differed by 40 C.177
The probable reason for this unusual
behaviour could be caused by the introduction of unfavorable conformational
discontinuity at the modification site.
The complexes formed by fully modified homo-oligomeric, homochiral cp-PNAs (1S,
2R)-H-(tSR)8-LysNH2 and (1R, 2S)-H-(tRS)8-LysNH2 (where tSR = thymine monomer of
cis-(1S, 2R)-cyclopentyl PNA and tRS = thymine monomer of cis-(1R, 2S)-cyclopentyl
PNA) exhibited a high thermal stability (ΔTm = +21 and +27 C, respectively) with
complementary ss-DNA compared to the analogous unmodified PNA oligomers. Their
complexes with poly rA likewise showed unprecedented thermal stabilities with no melting
detected up to 85 C. Binding was also shown to be specific as these fully modified cpPNA oligomers failed to hybridise to mismatch DNA.177 However, in case of the fully
modified oligomers with the same stereochemistry, the stability is maintained perhaps
due to a uniform conformational change over the entire backbone, without any sharp
discontinuities
(1R, 2S)- or (1S, 2R)-cyclopentyl monomers were incorporated within an original PNA
mixed oligomer to give corresponding oligomers (gtRSagatRScactRS) or (gtSRagatSRcactSR).
The resulting duplexes of PNA sequence (gtRSagatRScactRS) with complementary target antiparallel ss-DNA or anti-parallel RNA gave thermal stabilities (∆Tm = +23.8 C or ∆Tm = > +
29.6 C respectively) in comparison to unmodified PNA:ss-DNA or unmodified
PNA:RNA.177 Similarly, for oligomer (gtSRagatSRcactSR) the resulting hybrids with target
anti-parallel ss-DNA or anti-parallel RNA gave thermal stabilities (∆Tm = +22.1 C or ∆Tm =
58
Introduction
> + 15.4 C respectively) in comparison to unmodified PNA:ss-DNA or unmodified
PNA:RNA177 likewise cp-PNA:RNA hybrid adopts a more RNA like structure with
higher stability, cp-PNA:DNA forms similar conformation due to the flexible
cyclopentane ring puckering that allows better torsional adjustments to attain
hybridization competent conformation. Thus, there is a lack of preferential binding of cpPNA:RNA over cp-PNA:DNA.8
1.5.10.3. Backbone extended pyrrolidine PNA (bep-PNA)
Backbone extended pyrrolidine PNAs (bep-PNA) have been examined in which an α’-βmethylene bridge has been introduced into aep-PNA which thereby increases the length
of the glycyl fragment to homoglycine. Kumar et al.169,178 have synthesized the required
(2S, 4S)-bep PNA monomers (Figure 37). Studies of the abilities of both an alternating 8mer oligothymine (2S, 4S)-bep-PNA-PNA chimera and an 8-mer homothymine (2S, 4S)bep-PNA oligomer to hybridise to complementary ss-DNA revealed that neither formed
complexes, unlike the analogous unmodified original PNA (Tm = 51.5 C). However, the
alternating bep-PNA-PNA chimera was found to bind to poly(rA) with high affinity and
the stability of the resulting complex (Tm = 84.4 C) was far greater than those formed
either with the corresponding fully (2S, 4S)-bep-PNA (Tm = 58.9 C) or original PNA (Tm
= 65.8 C). The high binding affinity of the alternating (2S, 4S)-bep-PNA-PNA chimera
with RNA suggested the uniform spacing of the (2S, 4S)-bep-PNA monomers produced a
balanced conformation which enhanced recognition of RNA. In the case of the fully
modified (2S, 4S)-bep-PNA oligmer, the binding affinity may not have been as good as
for the alternating chimera due to over preorganization. This has been reported for fully
modified LNA.178,179
O
O
O
H
Base
N
O
Base
Base
H
H
H
N
H
H
NH
Figure 37
NH
H
H N
N
H
H
H
H
H N
N
Base
H
Four diastereoisomers of backbone extended pyrrolidine-PNA (bep-
PNA) monomer
59
Introduction
1.6. Conclusion
The potential of PNA is evident as it is the only oligonucleotide analogue to date that can
cause transcriptional arrest due to a strand invasion mechanism, which leads to antigene
behaviour. The drug discovery process taking the original PNA into the market has been
hampered due to several poor aspects in regard to its cellular uptake, orientational
selectivity, and target specificity.
In order to modify any properties of PNA, it is a pre-requisite to synthesize new
monomers, unless alterations can be made at either the N- or C-terminii of the PNA
oligomers.
L-α-PNA
was designed to overcome this issue. In the literature, cellular uptake has been
addressed by introduction of a D-amino acid at the α-position of glycine in the original
PNA (Section 1.5.7. PNA backbone modification) as these modifications were tolerable
from binding affinity point of view. Similarly, L-α-PNA (Section 1.5.8.) allows
introduction of D-amino acid at the α-position of homoserine, hence these could possibly
address cellular uptake issues too. The unsuccessful binding of L-α-PNA with ss-DNA
was reasoned to be due to the flexibility of the ethyl linkage between the backbone and
base.
To address the above issue, Howarth et al. have embarked upon an investigation to
replace the flexible ethyl linkage in L-α-PNA with a constrained cyclopentane ring, It
was envisaged that the resulting oligomers designated α-cycloPNA, may be able to
surmount other difficulties associated with the original PNA such as target specificity,
sequence specificity and orientational selectivity. These points were discussed previously
in section 1.5.9.
Thus, the specific aims and objectives of the work reported in this thesis were:
i)
To investigate the reproducibility of the route of Walker et al. (Section 2.3.) for the
preparation of all four diastereoisomers of the thymine-containing α-cycloPNA
monomer (Figure 39).
ii)
To explore viable alternative pathways for the synthesis of all four diastereoisomers
of the thymine-containing α-cycloPNA monomer
60
Introduction
iii)
To examine the versatility of the optimised route, identified from (ii) for the
preparation of the α-cycloPNA monomers bearing other nucleobases (Figure 38).
NH2
N
R1
N
N
N
HN
O
O
NH
N
NH
O
N
N
NH2
HN
NH2
O
NH
N
O
N
O
HN
O
O
NH
NH
O
N
O
O
NH
O
R
Figure 38
The four nucleobase derivatives of α-cycloPNA
61
Results and Discussion
2. Results and Discussion
Polyamide nucleic acids (PNA), or peptide nucleic acids, were first reported by Nielsen
et al. in 1991,7 and unprecedented interest has developed especially due to its mode of
binding to double stranded DNA by strand invasion mechanism. The work reported in
this thesis is concerned with one such analogue of PNA, designated as α-cycloPNA,
arising from the development of L-α-PNA oligomers, as reported by Howarth et al.
(Section 1.5.10.).161,180
Howarth et al.161 have reported the development of L-α-PNA which were prepared from
L-homoserine,
and are interspaced with other α-amino acids e.g. lysine. Thus, L-α-PNA
oligomers were designed to overcome the requirement to prepare new monomers for each
modification which was to be examined, as would have been the case when working with the
original PNA oligomers. The L-α-PNA oligomer was also designed to facilitate
exploration of different factors that may improve binding affinity and expand the
sequence selectivity exhibited by the original PNA through the incorporation of different
appropriate α-amino acid spacers during peptide synthesis. Unfortunately, subsequent
binding studies using suitable ss-DNA targets and a range of L-α-PNA oligomers proved to
be unsuccessful.160 The failure of L-α-PNA to bind to DNA was attributed to the degree of
flexibility in the ethyl linkage between the backbone and nucleobase. This linkage gives the
side chain three degrees of freedom compared with two in the original PNA and one in
DNA. The “freezing out” of these extra bonds upon binding has an associated entropic cost
which may be significant, as supported by research reported previously for PNA (see section
1.5.10).
The overall aim of the work reported in this thesis is to investigate L-α-PNA analogues in
which the flexible ethyl side chain linkage in L-α-PNA has been replaced with a
cyclopentane ring, with the intention of “freezing out” an ideal conformation for
hybridization to nucleic acid targets. There have been few precedents in the literature of
conformationally constrained PNAs, which have shown binding selectivity between target
DNA/RNA due to preorganization of the structure by freezing it into a particular
conformation, and orientational selectivity (discriminating parallel and anti-parallel
orientation) due to chirality, as shown in Section 1.5.11.
62
Results and Discussion
CO2H
Nucleobase
CO2H
Nucleobase
CO2H
Nucleobase
NH2
NH2
NH2
NH2
102
101
100
CO2H
Nucleobase
103
The four possible isomers of α-cycloPNA, Nucleobase: 1-thyminyl, 1-
Figure A
cytosinyl, 9-guanyl, 9-adenyl
As already mentioned in Section 1.5.10, the focus of the research described here centres
on the development of viable synthetic routes to all the four diastereoisomers of the
constrained α-cycloPNA monomers (Figure A) required for construction of the
oligomers. Synthesis of all four diastereoisomers was required in order to fully explore
the ideal conformation of α-cycloPNA oligomers for binding to target DNA or RNA.
2.1.
Retrosynthetic analysis of target compound
CO2R
Cl
Cl
NHBoc
109
108
Base
CO2H
HO
HO
CO2R
CO2R
Base-H
104
CO2R
NHBoc
NHBoc
106
105
Base: : 1-thyminyl, 1-cytosinyl, 9-guanyl, 9-adenyl
O
OH
OMe
MeO
O
107
Scheme 1
Retrosynthesis analysis of the α-cycloPNA monomer
63
Results and Discussion
Retrosynthetic analysis was applied in order to identify suitable strategies for the
synthesis of all four diastereoisomers of the α-cycloPNA monomer described in Scheme
1. The first bond to be disconnected was that between C3 of the cyclopentane ring and
the nucleobase, as shown in Scheme 1. This yielded the cyclopent-3-ol derivative (105)
and the nucleobase as synthons (Base-H); the latter was commercially available. This
disconnection has been demonstrated to be viable by Walker et al.9 and numerous other
examples exist in the literature in which a nucleobase functionality has been introduced
using an alcohol as a starting material.181,182,183 We envisaged that the required key
alcohol (105) could be derived in three possible ways. Firstly, Walker et al.9 have
reported that alcohol (105) can be synthesized via dialkylation of commercially available
N-(diphenylmethylene)glycine ethyl ester (145) with 2(S)-1,4-diiodo-2-trityloxybutane.
The latter was prepared from (S)-dimethyl malate (107) as described in Scheme 1.
Secondly, it was also deemed possible that the alcohol (105) could be obtained from a
hydroboration reaction using alkene (108). This alkene could be obtained from
commercially available cis-1,4-dichlorobutene (109) and the appropriate Schiff’s base,
prepared from glycine ethyl ester, as described in the literature.199,200 Finally, Ma et
al.,184 have shown that the alcohol (105) can be prepared by a Curtius rearrangement
involving the monoacid ester (106), obtained from a cyclization reaction of commercially
available diethylmalonate (166) and (S)-dimethyl malate (107).
The following section describes our exploration of each of these strategies.
2.2.
Synthesis of key intermediate alcohol (123 and 124) via Curtius
rearrangement
CO2H
HO2C
CO2H
HO2C
Figure B
CO2H
HO2C
NH2
NH2
NH2
110
CO2H
HO2C
111
112
The four possible isomers of APCD
64
NH2
113
Results and Discussion
O
O
OH
OMe
i
MeO
OR
OMe
MeO
O
O
Ph2HC
O
90 %
iii
OH
HO
107
OR
OR
ii
95 %
95 %
116 R=CHPh2
115 R=CHPh2
OMs
MsO
117 R=CHPh2
CHPh2
P O
O
Ph2HC
O
iv
114
RO
CO2Et
RO
NHCO2Et +
CO2Et
CO2H
vi & vii RO
RO
CO2Et +
NHCO2Et
119 & 120 R=CHPh2
121 & 122 R=CHPh2
CO2H
CO2Et
v
RO
70 %
CO2Et
CO2Et
118 R=CHPh2
123 & 124 R = OH
Scheme 2184
Reagents and conditions: i) Phosphoric acid tribenzhydryl ester
[P(O)[ODPM]3] (114), TFA, CHCl3; ii) LiAlH4, THF, 0 → 25 C; iii) MsCl, TEA,
CH2Cl2, 0 → 25 C, overnight; iv) CH2(CO2Et)2, NaOEt, EtOH, Δ; v) NaOH, EtOH,
H2O, 0 → 25 C, 48 h; vi) (PhO)2P(O)N3, Et3N, benzene, Δ then EtOH; vii) H2, Pd/C, 30
atm, EtOH.184
Ma et al.184 demonstrated the first enantiospecific synthesis of a 1-aminocyclopentane-1,3dicarboxylic acid (APCD) (Figure B) derivative starting form the chiral building block (S)dimethyl malate (107), as detailed in Scheme 2. In this route, (S)-dimethyl malate (107)
was treated with phosphoric acid tri(benzyhydryl)ester (114) in the presence of a catalytic
amount of TFA to give the benzhydryl protected diester (115) in 90% yield. Following
reduction with lithium aluminum hydride, the resulting 1,4-diol (116) was next treated with
mesyl chloride in the presence of triethylamine to afford the dimesylate (117) in an 86%
yield over the two steps. The cyclization of dimesylate (117) was subsequently
accomplished by treatment with diethylmalonate (166) in the presence of sodium ethoxide
to give the corresponding cyclopentan-3-ol derivative (118) in 70% yield. As depicted in
the Scheme 2, selective hydrolysis of the diester (118) in the presence of sodium hydroxide
introduced a second stereogenic centre at the quaternary carbon, as indicated by the
diastereoisomers ratio formation of cis (1S) and trans (1R) isomers relative to the alcohol.
The ratio of isomers formed depends on the bulkiness of the protecting group used i.e.
when benzhydryl group is used to protect the alcohol function of diester (112) the ratio of
trans to cis isomers of diastereomeric monoacid esters (119) and (120) was found to be
97:3. The obvious explanation for this result is that the greater steric hindrance caused the
65
Results and Discussion
benzyhydryl group to prevent hydrolysis of the ester group on the same face.
Subsequently, the monoacid esters (119) and (120) underwent a Curtius rearrangement
upon addition of diphenylphosphoryl azide to give the protected amino acids (121) and
(122) which, after deprotection, gave the desired APCD alcohols (123) and (124) in an
88% yield over the last three steps (i.e. v-vii, [Scheme 2]).
The key step in the synthetic pathway reported by Ma et al.184 was the Curtius
rearrangement. This was successfully achieved using diphenylphosphoryl azide (126)
which first activated the carboxylic acid (125) and then generated the acyl azide (128), as
outlined in Scheme 5. Under thermal conditions, the acyl azide undergoes a
rearrangement to give the corresponding isocyanate (129), which in turn reacts with
ethanol to generate the ethyl carbamate.
OH
R
O
O
O
125
N3
P
OPh
OPh
126
R
N3
O
O
O
N
P
OPh
OPh
N
R
N
128
127

- N2
R
H
N
OR'
O
N
C O
R' OH
129
130
Scheme 3
R
Curtius rearrangement
Inspired by the findings of Ma et al. previous work in our group conducted by Walker et
al. focussed on utilising a modified strategy for preparation of the required key
cyclopentan-3-ol α-amino acid intermediate (143 and 144) (Scheme 6) needed for the
preparation of our nucleobase-containing α-cycloPNA monomers, as shown in Scheme 5.
Thus, (S)-dimethyl malate (107) was used as the chiral building block, and the first step
in this pathway involved protection of the 2-hydroxyl function with a benzyhydryl group
using identical reagents and conditions as described by Ma et al.184 This reaction proved
to be not trivial as it required the preparation of fresh phosphoric acid tribenzhydryl ester
(114). Oxidation of benzophenone hydrazone by the hypervalent iodine of iodobenzene
diacetate (132) gave diphenyl diazomethane (131), which reacted with phosphoric acid to
give phosphoric acid tribenzhydryl ester (114). This required careful addition of both
66
Results and Discussion
solid (131) and (132), in small portions to phosphoric acid, as upon addition, nitrogen gas
was liberated, which was found to have safety issues as it might cause explosion on large
scale, consequently resulted in the scale of synthesis of phosphoric acid tribenzhydryl
ester (114) (Scheme 4) being limited to 1.8 g. Despite this problem, the benzyhydryl
protected diester (114) was afforded in 90% yield.
Ph
H2N
AcO
N
Ph
I
OAc
O
Phosphoric acid
Ph
Ph
Ph
O
Ph
Ph
P
O
O
DCM, diethyl ether
Ph
131
132
Scheme 49
114
Synthesis of phosphoric acid tribenzhydryl ester 1149
The protected diester (115) was reduced to protected diol (116) in 97% yield. Subsequent
mesylation of diol (116) afforded protected dimesylate (117) in 91% yield. The next step
in the synthetic pathway involved condensation of the dimesylate (117) with
diethylmalonate to afford cyclised diester (118) in 51% yield, which was subjected to
mono-hydrolysis of the ethyl ester function to afford the desired monocarboxylic acid
product (119) and (120) in 92% yield. Thus, similar reagents and conditions as described
by Ma et al. were utilized for the preparation of monocarboxylic acid product (119) and
(120). The next step in the pathway involved the Curtius rearrangement. Walker et al.9
required the amino function to be protected with a Boc group in order to carry out the
solid phase peptide synthesis later, to prepare the α-cycloPNA oligomers, Thus, Ma et
al.’s approach was slightly altered such that the alcohol used was tBuOH rather than
ethanol; all other reagents and reaction condtions remained unchanged. However, in this
case the reaction was found to be unsuccessful and the desired products (133 and 134)
were not obtained. The failure of this reaction was attributed to the use of the sterically
bulky tBuOH.
CO2Et
Ph2HCO
i
Ph2HCO
Scheme 59
CO2Et
CO2Et Ph2HCO
Ph2HCO
ii
CO2Et
118
CO2Et
CO2Et Ph2HCO
CO2H
CO2H
+
NHBoc +
NHBoc
Minor
Major
Minor
Major
120
119
133
134
Reagents and conditions: i) NaOH, H2O, ethanol; 0 C → r.t., 48 h ii)
DPPA, Toluene, tBuOH, r.t. → reflux, 18 h9
67
Results and Discussion
2.3.
Synthesis of key intermediate alcohol (143 and 144) via Schiff’s base route
using N-(diphenylmethylene)glycine ethyl ester (145)
O
OH
MeO
O
i
OMe
OCPh3
OMe
MeO
107
OCPh3
ii
O
135
OH
HO
O
136
iii
CO2Et
Ph3CO
OCPh3
v
+
N
Ph
139
CO2Et
Ph3CO
N
I
Ph
Ph
2:1
OCPh3
iv
I
OMs
MsO
Ph
137
138
140
vi
CO2Et
HO
NH2.HCl
141
Scheme 69,165
CO2Et
HO
+
2:1
vii
CO2Et
HO
CO2Et
HO
NHBoc
NHBoc +
NH2.HCl
143
142
2:1
144
Reagents and conditions: i) Ph3CCl, DBU, DCM, 0 C → r.t., overnight;
ii) LiBH4, THF, B-methoxy 9-BBN, 0 C → r.t., overnight; iii) triethylamine, CH3SO3Cl,
DCM, 0 C → r.t., overnight; iv) NaI / acetone, , 48 h; v) LiHMDS, THF, Ndiphenylmethylene)glycine ethyl ester (145), - 78 C, 2 h, → r.t., overnight; vi) diethyl
ether, 1M HCl (aq); r.t., 2h vii) Boc2O, NaCl, Na2CO3, CHCl3, H2O,  75 C, 1.30 h9,165
(S)-Dimethyl malate (107) had been used in the Curtius rearrangement strategy. Walker
et al. recognised that construction of the cyclopentane ring and generation of the amino
function could be achieved through the use of the appropriate Schiff’s base derived from
glycine. Thus, Walker et al. devised an approach to the required Boc-protected
cyclopentan-3-ol intermediate as outlined in Scheme 6 below and these researchers
showed that this strategy was viable (unlike the Curtius rearrangement route). The first
step in this optimized pathway involved protection of the hydroxyl group of (S)-dimethyl
malate (107) with a trityl group by treatment with trityl chloride in the presence of DBU
to afford protected diester (135) in a 99% yield. The protected diester (135) was then
reduced quantitatively to the diol (136) using lithium borohydride in the presence of a
catalytic amount of B-methoxy-9-BBN. Mesylation of both hydroxyl groups followed by
heating the resulting dimesylate (137) with sodium iodide in acetone, gave the more
68
Results and Discussion
stable 1,4-diiodide product (138) in an overall yield of 84% for the two steps. The next
step was the key cyclization reaction employing the commercially available Schiff’s base,
N-(diphenylmethylene)glycine ethyl ester (145). Thus, a solution of the Schiff’s base in
anhydrous THF was treated with a slight excess of LiHMDS, followed by the diiodide
(138). Upon work-up and purification, the cyclized compounds (139 and 140) were
afforded in a 98% yield as an inseparable mixture of the cis:trans products (relative to
the protected alcohol) in a 2:1 ratio.
The final steps in this route involved first concomitant removal of the trityl protecting
group and hydrolysis of the imino function under acidic conditions followed by Bocprotection of the generated amino moieties to give the desired cyclopentan-3-ol
intermediates (143 and 144) in 55% yield over the two steps. The two isomers were
successfully separated using column chromatography and the stereochemistry of the
minor product (15%) was assigned as (1S, 3S)- (i.e. trans isomer) from NOE
experiments.
The success of this route prompted us to follow the same approach for the preparation of
the intermediate cyclopentan-3-ol required for synthesis of the α-cycloPNA monomers.
The first step involved protection of (S)-diethyl malate (107) with a trityl group using
trityl chloride and DBU as a base to give compound (135) (Scheme 6) in an 85% yield
after work-up and purification. This yield was slightly lower than the 100% yield
reported by Walker et al.9
Subsequently, the trityl-protected compound (135) was reduced using lithium
borohydride in the presence of a catalytic amount of B-methoxy-9-BBN.9 This reduction
is thought to occur by activation of the B-methoxy-9-BBN (146) to form its hydride
(147), which then acts as the reducing agent for the ester (Scheme 7). A few minor
problems were encountered with the published protocol which centred on purification of
the crude product by column chromatography. Upon using the eluent diethyl ether as
described by Walker et al.,9 the desired compound (136) was found to streak from the
column and so alternative eluting systems were investigated. The best results were
obtained when a gradient (from 100% DCM to 98% DCM: 4% methanol) was applied.
The yield of (136) was found to be further improved by using fresh catalyst and
69
Results and Discussion
maintaining rigorously anhydrous conditions. Using this optimized procedure, pure diol
(136) was obtained in the slightly reduced yield of an 83% yield in comparison to the
reported 100% yield.
OMe
H
B
B
OMe
LiBH4, THF
147
146
Scheme 7
Active reducing agent derived from B-methoxy-9-BBN
The structure of (136) was confirmed from the 1H-NMR and 13C-NMR spectra recorded.
Both showed an absence of the two methyl ester signals (at δ 3.37 and δ 3.62 in the 1HNMR and at δ 170.1 and δ 171.9 in the 13C-NMR), which had been previously present in
the NMR spectra of the starting diester (135). Furthermore, the multiplet signal at δ 1.67,
quartet doublet signal at δ 3.18, and multiplet signal at δ 3.80 in the 1H-NMR spectrum
verified the presence of the three methylene groups which also corroborated formation of
(136).
The third step in the synthetic pathway reported by Walker et al.9 required the conversion
of the diol (136) into the dimesylate (137) (Scheme 6). Thus, a solution of the diol (136)
in DCM was treated with methanesulfonyl chloride in the presence of triethylamine at
ambient temperature. The dimesylate (137) was obtained in a 71% yield after
purification, compared with the reported yield of 93%.9 The structure of dimesylate (137)
was verified by 1H-NMR and
13
C-NMR spectroscopy. In the 1H-NMR spectrum two
singlets were observed at δ 2.89 and 2.92 corresponding to the methanesulfonyl protons
while in the 13C-NMR spectrum, two new methyl carbon signals were apparent at δ 37.1
and 37.2 due to the methanesulfonyl carbons.
The fourth step in Walker et al.’s strategy to the desired cyclopentan-ol intermediate
involved displacement of the two mesylate groups of (137) with iodide to obtain the
corresponding diiodo derivative (138) (Scheme 6).9 This involved performing a
Finkelstein reaction9,184 whereby a solution of (137) and sodium iodide in acetone was
70
Results and Discussion
heated under reflux for 2 days. Unfortunately, although this reaction was attempted
several times, the yield of the resulting diiodide (138) was never greater than 10% in our
hands, after work-up and purification (cf. literature yield of (138) was 93%). This
reaction was repeated using a fresh source of sodium iodide but this failed to lead to an
improvement in the yield of diiodide (138). The formation of the desired diiodo product
was substantiated by the disappearance of the two singlets at δ 2.89 and 2.92
corresponding to the methanesulfonyl protons in the 1H-NMR spectrum which had been
present in the 1H-NMR spectrum recorded for the starting material (137). Furthermore, in
the
13
C-NMR spectrum of diiodide (138), the 1- and 4-methylene carbon signals had
moved significantly up-field to δ 0.7 and δ 11.2, relative to their positions in the
13
C-
NMR spectrum recorded for dimesylate (137), as an iodo group is less electronegative
than sulfonate group.
It was decided to repeat the reaction and monitor it closely by TLC to see whether the trityl
group was lost early in the reaction or after diiodide (138) had formed. Since the original
reaction had been left at reflux for 2 days, it was envisaged that the latter was possible.
However, from periodic TLC analysis of the reaction mixture, it became apparent that loss
of the trityl group occurred from dimesylate (137) almost instantly, i.e. within 30 mins.
I
HO
OMe
N
PPh3, DEAD, MeI
dry THF, 0 °C r.t
OMe
N
O
O
O
O
148
Scheme 8185
94 %
O
O
149
Literature precedence for direct conversion of hydroxyl to iodo group185
Due to the problems encountered with a loss of the trityl group from dimesylate (137)
under Finkelstein conditions, it was decided to explore alternative routes to the desired
diiodo derivative (138). Joulie et al. have shown that the alcohol (148) can be directly
converted into the epimeric iodide (149) in 94% yield utilising a Mitsunobu type reaction
(Scheme 8).185
71
Results and Discussion
PPh3, MeI, DIAD, dry THF
0 °C  r.t, 24 h, r.t 25 °C, 24 h
OCPh3
OCPh3
OH
HO
I
I
138
136
Scheme 9
Direct
conversion
of
(2S)-2-trityloxybutane-1,4-diol
to
(2S)-2-
trityloxybutane-1,4-diiodide
Therefore, we decided to examine the utility of this approach for transforming diol (136)
into diiodide (138), as outlined in Scheme 9. The equivalents of the reagents was doubled
compared to Joulie et al’s method. Thus, a solution of diol (136) in dry THF was treated
with triphenylphosphine, DIAD and methyl iodide at 0 C. The reaction temperature was
then raised to r.t. and the progress of the reaction was monitored periodically by TLC
analysis. After a day, TLC still did not show the presence of the desired diiodo derivative
(138) but showed unreacted starting material and so the reaction was left for another 24
h. Subsequently, the reaction was worked up and the crude product obtained was
analysed by NMR spectroscopy. Disappointingly, the spectra recorded did not show the
presence of the diiodide (138) and showed unreacted starting material.
O
O
I2, Imidazole, PPh3
DCM, Reflux
OH
O
O
I
H3C
H3C
63 %
I
OH
151
150
Scheme 10187
Literature precedence for direct conversion of diol to diiodo group187
Ramig et al have reported another approach for the direct conversion of a diol into the
corresponding diiodo derivative.187 This involved treatment of the diol (136) with
triphenylphosphine, iodine and imidazole in dry toluene or dry DCM at reflux, as
outlined in Scheme 10. This protocol had been successfully implemented using
sterically-hindered primary alcohols.
Garegg et al.
186
have postulated a mechanism for this reaction, which is highlighted in
Scheme 11. It has been proposed that triphenylphosphine (152), iodine (153) and
imidazole (154) in toluene at reflux temperature forms a biphasic system. Initially, the
72
Results and Discussion
partially-soluble complex of triphenylphospine:imidazole:iodide (155) is produced,
which then undergoes reaction with the alcohol (157) and imidazole (154) is displaced.
Finally, the triphenylphosphine:alcohol:iodide (155) complex breaks down to afford the
more stable triphenylphosphine oxide (159) and the desired alkyl iodide (160).
Ph3P + I2 +
152
Ph3P
N
2HN
Ph3P
154
153
N
N
Ph3P
OR I
N
RI
159
160
PPh3, I2, Imidazole
dry DCM, reflux, 9 h
OCPh3
OH
I
I
138
136
Scheme 12
HN
Mechanism of direct conversion of alcohol to halide
OCPh3
HO
+
154
O +
Ph3P
158
Scheme 11
OR I
158
157
155
.HI
156
Ph3P
ROH
N
HN
+
I
155
+
I
N
N
Direct
conversion
of
(2S)-2-trityloxybutane-1,4-diol
to
(2S)-2-
trityloxybutane-1,4-diiodide
It was therefore decided to investigate this method for converting diol (136) directly into
diiodo derivative (138). Disappointingly, this too only gave (138) in the low yield of 5%,
after work-up and purification by column chromatography.
Based on our previous findings with the Finkelstein reaction with dimesylate derivative
(137), it was reasoned that perhaps thermal decomposition of (136) was occurring here
too. To investigate this possibility the solvent was changed to anhydrous DCM which
has a lower boiling point (40 C) than toluene (120 C). It was envisaged that this should
not prevent the reaction from occurring as Garegg et al.186 had proposed in their
mechanism that the solvent (in their case toluene) was acting merely as an inert heattransfer medium which dissolves the product as it forms.
73
Results and Discussion
OCPh3
OCPh3
I
I
161
162
Scheme 13
Possible elimination side products during direct conversion of alcohol to
halide
Surprisingly, this approach proved to be successful with the desired diiodo derivative
(138) being obtained in a 56% yield after work-up and purification. The optimum length
of time for this reaction was found to be 10-12 h and optimum number of equivalents of
iodine was shown to be 6.5. It should be noted that the numbers of equivalents of
imidazole should be slightly higher than iodine in order to neutralise the hydroiodic acid
side product formed during the course of reaction. The presence of hydroiodic acid could
potentially pose a problem in this case because it may cause removal of the trityl
protecting group. However, excess imidazole may also present a problem with (138) as it
may lead to elimination of the iodide (good leaving group) and the formation of the
unwanted alkene side products (161 or 162) (Scheme 13) or may undergo alkylation by
the iodide product.
CO2Et
OCPh3
I
I
CO2Et
Ph3CO
Ph
Ph
145
CO2Et
Ph3CO
+
N
N
Ph
138
LiHMDS, dry THF -78 oC, 2h,  r.t, overnight
Ph
139
N
Ph
Ph
140
Scheme 149,165 Cyclization of (2S)-2-trityloxybutane-1,4-diiodide9,165
Having prepared diiodide (138), we were now in a position to investigate the fifth step in
Walker et al’s strategy to the desired key cyclopentan-3-ol intermediate needed for the
synthesis of the α-cycloPNA monomers. This involved an intramolecular cyclization
through dialkylation of the glycine Schiff’s base (145) with diiodide (138) to give the
cyclic derivatives (139 and 140) as shown in Scheme 14.9 Therefore a solution of the
commercially available N-(diphenylmethylene)glycine ethyl ester (145) in dry THF at 78 C was treated first with lithium hexamethyldisilylazide (2 eq) followed by the
diiodide (138). It was important that the reaction was carried out under rigorously
anhydrous conditions as N-(diphenylmethylene)glycine ethyl ester (145) and lithium
74
Results and Discussion
hexamethyldisilylazide are both very sensitive to moisture. This problem had been found
previously to limit the scale of the reaction.9,189 The strong base deprotonates the N(diphenylmethylene)glycine ethyl ester (145) to generate an anion, which then undergoes
an SN2 reaction with diiodide (138) to form the intermediate monoalkylated product. The
lithium hexamethyldisilylazide then removes the second acidic proton from the N(diphenylmethylene)glycine ethyl ester component and intramolecular cyclization
subsequently occurs. The key to this synthesis is control of the reaction temperature as it
decides both the rate of reaction and stereoselectivity.
Unfortunately, all attempts to repeat this procedure resulted in failure. Purification of the
crude product afforded from this reaction led to the recovery of 60% of unreacted
starting material (138) as the only identifiable component. It has been reported that a
problem with this type of reaction is that the reagent N-(diphenylmethylene)glycine ethyl
ester (145) decomposes very quickly once it has been exposed to air.190 However, even
when a fresh sample of (145) was used, this reaction was still unsuccessful. We also
investigated the effect of adding just 1 equivalent of lithium hexamethyldisilylazide
initially, allowing the temperature to warm from –78 C to – 40 C over 30 min and then
bringing it back down to -78 C before adding a second equivalent of the base, to ensure
that there was sufficient base present for both deprotonation reactions. However, once
again no cyclised products or monoalkylated intermediates could be observed after
purification of the crude reaction mixture by column chromatography.
CO2Et
N
-78°C, 2h r.t, overnight
N
Ph
CO2Et
MeI,LiHMDS, dry THF
Ph
145
Scheme 15
Ph
Ph
163
Dialkylation of N-(diphenylmethylene)glycine ethyl ester
Since our attempts to form products (139) and (140) had been unsuccessful, it was
decided to explore the criteria for performing dialkylation reactions with N(diphenylmethylene)glycine ethyl ester (145). To facilitate this study, a model reaction to
study the dialkyation of glycine Schiff’s base was selected in which our diiodo derivative
(138) was replaced with the inexpensive reagent methyl iodide. Thus, a solution of N75
Results and Discussion
(diphenylmethylene)glycine ethyl ester in dry THF was cooled to –78 C and 2.2
equivalents of lithium hexamethyldisilylazide were added. The reaction mixture was then
kept at this temperature for 45 min, after which time 2.2 equivalents of methyl iodide
were added. After work-up and purification by column chromatography, the desired
dialkylated product (163) was obtained in a poor yield of 14%. The structure of (163)
was confirmed by the appearance, in the 1H-NMR spectrum recorded, of two singlets at 
1.38 and 1.44 which corresponded to the protons of the two methyl groups in (163).
CO2Et
i
N
Ph
Ph
145
Scheme 16191
Ph
CO2Et
Ph
N
Ph
Ph
CO2Et
Ph
NH2
ii
step i and ii = 24 %
Ph
165
164
Reagents and conditions: i) 10 eq powdered KOH, 5 eq BnBr, 0.1 eq
tetra-n-butylammonium bromide, 0 C → r.t., 16 h, ii) 1M HCl (aq), r.t., 48 h.191
It was decided to examine the effect of adding excess of methyl iodide on the outcome of
the dialkylation reaction. This was inspired by the findings of Lopez et al.191, which had
demonstrated that dialkylation of (145) with less than 2 equivalents of benzyl bromide in
the presence of powdered potassium hydroxide failed to give dialkylated imine (164)
(Scheme 16). However, when this reaction was repeated using either 5.0 or 10.0
equivalents of benzyl bromide followed by hydrolysis, dialkylated amine (165) had been
afforded in 24% overall yield.
The reaction was repeated as described above but 4.5 equivalents of methyl iodide were added
this time. However, after work-up and purification by column chromatography, the desired
dialkylated product (163) was still only afforded in a low yield 16%. Thus, the presence
of an excess of methyl iodide did not seem to significantly improve the yield of the
desired dialkylated product (163).
Despite the low yields of the dialkyated product received, the model reactions using
methyl iodide showed that it was possible to dialkylate N-(diphenylmethylene)glycine
ethyl ester using the reagents and conditions reported by Walker et al. for the preparation
of the cyclised products (139 and 140). It is also possible that the yields of dialkyated
76
Results and Discussion
product (139 and 140) in the crude product were higher than those determined following
purification as it was observed that the dialkyated product was not stable to column
chromatography.
In order to gain more insight into intramolecular cyclisation reactions using diiodide (138),
dialkylation of diethyl malonate (166) in place of N-(diphenylmethylene)glycine ethyl ester
(145), with diiodide (138) was next examined.184 The principle advantages of diethyl
malonate (pKa 15.7 in DMSO) are that it is stable and can be deprotonated more readily
than a Schiff’s base like N-(diphenylmethylene)glycine ethyl ester (pKa 18.7 in DMSO).
Thus, a solution of diethyl malonate (166) (3 eq) in dry THF was treated with freshly
prepared solution of sodium ethoxide (5 eq) at 0 C followed by diiodide (138) (1 eq) and
the resulting solution was subsequently heated at reflux for 6 h (Scheme 17). After workup and purification the desired cyclised product (167) was obtained in 93% yield. The
structure of (167) was confirmed by both 1D NMR spectroscopy and a 2D NMR C-H
correlation experiment. The 1H-NMR spectrum recorded showed two triplet signals at 
1.18-1.22 and  1.11-1.16 respectively, which corresponds to the two sets of methyl
protons of the ester group. A multiplet at  7.32-7.44 was also observed which was
assigned to the fifteen protons of the trityl group. The
13
C-NMR spectrum recorded for
(167) showed a quaternary carbon signal at δ 55.3; which was attributed to the C1 ring
carbon. Additionally, the CH signal corresponding to the C3 carbon of the cyclopentane
ring had moved significantly downfield to δ 75.0 relative to the signal position for this
carbon in the 13C-NMR spectrum recorded for the starting diiodide (138).
Diethyl malonate (166), NaOEt, dry THF
OCPh3
CO2Et
Ph3CO
I
I
CO2Et
r.t reflux, 6 h
138
Scheme 17
167
Dialkylation of diethylmalonate
The above results clearly demonstrated
that intramolecular cyclisation, involving
dialkylation with the sterically-hindered and acid sensitive diiodide (138), to generate a
cyclopentane derivative could be accomplished in high yield when the one-carbon
component was diethyl malonate and the base was sodium hydride.
77
Results and Discussion
CO2Et
OCPh3
I
I
Scheme 18
CO2Et
Ph3CO
Ph
Ph
145
CO2Et
Ph3CO
+
N
N
Ph
138
NaH, dry THF, r.t, 2 h, , 1h
Ph
139
N
Ph
Ph
140
Dialkylation of Schiff’s base
Based on this finding, it was decided to explore whether this intramolceular cyclisation
could be achieved with diiodide (138) and N-(diphenylmethylene)glycine ethyl ester
(145) if the original base LiHMDS was replaced with sodium hydride. Sodium hydride is
a stronger than LiHMDS. Thus, following Kurth et al’s protocol,199,200 a solution of N(diphenylmethylene)glycine ethyl ester (145) (1.0 eq) in dry THF was added dropwise to
a suspension of sodium hydride (2.2 eq) in dry THF. After 15 min, 1,4-diiodide (138)
(1.0 eq) was added dropwise. The reaction was stirred for 2 h at r.t. before being heated
at reflux for a further 1.15 h. This time, after work-up and purification, the desired
cyclised products (139 and 140) (Scheme 18) were afforded in an approximate yield of
15-20%. It was not possible to obtain an exact yield for these cyclised products as they
were found to co-elute with other reaction impurities. The formation of the mixture of
cyclised products (139 and 140) (1:1.05 ratio based on 1H-NMR) was verified by
spectroscopy and the analytical data collected were consistent with those reported by
Walker et al.9 In the 1H-NMR spectrum additional aromatic CH protons were observed
which were assigned to the diphenyl group. The triplets at δ 0.94-1.02 (minor isomer) and
0.89-0.96 (major isomer) respectively corresponded to the CH3 protons of the ethyl ester
group. Furthermore, the protons of the CH2 groups of both the 1- and 4-positions had been
shifted up-field relative to their positions in the 1H-NMR spectrum recorded for the starting
material (138), to δ 1.2-3.6. The
13
C-NMR spectrum corroborated the presence of a
quaternary carbon at δ 72.2, which was attributed to the C1 ring carbon atom.
As highlighted above, although the intramolecular cyclisation using diiodide (138) and N(diphenylmethylene)glycine ethyl ester (145) had eventually met with some success when
sodium hydride had been employed as the base, we had encountered several problems with
this strategy. We reasoned that one factor which may have contributed to these difficulties
was the bulkiness of the trityl protecting group of diiodide (138). This might have
sterically-hindered attack on the methylene carbon adjacent to it. The trityl group had also
78
Results and Discussion
caused problems earlier on in the synthetic pathway because of its labile nature under
acidic conditions. Due to these considerations, it was decided to explore the use of
alternative hydroxyl protecting groups in this strategy and the benzyl group was identified
as being worthy of pursuit. The benzyl group is reported to be more stable than a trityl
group in both acidic and basic media,9 but it can be cleaved selectively and readily by
hydrogenation.9 The revised pathway to the key cyclopentan-3-ol intermediate (139 and
140) is shown in Scheme 19 above.
O
OH
OMe
MeO
O
i
OCH2Ph
MeO
107
OCH2Ph
ii
OMe
O
168
OH
HO
O
169
iii
CO2Et
PhH2CO
+
N
Ph
Scheme 19
OCH2Ph
iv
N
I
I
Ph
171
CO2Et
PhH2CO
Ph
172
Ph
170
Reagents and conditions: i) BnBr, Ag(I) oxide, EtOAc, r.t., overnight; ii)
LiBH4, THF, B-methoxy 9-BBN, 0 C → r.t., overnight; iii) PhP3, I2, Imidazole, dry
DCM, reflux, 9 h; iv) NaH, N-diphenylmethylene)glycine ethyl ester, dry THF, r.t., 2 h,
→ reflux, 1 h.
Therefore, the first step in the revised route involved protection of the hydroxyl group of
the commercially-available starting material (S)-dimethyl malate (107) with the benzyl
group. Thus, a solution of (107) in ethyl acetate was treated with benzyl bromide in the
presence of silver(I) oxide (Scheme 20). This procedure had been reported by Walker et
al.9 The desired benzyl-protected product (168) was obtained in 55% yield after work-up
and purification. It has been suggested that the silver(I) oxide facilitates the co-ordination
and enhances the rate of reaction. Since the reaction is thought to progress through an SN1
mechanism it requires driving conditions. Surprisingly, this yield of (168) was a
significant improvement on the 20% yield reported by Walker et al.9 The formation of
the benzyl-protected product (168) was confirmed by NMR spectroscopy.
79
Results and Discussion
O
OH
OMe
MeO
EtOAc, r.t, overnight
O
O
BnBr, Ag(I)Oxide
OBn
OMe
MeO
O
168
107
Scheme 20
Benzylation of (S)-dimethyl malate
The second step in the synthetic strategy involved reduction of the diester (168) using
lithium borohydride in the presence of a catalytic amount of fresh B-methoxy-9-BBN
under the same conditions as had been utilized earlier for the corresponding trityl
analogue (Scheme 21).9 The desired diol (169) was obtained in 85% yield after work-up
and purification.192,193
O
OBn
OMe
MeO
OBn
LiBH4, B-methoxy 9-BBN, dry THF
OH
HO
0°C,  r.t, 3 h
O
169
168
Scheme 21
Reduction of (S)-dimethyl 2-(benzyloxy)succinate
Subsequently, a solution of diol (169) in dry DCM was treated with triphenylphosphine,
iodine and imidazole (Scheme 22). Following work-up and purification, the desired
diiodide (170) was obtained in quantitative yield. The increase in the yield for iodination
of the benzyl-protected diol (169), in comparison with the analogous trityl protected diol
(169), may be due to the decrease in the steric hindrance offered by the benzyl group.
OBn
OH
HO
OBn
Imidazole, PPh3, I2, dry DCM
r.t  reflux, 5 h
169
Scheme 22
I
I
170
Iodination of (S)-2-(benzyloxy)butane-1,4-diol
The intramolecular cyclisation involving the benzyl-protected diiodide (170) and N(diphenylmethylene)glycine ethyl ester (145) was next examined.
Since formation of the analogous trityl-protected cyclized products (139 and 140) had
80
Results and Discussion
been successful when sodium hydride had been used, the same base was employed here.
Thus, a solution of N-(diphenylmethylene)glycine ethyl ester (145) in dry THF was first
treated with sodium hydride (2.2 eq) followed by addition of the diiodide (170). After
work-up and purification by column chromatography, unfortunately in this case, none of
the desired cyclised products (171 and 172) (Scheme 23) could be identified in any of the
fractions obtained and only 45% of the starting material (170) was recovered. The failure
of this reaction was attributed to the instability of the benzyl-protected diiodide (170) this
was found to quickly degrade on storage. Before it could be used in the above reaction, it
needed to be re-purified by column chromatography.
CO2Et
NaH, dry THF, r.t, 2 hrs, , 1 h
OBn
Ph
170
Ph
Ph
145
Scheme 23
N
N
I
I
CO2Et
BnO
CO2Et
BnO
+
Ph
171
N
Ph
Ph
172
Cyclization of (S)-2-(benzyloxy)butane-1,4-diiodide
Since numerous problem had been encountered with both the trityl and benzyl routes to
the key cyclopentan-3-ol intermediate required for synthesis of the α-cylcoPNA
monomers and as they had both met with limited success at the critical intramolecular
cyclization step, this approach using the commercially available Schiff’s base N(diphenylmethylene)glycine ethyl ester (145) was abandoned. Hence, alternative routes
to the constrained α-cycloPNA monomers were sought.194
2.4.
Synthetic routes to the key cyclopentan-3-ol via hydroboration of the
corresponding cyclopent-3-ene (108)
Retrosynthetic analysis of the key intermediate cylopentan-3-ol (105) described earlier in
section 2.1 suggested that it could be prepared from the corresponding alkene (108) by
asymmetric hydroboration. A similar strategy has been reported by Hodgson et al.
181,182,183
for the synthesis of key intermediate cylopentan-3-ol (105)
As shown in Scheme 1, the first step in the synthetic pathway to the key cyclopentan-3ol intermediate (105) involved the synthesis of alkene (108). There are three general
81
Results and Discussion
strategies which have been reported in the literature to date for preparing alkene (108).
These involve either:
1) bis-allylation of a glycine equivalent followed by ring-closing metathesis
(RCM);194,195,196
2) intramolecular dialkylation of dialkyl malonate with a dielectrophile followed by
partial ester hydrolysis and a Curtius rearrangement;181,182,183, 197,198 or
3) cycloalkylation of a glycine equivalent with a dielectrophile.199,200
These three approaches will be discussed in more detail in the following sections.
2.4.1.
Ring-closing metathesis (RCM)
O
O
O
i
HOOC
N
H
ii
O
Ph
N
O
i and ii = 90 %
N
Ph
Ph
174
173
175
iii
CO2H
NH2.HCl
178
Scheme 24194
CO2Me
v
99%
NHCOPh
iv
91 %
99 %
COOMe
NHCOPh
176
177
Reagents and conditions: i) DCC, dry THF, 0 C → r.t.; ii) DIPEA, NaI,
allyl bromide, dry DMF, r.t., 6 h; iii) NaOMe, MeOH, r.t., 2 h; iv) Grubbs' catalyst, r.t., 6
h; v) 6 N HCl, refluxed 24 h194
Cativiela et al.194 have recently described a large scale synthetic route to alkene (108)
(Scheme 24). These researchers overcame the issue of the reactivity of glycine by
transforming it into 2-phenyl-5(4H)-oxazolone (174), which can be easily deprotonated
since the resulting anion is stabilized by both the phenyl substituent and the ketone
functionality. Thus, oxazolone (174) was readily prepared from N-benzoyl protected
glycine (i.e. hippuric acid) (173) upon treatment with DCC. Subsequently, oxazolone
(174) was bis-allylated by treatment with allyl bromide in the presence of N,Ndiisopropylethylamine (DIPEA) and sodium iodide. This was followed by oxazolone ring
opening and subsequently with ring-closing metathesis of the α,α-diallyl glycine
82
Results and Discussion
derivative (176) using Grubbs catalyst to give cyclopent-3-ene (177). The equilibrium of
the Grubbscatalysed reaction was shifted by removal of the product from the reaction
mixture. The final step involved removal of both the amino and carboxylic acid
protecting groups to afford 1-aminocyclopent-3-ene-1-carboxylic acid hydrochloride
(178) in an overall yield of 80%.
CO2t-Bu
i
CO2t-Bu
NC
CO2t-Bu
ii
NH2
NC
179
181
180
iii
CO2t-Bu
iv
CO2t-Bu
NHBoc
NHBoc
183
Scheme 25196
182
Reagents and conditions: i) Allylbromide, K2CO3, TBHAS, 70 C, 12 h,
ii) 33% HCl, EtOH, 5 h; iii) (Boc)2O, CHCl3, reflux; overnight iv) Grubbs' catalyst (10
mol%), CHCl3, reflux, 12 h.196
Brunel J. M. et al.196 have reported another RCM strategy to a 1-aminocyclohex-3-ene-1carboxylic acid derivative, as shown in Scheme 25. Here tert-butyl isocyanoacetate (179)
is first bis-allylated to afford the dialkylated product (180) in 86% yield. Subsequent acid
hydrolysis of isocyanide function to give (181) followed by protection of the resulting
primary amino group using di-tert-butyl dicarbonate gave rise to the Boc-protected
derivative (182) in 69% yield. The last step in this pathway involved using the ring
closing metathesis (RCM) reaction and employing the Grubbs catalyst, to afford the
desired protected alkene (183) in 89% yield.
2.4.2.
Curtius Rearrangement
Hodgson et al. have reported a synthetic route to alkene (188) involving the Curtius reaction
or Curtius rearrangement, in which an acyl azide is thermally decomposed to the
corresponding isocyanate via loss of N2.181,182,183 This pathway began with the dialkylation
of diethyl malonate (166) by treatment with cis-1,4-dichlorobutene (109) in the presence of
lithium hydride, which gave the cyclopent-3-ene derivative (185) in 79% yield (Scheme 26).
83
Results and Discussion
Subsequent mono-ester hydrolysis with alcoholic potassium hydroxide afforded the monocarboxylic acid (186) in 66% yield. Compound (186) was then converted into the acyl
chloride (187) in almost quantitative yield upon addition of oxalyl chloride. Finally, acyl
chloride (187) was treated with sodium azide to generate the intermediate acyl azide (188),
which was then subjected to the reagents and conditions required to accomplish the key
Curtius rearrangement reaction. This resulted in the formation of the desired protected alkene
(189) in 71% yield.
i
EtO2C
CO2Et
CO2Et
CO2Et
79 %
166
CO2Et
ii
COOH
66 %
186
185
iii
v
CO2Et
100 %
CO2Et
iv
CO2Et
COCl
CON3
NHBoc
71 %
189
(Steps 4 & 5)
Scheme 26181,182,183
188
187
Reagents and conditions: i) LiH, DMF, cis-1,4-dichloro-2-butene,
0 C → r.t., 72 h; ii) KOH, EtOH, r.t., 20 h; iii) oxalyl chloride, toluene, Δ, 2 h; iv) NaN3 /
H2O, acetone, < 6 C, 30 min; v) Toluene, 3 Å MS, tBuOH Δ 2 h, then SnCl4 Δ 1 h, → r.t.,
17 h. 181,182,183
Larinov et al.197 have reported a similar approach to Hodgson et al. for the preparation of the
analogous t-butyl ester protected alkene (183) (Scheme 27). The first step again involved
dialkylation of a dialkyl malonate (166) with cis-1,4-dichlorobutene (109) in the
presence of lithium hydride, although this time the dialkyl malonate used was t-butyl
methyl malonate (190) and the solvent was DME/DMU rather than DMF.
The methyl ester (191) was next selectively hydrolysed using a mixture of sodium
hypobromite and sodium bromide in NaOH to give mono-carboxylic acid (192) in 75100% yield depending upon the number of equivalents of sodium hypobromite and
sodium bromide. It was found that the key Curtius degradation could be subsequently
achieved under two different reaction conditions. The mono-carboxylic acid (192) could
be either (i) activated with ethyl chloroformate before being treated with sodium azide, to
84
Results and Discussion
generate the intermediate acyl azide, and then heated in the presence of tin(II) chloride,
or (ii) heated together with diphenylphosphoryl azide (DPPA) and t-butanol. The desired
protected cyclopent-3-ene (183) was afforded in 53% and 29% yields, respectively.
Cl
O
t-BuO
ii
i
O
OMe
iii
CO2t-Bu
CO2t-Bu
CO2t-Bu
CO2Me
COOH
NHBoc
Cl
190
109
Scheme 27197
183
192
191
Reagents and conditions: i) LiH, DME/DMU, 65 C, 72 h, 61% ii)
NaOBr/NaBr (17-30 eq), aq NaOH, 0 C, 3 h,
75-100% iii) Curtius degradation:
Condition A: a) ClCO2Et, Et3N, acetone -5 C, 2.5 h b) NaN3/ H2O; 0 C, 2.5 h c)
t
BuOH, SnCl4, 85 C, 5 h. (Condition A: overall yield 53%) Condition B: DPPA, Et3N,
t
BuOH, 85 C, 10 h; (Condition B: yield 29%).197
i
MeO2C
COOH
ii
CO2Me
CO2t-Bu
CO2t-Bu
190
CO2t-Bu
1 & 2 = 79 %
191
192
iii
NHBoc
CO2t-Bu
183
Scheme 28198
v
3-5 = 75 %
6 kg scale
iv
CON3
COCl
CO2t-Bu
194
CO2t-Bu
193
Reagents and conditions: i) DMPU, LiH, THF, 65 C; ii) NaOH, H2O;
iii) SOCl2, Et3N, toluene, MTBE; iv) a) NaN3, H2O, cat n-Bu4NHSO4 b) 95 C, toluene
v) a) KOt-Bu, THF, 0 C.198
Varie et al.198 have used an almost identical strategy to Larinov et al. for the preparation
of alkene (183), with a few variations (Scheme 28). Firstly, the methyl ester of (191) was
selectively hydrolyzed using aqueous NaOH, to give mono-carboxylic acid (192) in 79%
overall yield. Compound (192) was then converted to the Boc-protected amine (183) via
a four-step sequence involving acyl chloride (193) formation using thionyl chloride,
phase transfer-catalyzed preparation of the intermediate acyl azide (194), a Curtius
85
Results and Discussion
rearrangement to give isocyanate intermediate, and finally reaction with potassium tertbutoxide / tert-butyl alcohol. The resulting desired alkene (183) was obtained in 56%
overall yield.
Varie et al.198 overcame one of the drawbacks reported by Hodgson et al. and Larinov et
al.,197 namely the use of the highly toxic and environmentally damaging compound
tin(IV) chloride as a Lewis acid. This had been introduced to improve the migration
aptitude of the migrating carbon at the α-position in the alkene, which is reduced because
it is adjacent to an electron-withdrawing group. However, Varie et al.198 realized that tin
salts were in fact not needed as it was shown that it was actually the lack of reactivity of
tert-butanol which was increased by using tert-potassium butoxide instead. In this
manner, not only the tin salt was avoided but also the overall yield of the product was
increased.
2.4.3.
Cycloalkylation of a glycine equivalent with a dielectrophile
CO2Et
NH2.HCl
Br
i
CHO
Cl
CO2Et
CO2Et
ii, iii, iv
N
Cl
NHBoc
95 %
Br
184
Scheme 29199,200
196
109
197
189
Reagents and conditions: i) MgSO4, TEA, dry DCM, r.t., 10 h; ii) NaH,
dry THF, r.t., 2 h, reflux, 1h iii) 1N HCl, ether, r.t., 2 h; iv) [(CH3)3COCO]2O, CHCl3,
reflux 5 h.199,200
Kurth et al.199,200 have reported a synthetic route to the cyclopent-3-ene derivative (189)
based on cycloalkylation of a glycine equivalent with a dielectrophile, as shown in
Scheme 29. There were a few disadvantages with Larinov et al.197 and Hodgson et
al.181,182,183 routes. It was difficult to modify functional group on the ester group and
amino group of alkene (108), which would be critical in terms of further exploration of
facial selectivity, steric influence and substrate directed hydroboration reaction, as
described in section 2.4.5. The use of SnCl4 as a Lewis acid was not desirable as tin salts
are both highly toxic and environmentally damaging. Another disadvantage with Larinov
et al. route was huge excess of reagent in the haloform reaction as shown in Scheme 27.
86
Results and Discussion
Thus we opted for Kurth et al.199,200 synthetic route to synthesize the cyclopent-3-ene
derivative (189), as it overcomes the above short comings.
The first step in the synthesis of intermediate alkene (189) involved condensation of
commercially available 4-bromobenzaldehyde and ethyl glycine hydrochloride to afford
bromo Schiff’s base (197) in 93% yield. The Schiff’s base was dialkylated with reactive
bis-alkylating agent, such as cis-1,4-dichlorobutene (109), when sodium hydride had been
employed as the base to give cyclised product (199) in 80% yield. Subsequent hydrolysis
of the imine with 1N hydrochloric acid afforded cyclised amine (201) in quantitative
yield. Finally, Boc protection of amine (201) with Boc-anhydride afforded the desired
alkene (189) in 93% yield as described in Scheme 29.
Using this route, Kurth et al.199,200 obtained a 56% overall yield for the intermediate
alkene (189).
2.4.4.
Preparation of intermediate alkene (108)
Due to the advantages outlined above in Section 2.4.3., we decided to employ the
approach described by Kurth et al.199,200 for the synthesis of intermediate alkene (108).
The first step involved preparation of the Schiff’s base (197 or 198). This was
successfully accomplished using the method described by Kurth et al.,199,200 in which a
solution of the commercially available p-bromobenzaldehyde (184) in dichloromethane
was reacted with either glycine ethyl or methyl ester hydrochloride (196 or 195) in the
presence of triethylamine and anhydrous magnesium sulphate (Scheme 30). This gave
the corresponding Schiff’s base (197 or 198) in 93% and 75% yields respectively, after
work-up and purification.9,199,200
COOR
dry TEA, MgSO4, dry DCM
CHO
Br
184
Scheme 30
HCl. H2N
CH2
COOR
0°C,  r.t, 10 h
N
Br
R = Et =196
R = Et =197
R = Me =195
R = Me =198
Preparation of ethyl 2-[(4-bromophenyl)methylidene]amino]acetate and
methyl 2-[(4-bromophenyl)methylidene]amino]acetate
87
Results and Discussion
The most intriguing aspect of Schiff’s base (198) was its stability in comparison to the
ethyl analogue (197); even after a week of storage in a fridge no obvious degradation was
detected.
COOR
N
Cl
Br
R = Et =197
NaH, Dry THF
COOR
r.t 2 h, Reflux, 1 h
N=CH-C6H4-Br
Cl
R = Et =199
109
R = Me =198
Scheme 31
R = Me =200
Cyclization of bromo Schiff’s base
The second step in the route to the required cyclopent-3-ene intermediate involved the
intramolecular cyclization reaction between the Schiff’s base (i.e. (197) or (198)) and
commercially available cis-1,4-dichlorobutene (109) (Scheme 31). Following Kurth et
al’s protocol,199,200 a solution of the appropriate Schiff’s base [i.e. (197) or (198)] (1.0 eq)
in dry THF was added dropwise to a suspension of sodium hydride (2.2 eq) in dry THF.
After 15 min, cis-1,4-dichlorobutene (109) (1.1 eq) was added dropwise. The reaction
was stirred for 2 h at r.t. before being heated at reflux for a further 1 ¼ h. After work-up
compounds (199) and (200) were afforded as impure products. Since Kurth et al.199,200
had reported that these products degrade upon performing column chromatography, we
decided not to purify (199) and (200) further, but to use them crude in the next step of our
synthetic strategy.
The third step in the strategy involved hydrolysis of the imine function of (199) or (200) to
generate the corresponding amino derivative (201) or (202) respectively (Scheme 32). This
was achieved using the procedure reported by Kurth et al.199,200 with the exceptions that
the number of equivalents of 1N HCl used was raised to 3, the reaction solvent was
changed from THF to diethyl ether and the reaction time was increased from 30 min to 2
h.199,200 These changes were inspired by the protocol described by Walker et al.9 for the
hydrolysis of cyclised products (139 and 140) with similar imino moiety. After work-up,
the desired products (201 and 202) were obtained in crude yields of 53% and 43%
respectively. The formation of amino derivative (201) was confirmed by 1H-NMR
spectroscopy. There was an absence of aromatic signals between δ 7.66-7.70 and δ 7.517.57, and an absence of the imine signal at δ 8.13. Furthermore, a new signal was apparent
88
Results and Discussion
at δ 5.68 which was assigned to the amino protons.
COOR
COOR
1N HCl, Ether
NH2
N=CH-C6H4-Br
r.t, 2 h
R = Et =199
R = Et =201
R = Me =200
R = Me =202
Scheme 32
Hydrolysis of Imines
The final step in the pathway to the key cyclopent-3-ene intermediate involved the Bocprotection of the amine functions of (201) or (202) (Scheme 33). This was achieved
following the protocol reported by Kurth et al.199,200 Thus, a solution of impure amine
(201) or (202) in chloroform was added to a solution of Boc-anhydride in chloroform and
the resulting mixture was heated at reflux for 5 h. The crude products obtained after
work-up were purified by column chromatography to afford pure Boc-protected amines
(189) and (222) in overall yields of 27% and 32% respectively for the last 3 steps.
Unfortunately, both these yields were lower than the overall yield of 67% which had
been reported by Kurth et al.199,200
COOR
COOR
[(CH3)3COCO]2O
NHBoc
NH2
CHCl3 reflux 5 h
R = Et = 201
R = Et = 189
R = Me = 202
R = Me = 222
Scheme 33
Boc protection of Amine
In conclusion, the desired Boc-protected alkene intermediates (189) and (222) were
successfully synthesized following the protocol described by Kurth et al.199,200 with minor
modifications, from the appropriate ethyl or methyl Schiff’s base (197) or (198)
respectively in three steps in overall yields of 27% and 32% respectively. As per the
proposed revised route to the key cyclopentan-3-ol intermediate (105) required for
synthesis of the α-cycloPNA monomers (see Scheme 33), alkene (189) was subjected to
hydroboration according to the protocol described by Hodgson et al. The outcome of these
studies will be discussed in the following section.
89
Results and Discussion
2.4.5.
Synthesis of key intermediate cis-alcohol (203 and 143) and trans-alcohol (204
and 144).
Hodgson et al. have reported two strategies to obtain the key intermediate cis-alcohol
(203 and 143) and trans-alcohol (204 and 144) from alkene (189): i) epoxidation of alkene
(189)183 and ii) hydroboration of alkene (189).181,182,183 The work reported in this thesis
relates to the latter approach. The hydroboration reaction has been studied in great depth
by many eminent chemists, notably Brown who received a Nobel Prize in 1979 in
recognition of his contribution to this area of chemistry.205 Space limitations mean that
discussion of this field has to be restricted to those areas which are of direct relevance to
the work carried out here.205 The factors which have been found to affect the outcome of
hydroboration reaction undertaken by Hodgson et al. are substrate-directed
hydroboration, solvent effects and facial selectivity. Each of these will be considered in
more detail below.
CO2Et
HO
HO
NHBoc
cis
203
HO
CO2Et
NHBoc
204
143
CO2Et HO
CO2Et
NHBoc
NHBoc
144
trans
i
CO2Et
HO
HO
CO2Et
NHBoc
203
Major
cis
CO2Et
ii
cis
203
189
143
HO
CO2Et
NHBoc
NHBoc
NHBoc
Minor
Minor
Scheme 34181,182,183
CO2Et
HO
iii
NHBoc
143
Major
Reagents and conditions: i) BH3.THF, dry THF, 0 C → r.t., 17
h, ii) Boron trifluoride etherate, (+)-IpcBH2.TMEDA, dry THF, -40 C, 72 h, iii) Boron
trifluoride etherate, (-)-IpcBH2.TMEDA , dry THF, -40 C, 72 h.181,182,183
Hodgson et al. obtained a mixture of cis-alcohols (203 and 143) and trans-alcohols (204
and 144) upon treatment of alkene (189) with borane-THF complex under the conditions
described in Scheme 34, followed by standard oxidation. Subsequent purification of the
crude mixture gave cis-alcohols (203 and 143) in a combined yield of 58% and transalcohols (204 and 144) in a 4% yield. However, when alkene (189) was treated with
boron
trifluoride
etherate
in
the
presence
of
either
(+)-
or
(-)-
monoisopinocampheylborane the mixture of cis-alcohols (203 and 143) were afforded in
90
Results and Discussion
a combined yield of 77% in the former case and 74% in the latter. Furthermore, addition
of (+)-monoisopinocampheylborane resulted in the formation of the (1S, 3R)diastereoisomer (203) in a 47% enantiomeric excess (e.e.) whereas addition of (-)monoisopinocampheylborane afforded the (1R, 3S)-cis-alcohol (143) in a 48% (e.e.).
The factors influencing this hydroboration reaction will be considered below.
i) Substrate directed hydroboration
Substrate-directed hydroboration is limited to a few specific examples in the literature.
Hydroboration of alkene (189) (Scheme 35) with BH3.THF followed by oxidation gave
products (203 and 143) and (204 and 144) in a cis:trans ratio of 94:6.183 Here the cis
products (203 and 143) refers to the compounds in which the hydroxyl group is located
on the same face as the NHBoc moiety and the trans products (204 and 144) denotes the
compounds where these groups reside on opposite sides. This finding implied that a
strong carbamate directing effect may be operating as it had been expected that the ethyl
ester would impede the approach of borane less than the NHBoc group. Hence,
hydroboration had been predicted to take place cis to the ester (i.e. trans to the NHBoc
group), on the less hindered face of the molecule.181,182,183
CO2Et
NHBoc
189
HO
1 BH3.THF, THF
2 water, NaOH, H2O2
63 %
203
CO2Et HO
CO2Et
NHBoc
NHBoc
HO
204
143
cis
CO2Et HO
CO2Et
NHBoc
NHBoc
trans
144
cis : trans = 94:6
Scheme
35181,182,183
Hydroboration
of
tert-butyl
1-(ethoxycarbonyl)-3-
hydroxycyclopentylcarbamate181,182,183
To investigate whether coordination between borane and the NHBoc group may be
directing hydroboration, the simpler alkene (205) (Scheme 36) was treated under similar
condition with BH3.THF, followed by oxidation.183 In this case, alcohols (206) and (207)
were afforded in a cis:trans ratio of 23:77. This result was in line with previous
expectations and indicated that hydroboration of (205) was not purely directed by the
NHBoc group.181,182,183
91
Results and Discussion
NHBoc
NHBoc
1 BH3.THF, Solvent
NHBoc
2 Water, H2O2, NaOH
56 %
HO
205
206
HO
23
Scheme 36183
207
77
Hydroboration of tert-butyl cyclopent-3-enylcarbamate183
The hydroboration of 4-methyl-cyclopent-3-ene (208) (Scheme 37) followed by
oxidation gave products (209 and 210) in a cis:trans ratio of 17:83.183 It was envisaged
that the approaching borane would encounter less steric hindrance from the methyl group
of (208) than from the NHBoc of (205), and so the proportion of the trans-alcohol
product had been predicted to significantly increase. However, since the increase
observed had not been dramatic, it was reasoned that the NHBoc groups of (205) and
(189) were involved to some extent in directing the approach of the hydroborating agent.
CH3
CH3
1 BH3.THF, Solvent
CH3
2 Water, H2O2, NaOH
56 %
208
Scheme 37183
HO
209
HO 210
17
83
Hydroboration of 4-methyl cyclopentene183
The hydroboration of alkene (211) (Scheme 38) has also been studied.183 Here, the
NHBoc group is one carbon atom further removed from the cyclopentane ring than in
alkene (205); this was expected to reduce the effect of any coordination between the
NHBoc group and the approaching borane. However, this spacer also reduced the steric
hindrance to borane attack on the substituted side of the cyclopentene ring. As a result,
these two effects were found to balance out with the ratio of the cis-:trans-alcohols (i.e.
212:213) being the same as for alkene (208).181,182,183
NHBoc
NHBoc
NHBoc
1 BH3.THF, Solvent
2 Water, H2O2, NaOH
211
63 %
HO
212
HO
213
22
Scheme 38183
78
tert-butyl (cyclopent-3-enyl)methylcarbamate183
92
Results and Discussion
Based on all these findings, it seems that an NHBoc group may be able to direct the
hydroboration of an alkene, leading to preferential formation of the cis-alcohol rather
than the corresponding trans-product. However, the extent to which this moiety
influences cis-product production is yet to be resolved.
ii) Solvent effects
A solvent study on the hydroboration of the alkene (189) with BH3.SMe2 has been
carried out to examine the influence of the reaction solvent on the facial selectivity of
this reaction.183
Treatment of alkene (189) with BH3.SMe2 in THF was found to give virtually the same
ratio of cis:trans products (203 and 143 : 204 and 144) as had been obtained with
BH3.THF. This indicated that the Lewis base, to which the borane is complexed, did not
influence the facial selectivity of this reaction. However, when the reaction was carried
out in diethyl ether, pentane or CH2Cl2 instead, a gradual increase in the production of
the cis-products was observed (Table 2). Unlike THF and diethyl ether, dichloromethane
and pentane do not coordinate to borane. Thus, the results obtained in this study imply
that in the absence of a solvent to which the borane can co-ordinate, the borane reagent is
free to interact with the NHBoc group of alkene (189) so leading to an increase in the
proportion of cis-products received.
No Solvent
Ratio
of
reaction
HO
products
trans-
cis-
alcohol
alcohol
1
THF
22
78
2
CH2Cl2
46
54
3
Pentane 37
63
4
Ether
71
29
CO2Et HO
CO2Et
NHBoc
NHBoc
203
HO
204
Figure C
Table 2183
Solvent effect on Hydroboration183
93
cis
143
CO2Et HO
CO2Et
NHBoc
NHBoc
trans
144
cis-alcohol and trans-alcohol
Results and Discussion
The above investigations suggest that the weak NHBoc directing effect operating in the
hydroboration of (189) can be enhanced by carrying out the reaction in non-coordinating
solvents like CH2Cl2.
iii) Facial selectivity
It was postulated that if the NHBoc group of alkene (189) was directing hydroboration
there should be liberation of hydrogen gas during the course of the reaction; this would
lead to the association of the boron with the NBoc group via formation of an ion pair or a
covalent bond. Based on this reasoning, the amount of hydrogen gas was recorded by
Hodgson et al. and the composition of the reaction mixture at various times was analysed
by HPLC analysis.181,182,183
OEt
O
H
N
OEt
O
O
H
N
O
O
O
B
H B
H
H
H
214
Figure D
215
Facial selectivity
The HPLC traces collected after 6 min, 30 min and 17 h showed that the ratio of cis: trans
products (203 and 143:204 and 144) changed from 77:23, to 84:16 to 94:6 respectively, and
that the reaction had gone to completion after 6 min. The increase in the proportion of cis
product over trans product with time was explained by the selective removal of the transalcohol products (204 and 144) through a second side reaction. This conclusion was
supported by the finding that the total yield of the reaction declined with time. The
preferential removal of the trans-alcohols (204 and 144) was attributed to the fact that the
ester group of the trans-organoborane intermediate was selectivity activated to undergo a
hydride reduction through the formation of a Lewis acid-base complex between the boron
and the carbonyl group of the ester function (215) (Figure D).181,182,183
In conclusion, the findings of Hodgson et al.181,182,183 provide evidence that the
hydroboration alkene (189) with BH3.THF is accompanied by two side reactions: (i)
94
Results and Discussion
deprotonation of the NHBoc group and (ii) possible selective reduction of the ester group
of the trans-organoborane intermediate. Although deprotonation of the NHBoc group does
not appear to influence the final outcome of the reaction, subsequent reduction of the transalcohols significantly enhances the relative amounts of cis products present in the reaction
mixture with time.
2.4.6.
Preparation of cis-alcohol (203 and 143) and trans-alcohol (204 and 144)
via hydroboration with borane-THF complex
Taking on board the findings described by Hodgson et al., 181,182,183 the hydroboration of
alkene (189) was attempted. Since, both the cis alcohols (203 and 143) and the transalcohols (204 and 144) (Scheme 39) were required for synthesis of all four
diastereoisomers of the α-cycloPNA monomers, this reaction was conducted for only 7
min in order that the trans-products may be retained. Thus, a solution of alkene (189) in
dry THF was treated with the BH3.THF complex at 0 C for 7 min. Subsequently, pH 7.0
buffer was added and the reaction mixture was left overnight. After work-up and
purification using column chromatography, the two diastereoisomer pairs of alcohols [i.e.
cis-diastereoisomers (203 and 143) and trans-diastereoisomers (204 and 144)] (68:32)
were isolated in a combined yield of 48% for the cis-products and 23% for the transproducts.181,182,183 Interestingly, when Hodgson et al. conducted similar hydroboration
reaction of alkene (189) for only 6 min, the observed diastereomeric ratio of cis-alcohols
and trans-alcohols [(203 and 143) : (204 and 144)] was 77:23 and total isolated yield of
alcohols was 84%.
CO2Et
NHBoc
1 BH3.THF, THF
0°C, 7 min
CO2Et
HO
CO2Et
HO
NHBoc
2 Water, H2O2, NaOH
CO2Et
HO
HO
CO2Et
NHBoc
NHBoc
NHBoc
71 %
189
203
cis
143
204
trans
144
cis : trans = 68: 32
Scheme 39
Hydroboration of alkene
The structures of both the mixture of cis-alcohols (203 and 143) and trans-alcohols (204
and 144) were confirmed by spectroscopy and the NMR spectra recorded were consistent
with those reported by Hodgson et al.181,182,183 The most distinguishing differences in the
95
Results and Discussion
1
H-NMR spectra obtained for the cis-alcohols (203 and 143) and trans-alcohols (204 and
144) were the broad chemical shifts of the signal assigned to the NH proton. This broad
signal was found to reside downfield at δ5.84 in the 1H-NMR spectrum recorded for the
mixture of cis-alcohols while the same signal appeared upfield at δ5.07 in the 1H-NMR
spectrum recorded for the mixture of trans-alcohols. These differences may be due to the
proximities of the NH group to the 3-OH group in each set of diastereoisomers.
Although, all the four desired intermediate alcohols were obtained by hydroboration of
alkene (189) by BH3-THF complex, it would be more convenient to isolate cis-alcohols
(203 and 143) by selective removal of the minor isomer i.e. trans-alcohols (204 and 144),
to proceed, since chromatographic separation of the two alcohols is difficult, especially
when the reaction is performed on large scale. Diastereoselective synthesis of cisalcohols (203 and 143) could be explored by asymmetric hydroboration of alkene (189),
as reported by Hodgson et al.183
2.4.7.
Asymmetric Hydroboration
2.4.7.1.
CO2Et
NHBoc
Hydroboration with [(+)-IpcBH2]2TMEDA or [(-)-IpcBH2]2TMEDA
1 (-)-IpcBH2/ THF, -36°C, 72 h
CO2Et
HO
NHBoc
203
Major
CO2Et
1 (+)-IpcBH2/ THF, -36 °C, 72 h
NHBoc
2 Water, H2O2, NaOH
NHBoc
143
cis
Minor
CO2Et
HO
HO
CO2Et
NHBoc
203
Minor
Scheme 40
CO2Et
2 Water, H2O2, NaOH
189
189
HO
cis
NHBoc
143
Major
Asymmetric hydroboration reported by Hodgson et al.
Hodgson et al. have demonstrated that cis alcohols (203 and 143) can be obtained
diastereoselectively via hydroboration of alkene (189) by using the chiral hydroborating
agent of monoisopinocampheylborane is used.181,182,183 These researchers showed that
hydroboration of alkene (189) with [(+)-IpcBH2]2TMEDA afforded the (1S, 3R) cisalcohol (203 and 143) with (203) as major diastereoisomer in a 77% yield with a 47%
96
Results and Discussion
e.e.. Similarly, hydroboration of alkene (189) with [(-)-IpcBH2]2TMEDA gave the other
diastereoisomer, (1R, 3S) cis-alcohol (203 and 143) with (143) as a major
diastereoisomer, in a 74% yield with a 48% e.e.. Inspired by these findings, it was
decided to employ the same method here for the preparation of the key cyclopentan-3-ol
intermediate required for synthesis of the -cycloPNA monomers.
BF3.Et2O was first added dropwise to a solution of either [(+)-IpcBH2]2TMEDA or (-)IpcBH2]2TMEDA in dry THF. After stirring for 1 h a white precipitate had formed. The
reaction mixture was filtered under nitrogen and the filtrate was subsequently cooled to 45 to -35 °C. A precooled solution of alkene (189) in dry THF then was added slowly
dropwise and the resulting reaction mixture was stirred at -45 to -35 °C for 2 h before
being stored in a freezer at -36 °C for 72 h. The reaction was quenched by standard
oxidation
and
the
crude
product
afforded
was
purified
by
column
chromatography.181,182,183
The reaction with [(+)-IpcBH2]2TMEDA was found to produce a mixture of the two cisalcohols (203 and 143) in a combined yield of 67% yield. However, trans-alcohols (204
and 144) were also formed in a 2% yield. The reaction with [(-)-IpcBH2]2TMEDA gave
cis-alcohols (203 and 143) in a combined yield of 44% and again with trans-alcohols
(204 and 144) in a 17% yield. Unfortunately, the results achieved by Hodgson et al. were
not reproduced here. The reason for this is unknown but the fact that we were unable to
use a cryostat to maintain the low reaction temperature required over a long period may
have contributed.181,182,183
Since we had been able to successfully obtain the key alcohol intermediate as a
diastereomeric mixure of cis-alcohols (203 and 143) and trans-alcohols (204 and 144) in
relatively good yields of 48% and 23% respectively through the hydroboration of alkene
(189) with BH3-THF complex in dry THF at 0 C for 7 min, no further work was
undertaken on the asymmetric hydroboration.
Walker et al. synthesized the key alcohol in an enantioselective manner by fixing the
chirality of the alcohol at 3-position. It was essential for us to separate these enantiomers
in order to get all the four diastereosiomers.
97
Results and Discussion
2.4.8.
Chiral Resolution
We decided to investigate the feasibility of separating the enantiomer at this stage of the
revised route by chiral resolution.
The conventional procedure is based on separation of enantiomer via formation of
diastereoisomers by employing a chiral auxiliary. This method is often used on both
large and small scales due to its simplicity and reliability; however the major drawback
to the resolution is its limitation to certain functional groups like acids, alcohols and
bases.
Another conventional approach which is often employed to achieve chiral resolution
centres on the formation of a diastereomeric salt. It has been reported that when a
solution of the racemic (±) acid (216) and (-)-(s)-brucine (217) (Scheme 41) in methanol
was refluxed overnight.214 After 24 h, the precipitated salts were collected by filtration
and three recrystallization gave (+) acid: (-)-(s)-brucine (217), respectively. The
corresponding complex was treated with 25% aqueous solution of ammonium hydroxide
followed by hydrochloric acid. This afforded the separated diastereoisomers (218) in
71.5%. The more soluble was isolated by combining methanol fraction and evaporating
to dryness invacuo gave salts, which on three recrystallization gave (-) acid: (-)-(s)brucine (217) respectively, which on similar treatment
as
above afforded
diastereoisomers (219) in 19.5% yields, respectively.214
COOH H
N
COOH H
N
O
ii) Recrystallisation (MeOH)
NH
NH
H
i) (-)-(S)-Brucine (217) / MeOH
O
H
O
O
H
iv) HCl
O
COOH H
N
O
O
NH
NH
iii) NH4OH (25 %)
216
Scheme 41214
COOH H
N
H
O
218
219
71.5 %
19.5 %
Resolution by Brucine (217) 214
Inspired by the above findings of Ezquerra et al.,214 it was decided to explore this strategy for
the resolution of the mixture of cis-alcohols (203 and 143). In order to do this, the ester
protecting group first had to be removed. This was successfully accomplished by treating a
98
Results and Discussion
solution of (203 and 143) in dioxane with a 1N aqueous solution of sodium hydroxide at
room temperature for 24 h. The 1H-NMR spectrum recorded for mixture of cis-acids (220
and 221) isolated from this reaction was comparable to that reported by Hodgson et al.183
HO
CO2Et HO
CO2Et
NaOH, Dioxane
NHBoc
NHBoc
pH 11.0, r.t, 24 h
203
Cis
Scheme 42
HO
220
143
COOH HO
COOH
NHBoc
NHBoc
Cis
221
Hydrolysis of ester
N
HO
COOH HO
COOH
NHBoc
NHBoc
HH
O
Ether: Hexane
H
O
N
O
H
220
cis
221
220:217 complex
221:217 complex
O
217
Scheme 43
Chiral resolution of (1S, 3R) and (1R, 3S)-acid (220 and 221) by brucine
(217).
Having synthesised the desired mixture of cis-acids (220 and 221), chiral resolution was
next attempted involving formation of the diastereomeric salts of brucine.214 Thus, a
solution of the mixture of cis-acids (220 and 221) in methanol was heated to reflux with
(S)-brucine dihydrate (217) overnight before being allowed to crystallize at r.t. for 24 h
(Scheme 43).215 Unfortunately, no crystals formed. Therefore, we preferred to look into
other approaches of resolving cis-alcohols (203 and 143).
An alternative approach which has been used for chiral resolution is based on
enantioselective separation via complex formation of hydrogen-bond donor-acceptor, which
unlike the conventional method can be used to separate compounds containing any
functional group.215,216,217,218 Enantioselective inclusion complex is the formation of complex
between a resolving agent (like taddol) and one enantiomer [like (-) menthol] of the racemate
(like ± menthol).219 The formation of complex is by the virtue of intramolecular and
intermolecular H-bond networks for inclusion crystal formation, and resulting through
selective diastereoisomer crystallization, in the successful enantioselective inclusion
99
Results and Discussion
complexes.
O
O
R
R
R OH HO R
Figure E
Taddol Derivative
Taddols are chiral auxiliaries derived from tartaric acid and contain two adjacent
diarylhydroxymethyl groups in a trans relationship on a 1,3-dioxolane ring.218 The taddol
auxiliary system has been shown to associate intermolecularly with H-bond acceptors in
solution (NMR chiral shift reagent) 220,221 or solid state (crystallization).223
The general protocol which is employed in the literature to resolve racemic mixture is as
follows: A solution of chiral host and racemic guest compounds in an appropriate solvent
is either kept at r.t. or heated to reflux before being allowed to crystallize at r.t. or colder
temperature. The inclusion complex formed is collected and purified by further
recrystallization once formed. The purified inclusion complex can be finally dissociated
into its components by application of an appropriate procedure such as distillation,220
chromatography,220 or extraction with base or acid.219 Diethyl ether215 and methanol222
are used widely for resolving polar compounds, while hexane219 and pentane220 are
widely used for resolving non-polar compounds with taddol.
Seebach et al.220 have successfully employed the use of taddol (224) to resolve racmenthol (225). A solution of rac-menthol (225) and taddol (224) in pentane was heated
to reflux, cooled to r.t. before being stored for 2 weeks at - 18 C (Scheme 44).
Following a single re-crystallization of the precipitate afforded, clathrates were obtained
composed of the (-)-menthol and taddol in a 1:1 ratio [(-)-225:224]. This complex was
subsequently degraded by column chromatography and bulb to bulb distillation in order
to release the (-)-menthol enantiomer (-)-225 from (224). This gave (-)-225 in 52% e.e.
as outlined in Scheme 64.220
100
Results and Discussion
O
O
O
OH HO
HO
223
Scheme 44220
OH HO
HO
225
224
O
224
Enantiomer enrichment by taddol220
Since our compounds were polar and 14 days of storage seemed to be impractical, we
preferred to employ the general protocol to resolve the mixture of trans-alcohols (204 and
144). Thus, a solution of the mixture of trans-alcohols (204 and 144) (Scheme 65) and
taddol (224) in diethyl ether, was heated to reflux. Thereupon, hexane was added dropwise
until the solution became slightly turbid in appearance. The solution was then cooled to r.t.
and it was kept at this temperature for 24 h. However, since no crystals had formed during
this time, the solution was cooled to 0 C and this temperature was maintained for another
24 h. As no crystals had again formed, the solution was stored at -36 C for a further 24 h.
Unfortunately, neither one of the host-guest complexes precipitated from the solution even
at this reduced temperature.
O
O
OH HO
HO
CO2Et HO
CO2Et
NHBoc
NHBoc
204:224 complex
224
204
trans
Scheme 45
144:224 complex
144
Chiral resolution of (1S, 3S) and (1R, 3R)- alcohol (204 and 144) by taddol
Ideally, it was envisaged that the key cis alcohols (203 and 143) or the trans alcohols
(204 and 144) could be separated at some stage in the synthetic pathway, without the
need to add extra steps concerning the introduction and removal of a chiral auxiliary. We
speculated that this could be achieved by using Alessandro et al. protocol to convert
racemic alcohol to separable diastereoisomers with the help of chiral auxiliary
camphorsulfonyl chloride, which has dual role i.e. separation of enantiomer as well as
101
Results and Discussion
working as good leaving group for a subsequent nucleophilic substitution reaction by a
nucleobase.
2.4.9. Sulfonation (introduction of a chiral auxiliary)
In the original route to the required α-cycloPNA monomers, the next step following
production of the cis-alcohol enantiomer (143) involved sulfonation with 4bromobenzenesulfonyl chloride (See Scheme 51). It was speculated that if brosyl
chloride was replaced with a chiral sulfonyl chloride reagent, then it may be possible to
separate the cis-sulfonate diastereoisomeric mixture (232 and 233) and a similar strategy
could be applied for the trans-isomers (236 and 237) [Scheme 47 & 49]. Furthermore, it
was proposed that it may be possible to directly displace the chiral sulfonyl moiety
during the next step with the appropriate nucleobase, thereby eliminating the requirement
to remove this chiral auxiliary following separation (Scheme 47).
HO
OH
Me
O
O
O
H
i
HO
(+)-ASCO
OCSA-(+)
Me
H
H
227
228
ii
ii
O
O
HO
B
B
Me
OH
H
H
Me
230
229
Scheme 46206
Me
(+) OR ( -)
(+) OR ( -)
226
OH
Where B indicates thymine or 6-chloropurine or adenine i) 10-
camphorsulfonyl chloride (231), pyridine, 16 h, 83%, ii) caesium carbonate, [bmim][BF4],
DMF, heterocyclic base (thymine or 6-chloropurine or adenine), r.t., 2 h, microwave
irradiation (4 min at 100 W, max temp. 205°C, max pres. 105 psi.), 58-70%.206
Alessandro et al.206 have reported a very elegant protocol for the conversion of the
racemic diol (226) into the chirally pure carbanucleosides (229 and 230) via the
formation of the two sulfonate diastereoisomers (227 and 228) as shown in Scheme 46.
102
Results and Discussion
The isolated yields of (227 and 228) were 35% (+) and 48% (-), respectively, after
separation by flash chromatography.206 Following resolution both chiral sulfonates (227
and 228) were individually treated with the appropriate heterocyclic base, in the presence
of caesium carbonate and 1-butyl-3-methylimidazolium tetrafluoroborate [bmim][BF4]
first at r.t. before being subjected to microwave irradiation for the final 4 min. After
work-up and purification the carbanucleoside diastereoisomers (229 and 230) were
afforded in overall yields of 58-70% depending upon the nucleobase (B) used.
Therefore, based on the above findings of Alessandro et al.,206 it was to decided to
explore whether a similar strategy could be employed for, initially, the resolution of the
mixture of either cis- or trans- alcohols [ i.e. (203 and 143) or (204 and 144)]. Once
resolved, it was envisaged that the camphorsulfonyl moiety of the chirally pure sulfonyl
derivatives (232 or 233) or (236 or 237) could be subsequently nucleophilically displaced
with the appropriate nucleobase to obtain the desired α-cycloPNA monomers (Scheme
47).
H
O
CO2Et HO
CO2Et 10-(+) Camphorsulfonyl chloride (231) O
NHBoc
NHBoc
203
cis
O
O
S
HO
H
O
DMAP,TEA, DCM, 0 0C
O
S
CO2Et
O
O
NHBoc
232
143
cis
CO2Et
NHBoc
233
NaH Nucleobase
dry DMF, 40 0C
Nucleobase
CO2Et
Nucleobase
CO2Et
NHBoc
NHBoc
234
Scheme 47
trans
235
(+)-10-camphorsulfonyl chloride (231) as a leaving group and chiral
auxiliary
Thus, a solution of the racemic mixture of cis-alcohol (203 and 143) in dry DCM was
treated with an excess of (+)-10-camphorsulfonyl chloride (231) in the presence of TEA
and DMAP (Scheme 47). After work-up the crude reaction mixture was purified by
column chromatography to give the mixture of camphorsulfonyl derivatives (232 and
233) in a combined yield of 92%.
103
Results and Discussion
The structures of (232 and 233) were confirmed by 1H-NMR spectroscopy, which
showed the presence of two singlet signals at δ 1.26 and δ 1.11, which were assigned to
the two bridgehead methyl groups of the camphor moiety. In the 13C-NMR spectrum, the
signal attributed to the 3-CH carbon of the cyclopentane ring was further downfield at δ
82.9, in comparison to its position in the 13C-NMR spectrum of the starting material (203
and 143). The 13C-NMR spectum also showed the presence of a keto group at δ 214 and
two sets of CH2 signals at δ 32.3/32.4 and δ 47.9/48.0 which suggested the existence of
both camphorsulfonyl diastereoisomers i.e. (232 and 233).
A range of mobile phases, centred around petroleum ether (40-60 C):ethyl acetate
mixtures, were examined in order to try and resolve this mixture of sulfonate
diastereoisomers by column chromatography. However, for all solvent systems
investigated, only one product spot was observed by TLC i.e. both diastereoisomers coeluted. Thus, resolution by column chromatography was abandoned and alternative
techniques were sought. Fractional recrystallisation has been reported extensively in the
literature as a viable method for separating diastereomeric derivatives of chiral
compounds. This approach was therefore explored here. The purified mixture of
camphorsulfonyl diastereoisomers (232 and 233) was recrystallized from hexane:ethyl
acetate and the resulting crystals were submitted for X-ray crystallography.
Disappointingly, the X-ray crystal structure afforded showed that both diastereoisomers
(232 and 233) were still present i.e. that no resolution had been achieved (Figure F).
Nevertheless, the X-ray crystal data corroborated the cis-conformation of these
diastereoisomers since both the C11-O4 and C13-N1 bonds were found to reside on the
same face, while the C13-C16 bond was located on the opposite face of the cyclopentane
ring. In addition, the formation of an intramolecular hydrogen bond between N1-H1...O3
(d = 2.30 Å) was observed.
We had attempted to resolve cyclopentanol diastereoisomers by forming CSA derivatives
but this had failed possibly due to minimum intra-molecular interaction between
camphor sulfonate and NHBoc group, which were on the same side of the molecules
(232 and 233) based on the X-Ray structure as shown below.
104
Results and Discussion
Figure F
X-ray crystal structure of camphorsulfonyl derivative of cis-alcohol (232
and 233)
H
O
CO2Et HO
HO
O
O
O
S
CO2Et
H
O
O
S
CO2Et
O
O
CO2Et
10-(+)-Camphorsulfonyl chloride (231)
NHBoc
204
trans
Scheme 48
NHBoc
NHBoc
144
DMAP,TEA, DCM, 0 °C
236
trans
NHBoc
237
(+)-10-camphorsulfonyl chloride (231) as a leaving group and chiral
auxiliary
Despite not being able to resolve the two cis-camphorsulfonate diastereoisomers (232
and 233), it was decided to examine the preparation of the corresponding transderivatives from the mixture of trans-alcohols i.e. (204 and 144). It was reasoned that, in
this case, separation may be possible as presumably these compounds would have
different hydrophobicities to the cis-analogues. Thus, a solution of the mixture of transalcohols (204 and 144) in dry DCM was treated with an excess of (+) 10camphorsulfonyl chloride (231) in the presence of TEA and DMAP under similar
reaction conditions to those that had been used previously for the cis-alcohols (Scheme
105
Results and Discussion
48). Surprisingly, this reaction was found not to go to completion and, after work-up, the
crude compound seemed to decompose on storage. The reaction was repeated with the
number of equivalents of the reagents was increased to 5.0 eq. However, this also failed
to push the reaction to completion. The overall yield of the crude product was 45%.
Also, there were stability issues when examining the composition of the crude reaction
mixture by TLC. It seemed that the trans-products degraded and two new spots appeared
on the TLC plate. The spot at Rf 0.26 corresponded to the cis-alcohols (203 and 143),
while the baseline material was probably due to the camphorsulfonic acid side-product.
These compounds had been produced as a result of nucleophilic displacement of the
trans-sulfonate diastereoisomers by water in the eluting solvent. These findings meant
that it was not possible to purify or resolve the crude reaction mixture by column
chromatography.
2.4.10. Cis-alcohol to Trans-alcohol
Although, it had not been possible to resolve the two cis-alcohols (203 and 143) at the
camphorsulfonate stage, this synthetic route to the α-cycloPNA monomers using the
diastereomeric mixture of camphorsulfonate derivatives (232 and 233) was continued, in
the hope that separation could be accomplished at a later stage. This will be discussed in
detail in the following section.
However, since significant amounts of the desired trans-alcohols intermediates had not
been afforded through either the achiral or chiral hydroboration reactions, it was decided
to explore their synthesis from the mixture of cis-alcohols (203 and 143), which had been
the major products of both reactions.
OH
MsCl, Et3N CH2Cl2
(R)-butan-2-ol
238
Scheme 49207
OMs
OH
H2O, NaHCO3
(R)-2-Butyl methanesulfonate
(S)-butan-2-ol
240
239
Inversion of alcohol207
A method has been reported in the literature for converting a cis-alcohol to the
corresponding trans-alcohol, and it is illustrated in Scheme 49.207 Here the (R)-alcohol
(238) was first converted into mesylate (239), before being treated with a mild base to
106
Results and Discussion
give the corresponding (S)-alcohol (240).207
H
H
O
O
O
O
S
O
O
S
O
CO2Et
O
pH 8.0, Reflux, overnight
HO
204
Scheme 50
cis
CO2Et
NHBoc
NHBoc
NHBoc
NHBoc
232
CO2Et HO
CO2Et
trans
144
233
Alternative route for the conversion of cis-alcohol to trans-alcohol
Inspired by this protocol, it was decided to investigate the use of a similar approach for
preparation of the trans-alcohols (204 and 144) (Scheme 50). The only difference was that
instead of preparing the mesylate derivative of cis-alcohols, we used the cis-CSA
derivatives (232 and 233) since these were already available. Thus, a solution of the
mixture of cis-CSA derivatives (232 and 233) in dioxane and sodium hydroxide was
heated at reflux. It was essential that the pH of the reaction mixture was maintained at
around 8.0 in order to obtain clean inversion. After completion of the reaction, the solvent
was removed and the crude material was purified by column chromatography to afford the
trans-alcohols (204 and 144) in a combined yield of 73%. The 1H-NMR spectrum
recorded confirmed that the trans-alcohols (204 and 144) had been formed exclusively.
2.4.11. Synthesis of thyminyl α-cycloPNA monomer
Walker et al.165,166 had established a synthetic route to all four diastereoisomers of the
thymine α–cycloPNA monomers (249, 250, 259 and 260) from the key intermediate
alcohols (143 or 144) as shown in Scheme 51. Walker et al.165,166 obtained two of the
diastereoisomers of the α–cyclo PNA monomers (249 and 250) by brosylation of (1S,
3S)-alcohol (144) and (1R, 3S)-alcohol (143) in presence of triethylamine and DMAP in
chloroform. Thus, treatment of the parent alcohols with 4-bromobenzenesulfonyl
chloride at ambient temperature overnight gave the corresponding (1S, 3S) and (1R, 3S)
brosylates (242 and 241). The brosylates (241 or 242) were than added dropwise to a
solution of N3-benzoylthymine (261) in the presence of sodium hydride at 40 C in DMF
solution. After work-up and purification the corresponding α–cyclo PNA (1S, 3R) and
107
Results and Discussion
(1R, 3R) monomers (244 and 243) each were obtained in 57% yield. Subsequent removal
of the benzoyl protecting group from thymine in α–cyclo PNA monomers (244 and 243)
using sodium ethoxide, followed by alkaline ester hydrolysis
gave (250 and 249)
respectively. This gave access to two of the four diastereoisomers of α–cyclo PNA
monomers.
BsO
CO2Et
iii) (1S,3R)
(1R,3R)
iv) (1S,3R)
(1R,3R)
O
= 244 (57%)
= 243 (57%)
= 246 (98%)
= 245 (93%)
HN
O
NHBoc
i
N
v) (1S,3R) = 248 (96%)
(1R,3R) = 247 ( 95%)
CO2H
NHBoc
1S, 3S = 242
1R, 3S = 241
HO
1S, 3R = 250
1R, 3R = 249
CO2Et
NHBoc
1S, 3S = 144
1R, 3S = 143
O
HN
ii
I
CO2Et
iii) (1S,3S)
(1R,3S)
iv) (1S,3S)
(1R,3S)
= 254
= 253
= 256
= 255
(49%)
(49%)
(98%)
(93%)
NHBoc
1S, 3R = 252
1R, 3R = 251
O
N
CO2H
NHBoc
v) (1S,3S) = 258 (96%)
(1R,3S) = 257 ( 95%)
1S, 3S - 260
1R, 3S - 259
Scheme 519,166 i) BSCl, Et3N, DMAP, CHCl3, r.t., ii) PPh3, DIAD, MeI, THF, 0 C iii) N3benzoyl thymine (261), NaH, DMF, 40 C, iv) NaOEt / EtOH, v) NaOH, dioxane, H2O.9,166
In order to obtain the other two diastereoisomers (259 and 260), the (1R, 3S)-alcohol
(143) was converted into the (1R, 3R)-iodide (251) via a Mitsunobu-type reaction using
triphenylphosphine and diisopropyl azodicarboxylate (DIAD) in anhydrous THF
followed by treatment with methyl iodide at 0 °C, as described by Schumacher et al.185
This produced the desired iodide as colourless oil in a 68% yield. The corresponding (1S,
3S)-alcohol (144) diastereoisomer was converted into the (1S, 3R)-iodide (252) and
obtained as a colourless oil in a 69% yield. This gave access to all the four
diastereoisomers α–cyclo PNA thymine monomers.
Therefore, it was decided to employ a similar strategy to Walker et al.9 for obtaining the
(1S, 3S)- and (1R, 3R)-diastereosiomers of the thyminyl α-cycloPNA monomer except
for the fact that the brosylate intermediates (1S, 3R) and (1R, 3S) were substituted for the
108
Results and Discussion
mixture of cis-camphorsulfonate diastereoisomers (1S, 3R) (232) and (1R, 3S) (233)
(Scheme 52). The use of this mixture of sulfonates was deemed worthy of investigation
in this reaction pathway because, as discussed earlier in Section 2.4.9, Alessandro et
al.206 had shown that the camphorsulfonate group could be displaced by nucleobases.
H
H
O
O
O
O
O
S
O
O
O
Scheme 52
cis
BzN
Dry DMF, 40 °C
CO2Et
O
N
CO2Et
NHBoc
NHBoc
232
BzN
O
S
CO2Et
O
NaH, Bz-Thymine
O
N
CO2Et
NHBoc
260
233
trans
NHBoc
249
Nucleophilic displacement by N3-benzoylthymine
Therefore, a solution of N3-benzoylthymine (261) in DMF was treated with sodium
hydride at 40 °C and the resulting mixture was left to stir at this temperature for ½ h.
After this time, a solution of the cis-CSA derivatives (232 and 233) in DMF at 40 C was
added dropwise and the reaction was then left to stir at this temperature overnight. After
the work-up and purification of the crude product by column chromatography,9,208 the
desired trans products (260 and 249) were isolated in a disappointingly low combined
yield of 22%. Unreacted starting material (232 and 233) was also recovered in a 49%
yield.
The nucleophilic displacement of the camphorsulfonyl moiety of (232 and 233) by N3benzoylthymine was confirmed by the absence of two camphor methyl bridgehead
signals and camphor keto signal at δ 19.7, δ 19.8 and δ 214.4 respectively in the
13
C-
NMR spectrum. The formation of the desired N1-alkylated product (260 and 249) was
confirmed by signals in the 1H-NMR spectrum at δ 7.45-7.94, which integrated to six
protons and was assigned to the aromatic region of the benzoyl protecting group of
thymine and the C6-CH proton of thymine. The regioselectivity was confirmed by the
presence of 3-CH signal at δ 54.3 in a 13C-NMR spectrum, which is a clear indication of
an N1-alkylated product (260 and 249); as in case of an O2-alkylated product it is around
δ 75-80. The spectra data collected for the mixture the N1-alkylated products (260 and
249) were comparable to those reported by Walker et al.9
109
Results and Discussion
Using camphorsulfonate as a leaving group the desired product (260 and 249) was
obtained in 22% yield and 49% of unreacted starting material (232 and 233) was
obtained, which could be reused. After taking this into consideration the yield was low
compared with 57% of the desired product (260 and 249) obtained from brosylate by
Walker et al. Initial NMR misinterpretation led us to believe that we were able to obtain
single isomer of camphorsulfonate derivative (232 and 233) as there was no separation of
signals in the
1
H-NMR spectrum at 200 MHz. Thus, we continued with the
camphorsulfonyl group in comparison to brosylate as a leaving group for nucleophilic
displacement.
2.4.11.1.
Nucleophilic substitution via Mitsunobu reaction
O
NBz
O
HO
O
NHBoc
N
NBz
DEAD, PPh3, benzene, r.t, 4 h
NHBoc
O
N
72%
N
N
H
COOEt
261
COOEt
263
262
Scheme 53213
Mitsunobu condition for conversion of alcohol (262) to monomer
(263).213
Based on the outcome of nucleophilic displacement of camphorsulfonate, we decided to
further investigate the methods of directly converting (1S, 3R) and (1R, 3S)-alcohols (203
and 143) into (1S, 3S)- and (1R, 3R)-diastereosiomers of the thyminyl α-cycloPNA
monomer (254 and 243) via Mitsunubo reaction. Since, the nucleophilic displacement of
camphorsulfonate gave moderate yields of thyminyl α-cycloPNA monomer (254 and
243), we thought it would be worthwhile investigating direct conversion of alcohols (203
and 143) under Mitsunubo conditions in order to improve the overall yield of the
thyminyl α-cycloPNA monomer (254 and 243), this would also reduce the number of
synthetic steps.
Govindraju et al.213 have demonstrated that secondary cyclic alcohol (262) could be
directly converted into protected thymine monomer (263) using N3-benzoylthymine
(261), PhP3, and DEAD in dry benzene under Mitsunobu conditions, with an overall
110
Results and Discussion
yield of 72% as shown in Scheme 53. Thus, we decided to explore whether the
Mitsunobu reaction could be employed to cis-alcohol (203 and 143) with N3benzoylthymine under conventional Mitsunobu condition to give access to two
diastereoisomers of α-cycloPNA monomers.
Based on these findings, a solution of DEAD and triphenylphosphine in DMF was
cooled to 0 C, whereupon the mixture of cis-alcohols (203 and 143) and N3-protected
thymine were added (Scheme 55). The reaction was allowed to warm to r.t. before being
left to stir for 24 h. After this time, as there was no sign of product formation from
analysis by TLC, it was decided to try and drive the reaction by heating it first at 40 C
for 6 h followed by 60 C for 24 h. After work-up and purification by column
chromatography, unfortunately only starting material was isolated. In a further attempt to
get this reaction to work, the more reactive and less sterically hindered tributylphosphine
was used in place of triphenylphosphine. However, as before, no product formation was
detected and only starting material was recovered.
O
O
DEAD, Bz-Thy, PBu3 / PPh3
HO
CO2Et HO
CO2Et
NHBoc
NHBoc
BzN
BzN
O
N
Scheme 54
2.4.12.
143
N
CO2Et
NHBoc
Dry DMF, 0 °C-r.t, -40 °C
cis
203
O
CO2Et
254
trans
NHBoc
243
Attempted synthesis of (1R, 3R) and (1S, 3S) protected thymine derivative
Synthesis of cytosine α-cycloPNA monomer
It was decided to explore whether a similar strategy could be applied to the preparation
of the analogous α-cycloPNA cytosine monomers (264 and 265) (Scheme 55). This
involved a nucleophilic displacement of the camphorsulfonyl group of (232 and 233)
with Cbz-protected cytosine (268), as outlined in Scheme 55. Before this could be
investigated, Cbz-protected cytosine (268) needed to be prepared. This was successfully
accomplished by treating a solution of cytosine (269) in DMF with benzyl chloroformate
(270) according to the protocol described by Thomson et al. (Scheme 56).209 After work-
111
Results and Discussion
up and purification by column chromatography, the desired Cbz-protected cytosine (268)
was obtained in 53% yield.
H
O
O
O
S
O
H
O
O
S
CO2Et
O
O
CO2Et
NHBoc
NHBoc
cis
232
233
CsCO3 , dry DMF, 40 °C
Cbz-Cytosine (268), 24 h
O
H
N
H
O
N
N
H
H
N
O
N
O
N
O
N
CO2Et
O
CO2Et
N
N
CO2Et
O
CO2Et
O
NHBoc
NHBoc
265
266
Desired product
Scheme 55
O
N
NHBoc
trans
N
N
NHBoc
264
O
O
O
trans
267
Undesired product
Nucleophilic displacement of camphorsulfonyl group by Cbz-cytosine
O
NH2
DMAP, dry Pyridine
N
N
H
269
HN
O
Cl
O
Scheme 56
N
0
0 C, 72 h
O
O
N
H
270
O
268
Synthesis of Cbz-cytosine
Having synthesised the required Cbz-protected cytosine (268), the preparation of the
cytosine -PNA monomers (264 and 265) could now be examined. Thus, a solution of
Cbz-protected cytosine (268) in anhydrous DMF was treated with sodium hydride at 40
°C and the resulting mixture was left to stir at this temperature for ½ h. After this time, a
solution of the cis-CSA derivatives (232 and 233) in DMF was added dropwise whilst
this temperature was maintained. After work-up and purification by column
chromatography, unfortunately none of the desired N1-alkylated cytosine α-cycloPNA
monomers (264 and 265) were isolated. However, one fraction was instead found to
112
Results and Discussion
contain a mixture of the corresponding O2-alkylated regioisomers (266 and 267) in 30%
yield. Unreacted starting material was also recovered in 9% yield.
The formation of the mixture of undesired O2-alkylated products (266 and 267) was
confirmed by 13C-NMR spectroscopy since the signal assigned to the cytosinyl C5 carbon
was found to reside downfield at δ 160.1. According to Meier et al.212 this carbon signal
would have been found further upfield at around δ 145 if the N1-alkylated products were
present. The regioselectivity was further confirmed by the presence of 3-CH signal at δ
77.6, which again was a clear indication of O2-alkylated products (266 and 267) as
substantiated by the findings of Meier et al. These researchers have reported that for the
N1-alkylated and O2-alkylated products (272 and 271) (Figure G) respectively the carbon
signal corresponding to C-1’ was located upfield at δ 56.0 in the
13
C-NMR spectrum
recorded for (271), while it resided downfield at δ 80.1 in the 13C-NMR spectrum recorded
for (272) (Table 3).
O
HN
O
C2
C4
HN
C2
O
N
C1'
C5
C6
N
C1'
C2'
C5'
C3'
C4'
C2'
C3'
C5
C6
C5'
C4'
1
Figure G Structure of N -alkylated
product (271) and O2-alkylated product
(272)
13
271 (δ)
152.3
164.2
111.2
137.4
C-7
12.9
12.8
C-1’
C-2’, C-5’
C-3’, C-4’
80.1
33.7
24.9
56.0
32.9
25.3
C7
272
271
Table 3212
C-2
C-4
C-5
C-6
272 (δ)
157.1
164.2
118.4
151.4
O
C7
C4
C-NMR signals of N1-alkylated product (271) and O2-alkylated product
(272) in DMSO.212
Thus, it could be concluded that only O2-alkylated regioisomers (266 and 267) had
formed during the above nucleophilic displacement reaction. The preference for O2alkylated regioisomer over N1-alkylated regioisomer could be due to the hardness of the
carbon connected to the hydroxyl group, which in an alcohol depends upon the nature of
substituents and their position in the molecule. A hard carbon atom appears at low
magnetic field as the electron density at the nucleus is low, hence it is hardly polarisable.
113
Results and Discussion
Thus, the harder the carbon atom, the more O2-alkylation should occur and vice
versa.210,211
Regioselectivity was deemed to be a major issue in the case of Cbz-cytosine derivative as O2alkylated (266 and 267) is preferred over N1-alkylated (264 and 265). It might be possible
to change the regioselectivity outcome with the introduction of a more reactive leaving
group. Thus we decided to convert cis CSA derivative (232 and 233) into trans iodo
derivative (273 and 274) as iodo group would be more reactive than CSA group, which in
turn could be reacted to Cbz-protected cytosine (268) as per Walker et al. protocol for
nucleophilic displacement to give the desired N1-alkylated cytosine α-cycloPNA
monomer (264 and 265) as described in Scheme 55.
2.4.12.1.
Iodo derivative
H
H
O
O
O
O
S
O
O
S
CO2Et
O
O
NHBoc
232
Scheme 57
cis
233
CO2Et
NaI, acetone
NHBoc
reflux 24 h
I
273
CO2Et I
CO2Et
NHBoc
NHBoc
trans
I
274
275
CO2Et I
CO2Et
NHBoc
NHBoc
cis
276
Nucleophilic displacement of CSA group by iodide
To gain access to the other desired N1-alkylated cytosine α-cycloPNA diastereoisomers,
it was decided to explore the use of the Finkelstein reaction to first convert the mixture of
cis-CSA derivatives (232 and 233) into their corresponding iodo derivatives (273 and
274). The first step in this pathway involved treatment of a solution of (232 and 233) in
dry acetone with sodium iodide and heated at reflux for 24 h. After work-up and
purification by column chromatography, the desired trans-iodides (273 and 274) were
obtained along with the unwanted cis-iodides (275 and 276) in a combined yield of 57%.
Unfortunately, the cis and trans diastereoisomers proved to be inseparable by column
chromatography. Furthermore, the epimerized iodides (273 and 274) and (275 and 276)
were found to decompose rapidly on column, which prevented any further exploration of
this route for the synthesis of the other α-cycloPNA diastereoisomers.
114
Results and Discussion
The iodo derivative (273 and 274) and (275 and 276) seems to be an extremely unstable
compound.
2.4.13. Ester Hydrolysis
H
H
N
O
O
O
O
H
N
O
N
O
H
O
N
O
NaOH, Dioxane
N
N
N
N
N
CO2Et
O
Scheme 58
trans
COOH
O
N
COOH
O
NHBoc
NHBoc
NHBoc
266
N
CO2Et
O
N
267
277
NHBoc
trans
278
Hydrolysis of ester
Based on the findings of Seebach et al.,219 it was decided to explore the host-guest
complex technique for resolution of enantiomers (266 and 267) as outlined in Scheme
58. Although, the O2-alkylated α-cycloPNA cytosine monomer (266 and 267) had not
been the desired monomer, it was deemed suitable for use as a model to assess the
feasibility of separating the two diastereoisomers of α-cycloPNA carboxylic acid
monomers. In order to do this, the ethyl ester group of the O2-alkylated α-cycloPNA
cytosine monomer (266 and 267) had to be first removed. This was successfully
accomplished by treatment of a solution of the mixure of (266 and 267) in dioxane with a
1N aqueous solution of sodium hydroxide as per the protocol of Walker et al.9 The
mixture of carboxylic acid diastereoisomers (277 and 278) was obtained in a combined
yield of 66% following an acid-base work-up.
We thought it would be worthwhile investigating the separation of enantiomer of O2alkylated α-cycloPNA cytosine monomer (277 and 278) by employing taddol (224).
Consequently, the chiral resolution of the racemic mixture of O2-alkylated α-cycloPNA
cytosine monomers (277 and 278) was attempted. Thus, a solution of the mixture of (277
and 278) and taddol (224) in methanol was heated to reflux and hexane was added
dropwise until the heated solution became slightly turbid in appearance. The solution was
then cooled to r.t. and it was kept at this temperature for 24 h. During this time, white
crystals precipated from the reaction mixture. These were collected and analyzed by X115
Results and Discussion
ray crystallography. Disappointingly, the X-ray structure obtained showed that the
crystals were not composed of a taddol complex. Instead the only compound observed
was the O2-alkylated α-cycloPNA cytosine enantiomers [i.e. (277 and 278)]. Hence, no
chiral resolution had been accomplished.
In conclusion, separation of the enantiomers of the cyclopentanol intermediate or PNA
monomers
by
chiral
resolution
with
the
aid
of
camphorsulfonyl
chloride
(diastereoisomeric derivative), brucine (diastereoisomeric salt formation) or taddol (EIC
technique) had all resulted in failure. Thus, it was proposed to investigate modifying the
strategy such that the mixture of cis-alcohols (203 and 143) formed during hydroboration
step could be separated at this stage itself, similarly trans-alcohol (204 and 144) could
also be separated in order to get access to all four diastereoisomers of key intermediate
alcohols as single isomer. To fulfil this objective, we aforethought to replace the methyl
ester group of alkene (189) with a sterically hindered chiral auxiliary such as L-menthyl
or D-menthyl group, giving corresponding ester alkenes (279) or (280) via a
transesterification reaction, which on hydroboration of alkene (279) or (280) would give
rise to the corresponding cis-alcohol diastereoisomers (287 and 288) and trans-alcohol
diastereoisomers (285 and 286). It was reasoned that these two products could then be
separated by either column chromatography or recrystallization, as these cis alcohols
would be diastereoisomers rather than enantiomers and same implies for trans-alcohol.
2.5.
Modified hydroboration route for the synthesis of α-cycloPNA cytosine
monomer
The revised route for the synthesis of the α-cycloPNA cytosine monomer is shown in
Scheme 59. This pathway starts with the methyl ester (222). This ester was selected over
the ethyl ester (189) in order to improve the rate of the subsequent transesterification
reaction, and overall yield, based on the findings of Meth-Cohn.224 The purpose of the
transesterification reaction was to introduce the chiral auxiliary, i.e. either a L- or Dmenthoxy-group to give 279 and 280 respectively. The hydroboration reaction of alkene
279 or 280 with the borane-tetrahedron complex, followed by standard oxidation would
give the key intermediate alcohol [287 + 288 (cis-alcohol) and 285 + 286 (transalcohol)]. Due to the introduction of chiral auxiliary (menthoxy group), the cis-alcohols
287 + 288 might be separated because of their diastereoisomeric relationship. Similarly
116
Results and Discussion
trans-alcohols 285 + 286 might also be separated as single isomers. Finally, it was
envisaged that the 3-hydroxy group of 285 or 286 or 287 or 288 could be converted into
a brosylate group, as had been used in Walker et al.’s strategy, prior to introduction of a
nucleobase.9 To ensure a regioselective outcome (i.e. N1-alkylation rather than O2alkylation) brosylate was worthy of investigation here because it is a better leaving group
than CSA and more reactive. Thus this may impact the regioselectivity issue and it could
improve the overall yield of this reaction. The sulfonate groups 290 or 291 or 292 or 293
would then be displaced by the protected cytosine to afford the desired monomers 294 or
295 or 296 or 297. Synthesis of methyl ester alkene (222) has been already described in
section 2.4.4. It was obtained in four steps starting from commercially available cis-1,4dichlorobutene (109).
O
R
i
COOMe
NHBoc
NHBoc
O
O
279
222
280
R=D-Menthoxy =281
R= L-Menthoxy = 282
ii
COR
B
B
COR
NHBoc
COR
B
296
Scheme 59
297
COR
HO
iii
NHBoc
292
286
COR
HO
NHBoc
293
COR
NHBoc
285
COR
R1O2SO
HO
NHBoc
NHBoc
291
COR
R1O2SO
iv, v
NHBoc
NHBoc
COR
iii
290
295
COR
R1O2SO
NHBoc
NHBoc
294
B
COR
R1O2SO
iv, v
COR
HO
NHBoc
287
NHBoc
288
Modified hydroboration route for the synthesis of α-cycloPNA cytosine
monomer. i) L-menthol (283) or D-menthol (284), BuLi, THF, 0 C; ii) BH3.THF, dry
THF, 0 C → r.t., 17 h; iii) BsCl (289), DMAP, TEA, DCM, 0 C, Overnight; iv) Cbzcytosine, NaH, DMF, 40 C, 24-48 h.
With the alkene methyl ester (222) to hand, the next step in the revised strategy involved
performing a transesterification reaction to yield the corresponding L-menthoxy (279)
and D-menthoxy (280) compounds.
117
Results and Discussion
Meth-Cohn demonstrated that transesterifications on various alkene methyl ester could
be accomplished using sterically hindered alcohols like ButOH, (+)-fenchol, (-)-menthol
and (-)-borneol in very good yields ranging from 82-100%.224 These transesterifcation
reactions involved first treatment of a solution of the appropriate alcohol in dry THF with
n-butyllithium at -10 C to r.t. followed by addition of the methyl ester.
Figure H
Simplified 2D representation of X-ray crystal structure of alkene (280)
Therefore, inspired by the findings of Meth Cohn224 the same protocol was employed
here to prepare the alkene L-menthyl ester alkene (279) and D-menthyl ester alkene (280)
from alkene methyl ester alkene (222). Thus, a 1.6 M solution of n-butyllithium in
hexane was added to D-menthol (284) in dry THF and the resulting mixture cooled to -10
C. Subsequently, a solution of (222) in THF was added dropwise and the reaction was
allowed to warm to r.t. before being left to stir overnight. After work-up and purification
by column chromatography, the desired D-menthyl product (280) was obtained in yield
of 69%. The formation of this product was confirmed by 1H-NMR spectroscopy, which
showed the absence of the singlet at δ 3.70 which had been present in the spectrum
118
Results and Discussion
recorded of (222) due to the methyl ester protons. Furthermore, three new signals
appeared between δ 0.70–0.87, which were assigned to the protons present in the three
methyl groups of the menthyloxy function. The transesterification was further supported
by the 13C-NMR spectrum recorded for (280) which showed the presence of a CH signal
at δ 75.7 which corresponded to the C1’-position of the menthol moiety. In addition, the
formation of (280) was further verified by X-ray crystallography. Analysis of the X-ray
structure showed that alkene (280) adopted 3 different conformations in the unit cell
(Figure H), which was confirmed by the presence of C9-N1 and C9-C8 bonds on the
same face. The basic difference in the three conformation lies with the facing of C7C71, C7- C72 and C15- C17 as shown in the 2D presentation in Figure 7.
O
O
O
NHBoc
279
Scheme 60
CO2Me
L-Menthol (283), BuLi
THF, O °C
D-Menthol (284), BuLi
O
THF, O °C
NHBoc
NHBoc
280
222
Transesterification by D-menthol and L-menthol
The same procedure was used to prepare L-menthyl ester alkene (279) from alkene
methyl ester (222).224 After work-up and purification by column chromatography, the
desired product (279) was obtained in an overall yield of 82%. Again the formation of
(279) was confirmed by 1H-NMR and 13C-NMR spectroscopy.
The next step in the synthesis of α-cycloPNA cytosine monomer involved hydroboration
of either L-menthyl ester (279) or D-menthyl ester (280) with borane-THF complex. We
speculated that the introduction of L- / D-menthyl esters in place of the ethyl ester would
offer a few advantages. Both L- / D-menthol have 3 chiral centres, as indicated by
asterisks in Figure I. One of these chiral centre is 3 bonds away from the quaternary
carbon chiral centre of alcohol, which has two chiral centres; this is an essential criterion
for a chiral auxiliary.225 It can be resolved under diastereoselective hydroboration
conditions as it will be able to generate two diastereoisomers of cis-alcohol, which could
be separated on recrystallization or by column chromatography. It may be possible to
monitor the ratio of the two diastereoisomers of cis-alcohol by NMR as it is not possible
119
Results and Discussion
in case of enantiomer, which could be monitored only when chiral NMR solvating agent
(which is expensive and time consuming) is used in case of NMR.
HO





O
BocHN
O
298
Figure I
Possible stereogenic centres after hydroboration (* shows the stereogenic
centre)
Thus, a solution of alkene D-menthyl ester (280) in dry THF was treated with boraneTHF complex at 0 C. The reaction mixture was allowed to warm to r.t. and stirred
overnight. After overnight oxidation with pH 7.0 buffer, the reaction was worked up as
per the procedure described by Hodgson et al.,182,183 and the crude material was purified
by column chromatography.
The desired cis-alcohols (299 and 300), as a 1:1 mixture were obtained in a combined yield
of 34%, whilst the two trans-alcohol products (301 and 302), as a 1:1 mixture were
received in a combined yield of 15.2%.
Although it had been hoped that it may be possible with this approach to further resolve
the mixtures of the two cis-alcohol diastereoiosmers and the two trans-alcohol
diastereoisomers through column chromatography, this unfortunately did not turn out to
be the case.
For each mixture, both diastereoisomers were found to co-elute in all the solvent systems
investigated. The structures of cis-alcohols (299 and 300) and trans-alcohol (301 and
302) were confirmed by both 1H and
13
C-NMR spectroscopy. The most distinguishing
feature between the mixture of cis-alcohols (299 and 300) and trans-alcohols (301 and
302) was the position of their NH signal in 1H-NMR spectrum. In case of the mixture of
cis-alcohols (299 and 300), this signal came at δ 5.94 while for the trans-alcohols (301
and 302) it was located slightly upfield at δ 5.10. The probable reason for the difference
in NH signal is due to the fact that in the cis-alcohols (299 and 300), the NH acts as a H-
120
Results and Discussion
bond donor and forms a H-bond to 3-OH. H atoms involved in H-bonding reside further
downfield in 1H-NMR spectra. Obviously, such intramolecular hydrogen bonding does
not happen in trans-alcohols (301 and 302) as the NH and OH are on opposite faces.
Unfortunately, it was not possible to establish the ratio of the two cis-alcohol
diastereoisomers (299 and 300) using the 1H-NMR spectrum recorded of the mixture as
the signals were not resolved distinctively.
CO2R
HO
CO2R
HO
NHBoc
HO
CO2R
NHBoc
NHBoc
NHBoc
cis
300
301
trans
302
cis
304
305
trans
306
299
303
CO2R
HO
HO
HO
(+) Menthol-D
(-) Menthol-L
R= 299, 300, 301, 302
R= 303, 304, 305, 306
i
O
O
O
HO
HO
iii
O
HO
NHBoc
300
cis
299
280
Scheme 61
HO
NHBoc
300
Minor
O
NHBoc
NHBoc
cis
299
CO2R
ii
NHBoc
O
O
O
Major
Major
Minor
Reagents and conditions: i) BH3.THF, dry THF, 0 C → r.t., 17 h, ii)
Boron trifluoride etherate, (+)-IpcBH2.TMEDA , dry THF, -40 C, 72 h, iii) Boron
trifluoride etherate, (-)-IpcBH2.TMEDA , dry THF, -40 C, 72 h
However, the two trans-alcohol diastereoisomers (301 and 302) were estimated to be
present in the mixture in an ca. 1:1.05 ratio from their 1H-NMR spectrum. This ratio of
(301 and 302) suggests that the bulky ester exerts little stereocontrol, which is in line
with the results from Hodgson et al..183
O
O
O
O
NHBoc
280
Scheme 62
O
HO
BH3.THF
NHBoc
THF 0 0C, 17 h
299
O
O
HO
300
O
HO
NHBoc
NHBoc
cis
O
O
HO
301
trans
NHBoc
302
Hydroboration of D-menthyl ester of alkene with borane.THF
121
Results and Discussion
The same protocol for the hydroboration reaction was applied to the alkene L-menthyl
ester alkene (279) (Scheme 63). After work-up, the crude material afforded was purified
by column chromatography. The desired mixtures of cis-alcohols (303 and 304) and
trans-alcohols (305 and 306) were afforded in similar combined yields to those received
for the analogous D-menthyl ester compounds. Again difficulties were encountered, akin
to those described above for the corresponding D-menthyloxy alcohols, when trying to
further resolve the mixtures of the two cis alcohol diastereoisomers (303 and 304) and the
two trans alcohol diastereoisomers (305 and 306).
From the 1H-NMR spectrum recorded for the mixture, the ratio of the two trans-alcohol
diastereoisomers (305 and 306) after isolation from the cis diastereoisomers was 1.1:1.05
calculated by measuring the integration of methyl groups of two isomers in the 1H-NMR
spectrum.
Given the formation of intractable isomer mixtures from D- and L-menthyl esters (280)
and (279), it was decided to explore combination with a chiral hydroboration reagent in
the hope of simplifying the product outcome.
O
HO
O
BH3.THF
NHBoc
dry THF, 0 °C, 17 h
279
O
HO
NHBoc
303
Scheme 63
2.5.1.
O
O
O
O
304
O
HO
NHBoc
NHBoc
cis
O
O
HO
305
trans
NHBoc
306
Hydroboration of L-menthyl ester of alkene with borane.THF
Hydroboration after introduction of chiral auxiliary on the ester group with (-)IpcBH2 and (+)-IpcBH2
As Hodgson et al. had shown that (-)-IpcBH2 or (+)-IpcBH2 could favour products cis to
the NHBoc group in ester (189), although enantioselectivity was an issue by using
sterically hindered alkene (183). We next explored hydroboration of alkenes (280) &
(279) with this hydroboration reagent in the hope of obtaining diastereoselective product
i.e. exclusively cis products, which in theory might be separated because of their
diastereoisomeric relationship
122
Results and Discussion
CO2Me
CO2t-Bu
NHCO2Me
NHBoc
307
CO2Et
NHBoc
189
183
Figure J
Different protecting groups on alkene
O
O
HO
NHBoc
299
Minor
CO2R
i
ii
O
HO
280
299
Major
Major
O
HO
NHBoc
NHBoc
NHBoc
300
O
O
O
O
HO
NHBoc
300
Minor
P.S: Assumption of minor or major isomer was based on Hodgson et al. work.
Scheme 64
Asymmetric hydroboration with (-)-IpcBH2 and (+)-IpcBH2
Reagant and condition: i) Boron trifluoride etherate, (+)-IpcBH2.TMEDA , dry THF, -40
C, 72 h, ii) Boron trifluoride etherate, (-)-IpcBH2.TMEDA , dry THF, -40 C, 72 h
Thus, the asymmetric hydroboration of the alkene D-menthyl ester (280) was explored.
This was carried out using chiral hydroborating reagent (+)-IpcBH2.TMEDA complex
with BF3.Et2O to give (+)-IpcBH2 in the filtrate followed by slow addition of D-menthyl
ester alkene (280) to pursue the protocol previously described in section 2.4.7.1. The
only modification made to this protocol was that once (+)-IpcBH2 had been prepared, the
reaction mixture was transferred, under nitrogen, via a cannula through a sinter, to
remove any precipitate, into a pre-cooled vessel (-45 to -35 °C) before alkene was added.
The hydroboration reaction was followed by standard oxidation, extraction and
purification by column chromatography. The combined yield of cis-alcohols (299 and
300) when (+)-IpcBH2 was employed was 82% and neither of the trans-alcohols (301 and
302) were detected. When (-)-IpcBH2TMEDA was used in place of (+)-IpcBH2TMEDA
in the above asymmetric hydroboration reaction, the mixture of cis-alcohol (299 and 300)
was afforded in a combined yield of 69%; again trans-alcohols (301 and 302) were not
observed.
Although, we were unable to separate D-menthyl derivatised cis-alcohols (299 and 300)
either by column chromatography or by recrystallization, we were able to synthesize
123
Results and Discussion
(299 and 300) using (+)-IpcBH2 or (-)-IpcBH2 in yields of 82% (59% e.e. based on
indirect 1H-NMR analysis) and 69% (62% e.e. based on indirect 1H-NMR analysis)
respectively, as described in section 2.5.2. Thus, asymmetric hydroboration reaction has
simplified the process of purification due to absence of trans-product. The preference for
cis-product has been already explained by Hodgson et al.182,183 (Section 2.4.5.) as being
possibly due to weak carbamate directed hydroboration and selective reduction of the ester
group of the trans-organoborane intermediates, which enables removal of trans-products.
The yields were comparable to Hodgson et al. asymmetric hydroboration of alkene (189)
i.e. (+)-IpcBH2 or (-)-IpcBH2 in 74% (48% e.e. based on1H-NMR analysis) and 77%
(47% e.e. based on 1H-NMR analysis) and the e.e. was relatively better in our case.
2.5.2.
Sulfonation
Despite not being able to separate the mixture of cis-alcohols (299 and 300), it was
decided to go ahead and examine the next step in the revised strategy to the cytosinyl cycloPNA monomers. It was hoped that resolution would be achieved at a later stage in
the pathway. The next step in this revised route involved transforming the 3-hydroxy
function of (299 and 300) into a good sulfonate leaving group which could then be
displaced by Cbz-protected cytosine. Walker et al.9 had previously explored the use of
various
sulfonate
groups
for
this
reaction
and
had
found
that
the
4-
bromobenzenesulfonate (brosylate) group gave the best results, due to a combination of
its greater reactivity than mesylate and better stability than nosylate. It was therefore
decided to use the same sulfonate group here. The mixture of cis-alcohols (299 and 300)
was treated with an excess of 4-bromobenzenesulfonyl chloride (289) in the presence of
dry TEA and DMAP (Scheme 65). After work-up, the crude reaction mixture was
purified by column chromatography. The diastereomeric mixture of cis-brosylates (308
and 309) was afforded in a 75% yield.
O
O
O
O
HO
O
HO
BsCl, DMAP, TEA, DCM
NHBoc
Scheme 65
cis
308
300
O
BsO
NHBoc
NHBoc
0 0C, Overnight
299
O
O
BsO
NHBoc
cis
309
4-bromobenezenesulfonyl chloride as a leaving group
124
Results and Discussion
All spectral data collected were consistent with the formation of the expected product
(308 and 309). Furthermore, the structures of these products were confirmed with the
help of X-ray crystallography (Figure K). The cis-configuration of these two
diastereoisomers was verified since the C9-N1 and C11-O5 bonds were found to reside
on the same face of the cyclopentane ring, while the C9-C8 bond lay on the opposite
face.
Figure K
X-ray crystal structure of cis-brosylate derivative (308 and 309)
From the 1H-NMR spectra recorded for the mixture of cis-brosylates, it was possible to
deduce the ratios of the two cis-alcohol diastereoisomers (299 and 300) present in the
starting mixtures obtained from the asymmetric hydroboration reactions with (-)-IpcBH2
or (+)-IpcBH2. The cis brosylates (308 and 309) were obtained in a ratio of 1:2.45
(41:100 = 59% e.e.) when starting from the mixture of cis-alcohol (299 and 300) derived
from the hydroboration reaction employing (+)-IpcBH2. In contrast, the cis-brosylates
(308 and 309) were obtained in a ratio of 2.65:1 (100:38 = 62% e.e.) when the mixture of
cis-alcohols (299 and 300) afforded from the hydroboration reaction using (-)-IpcBH2
was utilised. The cis brosylates (308 and 309) were obtained in a ratio of 1:0.94 when
starting from the mixture of cis-alcohol (299 and 300) derived from the hydroboration
reaction employing borane-tetrahedron complex.
125
Results and Discussion
2.5.3. Synthesis of cytosine α-cycloPNA monomer
Having successfully prepared the mixture of cis-brosylates (308 and 309) from borane
hydroboration, the final step in the synthetic pathway that required investigation
involved, nucleophilic displacement of the brosylate using Cbz-protected cytosine
(Scheme 66). Thus, sodium hydride was added to a stirred solution of Cbz-cytosine (268)
in dry DMF and the solution was heated to 40 °C. Subsequently, a solution of the
mixture of cis-brosylate derivatives (308 and 309) in dry DMF was added dropwise.
After work-up, the crude yellowish brown oil was purified by column chromatography.
Unfortunately, upon analysis of the spectral data collected for the isolated products, it
was determined that once again this reaction had produced the mixture of undesired O2alkylated products (312 and 313) instead of the required N1-alkylated analogues (310 and
311). The mixture of undesired O2-alkylated products (312 and 313) was afforded in a
combined yield of 25%. In addition, 39% of the starting material (308 and 309) was
recovered.
O
O
O
Bs
O
Bs
NHBoc
308
NHBoc
309
cis
Cbz-Cytosine, NaH
DMF, 40 °C, 24-48 h
O
H
N
O
H
N
N
N
O
O
H
O
N
O
O
N
O
O
trans
O
N
O
N
O
N
O
O
O
O
NHBoc
311
NHBoc
trans
312
Undesired product
Desired product
Scheme 66
N
O
NHBoc
NHBoc
310
O
N
N
O
O
H
Nucleophilic displacement by Cbz-cytosine
126
313
Results and Discussion
O
NBz
N3
N3
i
N
OCHO
CO2CH3
315
Scheme 67227,228
N3
OH
N
O
ii
N
N
CO2CH3
CO2CH3
314
316
Reagents and conditions: i) HCO2H, PPh3, DIAD, THF, −30 C
→r.t., 18 h; ii) N3-benzoylthymine, PPh3, DIAD, PhCO2Na, THF, r.t., 18 h227,228
The unsuccessful attempt to convert cis-brosylates (308 and 309) to N1-alkylated
analogues (310 and 311) prompted us to investigate alternative ways to obtain the desired
products (310 and 311). Cis-alcohols (299 and 300) could be directly converted into N1alkylated cytosine analogues (310 and 311) by Mitsunobu reaction. Thus, we decided to
explore two different orders of reagent addition for this, as reported by Micklefield et
al.,227,228 In the first method, they stirred a solution of alcohol (314) into dry THF, followed
by formic acid at -20 C. To the resulting solution triphenylphosphine was added followed
by dropwise addition of DIAD at -20 C, as described by Micklefield et al.227 After workup and purification, the desired product (315) was obtained in 71% yield. In the second
method, N3-benzoylthymine was added to a stirred solution of DIAD and alcohol (314) in
THF at r.t. followed by triphenylphosphine and finally by sodium benzoate. The addition
of sodium benzoate reportedly makes the alcohol more nucleophilic.214,230 After workup
and purification, the desired product (316) was obtained in 75% yield. Our previous
findings for conversion of cis-alcohols (203 and 143) to thyminyl α-cycloPNA monomer
(260 and 249) via Mitsunobu reaction prompted us to replace the conventional Mitsunobu
reagent (DIAD / PPh3 or DEAD / PPh3) with a modern Mitsunobu reagent (ADDP / PBu3),
which is more reactive than conventional Mitsunobu reagent and which also eliminates
side product (321) (Scheme 68) especially with compounds having pKa > 11 as reported
by Humphies et al.229 Hence, we decided to explore the order of addition (method i and
method ii) reported by Mickelfield et al.,214 and reagents as per Humphies et al.229
127
Results and Discussion
O
RO
N
Ph3P
N
OR
O
152
317
O
O
N
RO
N
H
RO
N
N
Desired
OR
O
OR
Side
PPh3
N
H
RO
Reaction
Reaction
319
HX
O
O
O
R1
PPh3
O
OH
HX
R1
pKa < 11
O
N
H
N
H
O
O
O
RO
OR
319
318
R1
N
pKa > 11
O
N
H
RO
OR
PPh3
N
OR
R1
320
R1
321
X
HX
O PPh3
Scheme 68
O PPh3
Proposed mechanism for the formation of side product in Mitsunobu
reaction
O
O
O
HO
O
HO
NHBoc
299
NHBoc
300
cis
Condition
i) ADDP, PBu3, Cbz-cytosine, Na-Benzoate, dry DMF, r.t, 18 h
ii) ADDP, PBu3, Cbz-cytosine, dry DMF, -10 °C r.t, 18 h
iii) ADDP, PBu3, Cbz-cytosine, dry DMF, Dry THF, 0 r.t  40 °C, 48 h
O
H
N
O
H
N
N
N
O
H
N
O
O
N
O
O
trans
O
N
O
O
O
N
O
O
O
O
NHBoc
NHBoc
312
311
trans
Undesired product
Desired product
Scheme 69
N
N
NHBoc
NHBoc
310
O
N
N
O
O
O
O
H
Mitsunobu condition
128
313
Results and Discussion
Thus, we decided to explore with 1.4 equivalents of ADDP added to 1.0 equivalent cis
menthyl derivative of alcohol (299 and 300) in dry THF. Subsequently, solution of N4benzyloxycarbonylcytosine (1.2 equivalents) in dry DMF was added to the above reaction
mixture. Finally, 1.2 equivalents of PBu3 in dry THF was added dropwise and the reaction
mixture was stirred for 20 h at r.t. After workup and purification, unfortunately, no desired
N1-alkylated product (310 and 311) was formed and only starting material was recovered.
Based on this finding, it was decided to repeat the reaction but to increase the number of
equivalents of Mitsunobu reagent present. Thus, 3.5 equivalents of ADDP were added
dropwise to a solution of the mixture of cis-alcohols (299 and 300) (obtained from borane
hydroboration
reaction)
in
dry
THF.
Subsequently,
3
equivalents
of
N4-
benzyloxycarbonylcytosine (268) in dry DMF were added. Followed by a solution of PBu3
in freshly distilled THF kept, under nitrogen at 0 C. Finally, 3 equivalents of sodium
benzoate were added. Unfortunately, still only unreacted starting material was recovered
from the reaction mixture.
In a final attempt to see if this reaction could work, the number of equivalents of
Mitsunobu reagent was raised to 6.0. The equivalents of all other reagents remained the
same.228 After work-up, the crude reaction mixture was purified by column
chromatography and this time, one of the fractions isolated contained the mixture of O2alkylated products (312 and 313) in 18% yield; unreacted starting material (299 and 300)
was also recovered in 43% yield.
The results obtained for this Mitsunobu reaction were intriguing as they too favoured
formation of the O2-alkylated cytosine products instead of the N1-alkylated regiomers.
Towards the end of this project, Micklefield et al.231 reported a similar regioselectivity
issue when performing a Mitsunubo reaction with alcohol (322) and Cbz-protected
cytosine (Scheme 70).
129
Results and Discussion
O
O
H
N
O
N3
N
N
N
N
H
O
H
N
N
N3
N3
O
N
N
O
N3
O
ii
i
N
N
N
N
OH
324
322
323
CO2CH3
CO2CH3
CO2CH3
CO2CH3
325
O
H
N
N
N
N3
N3
O
iii
N
N
OTs
CO2CH3
326
Scheme 70231
CO2CH3
327
Reagents and conditions: i) N4-benzyloxycarbonylcytosine, PPh3, DIAD,
THF, −25 C → r.t., 18 h; ii) N4-[p-(tert-butyl)benzoyl]cytosine, PPh3, DIAD, THF, −25 C
→ r.t., 18 h; iii) N4-[p-(tert-butyl)benzoyl]cytosine, K2CO3, 18-crown-6, DMF, 75 C, 16
h.231
It was reasoned that the N4-benzyloxycarbonyl protecting group in cytosine does not
decrease the delocalization of the lone pair of electrons from the N4-amino group onto the
O2-atom and thereby promotes O2-alkylation as it makes it soft nucleophile. These
researchers discovered though that this problem can be overcome by the use of a p-(tertbutyl)benzoyl protecting group instead. This group does decrease the delocalization of the
lone pair of electrons from the N4-amino group onto the O2-atom and thereby promotes
production of the N1-alkylated product.231 However, due to time constraints, application of
this strategy to the work reported in this thesis could not be investigated.
2.5.4.
Menthol Hydrolysis
Before concluding this route, it was essential to understand the ease of hydrolysis of the
D-menthyl
ester as this required removal at the end of a synthetic pathway to the 130
Results and Discussion
O
H
N
O
H
N
N
N
O
O
H
O
N
N
O
O
O
N
O
NHBoc
312
O
O
N
O
O
OH
N
O
O
OH
NHBoc
NHBoc
trans
N
N
N
O
O
O
H
313
277
NHBoc
trans
278
Scheme 71 Ester hydrolysis
cycloPNA monomers. Two interesting procedures have been reported in the literature for
the hydrolysis of the menthyl esters. Sunagawa et al.233 reported cleavage of the menthyl
ester of (328) was achieved in quantitative yield upon treatment with an aqueous solution
sodium hydroxide at r.t. for 24 h (Scheme 71). Therefore, this approach was applied to
the mixture of O2-alkylated products (312 and 313). However, after work-up, none of the
desired product (277 and 278) was obtained and only starting material was present
suggesting that it is difficult to hydrolyze this sterically hindered menthyl ester group.
Appela et al. encountered similar problems in hydrolyzing menthyl ester groups and they
employed rigorous condition by using methanolic solution of 1.5 N potassium hydroxide
under reflux232 in order to hydrolyse the menthyl ester. Unfortunately, when these
reagents and conditions were applied to the mixture of O2-alkylated products (312 and
313), again none of the desired carboxylic acid derivatives (277 and 278) (Scheme 71)
were afforded and only starting material was present.
OH
H
H
OH
H
COO
H
COOH
NaOH, r.t
HCl
N
O
DAM
328
N
O
DAM
329
Scheme 72232 Ester hydrolysis232
We came across a literature report by Timothy et al.234 which described a similar
problem in hydrolyzing menthol esters on Boc-containing substrates, as shown in
Scheme 73. They treated (330) with TFA, which cleaved the Boc and menthol groups
simultaneously. The resulting free amine was then treated with Boc-anhydride to give a
131
Results and Discussion
good yield of the hydrolyzed product (331). Our difficulty in removing the D-menthyl
group from the mixture of O2-alkylated product (312 and 313) might perhaps have been
overcome with the protocol of Timothy et al.234 protocol. Unfortunately, we came across
this approach too late and could not examine its utility with our compounds.
Ph
R
i) TFA
R
O
N
N
O
Boc
iii) (Boc)2O
Boc
330
Hydrolysis of Phenyl Menthol234
H
N
O
H
N
O
N
O
O
N
O
O
N
O
O
NHBoc
334
333
332
N
O
N
OH
NHBoc
Scheme 74
O
N
OH
2.6.
H
N
N
O
O
O
O
N
O
331
Scheme 73234
H
OH
ii) Aq NaOH
N
O
O
OH
OH
NHBoc
NHBoc
335
cis N1-alkylated (332 and 333) and trans N1-alkylated (334 and 335)
Final Conclusions
The inspiration for the synthetic strategy presented in this thesis was the continuation of
research carried out within our group by Walker et al. We continued the synthesis of key
intermediates (139 and 140) with the chiral building block (S)-dimethyl malate (107).
When we tried to repeat Walker’s approach for preparation of (2S)-2-trityloxybutane-1,4diiodide (138) from
(2S)-2-trityloxybutane-1,4-diol (136) via formation of (2S)-2-
trityloxy-1,4-methanesulfonyloxybutane (137), we managed to obtain only 5% yield in
comparison to Walker et al. 93% yield. This difficulty was overcome by directly
transforming (2S)-2-trityloxybutane-1,4-diol (136) into (2S)-2-trityloxybutane-1,4diiodide (138) upon treatment with iodine, triphenylphosphine and imidazole in 56%
yield. We were unable to reproduce the 99% yield reported by Walker et al. for the
cyclization
step,
which
involved
(2S)-2-trityloxybutane-1,4-diiodide
132
(138),
N-
Results and Discussion
(diphenylmethylene)glycine ethyl ester and lithium hexamethyldisilylazide as a nonnucleophilic base at -78 C initially for 2 h, then stirred overnight at r.t. When we
modified the reagent and conditions by using (2S)-2-trityloxybutane-1,4-diiodide (138),
N-(diphenylmethylene)glycine ethyl ester (145) and sodium hydride at r.t. for 2 h then 1
h reflux, it gave 16% yield of impure product (139 and 140) after column
chromatography. Given the work by Kurth et al.,200 the yields of the (139 and 140) might
have been improved improved, if the column chromatography had been avoided, as even
when using 10% of triethylamine as a mobile phase on silica column, degradation of
cyclised product (199) was observed. Also replacement of N-(diphenylmethylene)glycine
ethyl ester as Schiff’s base with methyl 2-{[(4-bromophenyl)methylidene]amino}acetate
might improve the reactivity of the dialkylation cyclization reaction based on the
findings of Donnell et al.203,204
The route pioneered by Ma et al.184 and developed by Walker et al.9 served as an
appropriate starting point for our purposes, considering the arduous task of dialkylation
(bis-electrophile) with Schiff’s base (glycine equivalent). We modified the dialkylation
reaction during the course of our investigation by employing (2S)-2-trityloxybutane-1,4diiodide (138) and the Schiff’s base was replaced by diethylmalonate (166). The overall
yield of the reaction after purification of the desired cyclised product (167) was 93%. The
use of a Lewis acid like tin chloride, which is highly toxic and environmentally
damaging, and the difficulty in imposing the Curtius rearrangement with tBuOH to
obtain
the
desired
ethyl
(1R,3S)
and
(1S,3S)-1-(tert-butoxycarbonyl)amino-3-
hydroxycyclopentane-1-carboxylic acid, prevented us from further pursuit of (167).
Our optimised pathway to (1S/R, 3S/R) and (1R/S, 3S/R)-1-(tert-butoxycarbonyl)amino3-hydroxycyclopentane-1-carboxylic acid (203 and 143) and (204 and 144) was attained
by dialkylating the bromobenzaldehyde Schiff’s base (197) with cis-1,4-dichlorobutene
(109), followed by hydrolysis of the imines and Boc protection to give the desired alkene
(189) with an overall yield of 27%. The alkene was then subjected to a hydroboration
reaction with borane-THF complex at 0 C for 7 min, with an overall yield of 48% for
the cis-products (203 and 143) and 23% for the trans-products (204 and 144).
Both the (1S/R, 3S/R) and (1R/S, 3S/R)-alcohols were suitably functionalized at the 3-
133
Results and Discussion
position, as per Walker's protocol, to enable subsequent preparation of the required
pyrimidine cyclo PNA monomers. Initially routes to the (1S/R, 3S/R) diastereoisomers of
1-(tert-butoxycarbonyl)amino-3-thyminyl-cyclopentane-1-carboxylic acid were examined.
Although, we were able to synthesize (232 and 233) by using cis-alcohol (203 and 143)
and (+)-10-camphorsulfonyl chloride (231) as a leaving group with overall yield of 92%,
we were unable to separate the cis-diastereoisomers.
Other attempts were made to separate the enantiomers, Firstly, treatment of trans-alcohol
(204 and 144) with taddol (enantioselective inclusion complexation). Unfortunately, no
crystals were formed between taddol (224) and (204 and 144). Secondly, we tried to
resolve the cis-acid alcohol (220 and 221) by the conventional resolution technique with
(S)-brucine dihydrate (217); this gave no crystals of diastereomeric salts of (220 and 221)
and (S)-brucine dihydrate (217). Finally, the alkene (222) was obtained in similar manner
as alkene (189) with overall yield of 32%. It was then subjected to transesterification
reaction with D- and L-menthol as a chiral auxiliary and butyl lithium as the base to give
corresponding D-menthyl ester alkene (280) and L-menthyl ester alkene (279) in 69% and
82% yields respectively. The corresponding alkene (280) on hydroboration reaction with
borane-THF gave cis-alcohol (299 and 300) (34%), a mixture of cis and trans-alcohol
(10%) and trans-alcohol (301 and 302) (15%). Alkene (279) under similar reagents and
conditions gave cis-alcohol (303 and 304) (31%), a mixture of cis and trans alcohol
(17%) and trans-alcohol (305 and 306) (16%). We were unable to separate the two cis
diastereoisomers and the two trans-diastereoisomers of above respective esters by
column chromatography or by recrystallization. Using the chiral hydroboration reagents
like (-)-IpcBH2 and (+)-IpcBH2 at – 40 C for 72 h, the overall yield of cis-alcohol (299
and 300) were 82% (59% e.e. based on indirect 1H-NMR analysis) and 69% (62% e.e.
based on indirect 1H-NMR analysis) respectively. In this manner, we manage to obtain
stereoselectively cis-isomer with no trace of trans-isomer.
Another significant outcome of this thesis is the discovery that trans-alcohol (204 and
144) is less reactive than cis-alcohol (203 and 143). When trans-alcohol (204 and 144)
was reacted with bulkier groups like bromobenzenesulfonyl chloride, as described by
Walker et al.. We obtained yields of around 25% for the desired product and recovered
mainly starting material. A similar difference in reactivity was observed, when cis-
134
Results and Discussion
alcohol (299 and 300) and trans-alcohol (301 and 302) were directly converted to the O2alkylated products with an overall yield of 18% for (312 and 313) and 3% for (336 and
337) respectively, when reacted with ADDP / PBu3 / Cbz cytosine.
The (1S/R, 3S/R) diastereoisomers of α-cycloPNA cytosine monomer were obtained from
tert-butyl (1R/S, 3S/R)-1-(ethoxycarbonyl)-3-hydroxycyclopentylcarbamate (203 and 143)
though conversion of the corresponding alcohols into their respective CSA derivatives
(232 and 233) in 92% yield. This was followed by nucleophilic displacement by
treatment of caesium carbonate and Cbz-cytosine, which gave O2-alkylated α-cycloPNA
cytosine derivative (266 and 267) in 30% yield instead of the desired N1-alkylated αcycloPNA cytosine derivative. An attempt to obtain the (1S/R, 3S/R) diastereoisomers of
α-cycloPNA cytosine monomer via cis-alcohol (299 and 300) led in the penultimate step
to O2-alkylated α-cycloPNA cytosine monomer instead of the desired N1-alkylated αcycloPNA cytosine monomer. This was achieved by converting the cis-alcohols (299 and
300) into their respective brosylate derivatives (308 and 309) with an in 75% yield,
followed by nucleophilic displacement using Cbz-cytosine and caesium carbonate,
resulting in O2-alkylated side products (312 and 313) in an overall yield of 25%.
The above results and literature precedent indicate that to promote N1-alkylation over O2alkylation, the protecting group for the nucleobase plays a pivotal role, especially in the
case of cytosine. The regioselectivity problem in cytosine could be overcome by using p(tert-butylbenzoyl) instead of Cbz protecting group, as the latter fails to decrease the
delocalization of the lone pair of electrons from the N4-amino group onto the O2-atom.
2.7.
Future Work
The research described in this thesis, identifies major obstacles for the synthesis of
homochiral α-cycloPNA oligomers for all four diastereoisomers and the means to
address them. The key to designing a successful strategy is the dialkylation reaction,
separation of diastereoisomers and regioselectivity (especially in case of Cbz-cytosine).
The route of Walker et al. could be pursued if the cyclization reaction is carried out using
pure and freshly prepared reagent. Instead of using N-(diphenylmethylene)glycine ethyl
ester (145) as a Schiff’s base, methyl 2-{[(4-bromophenyl)methylidene]amino}acetate
(197) should be used because aldimines (197) are better than ketimines (145) at
135
Results and Discussion
promoting dialkylation. The reason for this is that after 1st deprotonation by the base, the
pKa of ketimines (145) is increased by an additional 2.2 units in comparison to aldimines
(197) due to electronic affects. The cyclised product (139 and 140) should not be purified
by column chromatography, even with 1-5% triethylamine as the mobile phase. Instead,
the crude cyclised product (139 and 140) should be subjected directly to hydrolysis of the
imine and Boc protected to prevent degradation over the column as observed during the
preparation of alkene (189 and 222). These changes should improve the overall yield in
this route.
In Ma’s route, based on the bulkiness of protecting group, the diastereoselectivity could
be predicted, as observed during hydrolysis of diester (108) or (109) resulting in a
mixture of cis (1S) and trans (1R) isomers. When the diphenylmethyl group was used for
protection of the alcohol, the ratio was 3:97, while with a benzyl protecting group the
ratio was 1:3 ratio. When the trityl group is used for protection of alcohol (135), we
expect more diastereoselectivity as it is bulkier than the diphenylmethyl group. The best
dialkylation results were observed with diethylmalonate in the presence of freshly
prepared sodium ethoxide and (2S)-2-trityloxybutane-1,4-diiodide (138); this gave 93%
yield of alkylated product i.e. (S)-diethyl 3-(trityloxy)cyclopentane-1,1-dicarboxylate
(167). The Curtius rearrangement carried out on large scale by Varie et al. makes it an
attractive route as they succeeded in synthesizing alkene (167) without using highly toxic
tin salts by increasing the basicity of tert-butanol using tert-potassium butoxide, which in
turn increased the nucleophility of the tert-butanol. Hence, we believe that product (167)
should be pursued further by implementing monoester hydrolysis, followed by Varie's
Curtius rearrangement protocol. The protocol offers good overall yield, an
uncomplicated procedure, could be performed on a large scale, and diastereoselectivity is
more predictable than in other protocols. For initial exploration work, we would
recommend using the benzyl group because it gives access to all four diastereoisomers αcycloPNA.
From the above experiments and literature it can be envisaged that Ma’s route has better
control than Walker's route in terms of controlling stereoselectivity at the quaternary
carbon, and on hydrolysis of one ester an additional stereogenic centre is introduced with
good control. The preference for trans-isomer in Ma’s case is mainly due to the steric
hindrance provided by the protecting group, hence more diastereoselectivity is observed
136
Results and Discussion
in the bulkier group. In the case of Walker et al., the same kind of diastereoselectivity
was not induced even though the trityl group is much bulkier than the diphenylmethyl
group. This is probably due to some kind of co-ordination taking place between imines
and the protecting group, and possible π –π stacking between the phenyl group of trityl
group and Schiff’s base, which enhances cis-selectivity. While in Ma’s route selectivity
would be preferred on the least hindered side.
Selection of the protecting group plays a pivotal role in the regioselectivity of
nucleophilic with nucleobases. In the case of cytosine, Cbz-cytosine forms exclusively
O2-alkylated products instead of N1-alkylated products during nucleophilic substitution
with different substrates. For example: a) conversion of cis-CSA derivative (232 and
233) into O2-alkylated regioisomers (266 and 267), b) cis-alcohol (299 and 300) and
trans-alcohol (301 and 302) were directly converted into the O2-alkylated products (312
and 313) and (336 and 337) respectively, when reacted with ADDP/PBu3/ Cbz cytosine,
c) brosylate derivative (308 and 309) was converted to the O2-alkylated products (312
and 313). The above results suggest the Cbz protecting group promotes O2-alkylation.
Towards the end of our lab work, Micklefield et al. reported a similar regioselectivity
problem, which they resolved by protecting cytosine with p-(tert-butyl)benzoyl instead of
the Cbz group. This would provide a better protecting group to promote N1-alkylated
products.
137
Experimental
3. Experimental
Material Methods
All chemicals were purchased from Sigma, Aldrich, Avocado or Lancaster chemical
companies, UK. NMR spectra were recorded on Bruker AC 200 (200 MHz for 1H-NMR
and 50 MHz for 13C-NMR) and Bruker DPX 400 (400 MHz for 1H-NMR and 100 MHz
for 13C-NMR) spectrometers in CDCl3 unless otherwise stated. Spectra were calibrated
using chemical shift reported relative to solvent signals in CDCl3 [7.26 ppm for 1H
(singlet) and 77.0 for
13
C (central line of triplet)], DMSO-d6 [2.5 ppm for 1H (central
signal of quintuplet) and 39.7 ppm for 13C (central line of septuplet)] and CD3OD [3.31
ppm (central signal of quintuplet) and 4.84 ppm for 1H (singlet) and 49.05 ppm for 13C
(central line of septuplet)] respectively. Coupling constants (J values) are quoted in
Hertz (Hz). Infra-red spectra were recorded as thin films and obtained for samples
analysed as KBr discs on a Perkin Elmer 1600 FT-IR Spectrum spectrophotometer.
High resolution mass spectra (HMS) were obtained from Finnigan MAT 900 XLT high
resolution EI/CI/LSIMS/ESI and APcI capability at the National Mass Spectroscopy
Service Centre in Swansea. Low resolution Mass spectra (LRMS) were performed on
Kratos Concept 1S double sector mass spectrometer operated in Electron Impact mode
and Finnigan AutoMass Benchtop Single Quadrupole mass spectrometer operated in
Electrospray Ionisation mode (ESI) at Heriot-Watt University and Thermo LCQ
CLASSIC (ESI) and Thermo MAT 900 double focusing (EI) at Edinburgh University.
Optical Rotations were measured at r.t. using a Bendix-NPL 143D automatic
polarimeter (path length 1 cm) at room temperature. Concentrations used are cited in g /
100 mL. Units for []D values are quoted in degrees (). Melting points (mp) were
measured using Stuart Scientific SMP 10 Melting Point apparatus and are uncorrected.
Analytical thin-layer Chromatography (TLC) was performed on aluminium plates precoated with Kiesegel HF254 type 60 (Merck). Developed plates were visualized using
UV light (254 nm), and / or phosphomolybdic acid, potassium permagnate, vanillin,
anisaldehyde and ninhydrin dips.
Kiesegel H type 60 (Merck).
237
Flash chromatography was carried out using
X-RAY crystallography was performed by the
analytical services of Heirot-Watt University using a Bruker Nonius X8 Apex2 CCD
diffractometer running the Apex2 software. Elemental analyses were performed by the
138
Experimental
analytical service in the Department of Chemistry, School of Engineering and Physical
Sciences of Heriot-Watt Univerisity using a CE Exeter 440 Elemental Analyser.
Where required solvents were dried using conventional methods as described by Perrin
and Armarego.237 THF was pre-dried over Na wire and then distilled over Na /
benzophenone solution under dry N 2 .237 Ethyl acetate and DCM were dried over
CaH2 and stored over activated 4Å molecular sieves.
Acetone was first treated with successive small portions of potassium
permanganate at reflux until the violet colour persisted and then distilled. Distilled
acetone was dried with activated 4Å molecular sieve for 24 h. DMF was dried over
activated 4Å molecular sieves for 24 h, transferred via cannula onto a fresh batch of
activated 4Å molecular sieves for 1 h and then transferred again and stored over a
further fresh batch of activated 4Å molecular sieves. All reactions requiring anhydrous
conditions were conducted in heat-gun or overnight in oven at 100 C flame-dried
glassware under an inert atmosphere of dried spectroscopic grade nitrogen or argon.
Dimethyl (2S)-2-trityloxysuccinate (135)9
(S)-Dimethyl malate (107) (2.41 cm3, 18.2 mmol) was added at
O
r.t. under nitrogen to a stirred solution of triphenylmethyl
O 4
chloride (6.09 g, 21.8 mmol) and DBU (3.81 cm3, 25.5 mmol) in
3
OCPh3
1 O
2
O
135
3
dry DCM (15 cm ). The resulting mixture was left to stir at
r.t. for overnight. Subsequently, the reaction was quenched by addition of cold water
(20 cm3) and the two phases were separated and the organic layer was washed with
additional cold water (2 x 20 cm3) and the combined aqueous layers were re-extracted
with dichloromethane (2 x 30 cm3). The combined organic extracts were washed with
brine (20 cm3) and were dried over anhydrous sodium sulphate. Filtration followed by
solvent evaporation under reduced pressure gave crude yellow oil. This crude residue
was purified by column chromatography on silica using petroleum ether (40-60 C):
diethyl ether (8:2) as the eluent. The title compound (135) (6.22 g, 85%) was afforded
as colourless crystalline solid. mp 54-55 C (lit.1 54-55C); Rf 0.2. [Petroleum ether
(40-60 C): diethyl ether, (8:2)]; H (200 MHz; CDCl3) 2.48 (1H, dd, J 14.9 and 5.60,
139
Experimental
CH2), 2.61 (1H, dd, J 14.9 and 5.6, CH2), 3.37 (3H, s, CH3), 3.62 (3H, s, CH3), 4.46
(1H, apparent t, J 5.60, CH), 7.17-7.51 (15H, m, Ph-H).
(2S)-2-Trityloxybutane-1,4-diol (136)9
OCPh3
A solution of dimethyl (2S)-2-trityloxysuccinate (135) (5.45 g,
HO 4
13.47 mmol) in dry THF (15 cm3) was added at 0C under
OH
3
2
1
136
nitrogen dropwise to a stirred suspension of lithium borohydride
(LiBH4) (1.02 g, 46.8 mmol) in dry THF (45 cm3). A 1M
solution of B-methoxy 9-BBN in THF (1.25 cm3, 1.25 mmol) was then added dropwise
to the reaction mixture. The resulting mixture was allowed to warm to room
temperature, where upon it was left to stir for 3 h. Subsequently, the reaction was
quenched by addition of water (20 cm3) and the THF layer was separated. The aqueous
layer was re-extracted with ethyl acetate (3 20 cm3). The THF and ethyl acetate layers
were combined and the resulting organic solution was dried over anhydrous sodium
sulphate. Filtration followed by solvent evaporation under reduced pressure gave crude
colourless oil. This crude residue was purified by column chromatography on silica
using DCM:methanol (96:4) as the eluent. The title compound (136) (3.78 g, 80%) was
afforded as a colourless crystalline solid. mp 66-67C; Rf 0.22 [DCM:methanol (96:4)];
H (200 MHz; CDCl3) 1.63-1.71 (2H, m, CH2), 2.30 -2.66 (2H, br s, OH), 3.13-3.33
(2H, qd, J 4.1, 11.6, CH2), 3.55-3.66 (1H, m, CH), 3.74-3.86 (2H, m, CH2) and 7.237.57 (15H, m, Ph-H). C (100 MHz; CDCl3): 35.7 (CH2), 58.6 (CH2), 64.3 (CH2), 71.7
(CH), 87.1 (C), 127.2 (CH), 127.8 (CH), 128.7 (CH), 144.6 (C).
(2S)-2-Trityloxy-1,4-methanesulfonyloxybutane (137)9
Triethylamine
(0.2
cm3,
1.58
mmol)
followed
by
methanesulfonyl chloride (0.05 cm3, 0.79 mmol) were added
at 0C under nitrogen to a stirred solution of (2S)-2-
O
OCPh3
O
S
O 4
O
3
2
1
O
S
O
137
trityloxybutane-1, 4-diol (136) (0.12 g, 0.36 mmol) in dry
DCM (20 cm3). The reaction mixture was allowed to warm to r.t. and left to stir
overnight. The reaction was subsequently quenched by addition of (aq) solution of
saturated NaHCO3 (20 cm3) and the organic layer was separated. The resulting aqueous
140
Experimental
layer was subsequently extracted with DCM (3 x 20 cm3). The combined organic layers
were washed with brine (20 cm3) and dried over anhydrous sodium sulphate. Filtration
followed by solvent evaporation under reduced pressure gave crude yellow oil. This
crude residue was purified by column chromatography on silica using petroleum ether
(40-60 C): ethyl acetate (40:60) as the eluent. The title compound (137) was afforded
as colourless oil (0.13 g, 71%). Rf 0.22 [petroleum ether (40-60 C):ethyl acetate (4:6)];
H (200 MHz; CDCl3) 1.72-1.99 (2H, br m, CH2), 2.89 (3H, s, CH3), 2.92 (3H, s, CH3),
3.73-3.93 (3H, m, CH and CH2), 4.08-4.40 (2H, m, CH2) and 7.23-7.53 (15H, m, Ph-H);
C (50 MHz; CDCl3): 31.3 (CH2), 37.1 (CH3), 37.2 (CH3), 65.8 (CH2), 68.1 (CH), 69.5
(CH2), 87.7 (C), 127.4 (CH), 128.0 (CH), 128.6 (CH), 143.9 (C).
Attempted synthesis of (2S)-2-Trityloxybutane-1,4-diiodide (138)9
A solution of (2S)-2-trityloxy-1,4-methanesulfonyloxybutane
(137) (0.4 g, 0.79 mmol) in dry acetone (5 cm3) was added at
OCPh3
I
r.t. under nitrogen to a stirred solution of sodium iodide (0.66
I
4
3 2 1
138
g, 4.4 mmol) in dry acetone (20 cm3). The reaction mixture was
then heated to reflux, where upon it was left at 68 C for 48 h.
Subsequently, the reaction mixture was allowed to cool down to room temperature.
Evaporation of the solvent under reduced pressure gave dark brown oil, which was
subsequently re-dissolved in ethyl acetate (100 cm3). The resulting organic layer was
washed with water (100 cm3) and dried over anhydrous sodium sulphate. Filtration
followed by solvent evaporation under reduced pressure gave crude brown oil. This
crude residue was purified by column chromatography on silica using hexane:ethyl
acetate (20:1) as the eluent. However, none of the desired product (138) was could be
identified.
Attempted synthesis of (2S)-2-Trityloxybutane-1,4-diiodide (138)
OCPh3
OCPh3
OH
HO
136
I
I
138
Methyl iodide (0.06 cm3, 0.89 mmol) was added at 0 C under nitrogen to a stirred
141
Experimental
solution of triphenylphosphine (0.23 g, 0.89 mmol), diisopropyl azodicarboxylate
(DIAD) (0.18 cm3, 0.89 mmol), and (2S)-trityloxybutane-1,4-diol (0.13 g, 0.37 mmol)
in anhydrous THF (10 cm3). The reaction mixture was allowed to warm and left to stir
at r.t. for overnight. After this time TLC analysis implied that no product had formed
and so the reaction was gently heated at 25 C for another day. The reaction mixture
was allowed to cool down to room temperature. Subsequently, the reaction was
quenched by addition of water (10 cm3) and the mixture was left to stir for 10 min and
the organic layer was separated. The resulting aqueous layer was subsequently extracted
with ethyl acetate (2 x 20 cm3). The combined organic extracts were dried over
anhydrous sodium sulphate. Filtration followed by solvent evaporation under reduced
pressure gave a crude yellowish-red product. However, TLC analysis, 1H-NMR of
crude product, and 1H-NMR of fractions implied that none of the desired product (138)
was formed.
(2S)-2-Trityloxybutane-1,4-diiodide (138)9
A solution of (2S)-2-trityloxybutane-1,4-diol (136) (3.3 g, 9.46
mmol) in dry DCM (20 cm3) was added at r.t. under nitrogen to a
suspension of triphenylphosphine (19.85 g, 75.7 mmol), iodine
OCPh3
I
I
4
3 2 1
138
(15.61 g, 61.5 mmol) and imidazole (5.15 g, 75.7 mmol) in dry DCM (35 cm3). The
reaction mixture was then heated to reflux, whereupon it was left at 52 C for 9 h.
Subsequently, the reaction mixture was allowed to cool down to room temperature. The
reaction was quenched by addition of sodium bicarbonate (5.17 g, 61.5 mmol) in water
(20 cm3) and the resulting mixture was left to stir for 5 min. Subsequently, iodine was
added until the organic layer remained red in colour. Excess of iodine was removed by
addition of 2% aqueous solution of sodium thiosulphate to the reaction mixture until the
red colouration in the aqueous layer disappeared. The two layers were separated and the
organic layer was washed with water (20 cm3). The combined aqueous layers were reextracted with dichloromethane (2 x 20 cm3). The combined organic extracts were dried
over anhydrous sodium sulphate. Filtration followed by solvent evaporation under
reduced pressure gave crude yellowish powder. This crude residue was purified by
column chromatography on silica using diethyl ether as eluent. The pale yellowish oil
obtained was re-purified by column chromatography on silica using petroleum ether
142
Experimental
(40-60 C): ether (8.5:1.5) as the eluent. The title compound (138) was afforded as
colourless oil (3.0 g, 56%). Rf 0.72 [petroleum ether (40-60 C): ether, (8.5:1.5)]; [α]D25
-3.33 (c 0.9 in CHCl3) ( lit.19 [α]D25 -6.75 (c 1.93 in CHCl3); H (200 MHz; CDCl3)
2.03-2.35 (2H, m, CH2), 2.87 (1H, s, ½ CH2), 2.89-2.90 (1H, d , J 1.2, ½ CH2), 3.053.20 (2H, m, CH2), 3.34-3.42 (1H, m, CH) and 7.27-7.66 (15H, m, Ph-H); C (200
MHz; CDCl3) 0.7 (CH2), 11.2 (CH2), 38.9 (CH2), 72.2 (CH), 87.1 (C), 127.4 (CH),
128.0 (CH), 128.8 (CH) and 144.2 (CH); LRMS m/z (EI) (%): 568 ([M]+, 8), 450 (18),
423 (15), 409 (38), 364 (15), 361 (91), 335 (100), 312 (81); HMS (EI) found: 567.9763
([M]+, C23H22I2O requires 567.9755.
Atte mp t ed syn th esi s of eth yl (1 R , 3 S )- 1-(B en zh yd ryl i d en eami n o) -3 (triphenylmethoxy)cyclopentane-1-carboxylate (139) and ethyl (1S, 3S)-1(Benzhydrylideneamino)-3-(triphenylmethoxy)cyclopentane-1-carboxylate (140)9
LDA, dry THF -78 oC
OCPh3
Ph
CO2Et
1
3
N
I
I
2
Ph3CO
CO2Et
Ph
N
5
Ph
138
+
N
4
CO2Et
Ph3CO
Ph
Ph
Ph
145
139
140
Preparation of lithium diisopropylamide (LDA) in hexane [(1.0 M), (6.0 cm3, 6.0
mmol)]: A solution of 2.0 M n-butyl lithium in hexanes (3.0 cm3, 6.0 mmol) was added
dropwise at -70 C under nitrogen to a solution of diisopropylamine (0.85 cm3, 6.0
mmol) in hexane (2.15 cm3). After complete addition, the solution was gradually
brought up to -10 C and allowed to stir for 3 h at -10 C. The freshly prepared LDA 1.0
M, (4.67 cm3, 4.67 mmol) was added dropwise at -78 C under nitrogen to a stirred
solution of N-(diphenylmethylene)glycine ethyl ester (0.26 g, 0.97 mmol) in dry THF
(10 cm3). The reaction mixture was left to stir for half an h. Subsequently, a solution of
(2S)-2-trityloxybutane-1,4-diiodide (138) (0.61 g, 1.07 mmol) in dry THF (20 cm3) was
added dropwise at -78 C under nitrogen and the reaction was left to stir for 2 h before
being allowed to warm slowly to r.t. overnight. The reaction was then quenched by
addition of a saturated aqueous solution of ammonium chloride (30 cm3) and the
mixture was left to stir for ½ h. After the separation of THF and aqueous layers, the
THF layer was evaporated under reduced pressure and the crude oil was re-dissolved in
143
Experimental
the aqueous layer. Subsequently, the aqueous layer was extracted with ethyl acetate (3 x
30 cm3). The combined organic layers were washed with brine (30 cm3) and dried over
anhydrous sodium sulphate. Filtration followed by solvent evaporation under reduced
pressure gave crude dark red oil. This crude residue was purified by column
chromatography on silica using petroleum ether (40-60 C): diethylether: triethylamine
(8.9:1:0.1) as the eluent. Unfortunately, none of the fraction obtained showed the
presence of the desired compounds (139 and 140).
Atte mp t ed syn th esi s of eth yl (1 R , 3 S )- 1-(B en zh yd ryl i d en eami n o) -3 (triphenylmethoxy)cyclopentane-1-carboxylate (139) and ethyl (1S, 3S)1-(Benzhydrylideneamino)-3-(triphenylmethoxy)cyclopentane-1-carboxylate (140)9
LiHMDS, dry THF -78 oC
OCPh3
Ph
N
I
I
CO2Et
Ph3CO
CO2Et
CO2Et
Ph3CO
+
N
N
Ph
Ph
138
Ph
Ph
Ph
145
139
140
Method A - A 1.06M solution of lithium hexamethyldisilylazide (LiHMDS) in THF
(11.1 cm3, 11.09 mmol) was added dropwise at -78 °C under nitrogen to a stirred
solution of N-(diphenylmethylene)glycine ethyl ester (145) (1.45 g, 5.42 mmol) in dry
THF (5 cm3). The reaction mixture was left to stir for 45 min. Subsequently, a solution
of (2S)-2-trityloxybutane-1,4-diiodide (138) (2.8 g, 4.93 mmol) in dry THF (65 cm3)
was added dropwise at -78 °C under nitrogen and the reaction was left to stir for 45 min.
The reaction was allowed to warm slowly to r.t. and left to stir overnight. The reaction
was then quenched by addition of a saturated aqueous solution of ammonium chloride
(30 cm3) and the mixture was left to stir for half an h. After the separation of THF and
aqueous layers, the THF layer was evaporated under reduced pressure and the crude oil
was re-dissolved in the aqueous layer. Subsequently, the aqueous layer was extracted
with ethyl acetate (3 x 30 cm3). The combined organic layers were washed with brine
(30 cm3) and dried over anhydrous sodium sulphate. Filtration followed by solvent
evaporation under reduced pressure gave crude dark red oil. This residue was purified
by column chromatography on silica using Petroleum ether (40-60 C): ethyl acetate:
triethylamine (8.9:1:0.1) as the eluent. Again, no product was afforded.
144
Experimental
Method B - A 1.06 M solution of lithium hexamethyldisilylazide in THF ( 0.75 cm3,
0.78 mmol) was added dropwise to a stirred solution of N-(diphenylmethylene)glycine
ethyl ester (145) (0.2 g, 0.76 mmol) in dry THF (5 cm3) at -78 C under nitrogen, and
the reaction mixture left to stir for 45 min. Subsequently, a solution of (2S)-2trityloxybutane-1,4-diiodide (138) (0.4 g, 0.69 mmol) in dry THF (20 cm3) was added
dropwise and the reaction was left to stir at -78 C for 45 min. A second quantity of
1.06 M solution of lithium hexamethyldisilylazide in THF (0.75 cm3, 0.78 mmol) was
then added dropwise and the resulting mixture was left to stir for ½ h at -78 C. The
reaction was allowed to warm slowly to r.t. and left to stir overnight. Similar work-up
and purification protocol was followed as described in method A for (139 and 140). No
product was obtained.
Ethyl
(1R,
carboxylate
3S)-1-(Benzhydrylideneamino)-3-(triphenylmethoxy)cyclopentane-1(139)
and
ethyl
(1S,
3S)-1-(Benzhydrylideneamino)-3-
(triphenylmethoxy)cyclopentane-1-carboxylate (140)9
Method C - Sodium hydride (0.05 g, 1.27
mmol) was added at r.t. under nitrogen to a
Ph3CO
3
4
5
stirred
solution
(diphenylmethylene)glycine
of
ethyl
O
2
1
N
N-
Ph3CO
O
O
2
3
4
5
1
N
O
ester
(145) (0.14 g, 0.53 mmol) in dry THF (10
139
140
cm3). The reaction mixture was left to stir
for 15 min. Subsequently, a solution of (2S)-2-trityloxybutane-1,4-diiodide (138) (0.3 g,
0.53 mmol) in dry THF (20 cm3) was added dropwise and the reaction was left to stir
for 1 h at r.t. before being brought to reflux, whereupon it was left at 78 C for an hour.
The reaction was allowed to cool down to room temperature. The reaction was then
quenched with minimum amount of a methylated spirit and sufficient quantity of
crushed ice in order to destroy excess of sodium hydride. The insoluble solid in the
reaction mixture was filtered. After the separation of THF and aqueous layers, the THF
layer was evaporated under reduced pressure and the crude oil was re-dissolved in the
aqueous layer. Subsequently, the aqueous layer was extracted with ethyl acetate (3 x 30
cm3). The combined organic layers were washed with brine (30 cm3) and dried over
anhydrous sodium sulphate. Filtration followed by solvent evaporation under reduced
145
Experimental
pressure gave crude dark red oil. This crude residue was purified by column
chromatography on silica using petroleum ether (40-60 C): ethyl acetate: triethylamine
(8.9:1:0.1) as the eluent. The title compound (139 and 140) were afforded as pale
yellow oil (0.05 g, 16%). Rf 0.28 [petroleum ether (40-60 C): ethyl acetate:
triethylamine (8.9:1:0.1)]. H (200 MHz; CDCl3) 0.89-0.96 (3H, t, J 7.1, OCH2CH3
major isomer) and 0.94-1.02 .(3H, t, J 7.1, OCH2CH3 minor isomer), 1.19-2.33 (6H, br
m, 3 x CH2), 3.40-3.62 (2H, m, CH2), 3.89-4.03 (1H, q, J 6.4, CH major isomer) and
4.15-4.27 (1H, q, J 6.2, CH minor isomer) and 6.84-7.75 (25 H, m, Ph-H); C (50 MHz;
CDCl3) 13.9 (CH3), 32.4 (CH2 major isomer) and 32.8 (CH2 minor isomer), 37.8 (CH2
minor isomer) and 39.1 (CH2 major isomer), 46.6 (CH2 major isomer) and 48.2 (CH2
minor isomer), 60.4 (CH2), 72.2 (C), 75.1 (CH), 87.1 (C), 126.9 (CH), 127.7 (CH),
127.9 (CH), 128.4 (CH), 128.6 (CH), 129.0 (CH), 130.1 (CH), 130.4 (CH), 137.5 (C),
140.7 (C), 145.3 (CH) and 167.2 (CH), 174.1 (C).
Attempted
synthesis
of
ethyl
(1R,
(trityloxy)cyclopentane-1-carboxylate
3S)-1-(3-bromobenzylideneamino)-3-
and
ethyl
(1S,
3S)-1-(3-
bromobenzylideneamino)-3-(trityloxy)cyclopentane-1-carboxylate
CO2Et
OCPh3
N
I
I
NaH, Dry THF
CO2Et
Ph3CO
+
N
Br
CO2Et
Ph3CO
N
RT 2h, Reflux, 1h
H
H
138
197
Br
Br
338
339
Sodium hydride (0.08 g, 3.38 mmol) was added at r.t. under nitrogen to a stirred
solution of bromo-Schiff’s base (197) (0.34 g, 1.27 mmol) in dry THF (10 cm3). The
reaction mixture was left to stir for 15 min. Subsequently, a solution of (2S)-2trityloxybutane-1,4-diiodide (138) (0.6 g, 1.06 mmol) in dry THF (20 cm3) was added
dropwise and the reaction was left to stir for 1 h at r.t. before being brought to reflux,
where upon it was left at 78 C for an hour. Similar work-up and purification protocol
was followed as described in method C for (139 and 140). No product was obtained
(338 and 339).
146
Experimental
Model reaction for cyclisation
1)
Ethyl 2-(Benzhydrylideneamino)-2-methyl-1-propanoate (163)
A 1.06M solution of lithium hexamethyldisilylazide in
THF (3.04 cm3, 3.32 mmol) was added dropwise at -78 C
under
nitrogen
to
a
stirred
solution
of
N
N-
O
1
2
O
3
(diphenylmethylene) glycine ethyl ester (145) (0.41 g, 1.54
163
mmol) in dry THF (5 cm3). The reaction mixture was left
to stir for 45 min. Subsequently, a solution of methyl
iodide (1.0 g, 7.0 mmol) in dry THF (20 cm3) was added dropwise at -78 C under
nitrogen and the reaction was left to stir for 45 min. The reaction was allowed to
warm slowl y to room temperature and left to stir overnight. The reaction was then
quenched by addition of aqueous solution of saturated ammonium chloride (30 cm3) and
the mixture was left to stir for ½ h. After the separation of THF and aqueous layers, the
THF layer was evaporated under reduced pressure and the crude oil was re-dissolved in
the aqueous layer. Subsequently, the aqueous layer was extracted with ethyl acetate (3 x
30 cm3). The combined organic layers were washed with brine (30 cm3) and dried over
anhydrous sodium sulphate. Filtration followed by solvent evaporation under reduced
pressure gave crude dark red oil. This crude residue was purified by column
chromatography on silica using petroleum ether (40-60 C): ether (7.8:2.2) as the eluent.
The title compound (163) was afforded as pale yellow oil (77mg, 16%). Rf 0.28
[petroleum ether (40-60 C): ether (7.8:2.2)]; H (200 MHz; CDCl3) 1.38 (3H, t, J 7.1,
CH3), 1.54 (3H, s, CH3), 1.57 (3H, s, CH3), 4.25-4.35 (2H, m, CH2), 7.30-7.80 (10H, m,
Ph-H)
2)
Diethyl (3S)-3-(triphenylmethoxy)cyclopentane-1,1-dicarboxylate (167)
Sodium metal (0.41 g, 17.55 mmol) was carefully added
portionwise to anhydrous ethanol (20 cm3) at 0 C under
nitrogen. Subsequently, this ethanolic solution of sodium
2
CO2Et
Ph3CO
1
3
CO2Et
4
5
167
ethoxide (8.0 cm 3 , 7.02 mmol) was added dropwise to a
solution of diethylmalonate (166) (0.63 g, 3.96 mmol) in dry THF (10 cm3) at 0 C
under nitrogen and allowed to stir for 10 min. A solution of (2S)-2-trityloxybutane-1,4-
147
Experimental
diiodide (138) (1.2 g, 1.32 mmol) in dry THF (20 cm3) was added at 0 C under
nitrogen. Subsequently, the reaction mixture was then heated to reflux, where upon it
was left at 78 C for 6 h. The solvent was removed in vacuo and then purified by
column chromatography on silica using petroleum ether (40-60 C): ethyl acetate (8:2)
as the eluent. The title compound (167) was afforded as colourless oil (0.92 g, 93%). Rf
0.62 [petroleum ether (40-60 C): ethyl acetate (8:2)]; H (400 MHz; CDCl3) 1.11-1.16
(3H, t, J 7.1, CH3), 1.18-1.22 (3H, t, J 7.1, CH3), 1.40-1.46 (2H, dt, J 7.9 and 7.5 , C(5)H2), 1.74-1.85 (1H, dt, J 7.8 and 13.4, C(4)-H2), 1.87-1.95 (1H, dd, J 5.4 and 14.0,
C(2)-H2), 1.97-2.05 (1H, dd, J 6.5 and 14.0, C(2)-H2), 2.32-2.42 (1H, dt, J 7.6 and 13.4,
C(4)-H2), 3.99-4.22 (5H, m, 2 x CH2O and C(3)-H) and 7.32-7.44 (15H, m, Ph-H); C
(100 MHz, CDCl3) 13.9 (CH3), 14.0 (CH3), 31.5 (CH2), 32.7 (CH2), 41.1 (CH2), 58.5
(C), 61.2 (2 x CH2), 75.0 (CH), 87.1 (C), 126.9 (CH), 127.7 (CH), 128.9 (CH), 144.9
(C), 171.8 (C), 172.3 (C); LRMS m/z (EI) (%): 490 ([M+NH4]+, 100), 473 (10), 395
(28), 386 (10), 278 (16), 276 (14), HMS (ES) (Found: 490.2589 ([M+NH4]+,
C30H36NO5 requires 490.2588).
Dimethyl (2S)-2-(benzyloxy)succinate (168) 9,192,193
(S)-dimethyl malate (107) (1.0 g, 6.17 mmol) was added at r.t.
under argon to a stirred suspension of silver(I) oxide (2.29 g,
9.87 mmol) and benzyl bromide (1.58 g, 9.25 mmol) in dry
3
ethyl acetate (10 cm ). The resulting mixture was left to stir at
r.t. overnight. Subsequently, the inorganic precipitate was
removed by filtration on a celite pad and washed with ethyl
O
O 4
O
1 O
3
2
O
168
acetate (3  20 cm3). The filtrate was concentrated by removal of solvent in vacuo and
the resulting crude residue was purified by column chromatography on silica using
petroleum ether (40-60 C): ethyl acetate (8:2) as the eluent. The title compound (168)
was afforded as colourless oil (0.85 g, 55%). Rf 0.45 [petroleum ether 40-60 oC : ethyl
acetate (8:2)]; H (200 MHz; CDCl3) 2.77-2.78 (1H, d, J 2.2, ½ CH2), 2.80 (1H, s, ½
CH2) 3.66 (3H, s, CH3), 3.74 (3H, s, CH3), 4.35-4.41 (1H, dd, J 5.81 and 7.5, CH), 4.494.55 (1H, d, J 11.4, CH2), 4.73-4.78 (1H, d, J 11.4, CH2) and 7.28-7.34 (5H, m, Ph-H);
C (50 MHz; CDCl3) 38.1 (CH2), 52.3 (CH3), 52.6 (CH3), 73.4 (CH), 74.7 (CH2), 128.4
(CH), 128.7 (CH), 128.9 (CH), 137.4 (C), 170.8 (C) and 171.5 (C).
148
Experimental
(2S)-2-(benzoxy)butane-1,4-diol (169)192,193
A solution of dimethyl (2S)-2-benzyloxysuccinate (168) (0.79 g,
3.13 mmol) was added dropwise to a stirred solution of lithium
O
borohydride (0.3 g, 7.83 mmol) in dry THF (60 cm3) at 0C under
OH
HO
4
3
nitrogen. A 1M solution of B-methoxy 9-BBN in THF (0.38 cm ) was
2
3
1
169
then added dropwise to the above reaction mixture at 0C under
nitrogen. The resulting mixture was allowed to warm to r.t. and left to stir for 3 h.
Subsequently, the reaction was quenched slowly by addition of water (100 cm3).
After the separation of THF and aqueous layers, the THF layer was evaporated under
reduced pressure and the crude oil was re-dissolved in the aqueous layer. The aqueous
layer was re-extracted with ethyl acetate (5  20 cm3). The combined organic layers
were washed with brine (30 cm3) and dried over anhydrous sodium sulphate. Filtration
followed by solvent evaporation under reduced pressure gave crude colourless oil. This
crude residue was purified by column chromatography on silica using ethyl acetate as
eluent. The title compound (169) was afforded as colourless oil (0.52 g, 85%). Rf 0.51
(ethyl acetate); max (neat)/ cm-1 740 (m), 1054 (s), 1277 (m), 1453 (m), 1708 (m), 2863
(m), 2930 (m) and 3368 (s); H (200 MHz; CDCl3) 1.70-1.90 (2H, m, CH2), 2.93-3.30
(2H, brs, OH), 3.50-3.78 (5H, m, 2 x CH2 and 1 x CH), 4.55 (2H, d, J 1.3, CH2) and
7.21-7.38 (5H, m, Ph-H); C (50 MHz; CDCl3) 34.0 (CH2), 59.4 (CH2), 63.9 (CH2),
71.6 (CH2), 77.8 (CH), 127.9 (CH), 128.5 (CH), 138.1 (C); LRMS m/z (ES) (%): 197
([M+ H]+, 100), 192 (13), 104 (20), 91 (61); HMS m/z (ES) 197.1171 ([M+H]+
C11H17O3S requires 197.1172).
(S)-2-(benzoxy)butane-1,4-diiodide (170)
A solution of (S)-2-(benzoxy)butane-1,4-diol (169) (0.52 g, 3.0
mmol) in dry DCM (20 cm3) was added dropwise to a stirred
O
suspension of triphenylphosphine (5.61 g; 21.4 mmol), iodine
(4.75 g; 18.7 mmol) and imidazole (1.46 g; 21.4 mmol) in dry
DCM (35 cm3) at r.t. under argon. The reaction mixture was
I
I
4
3 2 1
170
then heated to reflux, whereupon it was left at 52 C for 5 h. The resulting mixture was
allowed to cool down to room temperature. Subsequently, the reaction was quenched by
149
Experimental
addition of sodium bicarbonate (0.73 g, 8.75 mmol) in water (20 cm3) and the resulting
mixture was left to stir for 5 min. Subsequently, iodine was added until the organic layer
remained red in colour. Finally, a 2% aqueous solution of sodium thiosulphate was
added to the reaction mixture until the red colouration in the aqueous layer disappeared.
Aqueous layer was separated and re-extracted with dichloromethane (2 x 20 cm3). The
combined organic extracts were extracted with brine (30 cm3) and dried over anhydrous
sodium sulphate. Filtration followed by solvent evaporation under reduced pressure
gave crude yellowish powder. This residue was purified by column chromatography on
silica using diethyl ether as eluent. The pale yellowish oil obtained was re-purified by
column chromatography on silica using petroleum ether (40-60 C): ethyl acetate (9:1)
as the eluent. The title compound (170) was afforded as colourless oil (1.11 g, 100%).
Rf 0.65 [petroleum ether (40-60 C): ethyl acetate (9:1)]; [α]D25 –56.09 (c 2.30 in
DCM); max (neat)/ cm-1 697 (s), 753 (s), 1027 (w), 1062 (s), 1207 (w), 1247 (w), 1333
(w), 1394 (w), 1423 (w), 1453 (m), 1495 (w), 1602 (w), 2863 (w), 3032 (w) and 3064
(w); δH (200 MHz, CDCl3) 2.10-2.15 (2H, q, J 6.2, CH2), 3.25-3.28 (2H, t, J 6.8, CH2),
3.31-3.32 (2H, dd, J 5.2 and 2.1, CH2O), 3.44-3.49 (1H, m, CH-O), 4.48-4.51 (1H, d, J
11.2 , Ph-CH2), 4.68-4.71 (1H, d, J 11.2 , Ph-CH2) and 7.32-7.45 (5H, m, Ph-H); δC(50
MHz, CDCl3) 2.0 (CH2), 8.6 (CH2), 38.7 (CH2), 71.8 (CH2), 77.0 (CH), 128.0 (CH),
128.5 (CH), 137.5 (C); LRMS m/z (EI) (%): 416 ([M+H]+, 40), 326 (36), 324 (100), 309
(42), 289 (32), 261 (12), 259 (64), 167 (28), 131 (12), 92 (32), 91 (100), 65 (10); HMS
(EI) 415.9126 ([M+H]+ C11H14OI2 requires 415.9129).
Attempted
synthesis
of
ethyl
amino)cyclopentane-1-carboxylate
(1R,
3S)-3-(benzyloxy)-1-(diphenylmethylene)
and
ethyl
(1S,
3R)-3-(benzyloxy)-1-
(diphenylmethylene) amino)cyclopentane-1-carboxylate (171 and 172)
Sodium hydride (0.05 g, 1.16
O
O
solution
of
O
O
mmol) was added to a stirred
N-
1
N
2
3
4
O
O
4
5
1
N
2
3
5
(diphenylmethylene)glycine ethyl
ester (145) (0.16 g, 0.58 mmol) in
171
172
3
dry THF (10 cm ) at r.t. under
nitrogen, and the reaction mixture was left to stir for 15 min. Subsequently, a solution
150
Experimental
of (2S)-2-benzyloxybutane-1,4-diiodide (170) (0.22 g, 0.53 mmol) in dry THF (20 cm3)
was added dropwise and the reaction was left to stir for 1 h at r.t. before being brought
to reflux for two h. The resulting mixture was allowed to cool down to room
temperature. Subsequently, the reaction was then quenched with minimum amount of a
methylated spirit and sufficient quantity of crushed ice in order to destroy excess of
sodium hydride. The insoluble solid in the reaction mixture was filtered. After the
separation of THF and aqueous layer, the THF layer was evaporated under reduced
pressure and the crude oil was re-dissolved in the aqueous layer. The aqueous layer was
re-extracted with ethyl acetate (3  30 cm3). The combined organic layers were washed
with brine (30 cm3) and dried over anhydrous sodium sulphate. Filtration followed by
solvent evaporation under reduced pressure gave crude dark red oil. This crude residue
was purified by column chromatography on silica using petroleum ether (40-60 C):
ethyl acetate (10:1) as the eluent. None of the desired product (171 and 172) was
formed.
Ethyl 2-[[(E)-1-(4-Bromophenyl)methylidene]amino]acetate (197)199,200,201
O
1
Anhydrous magnesium sulphate (5.0 g) and triethylamine (6.1
cm3, 43.2 mmol) were added at 0 C under argon to a solution of
5'
Br
4'
N
O
2
1'
3'
p-bromobenzaldehyde (184) (4.0 g, 21.6 mmol) and ethyl glycine
6'
2'
197
hydrochloride (196) (3.01 g, 21.6 mmol) in dry dichloromethane (100 cm3). The
reaction mixture was left to stir at r.t. for 10 h. The magnesium sulphate was removed
by filtration. The filtrate was evaporated under reduced pressure to give white solid,
which was re-dissolved in ether (100 cm3). The insoluble solid in the ether solution was
filtered. The ether layer was washed with water (50 cm3), brine (30cm3), and dried over
anhydrous sodium sulphate. Filtration followed by solvent evaporation under reduced
pressure gave the title compound (197) (5.74gm, 98.4%) as a colourless liquid. δH(200
MHz, CDCl3) 1.28-1.35 (3H, t, J 7.1, CH3), 4.19-4.30 (2H, q, J 7.1, CH2), 4.40 (2H, s,
CH2), 7.51- 7.66 (4H, q, J 8.7, Ph-H), 8.19 (1H, s, CH=N); δC (200 MHz, CDCl3) 13.7
(CH3), 60.5 (CH2), 61.3 (CH2), 125.0 (C), 129.3 (CH), 131.0 (CH), 134.0 (C), 163.4
(CH), 169.3 (C).
151
Experimental
Methyl 2-[[(E)-1-(4-Bromophenyl)methylidene]amino]acetate (198).
O
1
Anhydrous magnesium sulphate (6.24 g, 51.8 mmol) and
triethylamine (12.2 cm , 86.4 mmol) were added at 0 C under
5'
3
Br
argon to a solution of P-bromobenzaldehyde (184) (8.0 g, 43.2
6'
4'
N
O
2
1'
3'
2'
198
mmol) and methyl glycine hydrochloride (195) (5.96 g, 47.5
mmol) in dichloromethane (100 cm3). The reaction mixture was left to stir at r.t. for 10
h. Similar work-up protocol was followed as described for Schiff’s base (197). This
afforded as white solid crude product. This was purified by recrystallization with
hexane:ether to give the title compound (198) as white crystalline solid (8.2 g, 75%).
mp 51-53 C (Hexane:ether); (Found: C, 46.63; H, 3.77; N, 5.25% C10H10BrNO2
requires C, 46.9; H, 3.94; N, 5.47%); max (KBr)/ cm-1 821 (s), 851 (m), 961 (m), 1010
(s), 1069 (s), 1099 (m), 1198 (s), 1296 (w), 1349 (m), 1403 (m), 1435 (m), 1487 (m),
1568 (w), 1589 (m), 1647 (s), 1754 (s), 2866 (w), 2950 (m), 2997 (w) and 3480 (w);
δH(200 MHz, CDCl3) 3.73 (3H, s, CH3), 4.37 (2H, s, CH2), 7.48-7.62 (4H, q, J 8.7 , PhH), 8.19 (1H, s, CH=N); δC(50 MHz, CDCl3) 52.0 (CH3), 61.6 (CH2), 125.6 (C), 129.7
(CH), 131.7 (CH), 134.3 (C), 164.0 (CH), 170.2 (C). LRMS m/z (EI) (%): 255 ([M]+,
100).
Ethyl 1-[[(E)-1-(4’-Bromophenyl)methylidene]amino]-cyclopent-3-ene-1-carboxylate
(199) 199,200,201
Schiff’s base (197) (5.4 g, 20.0 mmol) was dissolved in dry THF (20
cm3) and was added dropwise to a stirred solution of sodium hydride
(1.76 g, 44.0 mmol) in dry THF (60 cm3) at r.t. under nitrogen, and
2
COOEt
1
N=CH-C6H4-Br
3
4
5
199
the reaction mixture was left to stir for 15 min. To
this reaction mixture cis-1,4-dichloro-2-butene (109) (2.75 g, 22.0 mmol) was added
dropwise, and the reaction mixture was stirred at r.t. for 2 h. Subsequently, the reaction
mixture was heated to reflux, where upon it was left at 78 C for 1 h. Subsequently, the
reaction mixture was allowed to cool down to room temperature. The reaction was
quenched with minimum amount of a methylated spirit and sufficient quantity of
crushed ice in-order to destroy excess of sodium hydride. The insoluble solid in the
reaction mixture was filtered. After the separation of THF and aqueous layers, the THF
152
Experimental
layer was evaporated under reduced pressure and the crude oil was re-dissolved in the
aqueous layer. The aqueous layer was re-extracted with ethyl acetate (3  50 cm3). The
combined organic layers were washed with brine (50 cm3) and dried over anhydrous
sodium sulphate. Filtration followed by solvent evaporation under reduced pressure
gave crude dark red oil (199) (6.18 g, 96%). δH(200 MHz, CDCl3) 1.21-1.28 (3H, t, J
7.1, CH3), 2.62-2.70 (2H, d, J 15.8, CH2), 3.12-3.20 (2H, d, J 15.4, CH2), 4.15-4.25
(2H, q, J 7.1, CH2), 5.68 (2H, s, CH=CH), 7.51-7.57 (2H, d, J 12.9, Ph-H), 7.66-7.70
(2H, d, J 6.2, Ph-H), 8.13 (1H, s, CH=N).
Methyl 1-[[(E)-1-(4’-Bromophenyl)methylidene]amino]-cyclopent-3-ene-1-carboxylate
(200).202
Schiff’s base (198) (6.7 g, 26.1 mmol) was dissolved in dry THF
(20 cm3) and was added dropwise to a stirred suspension of
sodium hydride (2.3 g, 57.6 mmol) in dry THF (40 cm3) at r.t.
2
COOMe
1
N=CH-C6H4-Br
3
4
5
200
under nitrogen. The reaction mixture was left to stir for 15 min.
To this reaction mixture cis-1,4-dichloro-2-butene (109) (3.30 cm3, 31.4 mmol) was
added dropwise, and the reaction mixture was stirred at r.t. for 2 h. Subsequently, the
reaction mixture was heated to reflux, whereupon it was left at 78 C for 1 h. Similar
work-up protocol was followed as described for cyclized product (199). The title
compound (200) was afforded as crude red oil (8.52 g). δH(200 MHz, CDCl3) 2.63-2.71
(2H, d, J 15.4, CH2), 3.12-3.20 (2H, d, J 15.4, CH2), 3.74 (3H, s, CH3), 5.69 (2H, s,
CH=CH), 7.52-7.58 (2H, d, J 12.0, Ph-H), 7.68-7.71 (2H, d, J 6.2, Ph-H) and 8.12 (1H,
s, CH=N).
Ethyl 1-(aminocyclopent-3-ene)-1-carboxylate (201)199,200,201
Crude cyclized product (199) (6.18 g 19.1 mmol) was dissolved in
diethylether (60 cm3). To this, aqueous 1N hydrochloric acid solution
(57.50 cm3, 57.5 mmol) was added and the reaction mixture was stirred at
2
COOEt
1
NH2
3
4
5
201
r.t. for 2 h. Water (30 cm3) was added to the reaction mixture. The ether
layer was separated from the aqueous layer. Subsequently, the aqueous layer was
extracted with diethylether (2 x 20 cm3). The aqueous layer was basified with 1N (aq)
153
Experimental
sodium hydroxide until the solution was adjusted to pH 9.0. The resulting reaction
mixture was extracted with ethyl acetate (5 x 30 cm3). The combined ethyl acetate layer
was dried over magnesium sulphate. Filtration followed by solvent evaporation under
reduced pressure afforded the title compound (201) (1.58 g, 53.1%) as a crude
yellowish brown liquid. δH(200 MHz, CDCl3) 1.00-1.07 (3H, t, J 7.5, CH3), 1.84 (2H, s,
NH2), 2.03-2.11 (2H, d, J 15.4, CH2), 2.69-2.76 (2H, d, J 15.4, CH2), 3.89-4.00 (2H, q,
J 7.1, CH2), 5.42 (2H, s, CH=CH).
Methyl 1-(aminocyclopent-3-ene)-1-carboxylate (202)202
Crude cyclized product (200) (8.52 g, 27.6 mmol) was dissolved
in diethylether (90 cm 3). To this, aqueous 1N hydrochloric acid
solution (70.0 cm3, 70.0 mmol) was added and the reaction
2
COOMe
1
NH2
3
4
5
202
mixture was stirred at r.t. for 4 h. Similar work-up protocol
was followed as described for cyclized amine (201). The title
compound (202) was afforded as crude yellowish brown oil (1.58 g, 42.8%).
Ethyl 1-[(tert-Butoxycarbonyl)amino]-cyclopent-3-ene-1-carboxylate (189)199,200,201
Crude ethyl-1-aminocyclopent-3-enecarboxylate (201) (1.58 g,
10.19 mmol) was treated with di-tert-butyl dicarbonate (2.67 g,
12.23 mmol) in chloroform (20 cm3) at r.t. under nitrogen. The
2
COOEt
1
NHBoc
3
4
5
189
reaction mixture was then heated to reflux, where upon it was left
at 73 C for 4.5 h. Subsequently, the reaction mixture was allowed
to cool down to room temperature. The solvent was removed in vacuo. Purification of
the crude residue obtained was performed by column chromatography on silica using a
gradient system of petroleum ether 40-60 C: ethyl acetate (9:1 to 8:2) as an eluent. The
title compound (189) was afforded as a white solid (1.4 g, 54%). mp 79-81 C, (lit.182
mp 76-78 C and lit.202 mp 82 C); Rf 0.4 [petroleum ether (40-60 C): ethyl acetate
(8.0:2.0)]. δH(200 MHz, CDCl3) 1.18 (3H, t, J 7.1 Hz, CO2CH3), 1.36 (9H, s, 3 x CH3),
2.53 (2H, d, J 16.1, CH2), 2.97 (2H, d, J 15.9, CO2CH2), 4.12 (2H, q, J 7.1), 5.24 (1H, s,
br, NH) and 5.57 (2H, s, CH=CH).
154
Experimental
Methyl 1-[(tert-Butoxycarbonyl)amino]-cyclopent-3-ene-1-carboxylate (222)202
Crude methyl-1-aminocyclopent-3-ene-1-carboxylate (202) (1.7
g, 12.1 mmol) was added at r.t. under nitrogen to a solution of
di-tert-butyl dicarbonate (1.13 g, 5.16 mmol) in chloroform (20
2
COOMe
1
NHBoc
3
4
5
222
cm3). The reaction mixture was then heated to reflux, where
upon it was left at 73 C for 4.5 h. Subsequently, the reaction
mixture was allowed to cool down to room temperature. The solvent was removed in
vacuo. Purification of the crude residue obtained was performed by column
chromatography on silica using a gradient system of petroleum ether 40-60 oC: ethyl
acetate (9:1 to 8:2). The title compound (222) was afforded as a white solid (2.0 g,
74%). mp 112-113 C; Rf 0.32 [petroleum ether (40-60 oC): ethyl acetate (8:2)]; (Found:
C, 59.45; H, 8.10; N, 5.63% C12H19NO4 requires C, 59.73; H, 7.94; N, 5.81%); max
(KBr)/ cm-1 1065 (m), 1086 (m), 1165 (m), 1225 (m), 1295 (m), 1365 (m), 1391 (m),
1439 (m), 1702 (s), 1736 (s), 2951 (w), 2977 (m), 3150 (w) and 3264 (m); δH(200 MHz,
CDCl3) 1.39 (9H, s, 3 x CH3), 2.57 (2H, d, J 16.1, CH2), 3.01 (2H, d, J 16.0, CH2), 3.70
(3H, s, CO2CH3), 5.21 (1H, br, s, NH), 5.61 (2H, s, CH=CH); δC(50 MHz, CDCl3) 28.3
(3 x CH3), 44.9 (2 x CH2), 55.6 (CH3), 64.3 (C), 80.0 (C), 127.7 (CH), 154.9 (C), 174.1
(C). LRMS m/z (EI) (%): 242 ([M]+, 20), 199 (32), 136 (38), 114 (40), 95 (43), 76 (30),
47 (52), 38 (100).
Ethyl
(1R/1S,
3S/3R)-1-(tert-Butoxycarbonyl)amino-3-hydroxycyclopentane-1-
carboxylic acid (203 and 143) and Ethyl (1R/1S, 3R/3S)-1-(tert-Butoxycarbonyl)amino3-hydroxycyclopentane-1-carboxylic acid (204 and 144). 9 165,166.181,182,183
2
HO
4
CO2Et
CO2Et HO
1
NHBoc
3
NHBoc
5
203
cis
HO
143
204
CO2Et HO
CO2Et
NHBoc
NHBoc
trans
144
Borane.tetrahydrofuran complex (3.1 cm3; 3.1 mmol) was added dropwise to a solution
of alkene (189) (0.75 g, 2.94 mmol) in dry THF (8 cm3) at 0 C under nitrogen. The
reaction was stirred for 7 min at 0 C. The reaction mixture was quenched with
155
Experimental
dropwise addition of water (5 cm3), followed by aqueous hydrogen peroxide (35% w/w;
1.43 cm3, 14.7 mmol) and 1N (aq) sodium hydroxide was used to adjust the pH of the
reaction mixture to pH 9.0. The reaction mixture was left to stir at r.t. for 2 h. The
reaction mixture was extracted with ethyl acetate (4 x 15 cm3), and then the aqueous
layer was saturated with potassium carbonate and re-extracted with THF (2 x 10 cm3).
The combined organic extracts were washed with brine (20 cm3). Subsequently, the
organic layer was dried over magnesium sulphate. Filtration was followed by solvent
evaporation under reduced pressure gave the crude colourless oil. The crude residue,
which was pre-absorbed onto silica and purified by column chromatography using a
gradient system of petroleum ether 40-60 C: ethyl acetate (8:2 to 5:5) as an eluent to
give starting material (189) (12%), cis-alcohol (203 and 143) (48%) and trans-alcohol
(204 and 144) (23%). The first to elute was the cis-alcohol (0.38 g, 48%) as a white
solid. mp 66-68 C; Rf 0.26 [petroleum ether (40-60 C): ethyl acetate (5:5)]; [α]D25
+7.69 (c 0.65 in DCM); 1H-NMR (200 MHz, CDCl3) δ 1.25-1.29 (3H, t J 7.1, CH3),
1.43 (9 H, s, 3 x CH3), 1.86-2.17 (4H, br m, 2 x CH2), 2.30 (1H, brs, CH2) 2.43-2.48
(1H, dd J 6.0 and 15.0, CH2), 4.18-4.23 (2H, q J 7.1, CH2O), 4.34 (1H, brs, CH-OH),
4.38 (1H, brs, OH), 5.84 (1H, brs, NH), δH(400 MHz, CDCl3) 13.95 (CH3), 28.2 (3 x
CH3), 34.9 (CH2), 35.6 (CH2), 46.6 (CH2), 61.7 (CH2), 64.6 (C), 73.0 (CH), 80.1 (C),
154.8 (C), 174.8 (C); LRMS m/z (ES) (%): 296 ([M+Na]+, 100), 282 (15), 195 (15);
HMS (ES): 296.1468 ([M+Na]+ C13H23NO5Na requires 296.1468. Second to elute was
the trans-alcohol (0.19 g, 23%). mp 85-86 C; Rf 0.18 [petroleum ether (40-60 C):
ethyl acetate (5:5)]; (Found: C, 57.45; H, 8.69; N, 4.98 C13H23NO5 requires C, 57.13; H,
8.48; N, 5.12%); max (KBr)/ cm-1 1043 (w), 1090 (w), 1167 (w), 1242 (w), 1292 (w),
1367 (m), 1383 (w), 1688 (s), 1729 (m), 2981 (m) and 3392 (s); δH(400 MHz, CDCl3)
1.28-1.33 (3H, t, J 7.1, CH3), 1.46 (9 H, s, 3 x CH3), 1.71 (1H, m, ½ CH2), 1.80 (1H, m,
½ CH2), 2.0 (1H, m, ½ CH2), 2.11 (1H, m, ½ CH2), 2.25-2.31 (1H, m, ½ CH2), 2.332.37 (1H, dd, J 3.4 and 14.6, ½ CH2), 2.90 (1H, brs, OH), 4.20-4.27 (2H, m, CH2O),
4.46 (1H, brs, CH-OH), and 5.07 (1H, brs, NH); δC(100 MHz, CDCl3) 13.9 (CH3), 28.1
(3 x CH3), 34.4 (CH2), 35.9 (CH2), 46.7 (CH2), 61.6 (CH2), 64.8 (C), 72.6 (CH), 79.7
(C) 155.1 (C), 175.6 (C); LRMS m/z (ES) (%): 296 ([M+Na]+, 100), 282 (10), 239 (31),
195 (70), 82 (12); HMS (ESI) 296.1468 ([M+Na]+ C13H23NO5Na requires 296.1468.
156
Experimental
Ethyl
(1S/1R,
3R/3S)-1-[(tert-Butoxycarbonyl)amino]-3-[{(camphor)-10'-
sulfonyl}oxy]cyclopentane-1-carboxylate (232 and 233)
DMAP (0.59 g, 4.85 mmol), dry TEA (0.68 cm3,
9'
4.85 mmol), (+) 10-camphorsulfonyl chloride (1.22
5'
g, 4.85 mmol) were added at 0 C under nitrogen to
6'
a stirred solution of cis-alcohol (203 and 143) (0.27
3
g, 0.97 mmol) in dry DCM (20 cm ). The resulting
mixture was allowed to stir at r.t. for overnight.
7'
H
4'
1'
O
8'
H
3'
2'
10' O
S
O
O 2
CO2Et
1
3
NHBoc
4
5
cis
232
O
O
S
O
O
CO2Et
NHBoc
233
Subsequently the reaction was diluted with DCM
(40 cm3), then quenched by addition of a cold solution of 1N (aq) citric acid (30 cm3). The
two phases were separated and the organic layer was washed with a further quantity of 1N
aqueous citric acid (30 cm3) followed by a saturated aqueous solution of NaHCO3 (25
cm3), water (20 cm3) and brine (20 cm3). The organic layer was then dried over
magnesium sulphate. Filtration was followed by removal of the solvent under reduced
pressure afforded the crude product. The crude residue, which was pre-absorbed onto
silica and purified by column chromatography using a gradient system of petroleum ether
40-60 C: ethyl acetate (8:2 to 5:5) as an eluent. The title compound (232 and 233) was
afforded as white crystalline solid (0.44 g, 92%). mp 79-81 C; Rf = 0.31 [Petroleum ether
(40-60 C): ethyl acetate (6.7:3.3)]; [α]D25 +22.95 (c 0.92 in DCM); (Found: C, 56.54; H,
7.82; N, 2.65% C23H37BrNO8S requires C, 56.65; H, 7.65; N, 2.87%); max (KBr)/ cm-1
892 (s), 940 (m), 1055 (m), 1167 (s), 1252 (m), 1284 (m), 1392 (m), 1417 (w), 1455 (m),
1504 (m), 1713 (s), 1745 (s), 2975 (s) and 3382 (m); δH(400 MHz, CDCl3) 0.88 (3H, s,
CH3), 1.11 (3H, s, CH3), 1.24-1.27 (3H, t, J 7.1, CH3), 1.43 (9 H, s, 3 x CH3), 1.45-1.51
(1H, br m, ½ CH2), 1.63-1.73 (1H, m, ½ CH2), 1.96-2.00 (1H, d, J 18.5, ½ CH2), 2.022.40 (8H, br m, 3 ½ x CH2 and 1 x CH), 2.42-2.54 (1H, m, ½ CH2), 2.73-2.82 (1H, m, ½
CH2), 2.97-3.01 (1H, d, J 15.1, ½ CH2), 3.57-3.61 (1H, d, J 15.1, ½ CH2), 4.15-4.21 (2H,
q, J 6.9, O-CH2), 5.18 (1H, brs, NH), 5.32 (1H, m, CH-O-S); δC(100 MHz, CDCl3) 14.1
(CH3), 19.7 (CH3), 19.8 (CH3), 24.7 (CH2), 24.9 (CH2), 26.9 (CH2), 28.3 (3 x CH3), 32.3
(CH2 (major), 32.4 (CH2 (minor), 35.4 (CH2), 42.5 (CH2), 42.7 (CH), 43.9 (CH2), 47.9
(CH2), 48.0 (C), 58.0 (C), 61.6 (CH2), 64.5 (C), 79.9 (C), 82.9 (CH), 155.0 (C), 173.6 (C)
and 214.4 (C); LRMS m/z (ES) (%): 488 ([M+ H]+, 100), 482 (10), 463 (10), 454 (10);
HMS (ES) 488.2308 ([M+H]+ C13H23NO8S requires 488.2313).
157
Experimental
Ethyl
(1S/1R,
3S/3R)-1-[(tert-butoxycarbonyl)amino]-3-[{(camphor)-10'-
sulfonyl}oxy]cyclopentane-1-carboxylate (236 and 237)
DMAP (0.36 g, 2.96 mmol), dry TEA (0.4
9'
cm3, 2.96 mmol), (+) 10-camphorsulfonyl
5'
chloride (0.75 g, 2.96 mmol) were added to a
6'
8'
7'
H
4'
1'
stirred solution of trans-alcohol (204 and
144) (0.27 g, 0.97 mmol) in dry DCM (40
cm3) at 0 C under nitrogen. The resulting
H
3'
2'
O
O
10' O
S
S
O
O
O 2
CO2Et
O
1
3
NHBoc
4
5
237
trans
236
O
CO2Et
NHBoc
mixture was allowed to stir at r.t. overnight.
Similar work-up protocol was followed as described for CSA derivative of cis-alcohol
(232 and 233). The crude product from the work-up was purified by column
chromatography on silica using a gradient system of petroleum ether 40-60 C: ethyl
acetate (8.5:1.5 to 5:5). This gave (0.22 g, 45%) of the desired product (236 and 237). Rf
= 0.29 [Petroleum ether 40-60 C: ethyl acetate (6.7:3.3)]; max (film)/ cm-1 754 (w),
899 (w), 1042 (s), 1084 (w), 1167 (s), 1206 (s), 1287 (m), 1360 (m), 1416 (w), 1454
(w), 1531 (w), 1743 (s) and 2958 (s); δH(200 MHz, CDCl3) 0.85 (3H, s, CH3), 1.08 (3H,
s, CH3), 1.21-1.28 (3H, t, J 7.5, CH3), 1.39 (9H, s, 3 x CH3), 1.54-1.70 (2H, m), 1.872.60 (11H, m), 2.94-3.01 (1H, d, J 15.4, CH2), 3.53-3.61 (1H, dd, J 2.1 and 15.1, ½
CH2), 4.12-4.27 (2H, q, J 7.1, O-CH2), 5.22-5.33 (2H, m, CH-O-S and NH). LRMS m/z
(ES) (%): 510 ([M+ Na]+, 6), 410 (12), 388 (100)
Attempted synthesis of Ethyl (1R/1S, 3S/3R)-1-{[tert-butoxycarbonyl]amino}-3iodocyclopentane-1-carboxylate (273 and 274)9
2
I
CO2Et
4
I
CO2Et
1
NHBoc
3
CO2Et
NHBoc
5
273
I
trans
Desired product
274
I
NHBoc
275
CO2Et
NHBoc
cis
276
Undesired product
A stirred solution of CSA derivative of cis-alcohol (232 and 233) (0.34 g, 0.69 mmol)
and sodium iodide (0.26 g, 1.72 mmol) in dry acetone (85 cm3) was heated at reflux for
24 h. The resulting reaction mixture was allowed to cool down to room temperature.
158
Experimental
The solvent was removed in vacuo, which gave dark brown oil. The resulting crude
residue was then re-dissolved in ethyl acetate (50 cm3). Subsequently, the organic
extract was washed with water (40 cm3); the organic layer was then separated and dried
over anhydrous sodium sulphate. Filtration was followed by evaporation of the solvent
under reduced pressure gave crude brown oil, which was purified by column
chromatography on silica gel using petroleum ether 40-60 C: ether (80:20) as the
eluent. This yielded colourless oil (273, 274, 275 and 276) (0.15 g, 57%). Rf = 0.33
[petroleum ether 40-60 C: ether (8:2)]; H(200 MHz; CDCl3) 1.24-1.29 (3H, m, CH3),
1.39 (9H, s, 3 x CH3 ), 1.41 (9H, s, 3 x CH3), 1.20-2.75 (11H, br m, 5.5 x CH2), 2.923.03 (1H, dd, J 14.7and 7.2, ½ CH2), 4.13-4.29 (6H, br m, 2xCH and 2xCH2), 5.05 (1H,
s, NH 1R,3R and 1S,3S) and 5.18 (1H, s, NH 1R,3S and 1S,3R); C(50 MHz; CDCl3)
14.2 (CH3), 19.4 (CH 1R,3R and 1S,3S), 20.8 (CH 1R,3S and 1S,3R ), 28.4 (3 x CH3),
38.9 (CH2 1R,3S and 1S,3R), 39.2 (CH2 1R,3R and 1S,3S), 50.2 (CH2), 61.8 (CH2 1R,3S
and 1S,3R), 61.9 (CH2 1R,3R and 1S,3S), 65.1 (C 1R,3S and 1S,3R), 65.7 (C 1R,3R and
1S,3S), 80.2 (C), 155.0 (C), 173.4 (C 1R,3R and 1S,3S), 174.4 (C 1R,3S and 1S,3R).9
Ethyl
(1R/1S,
3R/3S)-1-(tert-butoxycarbonyl)amino-3-hydroxycyclopentane-1-
carboxylic acid (204 and 144).9 165,166.181,182,183
A stirred solution of CSA derivative
2
HO
3
of cis-alcohol (232 and 233) (0.2 g,
4
0.72 mmol) in dioxane and 1N
5
204
CO2Et
1
HO
CO2Et
NHBoc
trans
NHBoc
144
aqueous sodium hydroxide was used
to adjust the pH to 8.0 at 80 C. The pH needs to be maintained at pH 6.5-8.0, allowed
to stir overnight and then the pH was brought to 7.0 and the solution was evaporated
and re-dissolved in ethyl acetate (30 cm3): water (60 cm3). The aqueous phase was
separated and extracted twice with ethyl acetate (2 x 30 cm3). The combined organic
layer was washed with brine solution. The organic layer was then dried with anhydrous
magnesium sulphate. This was followed by filtration and evaporation of the solvent
under reduced pressure which gave pure trans-alcohol (204 and 144) (0.12 g, 73%). 1HNMR matched with trans-alcohol (204 and 144). LRMS m/z (ES) (%): 569 ([2M+Na]+,
100), 369 (24), 296 ([M+Na]+, 67), 274 ([M+H]+, 28), 169 (69).
159
Experimental
N3-benzoylthymine (261)9,238
Thymine (3.0 g, 24.0 mmol), benzoyl chloride (6.12 cm3, 52.8
mmol), dry acetonitrile (40 cm3) and dry pyridine (16 cm3) were
O
4
stirred together at room temperature. After 16 h, the solvent was
N 3
5
6
evaporated till dryness and the residue dissolved in dioxane
N 2 O
H
1
(100 cm3) and 0.5M potassium carbonate (40 cm3) and stirred
261
O
for 1.5 h approx. The crude product was purified on a short
column by using DCM: methanol 9.3:0.7 as eluent system and crystallized from
acetonitrile to give the title compound (261) as white needles (3.0 g, 54%). Rf = 0.37
[DCM: methanol (9.3:0.7)]; δH(200 MHz, [(CD3)2SO]) 1.82 (3H, s, CH3), 7.55-7.64
(3H, J 7.89, 3 x CH), 7.74-7.83 (1H, m, CH), 7.92- 7.97 (2H, m, 2 x CH), 11.42 (1H, br,
NH); δC(100 MHz, [(CD3)2SO]) 12.4 (CH3), 108.5 (CH), 130.1 (CH), 130.9 (CH), 132.0
(C), 136.0 (CH), 139.5 (CH), 150.6 (C), 164.2 (C), 170.8 (C).
N4-benzyloxycarbonylcytosine (268)209
O
To a suspension of cytosine (269) (6.0 g, 5.23 mmol) in
HN
4
anhydrous pyridine (120 cm3), DMAP (1.32 g, 10.8 mmol)
was added at 0 C under nitrogen, followed by dropwise
addition of benzyl chloroformate (270) (16.92 cm3, 118.8
mmol). The reaction mixture was stirred for 3 days, and then
O
N 3
5
6
N 2
H
1
O
28
poured into ice water (250 cm3) and stirred for 15 min. The
resulting solid was collected by filtration. Upon standing, additional material
precipitated from the mother liquor and was also collected by filtration. The combined
crude product was washed with water (2 x 100 cm3), followed with cold diethylether (3
x 30). The crude product was triturated and dried in vacuum for 48 h, which gave (268)
(7.034 g, 53%) as a white solid. δH (200 MHz, [(CD3)2SO]) 5.17 (2H, s. CH2), 6.90-6.94
(1H, d, J 7.1, CH=CH), 7.39 (5H, m, Ph-H), 7.78-7.82 (1H, d, J 7.1, CH=CH).
160
Experimental
Attempted synthesis of ethyl 1S/R, 3R/S)-3-(3’-benzoyl-thymin-1’-yl)-1-{[(tertbutoxy)carbonyl]amino}cyclopentane-1-carboxylate (260 and 249)9
O
2
HO
3
4
CO2Et
CO2Et HO
1
NHBoc
NHBoc
5
203
DEAD, Bz-Thy, PBu3/ PPh3
4
cis
O
4'
5'
BzN 3'
1'
6'
O 2' N 2
CO2Et
3
1
143
0
0
Dry DMF,O C -RT-40 C
5
254
BzN
O
N
CO2Et
NHBoc
NHBoc
trans
243
Method A: DEAD (0.12 cm3 0.26 mmol) was added dropwise to a solution of PBu3
(0.07 cm3, 0.26 mmol) in dry DMF (5 cm3) kept under nitrogen at 0 C. The mixture
was stirred for 30 min and then a solution of (1R/S, 3S/R) alcohol ethyl ester (203 and
143) (0.05 g, 0.19 mmol) in dry DMF (5 cm3) was added slowly. The mixture was
stirred for another 30 min and then N3-benzoylthymine (261) (0.06 g, 0.26 mmol)
solution in dry DMF (5cm3) was added. The resulting mixture was allowed to stir at r.t.
overnight. The solvent was removed under reduce pressure and re-dissolved in ethyl
acetate (30 cm3): water (30 cm3). The aqueous phase was separated and extracted twice
with ethyl acetate (2 x 15 cm3). The combined organic layer was washed with brine (20
cm3). The organic layer was then dried with anhydrous magnesium sulphate. Filtration
was followed by evaporation of solvent under reduce pressure gave crude product,
which was purified by column chromatography using silica gel. No product (260 and
249) was obtained and only starting material was recovered.
Method B: DEAD (0.08 cm3 0.44 mmol) was added dropwise to a solution of PPh3
(0.12 g, 0.44 mmol) in dry DMF (5cm3) kept under nitrogen at 0 C. The mixture was
stirred for an additional 30 min and then a solution of (1R/S, 3S/R) alcohol ethyl ester
(203 and 143) (0.05 g, 0.16 mmol) in dry DMF (5 cm3) was added slowly. The mixture
was stirred for 30 min and then N3-benzoylthymine (261) (0.1 g, 0.44 mmol) solution in
dry DMF (5cm3) was added. The same procedure was followed as in Method A. No
product (260 and 249) was obtained and only starting material was recovered.
161
Experimental
Ethyl (1S/R, 3R/S)-3-(3’-benzoyl-thymin-1’-yl)-1-{[(tert-butoxy)carbonyl]amino}cyclopentane1-carboxylate (260 and 249)9
Sodium hydride (60% dispersion in mineral
oil) (0.03 g, 0.84 mmol) was added to a stirred
solution of N3-benzoylthymine (261) (0.22 g,
0.93 mmol) in dry DMF (5 cm3) under nitrogen
at room temperature. The solution was then
O
4'
5'
BzN 3'
O
BzN
1'
6'
O 2' N
2
CO2Et
3
1
4
5
260
O
N
CO2Et
NHBoc
NHBoc
249
trans
heated to 40°C and left to stir for ½ h. To this
solution a solution of CSA derivative of cis-alcohol (232 and 233) (0.23 g, 0.47 mmol)
in dry DMF (15 cm3) was added dropwise. The reaction was then stirred overnight at 40
C. Evaporation of the solvent under reduced pressure gave a yellow solid, which was
re-dissolved in ethyl acetate (40 cm3) and water (20 cm3) and filtered on a celite pad.
The organic layer was separated from the filtrate and the aqueous layer was re-extracted
with ethyl acetate (4 x 20 cm3). The organic layer was combined and washed with water
(20 cm3) and brine (20 cm3), then dried over anhydrous sodium sulphate. Filtration was
followed by solvent evaporation under reduced pressure gave yellowish brown oil. This
residue was purified by column chromatography on silica using a gradient system of
petroleum ether (40-60 C): ethyl acetate (7.5:2.5 to 5:5). The title compound (260 and
249) was afforded as colourless oil (0.05 g, 22%) and (232 and 233) (0.11 g, 49%). Rf =
0.18 [petroleum ether (40-60 C): ethyl acetate (6.2:3.8)]; δH(400 MHz; CDCl3) 1.271.30 (3H, t, J 7.1, CH3), 1.43 (9H, s, CH3), 1.83-1.94 (2H, br m, CH2), 2.00 (3H, s,
CH3), 2.17-2.33 (2H, br m, CH2), 2.47-2.52 (1H, br m, ½ CH2), 2.61-2.70 (1H, br m, ½
CH2), 4.13-4.31 (2H, m, CH2), 5.18-5.27 (1H, br m, CH), 7.45 (2H, t, J 7.9, Ph-H),
7.61-7.65 (2H, m, Ph-H and Thymine C6-CH), 7.90-7.94 (2H, d, J 7.1, Ph-H); δC(50
MHz; CDCl3) 12.7 (CH3), 14.1 (CH3), 28.2 (3 x CH3), 29.8 (CH2), 35.8 (CH2), 40.8
(CH2), 54.3 (CH), 62.0 (CH2), 63.9 (C), 80.5 (C), 111.4 (C), 129.1 (CH), 130.4 (CH),
131.6 (C), 134.9 (CH), 136.9 (CH), 150.0 (C), 155.1 (C), 162.7 (C), 169.1 (C), 174.4
(C).
162
Experimental
Attempted synthesis of ethyl (1R/S, 3R/S)-3-[(4’-{[(benzyloxy)carbonyl]-N4-cytosin1-yl)-1-{[(tert-butoxy)carbonyl]amino}cyclopentane-1-carboxylate (266 and 267)
O
HN
4'
5'
6'
H
O
H
N
N 3'
4
O
HN
4'
N
N 2' O 2
1'
3
CO2Et
1
NHBoc
N
O
CO2Et
NHBoc
O
2
N 2' O
1'
3
265
N
CO2Et
CO2Et
O
1
NHBoc
NHBoc
5
266
No Desired product obtained
O
N
4
trans
N
N 3'
5'
6'
5
264
O
O
O
trans
267
Undesired product obtained
Caesium carbonate (0.18 g, 0.55 mmol) was added to a stirred solution of N4benzyloxycarbonylcytosine (268) (0.14 g, 0.55 mmol) in dry DMF (5 cm3) under
nitrogen at room temperature. The solution was then heated to 40 °C and left to stir for
half an h. To this a solution of CSA derivative of cis-alcohol (232 and 233) (0.18 g,
0.37 mmol) in dry DMF (5 cm3) was added dropwise. The reaction was then stirred
overnight at 40-50 °C. Evaporation of the solvent under reduced pressure gave a yellow
solid, which was re-dissolved in ethyl acetate (40 cm3) and water (20 cm3) and filtered
on a celite pad. The filtrate was separated and the aqueous layer was washed (4 x 20
cm3) with ethyl acetate. The organic layer was combined, washed with water (20 cm3)
and brine (20cm3), and then dried over anhydrous sodium sulphate. Filtration was
followed by solvent evaporation under reduced pressure gave yellowish brown oil. This
residue was purified by column chromatography on silica using a gradient system of
petroleum ether (40-60 C): ethyl acetate (7.5:2.5 to 5:5). This afforded unreacted CSA
derivative of ethyl ester (232 and 233) (0.02 g, 8.3%) and the title compound (266 and
267) as a colourless oil, which solidified to white solid (0.06 g, 30%). mp 48-50 C; Rf
0.71 [petroleum ether (40-60 C): ethyl acetate (4.5:5.5)]; [α]D25 +10.00 (c 1.7 in
DCM); max (KBr)/ cm-1 1038 (m), 1098 (m), 1167 (s), 1202 (s), 1237 (s), 1296 (s),
1402 (s), 1438 (m), 1529 (s), 1586 (s), 1742 (s), 2978 (m) and 3382 (m); δH (200 MHz,
CDCl3) 1.28 (3H, t, J 7.2, CH3), 1.43 (9 H, s, 3 x CH3), 2.02-2.09 (2H, 2 x ½ CH2),
2.29-2.36 (1H, m, ½ CH2), 2.42-2.48 (1H, m, ½ CH2), 2.50-2.55 (1H, m, CH2), 2.65
(1H, br s, ½ CH2), 4.19-4.23 (2H, m, CH2O), 5.06 (1H, s, NH), 5.25 (2H, s, CH2), 5.445.47 (1H, m, CH-OH), 7.38-7.43 (5H, m, Ph-H), 7.54 (1H, s, NH), 7.59-7.57 (1H, d, J
5.7, CH=CH), 8.36-8.37 (1H, d, J 5.7, CH=CH); δC(100 MHz, CDCl3) 14.1 (CH3), 28.3
163
Experimental
(3 x CH3), 31.3 (CH2), 35.3 (CH2), 43.4 (CH2), 61.5 (CH2), 64.9 (C), 67.7 (CH2), 77.6
(CH), 80.0 (C), 102.2 (CH=CH), 128.3 (CH), 128.6 (CH), 128.7 (CH), 135.2 (C.), 152.3
(C), 155.1 (C), 159.2 (C), 160.1 (CH=CH), 164.2 (C), 173.6 (C); LRMS m/z (ES) (%):
501 ([M+H]+, 28), 488 (12), 257 (15), 256 (100), 200 (92), 79 (10), 74 (28); HMS (ESI)
501.2343 ([M+H]+ C25H33O7N4 requires 501.2344).
(1R/S,
3R/S)-3-[(4’-{[(benzyloxy)carbonyl]amino}pyrimidin-2’-yl)oxy]-1-
{[(tert-butoxy)carbonyl]amino}cyclopentane-1-carboxylic acid (277 and 278)
A 1.0M aqueous solution of sodium
O
O
H
hydroxide (5 cm3) was added at r.t. to a
HN
4'
5'
6'
stirred solution of amino acids (266 and
O
cm3). The reaction mixture was left to
2
COOH
N
COOH
O
1
NHBoc
4
NHBoc
5
277
stir at r.t. for 24 h. Subsequently, the pH
O
N
N 3'
N 2' O
1'
3
267) (0.08 g, 0.16 mmol) in dioxane (5
N
trans
278
of the aqueous solution was brought to pH 7.0 with dilute 0.2 N aqueous solution of
HCl. The solvent was evaporated under reduced pressure and the crude residue obtained
was re-dissolved in water (20 cm3). The pH was re-adjusted to 12 with 1.0 N aqueous
solution of sodium hydroxide. The resulting reaction mixture was washed with DCM (3
x 10 cm3). The pH of the aqueous layer was re-adjusted to 3.0 with 0.6 M (aq) solution
of citric acid and then extracted with ethyl acetate (5x 10 cm3). The combined ethyl
acetate extracts were dried over anhydrous sodium sulphate. Filtration was followed by
solvent evaporation under reduced pressure gave a mixture of the title compounds (277
and 278) (0.05 g, 66%) as a white solid. mp 145 C; Rf 0.36 [DCM:methanol (9:1)];
[α]D25 +11.03 (c 0.73 in MeOH); max (KBr)/ cm-1 1020 (w), 1103 (w), 1204 (s),
1237(s), 1297 (m), 1348 (m), 1402 (m), 1441 (w), 1527 (m), 1586 (s), 1712 (s), 2976
(w) and 3421 (w); δH(400 MHz, CD3OD) 1.45 (9 H, s, 3 x CH3), 1.92-2.00 (1H, m, ½
CH2), 2.02-2.10 (1H, br s, ½ CH2), 2.24-2.46 (3H, m, 1½ x CH2), 2.64 (1H, br s, ½
CH2), 5.24 (2H, s, CH2), 5.42-5.49 (1H, m, CH-OH), 7.34-7.45 (5H, m, Ph-H), 7.577.59 (1H, d, J 5.7, CH=CH), 8.32 (1H, brs, CH=CH); δC(100 MHz, CDCl3) 29.0 (3 x
CH3), 32.7 (CH2), 36.1 (CH2), 44.6 (CH2), 61.8 (C), 68.6 (CH2), 79.5 (CH), 80.7 (C),
103.5 (CH=CH), 129.5 (CH), 129.6 (CH), 129.8 (CH), 137.7 (C), 154.9 (C), 157.8 (C),
160.9 (CH=CH), 162.3 (C), 165.8 (C), 173.2 (C); LRMS m/z (ES) (%) 473 ([M+H])+,
164
Experimental
58) 454 (13), 427 (12), 189 (100), 104 (18), HMS (ES) 473.2031 ([M+H]+ C23H29N4O7
requires 473.2037).
(1’R,
2’S,
5’R)-1-menthol
1-([(tert-Butoxycarbonyl)amino]cyclopent-3-ene-1-
carboxylate (280)
1.6 M n-butyl-lithium in hexane (2.44 cm3, 3.9 mmol) was
4'
added dropwise to a stirred solution of D-menthol (284)
2
'6
2'
O
1
NHBoc
3
4
was added to the reaction mixture. The reaction mixture
3'
O
(0.67 g, 4.3 mmol) in dry THF (20 cm3) at 0 C under
nitrogen. After 5 min, alkene (222) (0.85 g, 3.52 mmol)
5'
1'
5
280
was left to stir for overnight at room temperature.
The solvent was evaporated under reduced pressure. Water and sat. NH4Cl were added
to the reaction mixture. Subsequently, the reaction mixture was washed with cold
hexane. The aqueous layer was extracted with ethyl acetate (3 x 20 cm3). The combined
organic layer was dried with MgSO4. Filtration was followed by solvent evaporation
under reduced pressure gave crude solid which was further purified by column
chromatography using silica and a gradient system of petroleum ether (40-60 C): ethyl
acetate (80:20 to 50:50)]. This gave the starting material (222) (0.11 g, 13%) and the
title compound (280) was afforded as white crystalline solid (1.01 g, 82%). mp 84-85
C; Rf 0.35 [petroleum ether (40-60 C): ethyl acetate (9.4:0.6)]; [α]D25 +37.78 (c 1.35
in DCM), (Found: C, 68.77; H, 9.70; N, 3.65% C21H35NO4 requires C, 69.01; H, 9.65;
N, 3.83%); max (KBr)/ cm-1 1042 (w), 1066 (w), 1169 (m), 1226 (m), 1299 (s), 1367
(m), 1391 (w), 1455 (w), 1507 (m), 1533 (m), 1674 (m), 1691 (s), 1707 (m), 1720 (s),
1738 (s), 2870 (s), 2958 (s) and 3343 (s); δH(200 MHz, CDCl3) 0.70-0.73 (3H, d, J 6.9,
CH3), 0.82-0.87 (1H, overlap, ½ CH2), 0.82-0.83 (3H, d, J 6.8, 1 x CH3), 0.86-0.87 (3H,
d, J 6.8, 1 x CH3), 0.93-1.30 (2H, m, 2 x ½ CH2 ), 1.38-1.41 (2H, overlap, 2 x CH), 1.40
(9H, s, 3 x CH3 ), 1.61-1.68 (2H, d, J 16.0, 1 x CH2), 1.81-2.91 (2H, m, 1 x CH and ½
CH2), 2.54-2.62 (2H, d, J 16.2, CH2), 2.96-3.06 (2H, d, J 16.0, CH2), 4.63-4.76 (1H, m,
CH-OH), 5.16 (1H, brs, NH), 5.61 (2H, s, CH=CH; δC(100 MHz, CDCl3) 16.4 (CH3),
21.4 (CH3), 22.5 (CH3), 23.6 (CH2), 26.4 (CH), 28.8 (3 x CH3), 31.8 (CH), 34.8 (CH2),
41.1 (CH2), 45.3 (CH2), 47.4 (CH), 64.7 (C), 75.7 (CH), 80.2 (C), 128.3 (CH=CH),
155.3 (C), 174.3 (C); LRMS m/z (ES) (%) 753 ([2M+Na]+,100), 388 ([M+Na]+,75) 366
165
Experimental
([M+H]+,55), 310 (40), 225 (20).
(1’S,
2’R,
5’S)-1-menthol-1-[(tert-Butoxycarbonyl)amino]cyclopent-3-ene-1-
carboxylate (279)
n-Butyl-lithium in hexane (1.6 M, 2.44 cm3, 3.9 mmol)
4'
was added dropwise to a stirred solution of L-menthol
2
'6
2'
O
1'
1
NHBoc
3
mmol) was added to the reaction mixture. The reaction
3'
O
(283) (0.67 g, 4.3 mmol) in dry THF (20 cm3) at 0 C
under nitrogen. After 5 min, alkene (222) (0.85 g, 3.52
5'
4
5
279
mixture was left to stir overnight at room
temperature. The same procedure for work-up was followed as described for (280). The
crude material was further purified by column chromatography using a gradient system
of petroleum ether (40-60 C): ethyl acetate (80:20 to 50:50). This gave the starting
material (222) (0.1 g, 13%) and the title compound (279) was afforded as white
crystalline solid (0.85 g, 69%). mp 83-84 C; Rf 0.35 [petroleum ether (40-60 C): ethyl
acetate (9.4:0.6)]; [α]D25 –38.95 (c 0.95 in DCM), (Found: C, 68.88; H, 9.63; N, 3.84%
C21H35NO4 requires C, 69.01; H, 9.65; N, 3.83%);max (KBr)/ cm-1 1042 (w), 1066 (w),
1169 (m), 1226 (m), 1299 (s), 1367 (m), 1391 (w), 1455 (w), 1507 (m), 1533 (m), 1674
(m), 1691 (s), 1707 (m), 1720 (s), 1738 (s), 2870 (s), 2958 (s) and 3343 (s); δH(200
MHz, CDCl3) 0.70-0.73 (3H, d, J 6.9, CH3), 0.82-0.87 (1H, overlap, ½ CH2), 0.82-0.83
(3H, d, J 6.8, 1 x CH3), 0.86-0.87 (3H, d, J 6.8, 1 x CH3), 0.93-1.30 (2H, m, 2 x ½ CH2
), 1.38-1.41 (2H, overlap, 2 x CH), 1.40 (9H, s, 3 x CH3 ), 1.61-1.68 (2H, d, J 16.0, 1 x
CH2), 1.81-2.91 (2H, m, 1 x CH and ½ CH2), 2.54-2.62 (2H, d, J 16.2, CH2), 2.96-3.06
(2H, d, J 16.0, CH2), 4.63-4.76 (1H, m, CH-OH), 5.16 (1H, brs, NH), 5.61 (2H, s,
CH=CH); δC(100 MHz, CDCl3) 16.4 (CH3), 21.4 (CH3), 22.5 (CH3), 23.6 (CH2), 26.4
(CH), 28.8 (3 x CH3), 31.8 (CH), 34.8 (CH2), 41.1 (CH2), 45.3 (CH2), 47.4 (CH), 64.7
(C), 75.7 (CH), 80.2 (C), 128.3 (CH=CH), 155.3 (C), 174.3 (C); LRMS m/z (ES) (%)
753 ([2M+Na]+,94), 429 (100), 388 ([M+Na]+,44), 366 ([M+H]+,80), 310 (40), 273
(20), 240 (14), 215 (24), 196 (17), 172 (14).
166
Experimental
(1’R, 2’S, 5’R)-1-menthol (1R/S, 3S/R)-1-{[(tert-butoxy)carbonyl]amino}-3-hydroxy1-cyclopentane-1-carboxylate (299 and 300) and (1’R, 2’S, 5’R)-1-menthol (1S/R,
3R/S)-1-{[(tert-butoxy)carbonyl]amino}-3-hydroxy-1-cyclopentane-1-carboxylate
(301
and 302)
4'
4'
HO
2
5'
3'
5'
3'
'6
2'
'6
2'
O
O
1'
O
O
HO
1
NHBoc
3
4
5
299
NHBoc
2
HO
O
300
5
301
O
1'
O
HO
1
NHBoc
3
4
cis
O
NHBoc
trans
302
Borane.tetrahedron complex (2.07 cm3; 2.07 mmol) was added dropwise to a solution of
alkene (280) (0.72 g, 1.97 mmol) in THF (5 cm3) at 0 C under nitrogen. The reaction
mixture was allowed to warm to r.t. and stirred overnight. The reaction mixture was
quenched with dropwise addition of water (5 cm3), followed by addition of aqueous
hydrogen peroxide (35% w/w; 0.96 cm3, 9.85 mmol). 1N (aq) sodium hydroxide was
used to adjust the pH of the reaction mixture to pH 9.0. The reaction mixture was left to
stir at r.t. for 2 h. The same procedure for work-up was followed as described for (203
and 143) and (204 and 144). The crude residue, was pre-absorbed onto silica and
purified by column chromatography using a gradient system of petroleum ether 40-60
C: ethyl acetate (9:1 to 6:4) to give cis-alcohol (299 and 300) (34%), a mixture of cis
and trans-alcohol (10%) and trans-alcohol (301 and 302) (15%). The first to elute was
the cis-alcohol (299 and 300) afforded as colourless oil (0.26 g, 34%). Rf = 0.40
[petroleum ether (40-60 C): ethyl acetate (6.6:3.4)]; [α]D25 +44.44 (c 1.13 in DCM);
(Found: C, 65.64; H, 9.82; N, 3.77% C21H37NO5 requires C, 65.77; H, 9.72; N, 3.65%);
max (KBr)/cm-1 981 (w), 1020 (m), 1079 (m), 1164 (s), 1272 (m), 1366 (m), 1457 (m),
1500 (m), 1694 (s), 1723 (s), 2870 (m), 2957 (s), 3133 (w) and 3374 (m); δH(400 MHz,
CDCl3) 0.75-0.81 (3H, d, J 6.9, CH3), 0.87-0.89 (3H, d, J 8.0, 1 x CH3), 0.88-0.90 (3H,
d, J 8.0 1 x CH3), 0.80-1.10 (3H, m, 1 ½ CH2), 1.36-1.55 (2H, m, 2 x CH), 1.40 (9H, s,
3 x CH3), 1.57-1.72 (2H, brs, m, CH2), 1.74-1.86 (1H, brs, m, CH), 1.88-2.07 (5H, brs,
m, 2 ½ x CH2), 2.42-2.56 (2H, m, CH2), 4.40 (1H, brs, O-CH), 4.71-4.81 (1H, m, CHOH), 4.89 (1H, brs, OH), 5.94 (1H, brs, NH); δC(100 MHz, CDCl3) 15.8 (CH3) (minor),
15.9 (CH3) (major), 20.9 (CH3), 22.0 (CH3), 23.0 (CH2) (minor), 23.1 (CH2) (major),
26.1 (CH) (minor), 26.2 (CH) (major), 28.3 (3 x CH3), 31.3 (CH), 34.1 (CH2), 34.7
167
Experimental
(CH2) (major or minor), 35.0 (CH2) (major or minor), 36.3 (CH2) (major or minor), 36.5
(CH2) (major or minor), 40.4 (CH2), 46.6 (CH2), 47.0 (CH), 64.6 (C), 73.4 (CH), 75.9
(CH), 80.3 (C), 154.7 (C) (minor), 154.8 (C) (major), 175.5 (C) (major), 175.6 (C)
(minor). Second to elute was trans-alcohol (301 and 302) afforded as white crystalline
solid (0.12 g, 15%). mp 111-112 C; Rf = 0.37 [petroleum ether (40-60 C): ethyl
acetate (6.6:3.4)]; [α]D25 +44.92 (c 0.94 in DCM); max (KBr)/ cm-1 990 (w), 1016 (m),
1096 (m), 1167 (s), 1247 (m), 1279 (s), 1385 (s), 1458 (m), 1690 (s), 1731 (s), 2871
(m), 2953 (s), 3273 (m), 3382 (m); δH(400 MHz, CDCl3) 0.75-0.82 (3H, dd, J 7.0 and
11.7, CH3), 0.85-1.16 (9H, m, 2 x CH3 and 1½ CH2), 1.40 (9H, s, 3 x CH3), 1.40-1.46
(2H, m, 2 x CH), 1.57-1.72 (2H, brs, m, CH2), 1.74-1.93 (3H, brs, m, CH2 and CH),
1.94-2.03 (1H, m, ½ CH2), 2.04-2.23 (2H, m, CH2), 2.26-2.40 (1H, m, ½ CH2), 2.432.57 (1H, m, ½ CH2), 2.84-293 (1H, d, J 37.2, OH), 4.46 (1H, brs, O-CH), 4.72-4.84
(1H, brs, CH-OH) and 5.10 (1H, brs, NH); δC(100 MHz, CDCl3) 15.8 (CH3) (minor),
16.0 (CH3) (major), 20.8 (CH3) (minor), 20.9 (CH3) (major), 22.0 (CH3), 23.0 (CH2)
(minor), 23.2 (CH2) (major), 25.7 (CH) (minor), 26.1 (CH) (major), 28.3 (3 x CH3),
31.4 (CH), 34.2 (CH2), 34.9 (CH2), 36.6 (CH2), 40.5 (CH2) (major), 40.7 (CH2) (minor),
46.9 (CH2), 47.0 (CH), 64.9 (C) (major or minor), 65.0 (C) (major or minor), 73.1 (CH)
(major), 73.2 (CH) (minor), 75.9 (CH), 79.9 (C), 154.8 (C) (minor), 155.0 (C) (major),
175.5 (C) (minor) and 175.6 (C) (major); LRMS m/z (ES) (%) 401 ([M+NH4]+, 100),
384 ([M+H]+, 55) 327 (27), 284 (14), 245 (39); HMS (ESI) 384.2748 ([M+H]+
C21H38NO5 requires 374.2744).
(1’S,
2’R,
5’S)-1-menthol
(1R/S,
3S/R)-1-{[(tert-butoxy)carbonyl]amino}-3-
hydroxy-1-cyclopentane-1-carboxylate (303 and 304) and (1’S, 2’R, 5’S)-1-menthol
(1S/R,
3R/S)-1-{[(tert-butoxy)carbonyl]amino}-3-hydroxy-1-cyclopentane-1-
carboxylate (305 and 306)
4'
4'
HO
2
5'
3'
'6
2'
O
O
1'
O
O
HO
1
NHBoc
3
4
5
303
NHBoc
2
HO
304
3'
'6
2'
O
O
5
305
O
1'
O
HO
1
NHBoc
3
4
cis
5'
NHBoc
trans
306
Borane.tetrahydrofuran complex (1.3 cm3; 1.3 mmol) was added dropwise to a solution
168
Experimental
of alkene (279) (0.44 g, 1.2 mmol) in THF (4 cm3) at 0 C under nitrogen. The reaction
mixture was allowed to warm to r.t. and stirred overnight. The reaction mixture was
quenched with dropwise addition of water (5 cm3), followed by addition of aqueous
hydrogen peroxide (35% w/w; 0.58 cm3, 6.0 mmol). 1N (aq) sodium hydroxide was
used to adjust the pH of the reaction mixture to pH 9.00. The reaction mixture was left
to stir at r.t. for 2 h. The same procedure for work-up was followed as described for
(203 and 143) and (204 and 144). The crude residue, was pre-absorbed onto silica and
purified by column chromatography using a gradient system of petroleum ether 40-60
C: ethyl acetate (9:1 to 6:4), gave cis-alcohol (303 and 304) (31%), mixture of cis and
trans alcohol (17%) and trans-alcohol (305 and 306) (16%). The first to elute was the
cis-alcohol (303 and 304) afforded as white crystalline solid (0.14 g, 31%). mp 108-109
C; Rf 0.51 [petroleum ether (40-60 C): ethyl acetate (6:4)]; [α]D25 -43.14 (c 1.02 in
DCM); max (KBr)/ cm-1 959 (w), 1020 (m), 1079 (m), 1164 (m), 1271 (m), 1367 (m),
1455 (m), 1500 (w), 1693 (s), 1723 (s), 2870 (m), 2956 (s), 3130 (w), 3342 (w) and
3361 (m); δH(400 MHz, CDCl3) 0.73-0.75 (3H, d, J 6.9, CH3), 0.87-0.89 (3H, d, J 8.0, 1
x CH3), 0.88-0.90 (3H, d, J 8.0, 1 x CH3), 0.80-0.90 (3H, m, 1 ½ CH2), 0.93-1.09 (2H,
m, CH2), 1.40-1.60 (2H, m, 2 x CH), 1.40 (9H, s, 3 x CH3), 1.61-1.71 (2H, m, CH2),
1.73-1.85 (1H, m, CH), 1.88-2.07 (5H, brs, m, 2 ½ x CH2), 2.33-2.55 (2H, m, CH2),
4.36 (1H, brs, O-CH), 4.73 (1H, m, CH-OH), 4.88 (1H, brs, OH) and 5.91 (1H, brs,
NH); δC(100 MHz, CDCl3) 15.8 (CH3) (minor), 15.9 (CH3) (major), 20.9 (CH3), 21.9
(CH3), 23.0 (CH2), 26.1 (CH) (major), 26.2 (CH) (minor), 28.3 (3 x CH3), 31.3 (CH),
34.1 (CH2), 34.7 (CH2) (major), 35.1 (CH2) (minor), 36.3 (CH2) (major), 36.5 (CH2)
(minor), 40.4 (CH2), 46.6 (CH2), 47.0 (CH), 64.5 (C) (major), 64.6 (C) (minor), 73.4
(CH), 75.9 (CH), 80.3 (C), 154.7 (C), 174.6 (C) (major) and 174.7 (C) (minor); LRMS
m/z (ES) (%) 384 ([M+H]+, 43) 327 (20), 310 (27), 285 (48), 284 (100); HMS (ESI)
384.2745 ([M+H]+ C21H38NO5 requires 374.2744). The second to elute was the transalcohol (305 and 306) afforded as white crystalline solid (0.075 g, 16%). mp 115-116
C; Rf 0.34 [petroleum ether (40-60 C): ethyl acetate (6.6:3.4)]; [α]D25 -32.26 (c 1.08
in DCM); IR (KBr) max (film)/ cm-1 980 (w), 1016 (m), 1096 (m), 1166 (m), 1279 (m),
1366 (m), 1460 (m), 1690 (s), 1731 (s), 2870 (m), 2958 (s ),3271 (m) and 3383 (m);
δH(400 MHz, CDCl3) 0.75-0.82 (3H, dd, J 4.60 and 6.90, CH3), 0.85-0.97 (7H, m, 2 x
CH3 and ½ CH2), 0.95-1.10 (2H, m, CH2), 1.40-1.55 (2H, brs, 2 x CH), 1.40 (9H, s, 3 x
CH3), 1.62-1.71 (2H, brs, m, CH2), 1.72-1.92 (3H, brs, m, CH2 and CH), 1.94-2.03 (1H,
169
Experimental
m, ½ CH2), 2.04-2.18 (2H, m, CH2), 2.24-2.38 (1H, m, ½ CH2), 2.42-2.55 (1H, m, ½
CH2), 2.88-2.98 (1H, d, J 38.9, OH), 4.44 (1H, brs, O-CH), 4.70-4.82 (1H, brs, CHOH), 5.11 (1H, brs, NH); δC(100 MHz, CDCl3) 15.9 (CH3) (minor), 16.0 (CH3) (major),
20.8 (CH3) (minor), 20.9 (CH3) (major), 21.9 (CH3), 22.9 (CH2) (minor), 23.1 (CH2)
(major), 25.7 (CH) (minor), 26.1 (CH) (major), 28.3 (3 x CH3), 31.3 (CH), 34.2 (CH2),
34.9 (CH2), 36.5 (CH2), 40.4 (CH2) (major), 40.6 (CH2) (minor), 46.9 (CH2), 47.0 (CH),
64.9 (C) (minor), 65.0 (C) (major), 73.0 (CHO) (major), 73.1 (CH) (minor), 75.9 (CH),
79.8 (C), 154.8 (C) (major), 155.0 (C) (minor), 175.4 (C) (minor), 175.6 (C) (major);
LRMS m/z (ES) (%) 384 ([M+H]+, 16), 285 (20), 284 (100), 189 (14), 146 (20); HMS
(ESI) 384.2749 ([M+H]+ C21H38NO5 requires 374.2744).
(1’S, 2’R, 5’S)-1-menthol (1R/S, 3S/R)-1-{[(tert-butoxy)carbonyl]amino}-3hydroxy-1-cyclopentane-1-carboxylate (299 and 300)
Boron trifluoride etherate (0.95 cm3, 7.53
4'
3'
5'
mmol) was added dropwise to a solution
'6
of (+)-isopinocampheylborane TMEDA
HO
2
4
5
299
cm3) at r.t. under nitrogen. After stirring
O
1'
O
O
HO
1
NHBoc
3
complex (1.57 g, 3.76 mmol) in THF (4
2'
O
NHBoc
cis
300
for 1 h, the white precipitate which had
formed during the course of the reaction was filtered with suction into a pre-cooled (-45
to -35 C) flask under a stream of nitrogen and then under vacuum. The precipitate was
washed with cold (-45 to -35 C) THF (3 x 1 cm3) via cannula under a stream of
nitrogen and then under vacuum. The washings were combined with the original filtrate.
The alkene (280) (0.4 g, 1.09 mmol) was dissolved in THF (3 cm3) at room temperature.
The alkene solution was gradually brought down to -35 to -45 C, and was added
dropwise to combined original filtrate via cannula under nitrogen. The reaction mixture
was stirred at -35 to -45 C for 2 h and then kept in a freezer at -36 C for 72 h. Water
was added (5 cm3), followed by aqueous hydrogen peroxide (30% v/v; 0.53 cm3, 5.45
mmol). 1N (aq) sodium hydroxide was used to adjust the pH of the reaction mixture to
pH 9.0. The reaction mixture was left to stir at r.t. for 2 h. The oxidised solution was
extracted with ethyl acetate (4 x 15 cm3), then saturated with potassium carbonate and
re-extracted with THF (2 x 15 cm3). The organic extracts were combined and then
170
Experimental
washed with water (30 cm3) and brine (20 cm3). The combined organic layer was dried
over magnesium sulphate. Filtration was followed by removal of solvent under reduced
pressure. The crude residue, was pre-absorbed onto silica and purified by column
chromatography using a gradient system of petroleum ether 40-60 C: ethyl acetate
(8.5:1.5 to 8.0:2.0) gave cis-alcohol (299 and 300) as a colourless oil, which formed
white solid on drying under vacuum, (0.31 g, 74%), with no trace of trans-alcohol (301
and 302) based on 1H-NMR (200 MHz, CDCl3) and TLC analysis.
(1’S,
2’R,
5’S)-1-menthol
(1S/R,
3R/S)--1-{[(tert-butoxy)carbonyl]amino}-3-
hydroxy-1-cyclopentane-1-carboxylate (299 and 300)
Following the procedure, work-up and
4'
purification for the preparation of cisalcohol (299 and 300) using boron
'6
2'
O
O
1'
O
HO
1
NHBoc
4
5
299
(-)-isopinocampheylborane
3'
O
3
trifluoride etherate (0.95 cm3, 7.53
mmol),
2
HO
5'
NHBoc
300
cis
TMEDA complex (1.57 g, 3.76 mmol)
and alkene (280) (0.4 g, 1.09 mmol) gave cis-alcohol (299 and 300) (0.34 g, 81%), with
no trace of trans-alcohol (301 and 302) based on 1H-NMR (200 MHz, CDCl3).
(1’R, 2’S, 5’R)-1-menthol (1S/R, 3R/S)-3-{[(4”-bromobenzene)sulfonyl]oxy}-1[(tert-butoxy)amino]cyclopentane-1-carboxylate (308 and 309)
Br
To a stirred solution of cis-alcohol (301
Br
and 302)) (0.23 g, 0.59 mmol) obtained
4''
5''
dry DCM (10 cm3) and dry TEA (0.29
2.08
mmol),
4'
2''
5'
1''
O
'6
6''
S
O
O
1'
O 2
O
1
3
NHBoc
4
5
from borane-tetrahydrofuran complex in
cm3,
3''
4-
308
bromobenzenesulfonyl chloride (0.5 g,
cis
3'
O
2'
O
S
O
O
O
NHBoc
309
2.08 mmol) and DMAP (0.25 g, 2.08
mmol) were added at 0 C under nitrogen. The reaction mixture was allowed to warm
to r.t. and stirred overnight. The mixture was subsequently washed with cold 1N (aq)
citric acid (3 x 30 cm3), saturated (aq) solution of sodium bicarbonate (25 cm3), water
171
Experimental
(20 cm3) and brine (20 cm3). The organic layer was dried over sodium sulphate.
Filtration was followed by solvent evaporation under reduced pressure gave crude
residue. This crude residue was purified by column chromatography using silica
gradient system of petroleum ether 40-60 C: ethyl acetate (8:2 to 6:4). This gave the
title compound (308 and 309) as a white crystalline solid (0.27 g, 75%). mp 103-105
C; Rf 0.25 [petroleum ether (40-60 C): ethyl acetate (8.8:1.2)]; C [α]D25 +30.85 (c
1.01 in DCM); (Found: C, 53.66; H, 6.80; N, 2.16% C27H40BrNO7S requires C, 53.82;
H, 6.69; N, 2.32%); max (KBr)/ cm-1 938 (w), 1010 (w), 1069 (w), 1095 (w), 1178 (m),
1279 (w), 1367 (m), 1456 (w), 1505 (m), 1577 (w), 1702 (s), 2869 (m), 2958 (m) and
3383 (s); δH(400 MHz, CDCl3) 0.75-0.82 (3H, dd, J 3.40 and 6.90, CH3), 0.85-0.90 (6H,
m, 2 x CH3), 0.75-1.13 (3H, m, 1 ½ CH2), 1.36-1.51 (2H, brs, 2 x CH),1.41 (9 H, s, 3 x
CH3), 1.63-1.76 (2H, m, CH2),1.73-1.87 (1H, brs, CH), 1.88-1.99 (1H, brs, ½ x CH2),
2.00-2.23 (5H, brs m, 2 ½ x CH2), 2.43-2.75 (1H, m, ½ CH2), 4.64-4.79 (1H, m, CHO),
5.10 (2H, brs, CHO and NH), 7.71-7.81 (4H, m, Ph-H); δC(100 MHz, CDCl3) 16.0
(CH3) (minor), 16.1 (CH3) (major), 21.1 (CH3), 22.2 (CH3) 23.3 (CH2), 26.3 (CH), 28.5
(3 x CH3), 31.6 (CH), 32.0 (CH2) (major), 32.1 (CH2) (minor), 34.4 (CH2), 35.5 (CH2),
40.7 (CH2) (major), 40.8 (CH2) (minor), 43.5 (CH2), 47.1 (CH) (major), 47.2 (CH)
(minor), 64.7 (C) (major), 64.8 (C) (major), 75.8 (CH), 78.0 (C), 84.1 (CH), 129.2
(CH), 129.5 (CH), 132.9 (CH), 136.2 (C), 155.0 (C), 173.2 (C) (minor), 173.3 (C)
(major); LRMS m/z (ES) (%) 1226 ([2M+Na]+, 58), 624 ([M+Na]+,100) 604 ([M+H]+,
78), 502 (40), 413 (30), 301 (25), 279 (38), 213 (25).
Attempted
Synthesis
of
(1’R,
2’S,
5’R)-1-menthol
(1R/S,
3R/S)-3-(4”-
{[(benzyloxy)carbonyl]-N4-cytosin-1-yl)-1-{[(tert-butoxy)carbonyl]amino}cyclopentane1-carboxylate (310 and 311)
O
H
5''
6''
N
4''
O
H
O
N 3''
N 2'' O 2
1''
3
4
H
N
5'
O
3'
O 1'
1
NHBoc
O
HN
4'
N
4'
6'
N
O
2'
5'
6'
O
O
N 3'
O
O
1 O 1'
NHBoc
4
5
312
311
Desired product
O
N
4'
6'
3'
NHBoc
trans
N
5'
N 2' O 2
1'
3
5
310
O
O
N
O
O
O
2'
NHBoc
trans
313
Undesired product
Sodium hydride (0.02 g, 0.49 mmol) was added at r.t. under nitrogen to a stirred
172
Experimental
solution of N4-benzyloxycarbonylcytosine (268) (0.13 g, 0.51 mmol) in dry DMF (5
cm3). The solution was then heated to 40 C and left to stir for half an h. To this
reaction mixture a solution of brosylate derivative of menthol ester (308 and 309) (0.16
g, 0.26 mmol) in dry DMF (5 cm3) was added dropwise. The reaction was then stirred
overnight at 40-50 C. Evaporation of the solvent under reduced pressure gave a yellow
solid, which was dissolved in ethyl acetate (40 cm3): water (20 cm3) and filtered though
a celite pad. The filtrate was separated and the aqueous layer was washed with ethyl
acetate (4 x 20 cm3). The combined ethyl acetate extracts were washed with water (40
cm3) and brine (30 cm3) and were dried over anhydrous sodium sulphate. Filtration was
followed by evaporation of the solvent under reduced pressure gave yellowish brown
oil. This crude residue was pre-absorbed onto silica and purified by column
chromatography using a gradient system of petroleum ether (40-60 C): ethyl acetate
(7.5:2.5 to 5:5) as an eluent. Unfortunately, there was no sign of desired N1-alkylated
product (310 and 311). Only O2-alkylated products were obtained (0.04 g, 24.8%) as a
colourless oil (312 and 313), along with unreacted starting material (0.06 g, 38.7%)
(308 and 309); mp 66-68 C; Rf 0.35 (Petroleum ether (40-60 C: ethyl acetate (7.5:2.5);
[α]D25 +16.67 (c 0.9 in DCM); max (KBr)/ cm-1 1018(s), 1098 (m), 1203 (s), 1237 (s),
1294 (s), 1402 (s), 1440 (m), 1526 (s), 1586 (s), 1719 (s), 2870 (w), 2957 (s) and 3382
(w); δH(400 MHz, CDCl3) 0.7-0.75 (3H, dd, J 4.8 and 6.9, CH3), 0.81-0.88 (6H, m, 2 x
CH3), 0.83-1.03 (3H, m, 1 ½ CH2), 1.40-1.47 (2H, overlap, 2 x CH), 1.42 (9H, s, 3 x
CH3), 1.66-1.69 (2H, brs, CH2), 1.81-2.09 (4H, m, 1 ½ x CH2 and 1 x CH), 2.26-2.67
(4H, m, 2 x CH2), 4.68-4.77 (1H, m, O-CH), 5.11 (1H, s, NH), 5.14 (1H, s, NH), 5.24
(2H, s, CH2), 5.39-5.49 (1H, m, CH-O), 7.34-7.41 (5H, m, Ph-H), 7.57-7.56 (1H, d, J
5.7, CH=CH), 8.34-8.35 (1H, d, J 5.7, CH=CH); δC(100 MHz, CDCl3) 15.8 (CH3)
(minor), 15.9 (CH3) (major), 20.8 (CH3), 21.9 (CH3), 22.9 (CH2) (major or minor), 23.0
(CH2) (major or minor), 25.8 (CH) (minor), 25.9 (CH) (major), 28.3 (3 x CH3), 31.3
(CH2), 34.2 (CH2), 35.1 (CH2), 40.5 (CH2), 43.0 (CH2), 46.8 (CH), 64.6 (C), 64.8 (C),
67.6 (CH2), 75.3 (CH), 77.2 (CH), 77.5 (C), 77.6 (C), 102.2 (CH=CH), 128.2 (CH),
128.5 (CH), 128.6 (CH), 128.6 (CH), 135.2 (C), 152.3 (C), 159.2 (C), 160.1 (CH=CH),
164.2 (C), 173.0 (C); LRMS m/z (ES) (%): 633 ([M+Na]+, 10), 611 ([M+H]+, 30), 366
(100), 311 (16), 310 (83), 279 (28), 217 (38), 199 (32), 145 (25), 129 (56), 113 (98), 77
(45); HMS (ES) 611.3449 ([M+H]+ C33H47O7N4 requires 611.3439).
173
Experimental
Attempted
Synthesis
of
(1’R,
2’S,
5’R)-1-menthol
(1R/S,
3S/R)-3-(4”-
{[(benzyloxy)carbonyl]-N4-cytosin-1-yl)-1-{[(tert-butoxy)carbonyl]amino}cyclopentane1-carboxylate (332 and 333)
O
H
5''
6''
N
4''
O
H
O
N 3''
N 2'' O 2
1''
3
4
H
N
5'
O
3'
O 1'
1
NHBoc
O
HN
4'
N
4'
6'
N
5'
O
2'
O
N 3'
N 2' O 2
1'
3
O
O
1 O 1'
NHBoc
4
Desired product
N
N
O
O
O
2'
NHBoc
5
336
333
O
4'
6'
3'
NHBoc
cis
N
5'
6'
O
5
332
O
O
cis
337
Side product
1,1’-(azodicarbonyl)dipiperidine (0.4 g, 1.6 mmol) was added dropwise to a solution of
tributylphosphine (0.4 cm3, 1.6 mmol) in dry THF (10 cm3) kept under nitrogen at 0 C.
The mixture was stirred for 30 min and then N4-benzyloxycarbonylcytosine (268) (0.2
g, 0.8 mmol) solution in dry DMF (2 cm3) was added. The mixture was stirred for an
additional 30 min and then a solution of trans-alcohol menthol ester (301 and 302) (0.11
g, 0.27 mmol) dry THF (5 cm3) was added slowly. The reaction mixture was allowed to
warm at r.t. and stirred for overnight. After this time TLC analysis implied that no
product had formed and so the reaction was gently heated at 40 C for another day. The
reaction mixture was allowed to cool down to room temperature. The solvent was
removed under reduced pressure and the crude reaction mixture was dissolved in ethyl
acetate (40 cm3): water (40 cm3) and filtered though the celite pad. The filtrate was
washed with hydrogen peroxide and then treated with sodium sulphite to remove excess
hydrogen peroxide. The aqueous layer was extracted with ethyl acetate (3 x 15 cm 3).
The combined ethyl acetate extracts were washed with water (40 cm3) and brine (30
cm3) and were dried over anhydrous sodium sulphate. Filtration was followed by
evaporation of solvent under reduced pressure gave crude reaction mixture. This crude
residue was pre-absorbed onto silica and purified by column chromatography using a
gradient system of petroleum ether (40-60 C): ethyl acetate (7.5:2.5 to 5:5) as an
eluent. This gave a mixture of mainly starting material (301 and 302) (0.08 g, 76%) and
trace amount of O2-alkylated product (336 and 337) (0.005 g, 3%) which was confirmed
by TLC and H1NMR.
174
Experimental
Attempted
Synthesis
of
(1’R,
2’S,
5’R)-1-menthol
(1R/S,
3R/S)-3-(4”-
{[(benzyloxy)carbonyl]-N4-cytosin-1-yl)-1-{[(tert-butoxy)carbonyl]amino}cyclopentane1-carboxylate (310 and 311)
O
H
5''
6''
O
N
4''
H
O
N 3''
N 2'' O 2
1''
3
4
H
N
5'
3'
O 1'
1
NHBoc
O
HN
4'
N
4'
6'
O
O
O
N
O
2'
5'
6'
O
O
N 3'
4
trans
4'
6'
3'
1 O 1'
NHBoc
NHBoc
5
310
O
N
O
O
O
2'
NHBoc
5
trans
312
311
O
N
5'
N 2' O 2
1'
3
O
N
Desired product
313
Undesired product
Method A: To a stirred solution of 1,1’-(azodicarbonyl)dipiperidine (0.46 g, 1.84 mmol)
and cis-alcohol menthol (299 and 300) (0.2 g, 0.51 mmol) in anhydrous DMF (25 cm3)
under argon was added N4-benzyloxycarbonylcytosine (268) (0.38 g, 1.53 mmol)
portion-wise over 10 min at r.t.. Tributylphosphine (0.044 cm3, 1.78 mmol) was then
added and the reaction mixture was left to stir for 5 min, followed by addition of sodium
benzoate (0.22 g, 1.53 mmol) and stirring at r.t. for 18 h. After this time the reaction
was concentrated under reduced pressure. The crude mixture was dissolved in ethyl
acetate (40 cm3): water (40 cm3) and filtered though a celite pad. The filtrate was
washed with hydrogen peroxide and then treated with sodium sulphite to remove excess
hydrogen peroxide. The aqueous layer was extracted with ethyl acetate (3 x 15 cm 3).
The combined ethyl acetate extracts were washed with water (30 cm3) and brine (30
cm3) and were dried over anhydrous sodium sulphate. Filtration was followed by
evaporation of solvent under reduced pressure gave crude reaction mixture. This crude
residue was pre-absorbed onto silica and purified by column chromatography using a
gradient system of petroleum ether (40-60 C): ethyl acetate (7.5:2.5 to 5:5) as an
eluent. Only starting material was recovered.
Method
B:
N4-benzyloxycarbonylcytosine
(268)
(0.06
g,
2.73
mmol)
and
tributylphosphine (0.064 cm3, 0.26 mmol) were added to a solution of cis-alcohol
menthol ester (299 and 300) (0.08 g, 0.22 mmol) in dry DMF (5 cm3) under nitrogen at
room temperature. The reaction mixture was cooled to −10 C and 1,1’(azodicarbonyl)dipiperidine (0.08 g, 0.3 mmol) was added dropwise. The mixture was
175
Experimental
left to stir at r.t. for 18 h. After this time the reaction was concentrated under reduced
pressure. The same procedure for work-up was followed as described in Method A (310
and 311). Only starting material was recovered.
Method C: 1,1’-(azodicarbonyl)dipiperidine (0.47 g, 1.88 mmol) was added dropwise to
a solution of PBu3 (0.47 cm3, 1.88 mmol) in freshly distilled dry THF (10 cm3) kept
under nitrogen at 0 C. The mixture was stirred for 30 min and then N4benzyloxycarbonylcytosine (268) (0.23 g, 0.94 mmol, 3.0 equi) solution in dry DMF (2
cm3) was added. The mixture was stirred for an additional 30 min and then a solution of
cis-alcohol menthol ester (299 and 300) (0.14 g, 0.36 mmol) in dry THF (5 cm3) was
added slowly. The reaction mixture was allowed to warm at r.t. and stirred for 20 h.
After this time TLC analysis implied some product had formed and starting material
was present. Inorder to push the reaction, it was gently heated at 40 C for another day.
The reaction mixture was allowed to cool down to room temperature. The reaction
mixture was stirred for 20 h at r.t. then 24 h at 40 C. After this time the reaction was
concentrated under reduced pressure. The same procedure for work-up was followed as
described in Method A (310 and 311). This gave a starting material (299 and 300) (0.06
g, 42.8%) and mixture of O2-alkylated product (312 and 313) (0.04 g, 18%) as white
solid, which was confirmed by TLC and H1NMR.
(1R/S,
3R/S)-3-[(4’-{[(benzyloxy)carbonyl]amino}pyrimidin-2’-yl)oxy]-1-
{[(tert-butoxy)carbonyl]amino}cyclopentane-1-carboxylic acid (277 and 278)
O
HN
4'
5'
6'
H
O
N 3'
H
N
5'
N 2' O
1'
3
2
O
4
5
312
5'
6'
N
3'
1 O 1'
NHBoc
HN
4'
O
4'
6'
O
O
O
O
N
O
Method A or B
O
2'
O
2
N 2' O
1'
3
O
N
O
O
OH
1 OH
NHBoc
4
313
O
N
N 3'
NHBoc
trans
N
NHBoc
5
277
trans
278
Method A: NaOH, Dioxane
Method B: Methanolic1N KOH reflux
Method A: A 1N (aq) solution of sodium hydroxide (5 cm3) was added at r.t. to a stirred
solution of amino acid ester (312 and 313) (0.09 g, 0.16 mmol) in dioxane (5 cm3). The
reaction mixture was left to stir for 24 h, followed by addition of 0.2 N HCl to adjust the
176
Experimental
pH to 7.0. The solvent was removed in vacuo. It was re-dissolved in water (20 cm3) and
the pH adjusted again to 14. The aqueous layer was washed with DCM (3 x 10 cm3) and
the pH adjusted to 3.0 with 0.6M aqueous citric acid. The aqueous solution was then
extracted with ethyl acetate (5 x 10 cm3). The combined ethyl acetate extracts were
washed with brine (20 cm3) and were dried over anhydrous sodium sulphate. Filtration
was followed by evaporation of the solvent under reduced pressure. No product was
isolated.
Method B: Amino acid ester (312 and 313) (0.09 g, 0.15 mmol) was stirred in
methanolic 1 N potassium hydroxide solution (10 cm3). The reaction mixture was then
heated, whereupon it was left at 80 C for 1.5 h. Subsequently, the reaction mixture was
allowed to cool down to room temperature. To this 0.2 N HCl was added to adjust the
pH to 7.0. The solvent was removed in vacuo. To the reaction mixture 0.2 N aqueous
potassium hydroxide was added to basify the solution to pH 10.0. The mixture was
washed with hexane (2 x 10 cm3) to remove the menthol. The pH of the aqueous layer
was readjusted to pH 2.0. The aqueous solution was then extracted with ethyl acetate
(5x 10 cm3). The combined ethyl acetate extracts were washed with brine (20 cm3) and
were dried over anhydrous sodium sulphate. Filtration to remove sodium sulphate was
followed by evaporation of the solvent under reduced pressure. No product was
isolated.
Attempted resolution of Ethyl (1R/1S, 3S/3R)-1-(tert-Butoxycarbonyl)amino-3hydroxycyclopentane-1-carboxylic acid (204 and 144) with taddol
O
O
OH HO
HO
CO2Et HO
CO2Et
NHBoc
NHBoc
204:224 complex
224
204
trans
144:224 complex
144
Inseparable mixture of trans-alcohol (204 and 144) (0.07 g, 0.25 mmol) and taddol
(224) (0.17 g, 0.25 mmol) was dissolved in minimum amount of hot diethyl ether, the
hexane solution was added till the heated solution was slightly turbid. Subsequently, the
177
Experimental
reaction was cooled to r.t. and the solution was concentrated at reduced pressure. The
concentrated solution was left to stand at room temperature. Unfortunately, no crystals
were formed. Therefore the solution was cooled to 0 C for another 24 h and then to -36
C for a further 24 h. However, still no crystals were formed so the reaction was
abandoned.
(1S/1R,
3R/3S)-1-{[(tert-butoxy)carbonyl]amino}-3-hydroxycyclopentane-1-
carboxylic acid (220 and 221)
Cis-alcohol (203 and 143) (0.25 g, 0.92
2
HO
3
mmol) was stirred with aq 1N sodium
4
3
hydroxide (3.67 cm ) and dioxane (15
COOH
COOH HO
1
NHBoc
NHBoc
5
220
cis
221
3
cm ) solution at r.t. for 24 hrs. This was
followed by addition of 0.2 N HCl to
layer was re-adjusted to pH 1.5-2.0. The hydrolysed cis-acid alcohol (220 and 221) was
extracted with ethyl acetate (6 x 10 cm3). The organic layers were combined and dried
over anhydrous magnesium sulphate. Filtration to remove sodium sulphate was
followed by evaporation of the solvent under reduced pressure. The desired product
(220 and 221) was dried over phosphorus pentoxide for 6 h under strong vacuum to give
white solid (0.16 g, 71%). δH(200 MHz, CD3OD) 1.19 (9 H, S, 3 x CH3), 1.45-1.94 (5H,
br m, 2 x CH2), 2.33-2.43 (1H, dd, J 6.6 and 14.11, CH2), 4.05-4.16 (1H, brs, CH-OH)
and 4.76 (2H, brs, NH and OH).
Attempted
resolution
of
(1S/1R,
3R/3S)-1-{[(tert-butoxy)carbonyl]amino}-3-
hydroxycyclopentane-1-carboxylic acid by brucine
N
HO
COOH HO
COOH
NHBoc
NHBoc
HH
Ether: Hexane
H
O
220
cis
221
O
N
O
H
220:217 complex
221:217 complex
O
217
Inseparable mixture of cis-acid alcohol (220 and 221) (0.16 g, 0.64 mmol) was added to
a solution of the methanol (5 cm3) with (S)-brucine dihydrate (217) (0.28 g, 0.64 mmol)
178
Experimental
at r.t. and the mixture was heated to reflux. The resulting solution was left at this
temperature overnight. Subsequently, the reaction was cooled to r.t. and the solution
was concentrated at reduced pressure. The concentrated solution was left to stand at
room temperature. Unfortunately, no crystals were formed. Therefore the solution was
cooled to 0 C for another 24 h and then to -36 C for a further 24 h. However, still no
crystals were formed so the reaction was abandoned.
179
Appendix A
4. Appendix
4.1. Appendix A
Ethyl
(1R/1S,
3R/3S)-1-(tert-Butoxycarbonyl)amino-3-hydroxycyclopentane-1-
carboxylic acid (204 and 144).
1
H 400.1MHz Biyani M R 5NT5 gHSQC CDCl3 25 C.
180
Appendix A
Ethyl
(1R/1S,
3R/3S)-1-(tert-Butoxycarbonyl)amino-3-hydroxycyclopentane-1-
carboxylic acid (204 and 144).
1H 400.1MHz Biyani M R 5NT5 gNOESY CDCl3 25 C
181
Appendix B
4.2. Appendix B
Single crystal data collection at Heriot Watt University since 2004
A single crystal was coated in Paratone-N heavy oil then mounted on a Hampton
Research Cryoloop and placed in a cold stream of nitrogen gas (100K) on a Bruker
Nonius X8 Apex2 CCD diffractometer running the Apex2 software. Data collection
consisted of, on average, 3000 frames from a combination of phi and omega scans.
After unit cell determination, the data were integrated using SAINT then scaled with
SADABS (TWINABS if the crystal was not single) before space group determination
with XPREP, structure solution with XS and structure refinement with XL. Pictures are
prepared using the graphics program XP. The quality of the datasets was checked using
the IUCr checkcif facility.239-240
Program reference:
APEX2 (SMART, SAINT, SADABS, TWINABS, XPREP, XS, XL, XP) Bruker AXS
Inc., Madison, Wisconsin, USA. 2004-2006
View down the a axis, showing the molecular packing arrangement and intermolecular
hydrogen bonding
182
Appendix B
183
Appendix B
184
Appendix B
There are three crystallographically independent molecules in the crystal lattice which
differ according to the relative arrangements of the menthol and boc groups
185
Appendix B
2D representation of X-RAY crystal of tert-butyl 1-(((1S, 2R, 5S)-2-isopropyl-5methylcyclohexyloxy)methyl)cyclopent-3-enylcarbamate
186
Appendix B
Table 1. Crystal data and structure refinement for X81638.
Identification code
x81638
Empirical formula
C21 H35 N O4
Formula weight
365.50
Temperature
100(2) K
Wavelength
0.71073 Å
Crystal system
Monoclinic
Space group
P2(1)
Unit cell dimensions
a = 10.731(2) Å
= 90°.
b = 10.450(2) Å
= 91.76(3)°.
c = 29.617(6) Å
= 90°.
Volume
3319.5(11) Å3
Z
6
Density (calculated)
1.097 Mg/m3
Absorption coefficient
0.075 mm-1
F(000)
1200
Crystal size
1.20 x 0.22 x 0.10 mm3
Theta range for data collection
2.00 to 26.38°.
Index ranges
-12=h<=13, -13k<=13, -36<=l<=36
Reflections collected
54016
Independent reflections
6884 [R(int) = 0.0998]
Completeness to theta = 25.00°
97.0%
Absorption correction
Semi-empirical from equivalents
Max. and min. transmission
0.9926 and 0.9158
Refinement method
Full-matrix least-squares on F2
Data / restraints / parameters
6884 / 1 / 721
Goodness-of-fit on F2
1.044
Final R indices [I>2sigma(I)]
R1 = 0.0556, wR2 = 0.1288
R indices (all data)
R1 = 0.0953, wR2 = 0.1524
Largest diff. peak and hole
0.263 and -0.366 e.Å-3
187
Appendix B
Table 2. Bond lengths [Å] and angles [°] for X81638.
C1A—O1A
1.469
(5)
C12B—C13B
1.503
C12A—H12A
0.9500
C1A—C6A
1.511
(6)
C12B—H12B
0.9500
C13A—H13A
0.9900
C1A—C2A
1.543
(6)
C13B—H13C
0.9900
C13A—H13B
0.9900
C1A—H1A
1.0000
C13B—H13D
0.9900
C14A—O3A
1.232
(5)
C2A—C3A
1.535
(6)
C14B—O3B
1.231
(5)
C14A—N1A
1.346
(5)
C2A—C7A
1.542
(6)
C14B—N1B
1.354
(5)
C14A—O4A
1.361
(5)
C2A—H2A
1.0000
C14B—O4B
1.358
(5)
C15A—O4A
1.480
(5)
C3A—C4A
1.526
C15B—O4B
1.486
(5)
C15A—C17A
1.517
(7)
C3A—H3A1
0.9900
C15B—C18B
1.519
(6)
C15A—C18A
1.524
(6)
C3A—H3A2
0.9900
C15B—C17B
1.530
(7)
C15A—C16A
1.525
(7)
C4A—C5A
1.542
C15B—C16B
1.533
(6)
C16A—H16D
0.9800
C4A—H4A1
0.9900
C16B—H16A
0.9800
C16A—H16E
0.9800
C4A—H4A2
0.9900
C16B—H16B
0.9800
C16A—H16F
0.9800
C5A—C6A
1.539
(6)
C16B—H16C
0.9800
C17A—H17D
0.9800
C5A—C51A
1.543
(6)
C17B—H17A
0.9800
C17A—H17E
0.9800
C5A—H5A
1.0000
C17B—H17B
0.9800
C17A—H17F
0.9800
C6A—H6A1
0.9900
C17B—H17C
0.9800
C18A—H18D
0.9800
C6A—H6A2
0.9900
C18B—H18A
0.9800
C18A—H18E
0.9800
C7A—C71A
1.544
(6)
C18B—H18B
0.9800
C18A—H18F
0.9800
C7A—C72A
1.544
(6)
C18B—H18C
0.9800
C51A—H51A
0.9800
C7A—H7A
1.0000
C51B—H07A
0.9800
C51A—H51B
0.9800
C8A—O2A
1.226
(5)
C51B—H07B
0.9800
C51A—H51C
0.9800
C8A—O1A
1.351
(5)
C51B—H07C
0.9800
C2C—C3C
1.538
(6)
C8A—C9A
1.521
(6)
C71B—H06A
0.9800
C2C—C7C
1.547
(7)
C9A—N1A
1.465
(5)
C71B—H06B
0.9800
C2C—H2C
1.0000
C9A—C13A
1.557
(6)
C71B—H06C
0.9800
C3C—C4C
1.526
C9A—C10A
1.565
(6)
C72B—H07D
0.9800
C3C—H3C1
0.9900
C10A—C11A
1.519
(6)
C72B—H07E
0.9800
C3C—H3C2
0.9900
C10A—H10E
0.9900
C72B—H07F
0.9800
C4C—C5C
1.527
C10A—H10F
0.9900
C1C—O1C
1.473
(5)
C4C—H4C1
0.9900
C11A—C12A
1.325
C1C—C6C
1.522
(6)
C4C—H4C2
0.9900
C11A—H11A
0.9500
C1C—C2C
1.531
(6)
C5C—C51C
1.530
(7)
C12A—C13A
1.501
C1C—H1C
1.0000
C5C—C6C
1.535
(6)
C5C—H5C
1.0000
C7B—C71B
1.537
(7)
C51C—H51E
0.9800
C6C—H6C1
0.9900
C7B—C72B
1.539
(7)
C51C—H51F
0.9800
C6C—H6C2
0.9900
C7B—H7B
1.0000
C71C—H71D
0.9800
C7C—C72C
1.525
C8B—O2B
1.215
C71C—H71E
0.9800
(6)
(6)
(7)
(6)
(8)
188
(6)
(5)
(7)
(7)
Appendix B
C7C—C71C
1.538
C8B—O1B
1.350
(5)
C71C—H71F
0.9800
C7C—H7C
1.0000
C8B—C9B
1.533
(6)
C9B—C13B
1.566
(6)
C8C—O2C
1.218
(5)
C9B—N1B
1.471
(5)
C10B—C11B
1.506
(6)
C8C—O1C
1.343
(5)
C9B—C10B
1.560
(6)
C10B—H10C
0.9900
C8C—C9C
1.527
(6)
C9C—C13C
1.561
(6)
C10B—H10D
0.9900
C9C—N1C
1.468
(5)
C10C—C11C
1.512
(6)
C11B—C12B
1.336
C9C—C10C
1.541
(6)
C10C—H10A
0.9900
C11B—H11B
0.9500
C71A—H71A
0.9800
C10C—H10B
0.9900
C72C—H72D
0.9800
C71A—H71B
0.9800
C11C—C12C
1.328
C72C—H72E
0.9800
C71A—H71C
0.9800
C11C—H11C
0.9500
C72C—H72F
0.9800
C72A—H72A
0.9800
C12C—C13C
1.511
N1A—H1A1
0.8800
C72A—H72B
0.9800
C12C—H12C
0.9500
N1B—H1B1
0.8800
C72A—H72C
0.9800
C13C—H13E
0.9900
N1C—H1C1
0.8800
C1B—O1B
1.465
(5)
C13C—H13F
0.9900
C1B—C6B
1.527
(6)
C14C—O3C
1.230
(5)
O1A—C1A—C6A
110.5
(3)
C1B—C2B
1.536
(6)
C14C—O4C
1.352
(5)
O1A—C1A—C2A
106.5
(3)
C1B—H1B
1.0000
C14C—N1C
1.359
(6)
C6A—C1A—C2A
113.4
(4)
C2B—C3B
1.536
(6)
C15C—O4C
1.483
(5)
O1A—C1A—H1A
108.8
C2B—C7B
1.553
(7)
C15C—C16C
1.512
(7)
C6A—C1A—H1A
108.8
C2B—H2B
1.0000
C15C—C18C
1.525
(8)
C2A—C1A—H1A
108.8
C3B—C4B
1.542
C15C—C17C
1.529
(7)
C3A—C2A—C7A
114.7
(3)
C3B—H3B1
0.9900
C16C—H16G
0.9800
C3A—C2A—C1A
108.4
(3)
C3B—H3B2
0.9900
C16C—H16H
0.9800
C7A—C2A—C1A
112.8
(4)
C4B—C5B
1.525
C16C—H16I
0.9800
C3A—C2A—H2A
106.8
C4B—H4B1
0.9900
C17C—H17G
0.9800
C7A—C2A—H2A
106.8
C4B—H4B2
0.9900
C17C—H17H
0.9800
C1A—C2A—H2A
106.8
C5B—C51B
1.529
(7)
C17C—H17I
0.9800
C4A—C3A—C2A
112.3
C5B—C6B
1.534
(6)
C18C—H18G
0.9800
C4A—C3A—H3A1
109.2
C5B—H5B
1.0000
C18C—H18H
0.9800
C2A—C3A—H3A1
109.2
C6B—H6B1
0.9900
C18C—H18I
0.9800
C4A—C3A—H3A2
109.2
C6B—H6B2
0.9900
C51C—H51D
0.9800
C2A—C3A—H3A2
109.2
H3A1—C3A—H3A2
107.9
C4A—C5A—H5A
108.1
H07A—C51B—H07B
109.5
C3A—C4A—C5A
111.8
C51A—C5A—H5A
108.1
C5B—C51B—H07C
109.5
C3A—C4A—H4A1
109.3
C1A—C6A—C5A
110.2
H07A—C51B—H07C
109.5
C5A—C4A—H4A1
109.3
C1A—C6A—H6A1
109.6
H07B—C51B—H07C
109.5
C3A—C4A—H4A2
109.3
C5A—C6A—H6A1
109.6
C7B—C71B—H06A
109.5
C5A—C4A—H4A2
109.3
C1A—C6A—H6A2
109.6
C7B—C71B—H06B
109.5
H4A1—C4A—H4A2
107.9
C5A—C6A—H6A2
109.6
H06A—C71B—H06B
109.5
C6A—C5A—C4A
109.1
H6A1—C6A—H6A2
108.1
C7B—C71B—H06C
109.5
(8)
(7)
(7)
(4)
(4)
189
(7)
(6)
(3)
(7)
(4)
Appendix B
C9B—C13B—H13C
111.0
C2A—C7A—C71A
113.1
(4)
H06A—C71B—H06C
109.5
C12B—C13B—H13D
111.0
C2A—C7A—C72A
110.9
(4)
H06B—C71B—H06C
109.5
C9B—C13B—H13D
111.0
C71A—C7A—C72A
110.9
(3)
C7B—C72B—H07D
109.5
H13C—C13B—H13D
109.0
C2A—C7A—H7A
107.2
C7B—C72B—H07E
109.5
O3B—C14B—N1B
124.5
(4)
C71A—C7A—H7A
107.2
H07D—C72B—H07E
109.5
O3B—C14B—O4B
124.8
(4)
C72A—C7A—H7A
107.2
C7B—C72B—H07F
109.5
N1B—C14B—O4B
110.7
(4)
O2A—C8A—O1A
123.0
(4)
H07D—C72B—H07F
109.5
O4B—C15B—C18B
111.0
(3)
O2A—C8A—C9A
124.2
(4)
H07E—C72B—H07F
109.5
O4B—C15B—C17B
101.7
(4)
O1A—C8A—C9A
112.4
(4)
O1C—C1C—C6C
108.0
(3)
C18B—C15B—C17B
110.4
(4)
N1A—C9A—C8A
111.2
(3)
O1C—C1C—C2C
106.7
(3)
O4B—C15B—C16B
109.4
(3)
N1A—C9A—C13A
110.5
(3)
C6C—C1C—C2C
113.0
(4)
C18B—C15B—C16B
113.1
(4)
C8A—C9A—C13A
113.0
(3)
O1C—C1C—H1C
109.7
C17B—C15B—C16B
110.8
(4)
N1A—C9A—C10A
107.7
(3)
C6C—C1C—H1C
109.7
C15B—C16B—H16A
109.5
C8A—C9A—C10A
109.7
(3)
C2C—C1C—H1C
109.7
C15B—C16B—H16B
109.5
C13A—C9A—C10A
104.4
(3)
C1C—C2C—C3C
109.1
H16A—C16B—H16B
109.5
C11A—C10A—C9A
101.6
(3)
H10E—C10A—H10F
109.3
C15B—C16B—H16C
109.5
C11A—C10A—H10E
111.4
C12A—C11A—C10A
112.3
H16A—C16B—H16C
109.5
C9A—C10A—H10E
111.4
C12A—C11A—H11A
123.8
H16B—C16B—H16C
109.5
C11A—C10A—H10F
111.4
C10A—C11A—H11A
123.8
C15B—C17B—H17A
109.5
C9A—C10A—H10F
111.4
C11A—C12A—C13A
112.2
C15B—C17B—H17B
109.5
C15B—C18B—H18A
109.5
C11A—C12A—H12A
123.9
H17A—C17B—H17B
109.5
C15B—C18B—H18B
109.5
C13A—C12A—H12A
123.9
C15B—C17B—H17C
109.5
H18A—C18B—H18B
109.5
C12A—C13A—C9A
102.7
H17A—C17B—H17C
109.5
C15B—C18B—H18C
109.5
C12A—C13A—H13A
111.2
H17B—C17B—H17C
109.5
H18A—C18B—H18C
109.5
C9A—C13A—H13A
111.2
C6A—C5A—C51A
111.9
(4)
H18B—C18B—H18C
109.5
C12A—C13A—H13B
111.2
C4A—C5A—C51A
111.4
(4)
C5B—C51B—H07A
109.5
C9A—C13A—H13B
111.2
C6A—C5A—H5A
108.1
C5B—C51B—H07B
109.5
H13A—C13A—H13B
109.1
O3A—C14A—N1A
123.3
(4)
C5C—C4C—H4C1
109.3
O2C—C8C—C9C
124.6
O3A—C14A—O4A
125.7
(4)
C3C—C4C—H4C2
109.3
H71A—C71A—H71B
109.5
N1A—C14A—O4A
110.9
(4)
C5C—C4C—H4C2
109.3
C7A—C71A—H71C
109.5
O4A—C15A—C17A
110.3
(4)
H4C1—C4C—H4C2
108.0
H71A—C71A—H71C
109.5
O4A—C15A—C18A
101.9
(3)
C4C—C5C—C51C
112.7
(4)
H71B—C71A—H71C
109.5
C17A—C15A—C18A
111.0
(4)
C4C—C5C—C6C
109.5
(4)
C7A—C72A—H72A
109.5
O4A—C15A—C16A
110.2
(4)
C51C—C5C—C6C
110.9
(4)
C7A—C72A—H72B
109.5
C17A—C15A—C16A
112.6
(4)
C4C—C5C—H5C
107.8
H72A—C72A—H72B
109.5
C18A—C15A—C16A
110.3
(4)
C51C—C5C—H5C
107.8
C7A—C72A—H72C
109.5
C15A—C16A—H16D
109.5
C6C—C5C—H5C
107.8
H72A—C72A—H72C
109.5
C15A—C16A—H16E
109.5
C1C—C6C—C5C
111.2
H72B—C72A—H72C
109.5
190
(4)
(4)
(4)
(4)
(4)
(4)
Appendix B
H16D—C16A—H16E
109.5
C1C—C6C—H6C1
109.4
O1B—C1B—C6B
109.2
(3)
C15A—C16A—H16F
109.5
C5C—C6C—H6C1
109.4
O1B—C1B—C2B
105.3
(3)
H16D—C16A—H16F
109.5
C1C—C6C—H6C2
109.4
C6B—C1B—C2B
113.4
(4)
H16E—C16A—H16F
109.5
C5C—C6C—H6C2
109.4
O1B—C1B—H1B
109.6
C15A—C17A—H17D
109.5
H6C1—C6C—H6C2
108.0
C6B—C1B—H1B
109.6
C15A—C17A—H17E
109.5
C72C—C7C—C71C
110.7
(5)
C2B—C1B—H1B
109.6
H17D—C17A—H17E
109.5
C72C—C7C—C2C
113.9
(5)
C3B—C2B—C1B
109.5
(4)
C15A—C17A—H17F
109.5
C71C—C7C—C2C
110.8
(4)
C3B—C2B—C7B
113.5
(3)
H17D—C17A—H17F
109.5
C72C—C7C—H7C
107.0
C1B—C2B—C7B
112.3
(4)
H17E—C17A—H17F
109.5
C71C—C7C—H7C
107.0
C3B—C2B—H2B
107.0
C15A—C18A—H18D
109.5
C2C—C7C—H7C
107.0
O1C—C8C—C9C
110.7
(4)
C1C—C2C—C7C
113.2
(4)
O2C—C8C—O1C
124.5
N1C—C9C—C8C
111.5
(3)
C3C—C2C—C7C
114.0
(4)
H18D—C18A—H18E
109.5
N1C—C9C—C10C
110.2
(3)
C1C—C2C—H2C
106.7
C15A—C18A—H18F
109.5
C8C—C9C—C10C
114.3
(4)
C3C—C2C—H2C
106.7
H18D—C18A—H18F
109.5
N1C—C9C—C13C
106.9
(3)
C7C—C2C—H2C
106.7
H18E—C18A—H18F
109.5
C8C—C9C—C13C
109.8
(3)
C4C—C3C—C2C
111.8
C5A—C51A—H51A
109.5
C10C—C9C—C13C
103.6
(3)
C4C—C3C—H3C1
109.3
C5A—C51A—H51B
109.5
C11C—C10C—C9C
102.8
(4)
C2C—C3C—H3C1
109.3
H51A—C51A—H51B
109.5
C11C—C10C—H10A
111.2
C4C—C3C—H3C2
109.3
C5A—C51A—H51C
109.5
C9C—C10C—H10A
111.2
C2C—C3C—H3C2
109.3
H51A—C51A—H51C
109.5
C11C—C10C—H10B
111.2
H3C1—C3C—H3C2
107.9
H51B—C51A—H51C
109.5
C9C—C10C—H10B
111.2
C3C—C4C—C5C
111.6
C7A—C71A—H71A
109.5
H10A—C10C—H10B
109.1
C3C—C4C—H4C1
109.3
C7A—C71A—H71B
109.5
C12C—C11C—C10C
112.2
C12C—C11C—H11C
123.9
C11C—C12C—H12C
124.5
C12C—C13C—H13E
111.2
C10C—C11C—H11C
123.9
C13C—C12C—H12C
124.5
C9C—C13C—H13E
111.2
C11C—C12C—C13C
110.9
C12C—C13C—C9C
103.0
(3)
C12C—C13C—H13F
111.2
C9C—C13C—H13F
111.2
C71B—C7B—C2B
111.3
(4)
H72E—C72C—H72F
109.5
H13E—C13C—H13F
109.1
C72B—C7B—C2B
113.8
(4)
C5C—C51C—H51F
109.5
O3C—C14C—O4C
126.1
(4)
C71B—C7B—H7B
107.3
H51D—C51C—H51F
109.5
O3C—C14C—N1C
123.5
(4)
C72B—C7B—H7B
107.3
H51E—C51C—H51F
109.5
O4C—C14C—N1C
110.4
(4)
C2B—C7B—H7B
107.3
C7C—C71C—H71D
109.5
O4C—C15C—C16C
109.8
(4)
O2B—C8B—O1B
123.8
(4)
C7C—C71C—H71E
109.5
O4C—C15C—C18C
109.8
(4)
O2B—C8B—C9B
126.4
(4)
H71D—C71C—H71E
109.5
C16C—C15C—C18C
113.7
(5)
O1B—C8B—C9B
109.7
(4)
C7C—C71C—H71F
109.5
C1B—C2B—H2B
107.0
N1B—C9B—C8B
109.6
(3)
H71D—C71C—H71F
109.5
C7B—C2B—H2B
107.0
N1B—C9B—C10B
112.6
(3)
H71E—C71C—H71F
109.5
C2B—C3B—C4B
112.6
C8B—C9B—C10B
111.8
(3)
C7C—C72C—H72D
109.5
C2B—C3B—H3B1
109.1
O4C—C15C—C17C
101.5
(4)
C7C—C72C—H72E
109.5
(4)
(4)
(4)
(4)
191
(4)
(4)
Appendix B
C4B—C3B—H3B1
109.1
C16C—C15C—C17C
110.0
(5)
H72D—C72C—H72E
109.5
C2B—C3B—H3B2
109.1
C18C—C15C—C17C
111.3
(4)
C7C—C72C—H72F
109.5
C4B—C3B—H3B2
109.1
C15C—C16C—H16G
109.5
H72D—C72C—H72F
109.5
H3B1—C3B—H3B2
107.8
C15C—C16C—H16H
109.5
N1B—C9B—C13B
109.0
(3)
C5B—C4B—C3B
111.9
H16G—C16C—H16H
109.5
C8B—C9B—C13B
107.6
(3)
C5B—C4B—H4B1
109.2
C15C—C16C—H16I
109.5
C10B—C9B—C13B
106.0
(3)
C3B—C4B—H4B1
109.2
H16G—C16C—H16I
109.5
C11B—C10B—C9B
104.1
(4)
C5B—C4B—H4B2
109.2
H16H—C16C—H16I
109.5
C11B—C10B—H10C
110.9
C3B—C4B—H4B2
109.2
C15C—C17C—H17G
109.5
C9B—C10B—H10C
110.9
H4B1—C4B—H4B2
107.9
C15C—C17C—H17H
109.5
C11B—C10B—H10D
110.9
C4B—C5B—C51B
111.6
(4)
H17G—C17C—H17H
109.5
C9B—C10B—H10D
110.9
C4B—C5B—C6B
109.6
(4)
C15C—C17C—H17I
109.5
H10C—C10B—H10D
109.0
C51B—C5B—C6B
111.7
(4)
H17G—C17C—H17I
109.5
C12B—C11B—C10B
112.6
C4B—C5B—H5B
107.9
H17H—C17C—H17I
109.5
C12B—C11B—H11B
123.7
C51B—C5B—H5B
107.9
C15C—C18C—H18G
109.5
C10B—C11B—H11B
123.7
C6B—C5B—H5B
107.9
C15C—C18C—H18H
109.5
C11B—C12B—C13B
112.9
C1B—C6B—C5B
111.8
H18G—C18C—H18H
109.5
C11B—C12B—H12B
123.6
C1B—C6B—H6B1
109.3
C15C—C18C—H18I
109.5
C13B—C12B—H12B
123.6
C5B—C6B—H6B1
109.3
H18G—C18C—H18I
109.5
C12B—C13B—C9B
103.9
C1B—C6B—H6B2
109.3
H18H—C18C—H18I
109.5
C12B—C13B—H13C
111.0
C5B—C6B—H6B2
109.3
C5C—C51C—H51D
109.5
C14A—N1A—C9A
119.8
H6B1—C6B—H6B2
107.9
C5C—C51C—H51E
109.5
C14A—N1A—H1A1
120.1
C71B—C7B—C72B
109.6
(4)
H51D—C51C—H51E
109.5
C14A—O4A—C15A
120.5
(3)
C14A—N1A—C9A
119.8
(3)
C9B—N1B—H1B1
120.5
C8B—O1B—C1B
118.8
(3)
C14A—N1A—H1A1
120.1
C14C—N1C—C9C
119.7
C14B—O4B—C15B
120.1
(3)
C9A—N1A—H1A1
120.1
C14C—N1C—H1C1
120.2
C8C—O1C—C1C
118.1
(3)
C14B—N1B—C9B
118.9
C9C—N1C—H1C1
120.2
C14C—O4C—C15C
120.1
(4)
C14B—N1B—H1B1
120.5
C8A—O1A—C1A
117.6
(4)
(3)
(3)
(4)
(3)
Table 3. Torsion angles [°] for X81638.
O1A—C1A—C2A—C3A
−177.6
(3)
C7C—C2C—C3C—C4C
−177.5
(4)
C6A—C1A—C2A—C3A
−55.9
(4)
C2C—C3C—C4C—C5C
−57.6
(5)
O1A—C1A—C2A—C7A
54.2
(4)
C3C—C4C—C5C—C51C
−179.2
(4)
C6A—C1A—C2A—C7A
175.9
(3)
C3C—C4C—C5C—C6C
56.7
(5)
C7A—C2A—C3A—C4A
−179.0
O1C—C1C—C6C—C5C
174.1
(4)
C1A—C2A—C3A—C4A
53.9
C2C—C1C—C6C—C5C
56.3
(5)
C2A—C3A—C4A—C5A
−56.6
C4C—C5C—C6C—C1C
−55.7
(4)
(5)
(5)
192
(5)
(4)
(4)
(4)
(3)
Appendix B
C3A—C4A—C5A—C6A
56.6
C51C—C5C—C6C—C1C
179.3
(4)
C3A—C4A—C5A—C51A
−179.4
C1C—C2C—C7C—C72C
64.4
(6)
O1A—C1A—C6A—C5A
178.2
(3)
C3C—C2C—C7C—C72C
−61.0
(6)
C2A—C1A—C6A—C5A
58.7
(5)
C1C—C2C—C7C—C71C
−170.0
(4)
C4A—C5A—C6A—C1A
−57.0
(5)
C3C—C2C—C7C—C71C
64.6
(6)
C51A—C5A—C6A—C1A
179.3
(4)
O2C—C8C—C9C—N1C
142.6
(4)
C3A—C2A—C7A—C71A
−65.2
(5)
O1C—C8C—C9C—N1C
−42.6
(5)
C1A—C2A—C7A—C71A
59.6
(5)
O2C—C8C—C9C—C10C
16.8
C3A—C2A—C7A—C72A
60.1
(5)
O1C—C8C—C9C—C10C
−168.5
(4)
C1A—C2A—C7A—C72A
−175.2
(3)
O2C—C8C—C9C—C13C
−99.1
(5)
O2A—C8A—C9A—N1A
−142.3
(4)
O1C—C8C—C9C—C13C
75.6
(4)
O1A—C8A—C9A—N1A
44.0
N1C—C9C—C10C—C11C
88.4
(4)
O2A—C8A—C9A—C13A
−17.4
(6)
C8C—C9C—C10C—C11C
−145.1
(4)
O1A—C8A—C9A—C13A
168.9
(3)
C13C—C9C—C10C—C11C
−25.7
(4)
O2A—C8A—C9A—C10A
98.7
(5)
C9C—C10C—C11C—C12C
17.3
(5)
O1A—C8A—C9A—C10A
−75.0
C10C—C11C—C12C—C13C
−0.6
(6)
N1A—C9A—C10A—C11A
93.2
C11C—C12C—C13C—C9C
−16.2
(5)
C8A—C9A—C10A—C11A
−145.6
(4)
N1C—C9C—C13C—C12C
−90.9
(4)
C13A—C9A—C10A—C11A
−24.3
(4)
C8C—C9C—C13C—C12C
148.0
(4)
C9A—C10A—C11A—C12A
16.0
(5)
C10C—C9C—C13C—C12C
25.6
(4)
C10A—C11A—C12A—C13A
−0.3
(6)
O3A—C14A—N1A—C9A
2.0
(6)
C11A—C12A—C13A—C9A
−15.7
(5)
O4A—C14A—N1A—C9A
−178.9
N1A—C9A—C13A—C12A
−91.1
(4)
C8A—C9A—N1A—C14A
49.0
C8A—C9A—C13A—C12A
143.6
(4)
C13A—C9A—N1A—C14A
−77.3
(5)
C10A—C9A—C13A—C12A
24.4
(4)
C10A—C9A—N1A—C14A
169.2
(4)
O1B—C1B—C2B—C3B
−172.1
(3)
O3B—C14B—N1B—C9B
−10.6
(6)
C6B—C1B—C2B—C3B
−52.7
(5)
O4B—C14B—N1B—C9B
169.1
(3)
O1B—C1B—C2B—C7B
60.8
C8B—C9B—N1B—C14B
−60.7
(5)
C6B—C1B—C2B—C7B
−179.8
C10B—C9B—N1B—C14B
64.4
C1B—C2B—C3B—C4B
52.6
(5)
C13B—C9B—N1B—C14B
−178.2
C7B—C2B—C3B—C4B
179.0
(4)
O3C—C14C—N1C—C9C
2.5
C2B—C3B—C4B—C5B
−56.1
(5)
O4C—C14C—N1C—C9C
−177.4
(3)
C3B—C4B—C5B—C51B
−179.8
(4)
C8C—C9C—N1C—C14C
−57.5
(5)
C3B—C4B—C5B—C6B
56.0
C10C—C9C—N1C—C14C
70.5
(5)
(4)
(5)
(4)
(4)
(4)
(3)
(5)
193
(6)
(3)
(5)
(5)
(3)
(6)
(5)
Appendix B
O1B—C1B—C6B—C5B
172.6
(3)
C13C—C9C—N1C—C14C
−177.5
C2B—C1B—C6B—C5B
55.5
(5)
O2A—C8A—O1A—C1A
−3.1
(6)
C4B—C5B—C6B—C1B
−55.6
(5)
C9A—C8A—O1A—C1A
170.7
(3)
C51B—C5B—C6B—C1B
−179.7
(4)
C6A—C1A—O1A—C8A
97.1
(4)
C3B—C2B—C7B—C71B
59.0
C2A—C1A—O1A—C8A
−139.4
C1B—C2B—C7B—C71B
−176.1
(4)
O3A—C14A—O4A—C15A
−5.8
(6)
C3B—C2B—C7B—C72B
−65.4
(5)
N1A—C14A—O4A—C15A
175.1
(4)
C1B—C2B—C7B—C72B
59.5
(5)
C17A—C15A—O4A—C14A
−59.8
(5)
O2B—C8B—C9B—N1B
136.6
(4)
C18A—C15A—O4A—C14A
−177.7
(4)
O1B—C8B—C9B—N1B
−45.8
(5)
C16A—C15A—O4A—C14A
65.2
(5)
O2B—C8B—C9B—C10B
11.0
O2B—C8B—O1B—C1B
5.8
(6)
O1B—C8B—C9B—C10B
−171.4
(4)
C9B—C8B—O1B—C1B
−171.8
O2B—C8B—C9B—C13B
−105.0
(5)
C6B—C1B—O1B—C8B
79.2
O1B—C8B—C9B—C13B
72.6
(4)
C2B—C1B—O1B—C8B
−158.7
(4)
N1B—C9B—C10B—C11B
125.5
(4)
O3B—C14B—O4B—C15B
−12.7
(6)
C8B—C9B—C10B—C11B
−110.6
N1B—C14B—O4B—C15B
167.6
(3)
C13B—C9B—C10B—C11B
6.4
C18B—C15B—O4B—C14B
64.9
(5)
C9B—C10B—C11B—C12B
−4.0
(5)
C17B—C15B—O4B—C14B
−177.7
(4)
C10B—C11B—C12B—C13B
−0.3
(6)
C16B—C15B—O4B—C14B
−60.5
(5)
C11B—C12B—C13B—C9B
4.5
O2C—C8C—O1C—C1C
5.4
N1B—C9B—C13B—C12B
−128.0
C9C—C8C—O1C—C1C
−169.4
C8B—C9B—C13B—C12B
113.2
(4)
C6C—C1C—O1C—C8C
88.4
C10B—C9B—C13B—C12B
−6.6
(4)
C2C—C1C—O1C—C8C
−149.9
O1C—C1C—C2C—C3C
−173.3
(4)
O3C—C14C—O4C—C15C
−1.1
(6)
C6C—C1C—C2C—C3C
−54.7
(5)
N1C—C14C—O4C—C15C
178.8
(3)
O1C—C1C—C2C—C7C
58.6
(5)
C16C—C15C—O4C—C14C
63.8
(6)
C6C—C1C—C2C—C7C
177.2
(4)
C18C—C15C—O4C—C14C
−61.9
(5)
C1C—C2C—C3C—C4C
54.9
(5)
C17C—C15C—O4C—C14C
−179.8
(4)
(5)
(6)
(4)
(4)
(5)
(4)
194
(3)
(3)
(3)
(5)
(6)
(3)
(4)
(4)
Appendix B
Table 4. Hydrogen bonds for X81638 [Å and °].
______________________________________________________________________
D-H...A
d(D-H)
d(H...A)
d(D...A)
<(DHA)
______________________________________________________________________
N(1A)-H(1A1)...O(3B)
0.88
2.06
2.928(5)
167.9
N(1B)-H(1B1)...O(2A)#1 0.88
2.33
2.954(5)
128.2
N(1C)-H(1C1)...O(2C)#2 0.88
2.26
3.116(5)
165.6
______________________________________________________________________
Symmetry transformations used to generate equivalent atoms:
#1 x,y-1,z
#2 -x,y+1/2,-z+1
195
Appendix B
Table 5. Crystal data and structure refinement for X81362M.
Identification code
x81362m
Empirical formula
C23 H37 N O8 S
Formula weight
487.60
Temperature
100(2) K
Wavelength
0.71073 Å
Crystal system
Monoclinic
Space group
P2(1)
Unit cell dimensions
a = 8.4463(13) Å
= 90°.
b = 9.5466(17) Å
= 90.200(7)°.
c = 15.497(3) Å
 = 90°.
Volume
1249.6(4) Å3
Z
2
Density (calculated)
1.296 Mg/m3
Absorption coefficient
0.176 mm-1
F(000)
524
Crystal size
0.18 x 0.12 x 0.08 mm3
196
Appendix B
Theta range for data collection
1.31 to 23.34°.
Index ranges
-9<=h<=9, -10<=k<=10, -17<=l<=17
Reflections collected
13375
Independent reflections
3362 [R(int) = 0.1213]
Completeness to theta = 23.34°
92.3%
Absorption correction
Semi-empirical from equivalents
Max. and min. transmission
0.9861 and 0.630
Refinement method
Full-matrix least-squares on F2
Data / restraints / parameters
3362 / 25 / 303
Goodness-of-fit on F2
0.882
Final R indices [I>2sigma(I)]
R1 = 0.0583, wR2 = 0.1246
R indices (all data)
R1 = 0.1375, wR2 = 0.1712
Absolute structure parameter
-0.1(2)
Largest diff. peak and hole
0.382 and -0.427 e.Å-3
Table 6. Bond lengths [Å] and angles [°] for X81362M.
N1—C19
1.330
(11)
C11—H11
1.0000
C23—H23B
0.9800
N1—C13
1.453
(11)
C12—C13
1.551
C23—H23C
0.9800
N1—H1
0.8800
C12—H12A
0.9900
C19—N1—C13
119.9
C1—C3
1.510
C12—H12B
0.9900
C19—N1—H1
120.0
C1—H1A
0.9800
C13—C14
1.516
(11)
C13—N1—H1
120.0
C1—H1B
0.9800
C13—C16
1.548
(11)
C3—C1—H1A
109.5
C1—H1C
0.9800
C14—C15
1.494
(12)
C3—C1—H1B
109.5
O1—C6
1.214
(10)
C14—H14A
0.9900
H1A—C1—H1B
109.5
S1—O3
1.436
(6)
C14—H14B
0.9900
C3—C1—H1C
109.5
S1—O2
1.450
(7)
C15—H15A
0.9900
H1A—C1—H1C
109.5
S1—O4
1.567
(6)
C15—H15B
0.9900
H1B—C1—H1C
109.5
S1—C10
1.770
(8)
C17—C18
1.347
O3—S1—O2
121.7
(4)
C2—C3
1.543
(12)
C17—H17A
0.9900
O3—S1—O4
109.7
(3)
C2—H2A
0.9800
C17—H17B
0.9900
O2—S1—O4
103.8
(3)
C2—H2B
0.9800
C18—H18A
0.9800
O3—S1—C10
109.1
(4)
C2—H2C
0.9800
C18—H18B
0.9800
O2—S1—C10
108.9
(4)
C3—C4
1.533
(11)
C18—H18C
0.9800
O4—S1—C10
101.8
(4)
C3—C7
1.554
(11)
C20—C22
1.502
(11)
C3—C2—H2A
109.5
C4—C5
1.532
(11)
C20—C21
1.526
(10)
C3—C2—H2B
109.5
C4—C9
1.553
(11)
C20—C23
1.527
(11)
H2A—C2—H2B
109.5
(10)
197
(11)
(16)
(7)
Appendix B
C4—H4
1.0000
C21—H21A
0.9800
C3—C2—H2C
109.5
O4—C11
1.513
(10)
O6—C17
1.422
(11)
H2A—C2—H2C
109.5
C5—C6
1.525
(12)
C7—C10
1.548
(11)
H2B—C2—H2C
109.5
C5—H5A
0.9900
C7—C8
1.561
(10)
C1—C3—C4
114.0
(7)
C5—H5B
0.9900
O7—C19
1.212
(10)
C1—C3—C2
107.5
(7)
O5—C16
1.215
(10)
C8—C9
1.555
(10)
O4—C11—C12
105.0
(6)
C6—C7
1.519
(11)
C8—H8A
0.9900
C15—C11—H11
111.2
O6—C16
1.350
(11)
C8—H8B
0.9900
O4—C11—H11
111.2
O8—C20
1.481
(9)
O8—C19
1.380
C12—C11—H11
111.2
C9—H9A
0.9900
C21—H21B
0.9800
C13—C12—C11
102.8
C9—H9B
0.9900
C21—H21C
0.9800
C13—C12—H12A
111.2
C10—H10A
0.9900
C22—H22A
0.9800
C11—C12—H12A
111.2
C10—H10B
0.9900
C22—H22B
0.9800
C13—C12—H12B
111.2
C11—C15
1.487
(11)
C22—H22C
0.9800
C11—C12—H12B
111.2
C11—C12
1.591
(11)
C23—H23A
0.9800
H12A—C12—H12B
109.1
N1—C13—C14
112.3
(7)
C10—C7—C3
117.9
(7)
H21A—C21—H21B
109.5
N1—C13—C16
111.3
(7)
C6—C7—C8
103.7
(7)
C21—C20—C23
110.7
C14—C13—C16
111.9
(8)
C10—C7—C8
118.5
(6)
C20—C21—H21A
109.5
N1—C13—C12
111.1
(7)
C3—C7—C8
103.2
(6)
C20—C21—H21B
109.5
C14—C13—C12
102.6
(6)
C9—C8—C7
103.4
(6)
C7—C8—H8B
111.1
C16—C13—C12
107.2
(6)
C9—C8—H8A
111.1
H8A—C8—H8B
109.0
C15—C14—C13
103.8
(7)
C7—C8—H8A
111.1
C19—O8—C20
120.9
(7)
C15—C14—H14A
111.0
C9—C8—H8B
111.1
C4—C9—C8
102.2
(6)
C13—C14—H14A
111.0
C11—C15—H15A
111.3
C4—C9—H9A
111.3
C15—C14—H14B
111.0
C14—C15—H15A
111.3
C8—C9—H9A
111.3
C13—C14—H14B
111.0
C11—C15—H15B
111.3
C4—C9—H9B
111.3
H14A—C14—H14B
109.0
C14—C15—H15B
111.3
C8—C9—H9B
111.3
C11—C15—C14
102.2
(7)
H15A—C15—H15B
109.2
H9A—C9—H9B
109.2
C4—C3—C2
114.6
(7)
O5—C16—O6
125.2
(8)
C7—C10—S1
115.6
C1—C3—C7
114.4
(7)
O5—C16—C13
123.2
(9)
C7—C10—H10A
108.4
C4—C3—C7
93.5
O6—C16—C13
111.4
(8)
S1—C10—H10A
108.4
C2—C3—C7
112.6
(7)
C18—C17—O6
111.7
(12)
C7—C10—H10B
108.4
C5—C4—C3
103.6
(7)
C18—C17—H17A
109.3
S1—C10—H10B
108.4
C5—C4—C9
106.3
(6)
O6—C17—H17A
109.3
H10A—C10—H10B
107.4
C3—C4—C9
102.9
(6)
C18—C17—H17B
109.3
C15—C11—O4
111.0
(7)
C5—C4—H4
114.2
O6—C17—H17B
109.3
C15—C11—C12
106.9
(6)
C3—C4—H4
114.2
H17A—C17—H17B
107.9
C20—C21—H21C
109.5
C9—C4—H4
114.2
C17—C18—H18A
109.5
H21A—C21—H21C
109.5
C11—O4—S1
120.4
C17—C18—H18B
109.5
H21B—C21—H21C
109.5
(6)
(5)
198
(10)
(6)
(7)
(5)
Appendix B
C6—C5—C4
101.6
H18A—C18—H18B
109.5
C20—C22—H22A
109.5
C6—C5—H5A
111.5
C17—C18—H18C
109.5
C20—C22—H22B
109.5
C4—C5—H5A
111.5
H18A—C18—H18C
109.5
H22A—C22—H22B
109.5
C6—C5—H5B
111.5
H18B—C18—H18C
109.5
C20—C22—H22C
109.5
C4—C5—H5B
111.5
O7—C19—N1
124.8
(8)
H22A—C22—H22C
109.5
H5A—C5—H5B
109.3
O7—C19—O8
124.8
(8)
H22B—C22—H22C
109.5
O1—C6—C7
127.9
(8)
N1—C19—O8
110.3
(8)
C20—C23—H23A
109.5
O1—C6—C5
126.2
(7)
O8—C20—C22
110.1
(7)
C20—C23—H23B
109.5
C7—C6—C5
105.9
(7)
O8—C20—C21
109.8
(6)
H23A—C23—H23B
109.5
C16—O6—C17
113.4
(7)
C22—C20—C21
112.1
(6)
C20—C23—H23C
109.5
C6—C7—C10
110.7
(7)
O8—C20—C23
101.8
(6)
H23A—C23—H23C
109.5
C6—C7—C3
100.5
(6)
C22—C20—C23
111.8
(8)
H23B—C23—H23C
109.5
(7)
Table 7. Torsion angles [°] for X81362M.
C1—C3—C4—C5
64.2
C8—C7—C10—S1
−10.1
(10)
C2—C3—C4—C5
−171.4
O3—S1—C10—C7
−70.4
(7)
C7—C3—C4—C5
−54.5
(7)
O2—S1—C10—C7
64.5
C1—C3—C4—C9
174.8
(7)
O4—S1—C10—C7
173.7
C2—C3—C4—C9
−60.8
(8)
S1—O4—C11—C15
−103.6
C7—C3—C4—C9
56.1
S1—O4—C11—C12
141.3
O3—S1—O4—C11
−20.7
C15—C11—C12—C13
3.5
O2—S1—O4—C11
−152.2
O4—C11—C12—C13
121.5
C10—S1—O4—C11
94.8
(6)
C19—N1—C13—C14
71.8
C3—C4—C5—C6
33.6
(8)
C19—N1—C13—C16
−54.5
(10)
C9—C4—C5—C6
−74.5
C19—N1—C13—C12
−173.9
(7)
C4—C5—C6—O1
−179.1
C11—C12—C13—N1
−96.3
C4—C5—C6—C7
2.3
C11—C12—C13—C14
23.9
O1—C6—C7—C10
19.6
C11—C12—C13—C16
141.9
C5—C6—C7—C10
−161.8
N1—C13—C14—C15
75.4
O1—C6—C7—C3
145.0
(8)
C16—C13—C14—C15
−158.6
C5—C6—C7—C3
−36.4
(7)
C12—C13—C14—C15
−44.0
O1—C6—C7—C8
−108.5
O4—C11—C15—C14
−144.0
C5—C6—C7—C8
70.1
C12—C11—C15—C14
−30.0
C1—C3—C7—C6
−64.1
C13—C14—C15—C11
46.3
(9)
C4—C3—C7—C6
54.3
C17—O6—C16—O5
−0.9
(12)
(8)
(6)
(7)
(7)
(6)
(7)
(8)
(8)
(11)
(7)
(9)
(7)
(8)
(7)
199
(7)
(6)
(7)
(6)
(9)
(7)
(9)
(8)
(8)
(7)
(9)
(7)
(8)
(7)
(9)
Appendix B
C2—C3—C7—C6
172.8
C17—O6—C16—C13
−176.0
C1—C3—C7—C10
56.3
N1—C13—C16—O5
147.4
(8)
C4—C3—C7—C10
174.6
(6)
C14—C13—C16—O5
20.9
(11)
C2—C3—C7—C10
−66.9
(9)
C12—C13—C16—O5
−90.8
(9)
C1—C3—C7—C8
−171.0
N1—C13—C16—O6
−37.4
(9)
C4—C3—C7—C8
−52.6
C14—C13—C16—O6
−163.9
C2—C3—C7—C8
65.9
C12—C13—C16—O6
84.4
C6—C7—C8—C9
−73.9
(8)
C16—O6—C17—C18
−149.2
C10—C7—C8—C9
163.0
(7)
C13—N1—C19—O7
2.4
C3—C7—C8—C9
30.6
(9)
C13—N1—C19—O8
−178.2
C5—C4—C9—C8
69.5
(8)
C20—O8—C19—O7
5.7
C3—C4—C9—C8
−39.1
C20—O8—C19—N1
−173.8
C7—C8—C9—C4
4.6
C19—O8—C20—C22
−63.5
C6—C7—C10—S1
−129.6
C19—O8—C20—C21
60.4
C3—C7—C10—S1
115.5
C19—O8—C20—C23
177.8
(7)
(9)
(7)
(7)
(8)
(8)
(9)
(6)
(7)
(7)
(7)
(8)
(13)
(13)
(6)
(12)
(6)
(9)
(9)
(7)
Table 8. Hydrogen bonds for X81362M [Å and °].
______________________________________________________________________
D-H...A
d(D-H)
d(H...A)
d(D...A)
<(DHA)
______________________________________________________________________
N(1)-H(1)...O(3)#1
0.88
2.30
3.177(9)
174.7
______________________________________________________________________
Symmetry transformations used to generate equivalent atoms:
#1 -x+2,y-1/2,-z+2
200
Appendix B
201
Appendix B
202
Appendix B
Table 9. Crystal data and structure refinement for x81660.
Identification code
x81660
Empirical formula
C27 H40 Br N O7 S
Formula weight
602.57
Temperature
100(2) K
Wavelength
0.71073 Å
Crystal system
Monoclinic
Space group
P2(1)
Unit cell dimensions
a = 18.2720(13) Å
= 90°.
b = 11.2746(9) Å
= 96.295(4)°.
c = 28.461(2) Å
 = 90°.
Å3
Volume
5827.8(8)
Z
8
Density (calculated)
1.374 Mg/m3
Absorption coefficient
1.526 mm-1
F(000)
2528
Crystal size
0.44 x 0.08 x 0.06 mm3
Theta range for data collection
0.72 to 23.30°.
Index ranges
-20<=h<=20, -12<=k<=12, -31<=l<=31
Reflections collected
70621
Independent reflections
16636 [R(int) = 0.1192]
Completeness to theta = 23.30°
99.6%
Absorption correction
Semi-empirical from equivalents
Max. and min. transmission
0.913 and 0.708
Refinement method
Full-matrix least-squares on F2
Data / restraints / parameters
16636 / 79 / 1359
Goodness-of-fit on
F2
1.019
Final R indices [I>2sigma(I)]
R1 = 0.0531, wR2 = 0.1025
R indices (all data)
R1 = 0.1352, wR2 = 0.1428
Absolute structure parameter
-0.022(9)
Largest diff. peak and hole
0.445 and -0.661 e.Å-3
203
Appendix B
Table 10. Bond lengths [Å] and angles [°] for x81660.
Br(1A)-C(22A)
1.897(9)
S(1A)-O(6A)
1.419(6)
S(1A)-O(7A)
1.440(6)
S(1A)-O(5A)
1.578(6)
S(1A)-C(19A)
1.749(10)
N(1A)-C(14A)
1.356(11)
N(1A)-C(9A)
1.433(11)
N(1A)-H(1AA)
0.8800
O(1A)-C(8A)
1.344(11)
O(1A)-C(1A)
1.468(10)
O(2A)-C(8A)
1.208(11)
O(3A)-C(14A)
1.207(10)
O(4A)-C(14A)
1.363(11)
O(4A)-C(15A)
1.470(10)
O(5A)-C(11A)
1.497(10)
C(1A)-C(6A)
1.516(12)
C(1A)-C(2A)
1.522(12)
C(1A)-H(1A)
1.0000
C(2A)-C(3A)
1.521(12)
C(2A)-C(7A)
1.547(12)
C(2A)-H(2A)
1.0000
C(3A)-C(4A)
1.523(12)
C(3A)-H(3A1)
0.9900
C(3A)-H(3A2)
0.9900
C(4A)-C(5A)
1.547(12)
C(4A)-H(4A1)
0.9900
C(4A)-H(4A2)
0.9900
C(5A)-C(6A)
1.521(12)
C(5A)-C(51A)
1.536(12)
C(5A)-H(5A)
1.0000
C(6A)-H(6A1)
0.9900
C(6A)-H(6A2)
0.9900
C(7A)-C(71A)
1.490(12)
C(7A)-C(72A)
1.541(12)
C(7A)-H(7A)
1.0000
C(8A)-C(9A)
1.529(12)
C(9A)-C(13A)
1.529(12)
C(9A)-C(10A)
1.570(12)
C(10A)-C(11A)
1.527(12)
C(10A)-H(10A)
0.9900
C(10A)-H(10B)
0.9900
C(11A)-C(12A)
1.513(11)
C(11A)-H(11A)
1.0000
C(12A)-C(13A)
1.537(12)
C(12A)-H(12A)
0.9900
C(12A)-H(12B)
0.9900
C(13A)-H(13A)
0.9900
C(13A)-H(13B)
0.9900
C(15A)-C(18A)
1.518(12)
C(15A)-C(17A)
1.520(12)
C(15A)-C(16A)
1.531(13)
C(16A)-H(16A)
0.9800
C(16A)-H(16B)
0.9800
C(16A)-H(16C)
0.9800
C(17A)-H(17A)
0.9800
C(17A)-H(17B)
0.9800
C(17A)-H(17C)
0.9800
C(18A)-H(18A)
0.9800
C(18A)-H(18B)
0.9800
C(18A)-H(18C)
0.9800
C(19A)-C(24A)
1.374(12)
C(19A)-C(20A)
1.394(12)
C(20A)-C(21A)
1.371(12)
C(20A)-H(20A)
0.9500
C(21A)-C(22A)
1.383(13)
C(21A)-H(21A)
0.9500
C(22A)-C(23A)
1.367(14)
C(23A)-C(24A)
1.379(12)
C(23A)-H(23A)
0.9500
C(24A)-H(24A)
0.9500
C(51A)-H(51A)
0.9800
C(51A)-H(51B)
0.9800
C(51A)-H(51C)
0.9800
C(71A)-H(71A)
0.9800
C(71A)-H(71B)
0.9800
C(71A)-H(71C)
0.9800
C(72A)-H(72A)
0.9800
C(72A)-H(72B)
0.9800
C(72A)-H(72C)
0.9800
S(1B)-O(6B)
1.426(7)
S(1B)-O(7B)
1.433(6)
S(1B)-O(5B)
1.568(6)
S(1B)-C(19B)
1.738(11)
Br(1B)-C(22B)
1.893(10)
N(1B)-C(14B)
1.344(11)
N(1B)-C(9B)
1.465(11)
N(1B)-H(1BB)
0.8800
C(1B)-O(1B)
1.480(10)
C(1B)-C(2B)
1.533(12)
C(1B)-C(6B)
1.536(12)
C(1B)-H(1B)
1.0000
C(2B)-C(7B)
1.526(12)
C(2B)-C(3B)
1.556(12)
C(2B)-H(2B)
1.0000
C(3B)-C(4B)
1.537(13)
C(3B)-H(3B1)
0.9900
C(3B)-H(3B2)
0.9900
C(4B)-C(5B)
1.541(13)
C(4B)-H(4B1)
0.9900
C(4B)-H(4B2)
0.9900
C(5B)-C(51B)
1.534(13)
C(5B)-C(6B)
1.542(13)
C(5B)-H(5B)
1.0000
204
Appendix B
C(6B)-H(6B1)
0.9900
C(6B)-H(6B2)
0.9900
C(7B)-C(71B)
1.524(12)
C(7B)-C(72B)
1.533(12)
C(7B)-H(7B)
1.0000
C(8B)-O(2B)
1.229(10)
C(8B)-O(1B)
1.338(10)
C(8B)-C(9B)
1.523(12)
C(9B)-C(13B)
1.545(11)
C(9B)-C(10B)
1.568(12)
C(10B)-C(11B)
1.515(11)
C(10B)-H(10C)
0.9900
C(10B)-H(10D)
0.9900
C(11B)-O(5B)
1.505(10)
C(11B)-C(12B)
1.519(12)
C(11B)-H(11B)
1.0000
C(12B)-C(13B)
1.535(12)
C(12B)-H(12C)
0.9900
C(12B)-H(12D)
0.9900
C(13B)-H(13C)
0.9900
C(13B)-H(13D)
0.9900
C(14B)-O(3B)
1.237(11)
C(14B)-O(4B)
1.335(12)
C(15B)-O(4B)
1.500(10)
C(15B)-C(18B)
1.511(12)
C(15B)-C(16B)
1.526(12)
C(15B)-C(17B)
1.532(12)
C(16B)-H(16D)
0.9800
C(16B)-H(16E)
0.9800
C(16B)-H(16F)
0.9800
C(17B)-H(17D)
0.9800
C(17B)-H(17E)
0.9800
C(17B)-H(17F)
0.9800
C(18B)-H(18D)
0.9800
C(18B)-H(18E)
0.9800
C(18B)-H(18F)
0.9800
C(19B)-C(20B)
1.393(12)
C(19B)-C(24B)
1.415(13)
C(20B)-C(21B)
1.383(12)
C(20B)-H(20B)
0.9500
C(21B)-C(22B)
1.384(13)
C(21B)-H(21B)
0.9500
C(22B)-C(23B)
1.397(13)
C(23B)-C(24B)
1.401(13)
C(23B)-H(23B)
0.9500
C(24B)-H(24B)
0.9500
C(51B)-H(51D)
0.9800
C(51B)-H(51E)
0.9800
C(51B)-H(51F)
0.9800
C(71B)-H(71D)
0.9800
C(71B)-H(71E)
0.9800
C(71B)-H(71F)
0.9800
C(72B)-H(72D)
0.9800
C(72B)-H(72E)
0.9800
C(72B)-H(72F)
0.9800
S(1C)-O(7C)
1.426(7)
S(1C)-O(6C)
1.437(7)
S(1C)-O(5C)
1.569(7)
S(1C)-C(19C)
1.764(9)
Br(1C)-C(22C)
1.877(9)
N(1C)-C(14C)
1.340(11)
N(1C)-C(9C)
1.467(11)
N(1C)-H(1CC)
0.8800
C(1C)-O(1C)
1.475(10)
C(1C)-C(6C)
1.533(12)
C(1C)-C(2C)
1.533(12)
C(1C)-H(1C)
1.0000
C(2C)-C(7C)
1.524(12)
C(2C)-C(3C)
1.534(12)
C(2C)-H(2C)
1.0000
C(3C)-C(4C)
1.529(12)
C(3C)-H(3C1)
0.9900
C(3C)-H(3C2)
0.9900
C(4C)-C(5C)
1.537(12)
C(4C)-H(4C1)
0.9900
C(4C)-H(4C2)
0.9900
C(5C)-C(6C)
1.521(12)
C(5C)-C(51C)
1.541(12)
C(5C)-H(5C)
1.0000
C(6C)-H(6C1)
0.9900
C(6C)-H(6C2)
0.9900
C(7C)-C(71C)
1.538(12)
C(7C)-C(72C)
1.540(12)
C(7C)-H(7C)
1.0000
C(8C)-O(2C)
1.219(10)
C(8C)-O(1C)
1.350(11)
C(8C)-C(9C)
1.540(13)
C(9C)-C(10C)
1.545(11)
C(9C)-C(13C)
1.570(12)
C(10C)-C(11C)
1.507(12)
C(10C)-H(10E)
0.9900
C(10C)-H(10F)
0.9900
C(11C)-O(5C)
1.521(10)
C(11C)-C(12C)
1.526(12)
C(11C)-H(11C)
1.0000
C(12C)-C(13C)
1.531(12)
C(12C)-H(12E)
0.9900
C(12C)-H(12F)
0.9900
C(13C)-H(13E)
0.9900
C(13C)-H(13F)
0.9900
C(14C)-O(3C)
1.234(10)
C(14C)-O(4C)
1.349(11)
C(15C)-O(4C)
1.485(10)
C(15C)-C(16C)
1.509(12)
C(15C)-C(17C)
1.519(12)
C(15C)-C(18C)
1.530(12)
C(16C)-H(16G)
0.9800
C(16C)-H(16H)
0.9800
C(16C)-H(16I)
0.9800
205
Appendix B
C(17C)-H(17G)
0.9800
C(17C)-H(17H)
0.9800
C(17C)-H(17I)
0.9800
C(18C)-H(18G)
0.9800
C(18C)-H(18H)
0.9800
C(18C)-H(18I)
0.9800
C(19C)-C(20C)
1.371(13)
C(19C)-C(24C)
1.389(13)
C(20C)-C(21C)
1.380(13)
C(20C)-H(20C)
0.9500
C(21C)-C(22C)
1.419(13)
C(21C)-H(21C)
0.9500
C(22C)-C(23C)
1.383(13)
C(23C)-C(24C)
1.394(13)
C(23C)-H(23C)
0.9500
C(24C)-H(24C)
0.9500
C(51C)-H(51G)
0.9800
C(51C)-H(51H)
0.9800
C(51C)-H(51I)
0.9800
C(71C)-H(71G)
0.9800
C(71C)-H(71H)
0.9800
C(71C)-H(71I)
0.9800
C(72C)-H(72G)
0.9800
C(72C)-H(72H)
0.9800
C(72C)-H(72I)
0.9800
S(1D)-O(6D)
1.422(6)
S(1D)-O(7D)
1.443(6)
S(1D)-O(5D)
1.568(6)
S(1D)-C(19D)
1.751(8)
Br(1D)-C(22D)
1.892(9)
C(1D)-O(1D)
1.470(10)
C(1D)-C(6D)
1.500(12)
C(1D)-C(2D)
1.527(12)
C(1D)-H(1D)
1.0000
C(2D)-C(7D)
1.535(12)
C(2D)-C(3D)
1.548(12)
C(2D)-H(2D)
1.0000
C(3D)-C(4D)
1.526(12)
C(3D)-H(3D1)
0.9900
C(3D)-H(3D2)
0.9900
C(4D)-C(5D)
1.521(12)
C(4D)-H(4D1)
0.9900
C(4D)-H(4D2)
0.9900
C(5D)-C(51D)
1.510(12)
C(5D)-C(6D)
1.529(12)
C(5D)-H(5D)
1.0000
C(6D)-H(6D1)
0.9900
C(6D)-H(6D2)
0.9900
C(7D)-C(72D)
1.534(13)
C(7D)-C(71D)
1.546(12)
C(7D)-H(7D)
1.0000
C(8D)-O(2D)
1.223(10)
C(8D)-O(1D)
1.327(10)
C(8D)-C(9D)
1.518(13)
C(9D)-N(1D)
1.462(11)
C(9D)-C(13D)
1.548(12)
C(9D)-C(10D)
1.551(12)
C(10D)-C(11D)
1.546(12)
C(10D)-H(10G)
0.9900
C(10D)-H(10H)
0.9900
C(11D)-O(5D)
1.496(10)
C(11D)-C(12D)
1.506(12)
C(11D)-H(11D)
1.0000
C(12D)-C(13D)
1.540(12)
C(12D)-H(12G)
0.9900
C(12D)-H(12H)
0.9900
C(13D)-H(13G)
0.9900
C(13D)-H(13H)
0.9900
C(14D)-O(3D)
1.197(11)
C(14D)-O(4D)
1.343(11)
C(14D)-N(1D)
1.364(11)
C(15D)-O(4D)
1.496(10)
C(15D)-C(18D)
1.498(12)
C(15D)-C(16D)
1.518(12)
C(15D)-C(17D)
1.527(12)
C(16D)-H(16J)
0.9800
C(16D)-H(16K)
0.9800
C(16D)-H(16L)
0.9800
C(17D)-H(17J)
0.9800
C(17D)-H(17K)
0.9800
C(17D)-H(17L)
0.9800
C(18D)-H(18J)
0.9800
C(18D)-H(18K)
0.9800
C(18D)-H(18L)
0.9800
C(19D)-C(24D)
1.374(12)
C(19D)-C(20D)
1.393(13)
C(20D)-C(21D)
1.383(13)
C(20D)-H(20D)
0.9500
C(21D)-C(22D)
1.372(15)
C(21D)-H(21D)
0.9500
C(22D)-C(23D)
1.374(14)
C(23D)-C(24D)
1.383(13)
C(23D)-H(23D)
0.9500
C(24D)-H(24D)
0.9500
C(51D)-H(51J)
0.9800
C(51D)-H(51K)
0.9800
C(51D)-H(51L)
0.9800
C(71D)-H(71J)
0.9800
C(71D)-H(71K)
0.9800
C(71D)-H(71L)
0.9800
C(72D)-H(72J)
0.9800
C(72D)-H(72K)
0.9800
C(72D)-H(72L)
0.9800
N(1D)-H(1DD)
0.8800
206
Appendix B
O(6A)-S(1A)-O(7A)
120.9(4)
C(4A)-C(3A)-H(3A1)
109.3
C(72A)-C(7A)-H(7A)
106.8
O(6A)-S(1A)-O(5A)
109.8(4)
C(2A)-C(3A)-H(3A2)
109.3
C(2A)-C(7A)-H(7A)
106.8
O(7A)-S(1A)-O(5A)
105.3(4)
C(4A)-C(3A)-H(3A2)
109.3
O(2A)-C(8A)-O(1A)
124.4(9)
O(6A)-S(1A)-C(19A)
108.0(4)
H(3A1)-C(3A)-H(3A2)
107.9
O(2A)-C(8A)-C(9A)
123.3(9)
O(7A)-S(1A)-C(19A)
109.0(4)
C(3A)-C(4A)-C(5A)
112.2(7)
O(1A)-C(8A)-C(9A)
112.3(8)
O(5A)-S(1A)-C(19A)
102.3(4)
C(3A)-C(4A)-H(4A1)
109.2
N(1A)-C(9A)-C(13A)
111.7(7)
C(14A)-N(1A)-C(9A)
119.2(8)
C(5A)-C(4A)-H(4A1)
109.2
N(1A)-C(9A)-C(8A)
111.8(7)
C(14A)-N(1A)-H(1AA)
120.4
C(3A)-C(4A)-H(4A2)
109.2
C(13A)-C(9A)-C(8A)
106.2(7)
C(9A)-N(1A)-H(1AA)
120.4
C(5A)-C(4A)-H(4A2)
109.2
N(1A)-C(9A)-C(10A)
113.1(7)
C(8A)-O(1A)-C(1A)
117.0(7)
H(4A1)-C(4A)-H(4A2)
107.9
C(13A)-C(9A)-C(10A)
103.7(6)
C(14A)-O(4A)-C(15A)
119.6(7)
C(6A)-C(5A)-C(51A)
110.8(8)
C(8A)-C(9A)-C(10A)
109.9(7)
C(11A)-O(5A)-S(1A)
117.9(5)
C(6A)-C(5A)-C(4A)
109.0(8)
C(11A)-C(10A)-C(9A)
105.6(7)
O(1A)-C(1A)-C(6A)
108.0(7)
C(51A)-C(5A)-C(4A)
112.5(7)
C(11A)-C(10A)-H(10A)
110.6
O(1A)-C(1A)-C(2A)
107.3(7)
C(6A)-C(5A)-H(5A)
108.2
C(9A)-C(10A)-H(10A)
110.6
C(6A)-C(1A)-C(2A)
113.5(7)
C(51A)-C(5A)-H(5A)
108.2
C(11A)-C(10A)-H(10B)
110.6
O(1A)-C(1A)-H(1A)
109.4
C(4A)-C(5A)-H(5A)
108.2
C(9A)-C(10A)-H(10B)
110.6
C(6A)-C(1A)-H(1A)
109.4
C(1A)-C(6A)-C(5A)
110.7(8)
H(10A)-C(10A)-H(10B)
108.8
C(2A)-C(1A)-H(1A)
109.4
C(1A)-C(6A)-H(6A1)
109.5
O(5A)-C(11A)-C(12A)
107.5(7)
C(3A)-C(2A)-C(1A)
106.3(7)
C(5A)-C(6A)-H(6A1)
109.5
O(5A)-C(11A)-C(10A)
105.9(7)
C(3A)-C(2A)-C(7A)
115.3(7)
C(1A)-C(6A)-H(6A2)
109.5
C(12A)-C(11A)-C(10A)
108.1(7)
C(1A)-C(2A)-C(7A)
113.1(7)
C(5A)-C(6A)-H(6A2)
109.5
O(5A)-C(11A)-H(11A)
111.7
C(3A)-C(2A)-H(2A)
107.2
H(6A1)-C(6A)-H(6A2)
108.1
C(12A)-C(11A)-H(11A)
111.7
C(1A)-C(2A)-H(2A)
107.2
C(71A)-C(7A)-C(72A)
109.0(8)
C(10A)-C(11A)-H(11A)
111.7
C(7A)-C(2A)-H(2A)
107.2
C(71A)-C(7A)-C(2A)
114.1(7)
C(11A)-C(12A)-C(13A)
103.8(7)
C(2A)-C(3A)-C(4A)
111.8(8)
C(72A)-C(7A)-C(2A)
112.9(7)
C(11A)-C(12A)-H(12A)
111.0
C(2A)-C(3A)-H(3A1)
109.3
C(71A)-C(7A)-H(7A)
106.8
C(13A)-C(12A)-H(12A)
111.0
C(11A)-C(12A)-H(12B)
111.0
H(17A)-C(17A)-H(17B)
109.5
C(19A)-C(24A)-H(24A)
120.3
C(13A)-C(12A)-H(12B)
111.0
C(15A)-C(17A)-H(17C)
109.5
C(23A)-C(24A)-H(24A)
120.3
H(12A)-C(12A)-H(12B)
109.0
H(17A)-C(17A)-H(17C)
109.5
C(5A)-C(51A)-H(51A)
109.5
C(9A)-C(13A)-C(12A)
104.2(7)
H(17B)-C(17A)-H(17C)
109.5
C(5A)-C(51A)-H(51B)
109.5
C(9A)-C(13A)-H(13A)
110.9
C(15A)-C(18A)-H(18A)
109.5
H(51A)-C(51A)-H(51B)
109.5
C(12A)-C(13A)-H(13A)
110.9
C(15A)-C(18A)-H(18B)
109.5
C(5A)-C(51A)-H(51C)
109.5
C(9A)-C(13A)-H(13B)
110.9
H(18A)-C(18A)-H(18B)
109.5
H(51A)-C(51A)-H(51C)
109.5
C(12A)-C(13A)-H(13B)
110.9
C(15A)-C(18A)-H(18C)
109.5
H(51B)-C(51A)-H(51C)
109.5
H(13A)-C(13A)-H(13B)
108.9
H(18A)-C(18A)-H(18C)
109.5
C(7A)-C(71A)-H(71A)
109.5
O(3A)-C(14A)-N(1A)
123.8(9)
H(18B)-C(18A)-H(18C)
109.5
C(7A)-C(71A)-H(71B)
109.5
O(3A)-C(14A)-O(4A)
126.4(9)
C(24A)-C(19A)-C(20A)
119.6(9)
H(71A)-C(71A)-H(71B)
109.5
207
Appendix B
N(1A)-C(14A)-O(4A)
109.7(8)
C(24A)-C(19A)-S(1A)
120.8(7)
C(7A)-C(71A)-H(71C)
109.5
O(4A)-C(15A)-C(18A)
102.5(7)
C(20A)-C(19A)-S(1A)
119.5(7)
H(71A)-C(71A)-H(71C)
109.5
O(4A)-C(15A)-C(17A)
110.9(7)
C(21A)-C(20A)-C(19A)
120.6(9)
H(71B)-C(71A)-H(71C)
109.5
C(18A)-C(15A)-C(17A)
110.9(8)
C(21A)-C(20A)-H(20A)
119.7
C(7A)-C(72A)-H(72A)
109.5
O(4A)-C(15A)-C(16A)
110.6(7)
C(19A)-C(20A)-H(20A)
119.7
C(7A)-C(72A)-H(72B)
109.5
C(18A)-C(15A)-C(16A)
109.3(8)
C(20A)-C(21A)-C(22A)
119.3(9)
H(72A)-C(72A)-H(72B)
109.5
C(17A)-C(15A)-C(16A)
112.1(8)
C(20A)-C(21A)-H(21A)
120.4
C(7A)-C(72A)-H(72C)
109.5
C(15A)-C(16A)-H(16A)
109.5
C(22A)-C(21A)-H(21A)
120.4
H(72A)-C(72A)-H(72C)
109.5
C(15A)-C(16A)-H(16B)
109.5
C(23A)-C(22A)-C(21A)
120.1(9)
H(72B)-C(72A)-H(72C)
109.5
H(16A)-C(16A)-H(16B)
109.5
C(23A)-C(22A)-Br(1A)
119.5(7)
O(6B)-S(1B)-O(7B)
120.5(4)
C(15A)-C(16A)-H(16C)
109.5
C(21A)-C(22A)-Br(1A)
120.3(8)
O(6B)-S(1B)-O(5B)
110.4(4)
H(16A)-C(16A)-H(16C)
109.5
C(22A)-C(23A)-C(24A)
120.9(9)
O(7B)-S(1B)-O(5B)
103.8(4)
H(16B)-C(16A)-H(16C)
109.5
C(22A)-C(23A)-H(23A)
119.5
O(6B)-S(1B)-C(19B)
107.8(4)
C(15A)-C(17A)-H(17A)
109.5
C(24A)-C(23A)-H(23A)
119.5
O(7B)-S(1B)-C(19B)
109.0(4)
C(15A)-C(17A)-H(17B)
109.5
C(19A)-C(24A)-C(23A)
119.4(9)
O(5B)-S(1B)-C(19B)
104.1(4)
C(14B)-N(1B)-C(9B)
119.3(8)
C(6B)-C(1B)-H(1B)
109.0
C(4B)-C(3B)-H(3B1)
109.1
C(14B)-N(1B)-H(1BB)
120.4
C(7B)-C(2B)-C(1B)
113.8(7)
C(2B)-C(3B)-H(3B1)
109.1
C(9B)-N(1B)-H(1BB)
120.4
C(7B)-C(2B)-C(3B)
112.7(7)
C(4B)-C(3B)-H(3B2)
109.1
O(1B)-C(1B)-C(2B)
108.3(7)
C(1B)-C(2B)-C(3B)
106.9(7)
C(2B)-C(3B)-H(3B2)
109.1
O(1B)-C(1B)-C(6B)
108.6(7)
C(7B)-C(2B)-H(2B)
107.8
H(3B1)-C(3B)-H(3B2)
107.8
C(2B)-C(1B)-C(6B)
112.8(7)
C(1B)-C(2B)-H(2B)
107.8
C(3B)-C(4B)-C(5B)
112.7(8)
O(1B)-C(1B)-H(1B)
109.0
C(3B)-C(2B)-H(2B)
107.8
C(3B)-C(4B)-H(4B1)
109.0
C(2B)-C(1B)-H(1B)
109.0
C(4B)-C(3B)-C(2B)
112.4(7)
C(5B)-C(4B)-H(4B1)
109.0
C(3B)-C(4B)-H(4B2)
109.0
C(9B)-C(10B)-H(10D)
110.6
C(15B)-C(16B)-H(16F)
109.5
C(5B)-C(4B)-H(4B2)
109.0
H(10C)-C(10B)-H(10D)
108.7
H(16D)-C(16B)-H(16F)
109.5
H(4B1)-C(4B)-H(4B2)
107.8
O(5B)-C(11B)-C(10B)
109.5(7)
H(16E)-C(16B)-H(16F)
109.5
C(51B)-C(5B)-C(4B)
113.0(8)
O(5B)-C(11B)-C(12B)
106.0(7)
C(15B)-C(17B)-H(17D)
109.5
C(51B)-C(5B)-C(6B)
110.6(8)
C(10B)-C(11B)-C(12B)
105.4(7)
C(15B)-C(17B)-H(17E)
109.5
C(4B)-C(5B)-C(6B)
107.3(8)
O(5B)-C(11B)-H(11B)
111.9
H(17D)-C(17B)-H(17E)
109.5
C(51B)-C(5B)-H(5B)
108.6
C(10B)-C(11B)-H(11B)
111.9
C(15B)-C(17B)-H(17F)
109.5
C(4B)-C(5B)-H(5B)
108.6
C(12B)-C(11B)-H(11B)
111.9
H(17D)-C(17B)-H(17F)
109.5
C(6B)-C(5B)-H(5B)
108.6
C(11B)-C(12B)-C(13B)
103.3(7)
H(17E)-C(17B)-H(17F)
109.5
C(1B)-C(6B)-C(5B)
109.6(8)
C(11B)-C(12B)-H(12C)
111.1
C(15B)-C(18B)-H(18D)
109.5
C(1B)-C(6B)-H(6B1)
109.8
C(13B)-C(12B)-H(12C)
111.1
C(15B)-C(18B)-H(18E)
109.5
C(5B)-C(6B)-H(6B1)
109.8
C(11B)-C(12B)-H(12D)
111.1
H(18D)-C(18B)-H(18E)
109.5
C(1B)-C(6B)-H(6B2)
109.8
C(13B)-C(12B)-H(12D)
111.1
C(15B)-C(18B)-H(18F)
109.5
208
Appendix B
C(5B)-C(6B)-H(6B2)
109.8
H(12C)-C(12B)-H(12D)
109.1
H(18D)-C(18B)-H(18F)
109.5
H(6B1)-C(6B)-H(6B2)
108.2
C(12B)-C(13B)-C(9B)
104.9(7)
H(18E)-C(18B)-H(18F)
109.5
C(71B)-C(7B)-C(2B)
113.4(7)
C(12B)-C(13B)-H(13C)
110.8
C(20B)-C(19B)-C(24B)
120.8(9)
C(71B)-C(7B)-C(72B)
109.9(8)
C(9B)-C(13B)-H(13C)
110.8
C(20B)-C(19B)-S(1B)
122.1(7)
C(2B)-C(7B)-C(72B)
113.5(8)
C(12B)-C(13B)-H(13D)
110.8
C(24B)-C(19B)-S(1B)
117.1(7)
C(71B)-C(7B)-H(7B)
106.5
C(9B)-C(13B)-H(13D)
110.8
C(21B)-C(20B)-C(19B)
120.5(9)
C(2B)-C(7B)-H(7B)
106.5
H(13C)-C(13B)-H(13D)
108.8
C(21B)-C(20B)-H(20B)
119.8
C(72B)-C(7B)-H(7B)
106.5
O(3B)-C(14B)-O(4B)
126.2(9)
C(19B)-C(20B)-H(20B)
119.8
O(2B)-C(8B)-O(1B)
122.5(8)
O(3B)-C(14B)-N(1B)
122.4(10)
C(20B)-C(21B)-C(22B)
118.9(9)
O(2B)-C(8B)-C(9B)
123.5(8)
O(4B)-C(14B)-N(1B)
111.4(9)
C(20B)-C(21B)-H(21B)
120.5
O(1B)-C(8B)-C(9B)
113.8(8)
O(4B)-C(15B)-C(18B)
101.4(7)
C(22B)-C(21B)-H(21B)
120.5
N(1B)-C(9B)-C(8B)
110.4(7)
O(4B)-C(15B)-C(16B)
111.1(7)
C(21B)-C(22B)-C(23B)
122.0(9)
N(1B)-C(9B)-C(13B)
109.2(7)
C(18B)-C(15B)-C(16B)
110.1(8)
C(21B)-C(22B)-Br(1B)
120.2(7)
C(8B)-C(9B)-C(13B)
108.5(7)
O(4B)-C(15B)-C(17B)
109.0(7)
C(23B)-C(22B)-Br(1B)
117.7(7)
N(1B)-C(9B)-C(10B)
113.4(7)
C(18B)-C(15B)-C(17B)
112.2(8)
C(22B)-C(23B)-C(24B)
119.4(10
)
C(8B)-C(9B)-C(10B)
109.6(7)
C(16B)-C(15B)-C(17B)
112.5(8)
C(22B)-C(23B)-H(23B)
120.3
C(13B)-C(9B)-C(10B)
105.6(7)
C(15B)-C(16B)-H(16D)
109.5
C(24B)-C(23B)-H(23B)
120.3
C(11B)-C(10B)-C(9B)
105.6(7)
C(15B)-C(16B)-H(16E)
109.5
C(23B)-C(24B)-C(19B)
118.3(9)
C(11B)-C(10B)-H(10C)
110.6
H(16D)-C(16B)-H(16E)
109.5
C(23B)-C(24B)-H(24B)
120.8
C(9B)-C(10B)-H(10C)
110.6
C(15B)-C(16B)-H(16F)
109.5
C(19B)-C(24B)-H(24B)
120.8
C(11B)-C(10B)-H(10D)
110.6
H(16D)-C(16B)-H(16F)
109.5
C(5B)-C(51B)-H(51D)
109.5
C(5B)-C(51B)-H(51E)
109.5
C(1C)-C(2C)-C(3C)
107.9(7)
O(2C)-C(8C)-O(1C)
124.3(9)
H(51D)-C(51B)-H(51E)
109.5
C(7C)-C(2C)-H(2C)
106.8
O(2C)-C(8C)-C(9C)
123.9(9)
C(5B)-C(51B)-H(51F)
109.5
C(1C)-C(2C)-H(2C)
106.8
O(1C)-C(8C)-C(9C)
111.5(8)
H(51D)-C(51B)-H(51F)
109.5
C(3C)-C(2C)-H(2C)
106.8
N(1C)-C(9C)-C(8C)
109.4(7)
H(51E)-C(51B)-H(51F)
109.5
C(4C)-C(3C)-C(2C)
112.5(8)
N(1C)-C(9C)-C(10C)
112.3(7)
C(7B)-C(71B)-H(71D)
109.5
C(4C)-C(3C)-H(3C1)
109.1
C(8C)-C(9C)-C(10C)
111.5(7)
C(7B)-C(71B)-H(71E)
109.5
C(2C)-C(3C)-H(3C1)
109.1
N(1C)-C(9C)-C(13C)
110.0(7)
H(71D)-C(71B)-H(71E)
109.5
C(4C)-C(3C)-H(3C2)
109.1
C(8C)-C(9C)-C(13C)
108.8(7)
C(7B)-C(71B)-H(71F)
109.5
C(2C)-C(3C)-H(3C2)
109.1
C(10C)-C(9C)-C(13C)
104.6(7)
H(71D)-C(71B)-H(71F)
109.5
H(3C1)-C(3C)-H(3C2)
107.8
C(11C)-C(10C)-C(9C)
106.9(7)
H(71E)-C(71B)-H(71F)
109.5
C(3C)-C(4C)-C(5C)
111.8(8)
C(11C)-C(10C)-H(10E)
110.3
C(7B)-C(72B)-H(72D)
109.5
C(3C)-C(4C)-H(4C1)
109.3
C(9C)-C(10C)-H(10E)
110.3
C(7B)-C(72B)-H(72E)
109.5
C(5C)-C(4C)-H(4C1)
109.3
C(11C)-C(10C)-H(10F)
110.3
H(72D)-C(72B)-H(72E)
109.5
C(3C)-C(4C)-H(4C2)
109.3
C(9C)-C(10C)-H(10F)
110.3
209
Appendix B
C(7B)-C(72B)-H(72F)
109.5
C(5C)-C(4C)-H(4C2)
109.3
H(10E)-C(10C)-H(10F)
108.6
H(72D)-C(72B)-H(72F)
109.5
H(4C1)-C(4C)-H(4C2)
107.9
C(10C)-C(11C)-O(5C)
107.9(7)
H(72E)-C(72B)-H(72F)
109.5
C(6C)-C(5C)-C(4C)
108.6(7)
C(10C)-C(11C)-C(12C)
104.0(7)
O(7C)-S(1C)-O(6C)
120.5(4)
C(6C)-C(5C)-C(51C)
110.6(8)
O(5C)-C(11C)-C(12C)
104.0(7)
O(7C)-S(1C)-O(5C)
104.2(4)
C(4C)-C(5C)-C(51C)
111.0(8)
C(10C)-C(11C)-H(11C)
113.4
O(6C)-S(1C)-O(5C)
109.9(4)
C(6C)-C(5C)-H(5C)
108.9
O(5C)-C(11C)-H(11C)
113.4
O(7C)-S(1C)-C(19C)
109.5(4)
C(4C)-C(5C)-H(5C)
108.9
C(12C)-C(11C)-H(11C)
113.4
O(6C)-S(1C)-C(19C)
107.1(4)
C(51C)-C(5C)-H(5C)
108.9
C(11C)-C(12C)-C(13C)
103.8(7)
O(5C)-S(1C)-C(19C)
104.5(4)
C(5C)-C(6C)-C(1C)
113.5(7)
C(11C)-C(12C)-H(12E)
111.0
C(14C)-N(1C)-C(9C)
119.8(8)
C(5C)-C(6C)-H(6C1)
108.9
C(13C)-C(12C)-H(12E)
111.0
C(14C)-N(1C)-H(1CC)
120.1
C(1C)-C(6C)-H(6C1)
108.9
C(11C)-C(12C)-H(12F)
111.0
(9C)-N(1C)-H(1CC)
120.1
C(5C)-C(6C)-H(6C2)
108.9
C(13C)-C(12C)-H(12F)
111.0
O(1C)-C(1C)-C(6C)
104.9(7)
C(1C)-C(6C)-H(6C2)
108.9
H(12E)-C(12C)-H(12F)
109.0
O(1C)-C(1C)-C(2C)
109.2(7)
H(6C1)-C(6C)-H(6C2)
107.7
C(12C)-C(13C)-C(9C)
105.8(7)
C(6C)-C(1C)-C(2C)
113.4(7)
C(2C)-C(7C)-C(71C)
109.8(8)
C(12C)-C(13C)-H(13E)
110.6
O(1C)-C(1C)-H(1C)
109.7
C(2C)-C(7C)-C(72C)
114.3(8)
C(9C)-C(13C)-H(13E)
110.6
C(6C)-C(1C)-H(1C)
109.7
C(71C)-C(7C)-C(72C)
108.7(8)
C(12C)-C(13C)-H(13F)
110.6
C(2C)-C(1C)-H(1C)
109.7
C(2C)-C(7C)-H(7C)
107.9
C(9C)-C(13C)-H(13F)
110.6
C(7C)-C(2C)-C(1C)
112.6(7)
C(71C)-C(7C)-H(7C)
107.9
H(13E)-C(13C)-H(13F)
108.7
C(7C)-C(2C)-C(3C)
115.4(7)
C(72C)-C(7C)-H(7C)
107.9
O(3C)-C(14C)-N(1C)
122.8(9)
O(3C)-C(14C)-O(4C)
125.7(9)
C(22C)-C(21C)-H(21C)
121.3
O(1D)-C(1D)-C(6D)
106.2(7)
N(1C)-C(14C)-O(4C)
111.5(8)
C(23C)-C(22C)-C(21C)
121.9(8)
O(1D)-C(1D)-C(2D)
107.9(7)
O(4C)-C(15C)-C(16C)
111.3(7)
C(23C)-C(22C)-Br(1C)
119.2(7)
C(6D)-C(1D)-C(2D)
114.1(7)
O(4C)-C(15C)-C(17C)
101.7(7)
C(21C)-C(22C)-Br(1C)
118.9(7)
O(1D)-C(1D)-H(1D)
109.5
C(16C)-C(15C)-C(17C)
109.9(8)
C(22C)-C(23C)-C(24C)
118.6(9)
C(6D)-C(1D)-H(1D)
109.5
O(4C)-C(15C)-C(18C)
109.0(7)
C(22C)-C(23C)-H(23C)
120.7
C(2D)-C(1D)-H(1D)
109.5
C(16C)-C(15C)-C(18C)
114.3(8)
C(24C)-C(23C)-H(23C)
120.7
C(1D)-C(2D)-C(7D)
113.6(7)
C(17C)-C(15C)-C(18C)
109.9(7)
C(19C)-C(24C)-C(23C)
119.8(10)
C(1D)-C(2D)-C(3D)
107.6(7)
C(15C)-C(16C)-H(16G)
109.5
C(19C)-C(24C)-H(24C)
120.1
C(7D)-C(2D)-C(3D)
114.4(8)
C(15C)-C(16C)-H(16H)
109.5
C(23C)-C(24C)-H(24C)
120.1
C(1D)-C(2D)-H(2D)
107.0
H(16G)-C(16C)-H(16H)
109.5
C(5C)-C(51C)-H(51G)
109.5
C(7D)-C(2D)-H(2D)
107.0
C(15C)-C(16C)-H(16I)
109.5
C(5C)-C(51C)-H(51H)
109.5
C(3D)-C(2D)-H(2D)
107.0
H(16G)-C(16C)-H(16I)
109.5
H(51G)-C(51C)-H(51H)
109.5
C(4D)-C(3D)-C(2D)
112.4(7)
H(16H)-C(16C)-H(16I)
109.5
C(5C)-C(51C)-H(51I)
109.5
C(4D)-C(3D)-H(3D1)
109.1
C(15C)-C(17C)-H(17G)
109.5
H(51G)-C(51C)-H(51I)
109.5
C(2D)-C(3D)-H(3D1)
109.1
C(15C)-C(17C)-H(17H)
109.5
H(51H)-C(51C)-H(51I)
109.5
C(4D)-C(3D)-H(3D2)
109.1
210
Appendix B
H(17G)-C(17C)-H(17H)
109.5
C(7C)-C(71C)-H(71G)
109.5
C(2D)-C(3D)-H(3D2)
109.1
C(15C)-C(17C)-H(17I)
109.5
C(7C)-C(71C)-H(71H)
109.5
H(3D1)-C(3D)-H(3D2)
107.9
H(17G)-C(17C)-H(17I)
109.5
H(71G)-C(71C)-H(71H)
109.5
C(5D)-C(4D)-C(3D)
114.3(8)
H(17H)-C(17C)-H(17I)
109.5
C(7C)-C(71C)-H(71I)
109.5
C(5D)-C(4D)-H(4D1)
108.7
C(15C)-C(18C)-H(18G)
109.5
H(71G)-C(71C)-H(71I)
109.5
C(3D)-C(4D)-H(4D1)
108.7
C(15C)-C(18C)-H(18H)
109.5
H(71H)-C(71C)-H(71I)
109.5
C(5D)-C(4D)-H(4D2)
108.7
H(18G)-C(18C)-H(18H)
109.5
C(7C)-C(72C)-H(72G)
109.5
C(3D)-C(4D)-H(4D2)
108.7
C(15C)-C(18C)-H(18I)
109.5
C(7C)-C(72C)-H(72H)
109.5
H(4D1)-C(4D)-H(4D2)
107.6
H(18G)-C(18C)-H(18I)
109.5
H(72G)-C(72C)-H(72H)
109.5
C(51D)-C(5D)-C(4D)
114.2(8)
H(18H)-C(18C)-H(18I)
109.5
C(7C)-C(72C)-H(72I)
109.5
C(51D)-C(5D)-C(6D)
112.3(8)
C(20C)-C(19C)-C(24C)
120.9(9)
H(72G)-C(72C)-H(72I)
109.5
C(4D)-C(5D)-C(6D)
107.2(8)
C(20C)-C(19C)-S(1C)
121.3(8)
H(72H)-C(72C)-H(72I)
109.5
C(51D)-C(5D)-H(5D)
107.6
C(24C)-C(19C)-S(1C)
117.8(8)
O(6D)-S(1D)-O(7D)
119.6(4)
C(4D)-C(5D)-H(5D)
107.6
C(19C)-C(20C)-C(21C)
121.4(9)
O(6D)-S(1D)-O(5D)
109.3(4)
C(6D)-C(5D)-H(5D)
107.6
C(19C)-C(20C)-H(20C)
119.3
O(7D)-S(1D)-O(5D)
105.8(4)
C(1D)-C(6D)-C(5D)
113.9(8)
C(21C)-C(20C)-H(20C)
119.3
O(6D)-S(1D)-C(19D)
109.5(4)
C(1D)-C(6D)-H(6D1)
108.8
C(20C)-C(21C)-C(22C)
117.4(9)
O(7D)-S(1D)-C(19D)
108.1(4)
C(5D)-C(6D)-H(6D1)
108.8
C(20C)-C(21C)-H(21C)
121.3
O(5D)-S(1D)-C(19D)
103.2(4)
C(1D)-C(6D)-H(6D2)
108.8
C(5D)-C(6D)-H(6D2)
108.8
H(12G)-C(12D)-H(12H)
109.2
C(24D)-C(19D)-C(20D)
121.5(8)
H(6D1)-C(6D)-H(6D2)
107.7
C(12D)-C(13D)-C(9D)
103.9(7)
C(24D)-C(19D)-S(1D)
118.5(7)
C(72D)-C(7D)-C(2D)
114.9(8)
C(12D)-C(13D)-H(13G)
111.0
C(20D)-C(19D)-S(1D)
119.9(7)
C(72D)-C(7D)-C(71D)
108.2(8)
C(9D)-C(13D)-H(13G)
111.0
C(21D)-C(20D)-C(19D)
118.4(9)
C(2D)-C(7D)-C(71D)
111.9(8)
C(12D)-C(13D)-H(13H)
111.0
C(21D)-C(20D)-H(20D)
120.8
C(72D)-C(7D)-H(7D)
107.2
C(9D)-C(13D)-H(13H)
111.0
C(19D)-C(20D)-H(20D)
120.8
C(2D)-C(7D)-H(7D)
107.2
H(13G)-C(13D)-H(13H)
109.0
C(22D)-C(21D)-C(20D)
120.4(10)
C(71D)-C(7D)-H(7D)
107.2
O(3D)-C(14D)-O(4D)
128.0(9)
C(22D)-C(21D)-H(21D)
119.8
O(2D)-C(8D)-O(1D)
123.4(8)
O(3D)-C(14D)-N(1D)
122.4(9)
C(20D)-C(21D)-H(21D)
119.8
O(2D)-C(8D)-C(9D)
122.8(8)
O(4D)-C(14D)-N(1D)
109.6(9)
C(21D)-C(22D)-C(23D)
120.4(9)
O(1D)-C(8D)-C(9D)
113.4(8)
O(4D)-C(15D)-C(18D)
109.5(7)
C(21D)-C(22D)-Br(1D)
119.2(9)
N(1D)-C(9D)-C(8D)
111.7(7)
O(4D)-C(15D)-C(16D)
107.9(7)
C(23D)-C(22D)-Br(1D)
120.4(8)
N(1D)-C(9D)-C(13D)
109.7(7)
C(18D)-C(15D)-C(16D)
113.2(8)
C(22D)-C(23D)-C(24D)
120.3(10)
C(8D)-C(9D)-C(13D)
106.4(7)
O(4D)-C(15D)-C(17D)
101.9(7)
C(22D)-C(23D)-H(23D)
119.8
N(1D)-C(9D)-C(10D)
112.4(7)
C(18D)-C(15D)-C(17D)
111.6(8)
C(24D)-C(23D)-H(23D)
119.8
C(8D)-C(9D)-C(10D)
110.4(8)
C(16D)-C(15D)-C(17D)
112.0(8)
C(19D)-C(24D)-C(23D)
118.9(9)
C(13D)-C(9D)-C(10D)
106.0(7)
C(15D)-C(16D)-H(16J)
109.5
C(19D)-C(24D)-H(24D)
120.6
C(11D)-C(10D)-C(9D)
104.5(7)
C(15D)-C(16D)-H(16K)
109.5
C(23D)-C(24D)-H(24D)
120.6
211
Appendix B
C(11D)-C(10D)-H(10G)
110.9
H(16J)-C(16D)-H(16K)
109.5
C(5D)-C(51D)-H(51J)
109.5
C(9D)-C(10D)-H(10G)
110.9
C(15D)-C(16D)-H(16L)
109.5
C(5D)-C(51D)-H(51K)
109.5
C(11D)-C(10D)-H(10H)
110.9
H(16J)-C(16D)-H(16L)
109.5
H(51J)-C(51D)-H(51K)
109.5
C(9D)-C(10D)-H(10H)
110.9
H(16K)-C(16D)-H(16L)
109.5
C(5D)-C(51D)-H(51L)
109.5
H(10G)-C(10D)-H(10H)
108.9
C(15D)-C(17D)-H(17J)
109.5
H(51J)-C(51D)-H(51L)
109.5
O(5D)-C(11D)-C(12D)
107.0(7)
C(15D)-C(17D)-H(17K)
109.5
H(51K)-C(51D)-H(51L)
109.5
O(5D)-C(11D)-C(10D)
106.7(7)
H(17J)-C(17D)-H(17K)
109.5
C(7D)-C(71D)-H(71J)
109.5
C(12D)-C(11D)-C(10D)
107.0(7)
C(15D)-C(17D)-H(17L)
109.5
C(7D)-C(71D)-H(71K)
109.5
O(5D)-C(11D)-H(11D)
111.9
H(17J)-C(17D)-H(17L)
109.5
H(71J)-C(71D)-H(71K)
109.5
C(12D)-C(11D)-H(11D)
111.9
H(17K)-C(17D)-H(17L)
109.5
C(7D)-C(71D)-H(71L)
109.5
C(10D)-C(11D)-H(11D)
111.9
C(15D)-C(18D)-H(18J)
109.5
H(71J)-C(71D)-H(71L)
109.5
C(11D)-C(12D)-C(13D)
102.0(7)
C(15D)-C(18D)-H(18K)
109.5
H(71K)-C(71D)-H(71L)
109.5
C(11D)-C(12D)-H(12G)
111.4
H(18J)-C(18D)-H(18K)
109.5
C(7D)-C(72D)-H(72J)
109.5
C(13D)-C(12D)-H(12G)
111.4
C(15D)-C(18D)-H(18L)
109.5
C(7D)-C(72D)-H(72K)
109.5
C(11D)-C(12D)-H(12H)
111.4
H(18J)-C(18D)-H(18L)
109.5
H(72J)-C(72D)-H(72K)
109.5
C(13D)-C(12D)-H(12H)
111.4
H(18K)-C(18D)-H(18L)
109.5
C(7D)-C(72D)-H(72L)
109.5
H(72K)-C(72D)-H(72L)
109.5
C(14B)-O(4B)-C(15B)
121.0(7)
C(11C)-O(5C)-S(1C)
119.5(5)
C(14D)-N(1D)-C(9D)
119.6(8)
C(11B)-O(5B)-S(1B)
119.9(5)
C(8D)-O(1D)-C(1D)
119.7(7)
C(14D)-N(1D)-H(1DD)
120.2
C(8C)-O(1C)-C(1C)
119.0(7)
C(14D)-O(4D)-C(15D)
119.1(7)
C(9D)-N(1D)-H(1DD)
120.2
C(14C)-O(4C)-C(15C)
120.3(7)
C(11D)-O(5D)-S(1D)
118.9(5)
C(8B)-O(1B)-C(1B)
116.4(7)
Table 11. Torsion angles [°] for x81660.
O(6A)-S(1A)-O(5A)-C(11A)
34.8(6)
N(1A)-C(9A)-C(10A)-C(11A)
-141.8(7)
O(7A)-S(1A)-O(5A)-C(11A)
166.4(6)
C(13A)-C(9A)-C(10A)-C(11A)
-20.7(9)
C(19A)-S(1A)-O(5A)-C(11A)
-79.7(6)
C(8A)-C(9A)-C(10A)-C(11A)
92.5(8)
C(8A)-O(1A)-C(1A)-C(6A)
77.2(9)
S(1A)-O(5A)-C(11A)-C(12A)
-105.5(7)
C(8A)-O(1A)-C(1A)-C(2A)
-160.1(7)
S(1A)-O(5A)-C(11A)-C(10A)
139.1(6)
O(1A)-C(1A)-C(2A)-C(3A)
-178.7(7)
C(9A)-C(10A)-C(11A)-O(5A)
112.5(7)
C(6A)-C(1A)-C(2A)-C(3A)
-59.5(9)
C(9A)-C(10A)-C(11A)-C(12A)
-2.5(9)
O(1A)-C(1A)-C(2A)-C(7A)
53.8(9)
O(5A)-C(11A)-C(12A)-C(13A)
-89.4(8)
C(6A)-C(1A)-C(2A)-C(7A)
173.0(7)
C(10A)-C(11A)-C(12A)-C(13A)
24.6(9)
C(1A)-C(2A)-C(3A)-C(4A)
57.7(9)
N(1A)-C(9A)-C(13A)-C(12A)
158.0(7)
C(7A)-C(2A)-C(3A)-C(4A)
-176.1(7)
C(8A)-C(9A)-C(13A)-C(12A)
-79.9(8)
212
Appendix B
C(2A)-C(3A)-C(4A)-C(5A)
-58.0(10)
C(10A)-C(9A)-C(13A)-C(12A)
36.0(8)
C(3A)-C(4A)-C(5A)-C(6A)
54.1(10)
C(11A)-C(12A)-C(13A)-C(9A)
-37.7(9)
C(3A)-C(4A)-C(5A)-C(51A)
177.4(8)
C(9A)-N(1A)-C(14A)-O(3A)
7.8(13)
O(1A)-C(1A)-C(6A)-C(5A)
178.5(7)
C(9A)-N(1A)-C(14A)-O(4A)
-173.1(7)
C(2A)-C(1A)-C(6A)-C(5A)
59.7(10)
C(15A)-O(4A)-C(14A)-O(3A)
-4.6(13)
C(51A)-C(5A)-C(6A)-C(1A)
-178.1(7)
C(15A)-O(4A)-C(14A)-N(1A)
176.2(7)
C(4A)-C(5A)-C(6A)-C(1A)
-53.9(10)
C(14A)-O(4A)-C(15A)-C(18A)
-179.6(7)
C(3A)-C(2A)-C(7A)-C(71A)
-72.4(10)
C(14A)-O(4A)-C(15A)-C(17A)
-61.2(10)
C(1A)-C(2A)-C(7A)-C(71A)
50.3(10)
C(14A)-O(4A)-C(15A)-C(16A)
63.9(10)
C(3A)-C(2A)-C(7A)-C(72A)
52.8(10)
O(6A)-S(1A)-C(19A)-C(24A)
163.6(7)
C(1A)-C(2A)-C(7A)-C(72A)
175.4(8)
O(7A)-S(1A)-C(19A)-C(24A)
30.6(9)
C(1A)-O(1A)-C(8A)-O(2A)
8.3(12)
O(5A)-S(1A)-C(19A)-C(24A)
-80.6(8)
C(1A)-O(1A)-C(8A)-C(9A)
-174.0(7)
O(6A)-S(1A)-C(19A)-C(20A)
-20.1(9)
C(14A)-N(1A)-C(9A)-C(13A)
174.5(7)
O(7A)-S(1A)-C(19A)-C(20A)
-153.1(7)
C(14A)-N(1A)-C(9A)-C(8A)
55.6(10)
O(5A)-S(1A)-C(19A)-C(20A)
95.7(8)
C(14A)-N(1A)-C(9A)-C(10A)
-69.1(10)
C(24A)-C(19A)-C(20A)-C(21A)
-1.4(14)
O(2A)-C(8A)-C(9A)-N(1A)
-157.2(8)
S(1A)-C(19A)-C(20A)-C(21A)
-177.8(7)
O(1A)-C(8A)-C(9A)-N(1A)
25.0(10)
C(19A)-C(20A)-C(21A)-C(22A)
-0.7(14)
O(2A)-C(8A)-C(9A)-C(13A)
80.8(10)
C(20A)-C(21A)-C(22A)-C(23A)
2.8(13)
O(1A)-C(8A)-C(9A)-C(13A)
-97.0(8)
C(20A)-C(21A)-C(22A)-Br(1A)
-177.6(7)
O(2A)-C(8A)-C(9A)-C(10A)
-30.8(12)
C(21A)-C(22A)-C(23A)-C(24A)
-2.7(13)
O(1A)-C(8A)-C(9A)-C(10A)
151.5(7)
Br(1A)-C(22A)-C(23A)-C(24A)
177.6(7)
C(20A)-C(19A)-C(24A)-C(23A)
1.5(13)
C(10B)-C(11B)-C(12B)-C(13B)
-39.1(9)
S(1A)-C(19A)-C(24A)-C(23A)
177.8(7)
C(11B)-C(12B)-C(13B)-C(9B)
35.6(9)
C(22A)-C(23A)-C(24A)-C(19A)
0.5(13)
N(1B)-C(9B)-C(13B)-C(12B)
-141.3(7)
O(1B)-C(1B)-C(2B)-C(7B)
56.8(10)
C(8B)-C(9B)-C(13B)-C(12B)
98.4(8)
C(6B)-C(1B)-C(2B)-C(7B)
177.0(7)
C(10B)-C(9B)-C(13B)-C(12B)
-19.0(9)
O(1B)-C(1B)-C(2B)-C(3B)
-178.1(7)
C(9B)-N(1B)-C(14B)-O(3B)
1.3(12)
C(6B)-C(1B)-C(2B)-C(3B)
-57.9(10)
C(9B)-N(1B)-C(14B)-O(4B)
-179.7(7)
C(7B)-C(2B)-C(3B)-C(4B)
179.5(7)
O(6B)-S(1B)-C(19B)-C(20B)
12.9(9)
C(1B)-C(2B)-C(3B)-C(4B)
53.8(10)
O(7B)-S(1B)-C(19B)-C(20B)
145.3(7)
C(2B)-C(3B)-C(4B)-C(5B)
-56.3(11)
O(5B)-S(1B)-C(19B)-C(20B)
-104.4(8)
C(3B)-C(4B)-C(5B)-C(51B)
179.2(8)
O(6B)-S(1B)-C(19B)-C(24B)
-165.7(7)
C(3B)-C(4B)-C(5B)-C(6B)
57.1(10)
O(7B)-S(1B)-C(19B)-C(24B)
-33.3(9)
O(1B)-C(1B)-C(6B)-C(5B)
-176.9(7)
O(5B)-S(1B)-C(19B)-C(24B)
77.1(8)
C(2B)-C(1B)-C(6B)-C(5B)
63.1(10)
C(24B)-C(19B)-C(20B)-C(21B)
-1.8(14)
C(51B)-C(5B)-C(6B)-C(1B)
177.3(8)
S(1B)-C(19B)-C(20B)-C(21B)
179.7(7)
213
Appendix B
C(4B)-C(5B)-C(6B)-C(1B)
-59.0(10)
C(19B)-C(20B)-C(21B)-C(22B)
1.4(13)
C(1B)-C(2B)-C(7B)-C(71B)
-178.4(8)
C(20B)-C(21B)-C(22B)-C(23B)
0.7(13)
C(3B)-C(2B)-C(7B)-C(71B)
59.7(10)
C(20B)-C(21B)-C(22B)-Br(1B)
178.8(6)
C(1B)-C(2B)-C(7B)-C(72B)
55.3(10)
C(21B)-C(22B)-C(23B)-C(24B)
-2.3(14)
C(3B)-C(2B)-C(7B)-C(72B)
-66.6(10)
Br(1B)-C(22B)-C(23B)-C(24B)
179.6(6)
C(14B)-N(1B)-C(9B)-C(8B)
-57.0(10)
C(22B)-C(23B)-C(24B)-C(19B)
1.8(13)
C(14B)-N(1B)-C(9B)-C(13B)
-176.2(7)
C(20B)-C(19B)-C(24B)-C(23B)
0.2(14)
C(14B)-N(1B)-C(9B)-C(10B)
66.4(10)
S(1B)-C(19B)-C(24B)-C(23B)
178.8(7)
O(2B)-C(8B)-C(9B)-N(1B)
148.6(8)
O(1C)-C(1C)-C(2C)-C(7C)
61.8(9)
O(1B)-C(8B)-C(9B)-N(1B)
-36.8(10)
C(6C)-C(1C)-C(2C)-C(7C)
178.4(7)
O(2B)-C(8B)-C(9B)-C(13B)
-91.8(10)
O(1C)-C(1C)-C(2C)-C(3C)
-169.7(7)
O(1B)-C(8B)-C(9B)-C(13B)
82.9(9)
C(6C)-C(1C)-C(2C)-C(3C)
-53.1(10)
O(2B)-C(8B)-C(9B)-C(10B)
23.1(12)
C(7C)-C(2C)-C(3C)-C(4C)
-177.2(8)
O(1B)-C(8B)-C(9B)-C(10B)
-162.3(7)
C(1C)-C(2C)-C(3C)-C(4C)
55.9(10)
N(1B)-C(9B)-C(10B)-C(11B)
114.9(8)
C(2C)-C(3C)-C(4C)-C(5C)
-59.4(10)
C(8B)-C(9B)-C(10B)-C(11B)
-121.3(8)
C(3C)-C(4C)-C(5C)-C(6C)
55.5(10)
C(13B)-C(9B)-C(10B)-C(11B)
-4.7(9)
C(3C)-C(4C)-C(5C)-C(51C)
177.2(8)
C(9B)-C(10B)-C(11B)-O(5B)
-86.6(8)
C(4C)-C(5C)-C(6C)-C(1C)
-53.2(10)
C(9B)-C(10B)-C(11B)-C(12B)
27.0(9)
C(51C)-C(5C)-C(6C)-C(1C)
-175.2(8)
O(5B)-C(11B)-C(12B)-C(13B)
77.0(8)
O(1C)-C(1C)-C(6C)-C(5C)
173.5(7)
C(2C)-C(1C)-C(6C)-C(5C)
54.5(10)
C(19C)-C(20C)-C(21C)-C(22C)
0.8(13)
C(1C)-C(2C)-C(7C)-C(71C)
-162.4(7)
C(20C)-C(21C)-C(22C)-C(23C)
-2.9(14)
C(3C)-C(2C)-C(7C)-C(71C)
73.1(10)
C(20C)-C(21C)-C(22C)-Br(1C)
178.7(7)
C(1C)-C(2C)-C(7C)-C(72C)
75.1(10)
C(21C)-C(22C)-C(23C)-C(24C)
2.5(14)
C(3C)-C(2C)-C(7C)-C(72C)
-49.4(11)
Br(1C)-C(22C)-C(23C)-C(24C)
-179.2(7)
C(14C)-N(1C)-C(9C)-C(8C)
52.7(10)
C(20C)-C(19C)-C(24C)-C(23C)
-2.2(13)
C(14C)-N(1C)-C(9C)-C(10C)
-71.6(10)
S(1C)-C(19C)-C(24C)-C(23C)
178.0(7)
C(14C)-N(1C)-C(9C)-C(13C)
172.3(7)
C(22C)-C(23C)-C(24C)-C(19C)
0.1(13)
O(2C)-C(8C)-C(9C)-N(1C)
-150.4(8)
O(1D)-C(1D)-C(2D)-C(7D)
61.2(9)
O(1C)-C(8C)-C(9C)-N(1C)
35.6(10)
C(6D)-C(1D)-C(2D)-C(7D)
178.9(7)
O(2C)-C(8C)-C(9C)-C(10C)
-25.5(12)
O(1D)-C(1D)-C(2D)-C(3D)
-171.2(7)
O(1C)-C(8C)-C(9C)-C(10C)
160.5(7)
C(6D)-C(1D)-C(2D)-C(3D)
-53.5(10)
O(2C)-C(8C)-C(9C)-C(13C)
89.3(10)
C(1D)-C(2D)-C(3D)-C(4D)
52.6(10)
O(1C)-C(8C)-C(9C)-C(13C)
-84.7(9)
C(7D)-C(2D)-C(3D)-C(4D)
179.8(8)
N(1C)-C(9C)-C(10C)-C(11C)
-105.6(8)
C(2D)-C(3D)-C(4D)-C(5D)
-56.5(11)
C(8C)-C(9C)-C(10C)-C(11C)
131.1(8)
C(3D)-C(4D)-C(5D)-C(51D)
179.0(8)
C(13C)-C(9C)-C(10C)-C(11C)
13.7(9)
C(3D)-C(4D)-C(5D)-C(6D)
53.9(10)
214
Appendix B
C(9C)-C(10C)-C(11C)-O(5C)
77.0(8)
O(1D)-C(1D)-C(6D)-C(5D)
175.6(7)
C(9C)-C(10C)-C(11C)-C(12C)
-33.0(9)
C(2D)-C(1D)-C(6D)-C(5D)
56.9(10)
C(10C)-C(11C)-C(12C)-C(13C)
39.3(9)
C(51D)-C(5D)-C(6D)-C(1D)
-179.7(8)
O(5C)-C(11C)-C(12C)-C(13C)
-73.6(8)
C(4D)-C(5D)-C(6D)-C(1D)
-53.5(10)
C(11C)-C(12C)-C(13C)-C(9C)
-30.7(9)
C(1D)-C(2D)-C(7D)-C(72D)
74.0(10)
N(1C)-C(9C)-C(13C)-C(12C)
131.5(7)
C(3D)-C(2D)-C(7D)-C(72D)
-50.1(11)
C(8C)-C(9C)-C(13C)-C(12C)
-108.6(8)
C(1D)-C(2D)-C(7D)-C(71D)
-162.1(7)
C(10C)-C(9C)-C(13C)-C(12C)
10.7(9)
C(3D)-C(2D)-C(7D)-C(71D)
73.8(10)
C(9C)-N(1C)-C(14C)-O(3C)
5.9(12)
O(2D)-C(8D)-C(9D)-N(1D)
152.7(8)
C(9C)-N(1C)-C(14C)-O(4C)
-173.4(7)
O(1D)-C(8D)-C(9D)-N(1D)
-33.6(10)
O(7C)-S(1C)-C(19C)-C(20C)
-155.3(7)
O(2D)-C(8D)-C(9D)-C(13D)
-87.7(10)
O(6C)-S(1C)-C(19C)-C(20C)
-23.0(8)
O(1D)-C(8D)-C(9D)-C(13D)
86.1(9)
O(5C)-S(1C)-C(19C)-C(20C)
93.5(8)
O(2D)-C(8D)-C(9D)-C(10D)
26.9(12)
O(7C)-S(1C)-C(19C)-C(24C)
24.5(8)
O(1D)-C(8D)-C(9D)-C(10D)
-159.4(7)
O(6C)-S(1C)-C(19C)-C(24C)
156.7(7)
N(1D)-C(9D)-C(10D)-C(11D)
124.9(8)
O(5C)-S(1C)-C(19C)-C(24C)
-86.7(7)
C(8D)-C(9D)-C(10D)-C(11D)
-109.7(8)
C(24C)-C(19C)-C(20C)-C(21C)
1.8(14)
C(13D)-C(9D)-C(10D)-C(11D)
5.1(9)
S(1C)-C(19C)-C(20C)-C(21C)
-178.5(7)
C(9D)-C(10D)-C(11D)-O(5D)
-93.9(8)
C(9D)-C(10D)-C(11D)-C(12D)
20.4(9)
C(16B)-C(15B)-O(4B)-C(14B)
60.5(10)
O(5D)-C(11D)-C(12D)-C(13D)
76.3(8)
C(17B)-C(15B)-O(4B)-C(14B)
-64.1(10)
C(10D)-C(11D)-C(12D)-C(13D)
-37.8(9)
C(10B)-C(11B)-O(5B)-S(1B)
-101.1(7)
C(11D)-C(12D)-C(13D)-C(9D)
40.2(8)
C(12B)-C(11B)-O(5B)-S(1B)
145.7(6)
N(1D)-C(9D)-C(13D)-C(12D)
-149.6(7)
O(6B)-S(1B)-O(5B)-C(11B)
-37.1(7)
C(8D)-C(9D)-C(13D)-C(12D)
89.5(8)
O(7B)-S(1B)-O(5B)-C(11B)
-167.5(6)
C(10D)-C(9D)-C(13D)-C(12D)
-28.0(9)
C(19B)-S(1B)-O(5B)-C(11B)
78.4(7)
O(6D)-S(1D)-C(19D)-C(24D)
-137.5(7)
O(2C)-C(8C)-O(1C)-C(1C)
12.6(12)
O(7D)-S(1D)-C(19D)-C(24D)
-5.6(8)
C(9C)-C(8C)-O(1C)-C(1C)
-173.4(7)
O(5D)-S(1D)-C(19D)-C(24D)
106.1(7)
C(6C)-C(1C)-O(1C)-C(8C)
119.3(8)
O(6D)-S(1D)-C(19D)-C(20D)
39.7(8)
C(2C)-C(1C)-O(1C)-C(8C)
-118.8(8)
O(7D)-S(1D)-C(19D)-C(20D)
171.6(7)
O(3C)-C(14C)-O(4C)-C(15C)
7.0(12)
O(5D)-S(1D)-C(19D)-C(20D)
-76.6(7)
N(1C)-C(14C)-O(4C)-C(15C)
-173.7(7)
C(24D)-C(19D)-C(20D)-C(21D)
-0.7(13)
C(16C)-C(15C)-O(4C)-C(14C)
-63.2(10)
S(1D)-C(19D)-C(20D)-C(21D)
-177.8(7)
C(17C)-C(15C)-O(4C)-C(14C)
179.9(7)
C(19D)-C(20D)-C(21D)-C(22D)
3.9(13)
C(18C)-C(15C)-O(4C)-C(14C)
63.8(9)
C(20D)-C(21D)-C(22D)-C(23D)
-5.2(14)
C(10C)-C(11C)-O(5C)-S(1C)
109.8(7)
C(20D)-C(21D)-C(22D)-Br(1D)
175.3(7)
C(12C)-C(11C)-O(5C)-S(1C)
-140.2(6)
C(21D)-C(22D)-C(23D)-C(24D)
3.2(14)
O(7C)-S(1C)-O(5C)-C(11C)
165.2(6)
215
Appendix B
Br(1D)-C(22D)-C(23D)-C(24D)
-177.3(7)
O(6C)-S(1C)-O(5C)-C(11C)
34.8(7)
C(20D)-C(19D)-C(24D)-C(23D)
-1.3(13)
C(19C)-S(1C)-O(5C)-C(11C)
-79.8(7)
S(1D)-C(19D)-C(24D)-C(23D)
175.9(7)
O(2D)-C(8D)-O(1D)-C(1D)
6.8(13)
C(22D)-C(23D)-C(24D)-C(19D)
0.0(13)
C(9D)-C(8D)-O(1D)-C(1D)
-167.0(7)
O(3D)-C(14D)-N(1D)-C(9D)
0.5(12)
C(6D)-C(1D)-O(1D)-C(8D)
117.6(8)
O(4D)-C(14D)-N(1D)-C(9D)
-177.1(7)
C(2D)-C(1D)-O(1D)-C(8D)
-119.7(8)
C(8D)-C(9D)-N(1D)-C(14D)
-60.0(10)
O(3D)-C(14D)-O(4D)-C(15D)
-1.3(13)
C(13D)-C(9D)-N(1D)-C(14D)
-177.7(7)
N(1D)-C(14D)-O(4D)-C(15D)
176.1(7)
C(10D)-C(9D)-N(1D)-C(14D)
64.7(10)
C(18D)-C(15D)-O(4D)-C(14D)
-59.7(10)
O(2B)-C(8B)-O(1B)-C(1B)
1.9(12)
C(16D)-C(15D)-O(4D)-C(14D)
63.9(9)
C(9B)-C(8B)-O(1B)-C(1B)
-172.8(7)
C(17D)-C(15D)-O(4D)-C(14D)
-178.0(7)
C(2B)-C(1B)-O(1B)-C(8B)
-157.6(7)
C(12D)-C(11D)-O(5D)-S(1D)
108.7(7)
C(6B)-C(1B)-O(1B)-C(8B)
79.6(9)
C(10D)-C(11D)-O(5D)-S(1D)
-137.0(6)
O(3B)-C(14B)-O(4B)-C(15B)
-7.2(13)
O(6D)-S(1D)-O(5D)-C(11D)
-27.9(7)
N(1B)-C(14B)-O(4B)-C(15B)
173.7(7)
O(7D)-S(1D)-O(5D)-C(11D)
-158.0(6)
C(18B)-C(15B)-O(4B)-C(14B)
177.5(7)
C(19D)-S(1D)-O(5D)-C(11D)
88.6(6)
Table 12. Hydrogen bonds for x81660 [Å and °].
______________________________________________________________________
D-H...A
d(D-H)
d(H...A)
d(D...A)
<(DHA)
______________________________________________________________________
N(1A)-H(1AA)...O(2B)#1 0.88
2.09
2.947(9)
165.2
N(1B)-H(1BB)...O(2A)#1 0.88
2.12
2.983(10)
168.2
N(1C)-H(1CC)...O(2D)
0.88
2.08
2.937(10)
165.1
N(1D)-H(1DD)...O(2C)#2 0.88
2.07
2.931(10)
165.0
______________________________________________________________________
Symmetry transformations used to generate equivalent atoms:
#1 -x+1,y-1/2,-z+1
#2 x,y+1,z
216
Appendix B
Table 13. Crystal data and structure refinement for mhb8trc
Identification code
mhb8trc
Empirical formula
C23 H28 N4 O7
Formula weight
472.49
Temperature
100(2) K
Wavelength
0.71073 Å
Crystal system
Triclinic
Space group
P-1
Unit cell dimensions
a = 10.380(5) Å
= 107.283(6)°.
b = 11.070(5) Å
= 100.037(15)°.
c = 11.952(4) Å
 = 113.268(13)°.
Volume
1136.1(8) Å3
Z
2
Density (calculated)
1.381 Mg/m3
Absorption coefficient
0.103 mm-1
F(000)
500
Crystal size
0.38 x 0.10 x 0.08 mm3
Theta range for data collection
2.19 to 21.50°.
Index ranges
-10<=h<=10, -11<=k<=10, 0<=l<=12
Reflections collected
24155
Independent reflections
2777 [R(int) = 0.0785]
217
Appendix B
Completeness to theta = 21.50°
96.1%
Absorption correction
Semi-empirical from equivalents
Max. and min. transmission
0.992 and 0.362
Refinement method
Full-matrix least-squares on F2
Data / restraints / parameters
2777 / 33 / 321
Goodness-of-fit on F2
1.572
Final R indices [I>2sigma(I)]
R1 = 0.1060, wR2 = 0.2485
R indices (all data)
R1 = 0.1829, wR2 = 0.2955
Largest diff. peak and hole
0.828 and -0.676 e.Å-3
218
Appendix B
Table 14. Bond lengths [Å] and angles [°] for TWIN5.
C1—N1
1.465
(10)
C9—H9
0.9500
C21—H21C
0.9800
C1—C6
1.496
(12)
N9—C10
1.335
(10)
C22—H22A
0.9800
C1—C2
1.527
(11)
C10—N10
1.333
(10)
C22—H22B
0.9800
C1—C5
1.528
(12)
N10—C11
1.412
(11)
C22—H22C
0.9800
N1—C19
1.336
(11)
N10—H10N
0.89
C23—H23A
0.9800
N1—H1N
0.88
C11—O11
1.177
(10)
C23—H23B
0.9800
O1—C7
1.376
(10)
C12—C13
1.445
(13)
C23—H23C
0.9800
O1—C3
1.584
(12)
C12—H12A
0.9900
O61—H61
0.89
C2—C3
1.552
(12)
C12—H12B
0.9900
C21—H21C
0.9800
C2—H2A
0.9900
C13—C18
1.370
(13)
C22—H22A
0.9800
C2—H2B
0.9900
C13—C14
1.402
(14)
C22—H22B
0.9800
C3—C4
1.391
C14—C15
1.350
(15)
C22—H22C
0.9800
C3—H3
1.0000
C14—H14
0.9500
C23—H23A
0.9800
C4—C5
1.506
C15—C16
1.351
C23—H23B
0.9800
C4—H4A
0.9900
C15—H15
0.9500
C23—H23C
0.9800
C4—H4B
0.9900
C16—C17
1.373
(17)
O61—H61
0.89
C5—H5A
0.9900
C16—H16
0.9500
C5—H5B
0.9900
C17—C18
1.374
(14)
N1—C1—C6
111.5
(7)
C6—O62
1.196
(10)
C17—H17
0.9500
N1—C1—C2
111.7
(7)
C6—O61
1.334
(10)
C18—H18
0.9500
C6—C1—C2
115.0
(7)
C7—N8
1.299
(10)
C19—O21
1.242
(9)
N1—C1—C5
105.3
(7)
C7—N9
1.322
(11)
C19—O20
1.325
(9)
C6—C1—C5
110.8
(7)
C8—C9
1.315
(12)
C20—O20
1.453
(11)
C2—C1—C5
101.7
(7)
C8—N8
1.378
(11)
C20—C21
1.480
(13)
C19—N1—C1
126.7
(7)
C8—H8
0.9500
C20—C22
1.502
(12)
C19—N1—H1N
116
(5)
O8—C11
1.329
(11)
C20—C23
1.505
(12)
C1—N1—H1N
117
(5)
O8—C12
1.453
(10)
C21—H21A
0.9800
C7—O1—C3
114.3
(6)
C9—C10
1.399
(12)
C21—H21B
0.9800
C1—C2—C3
106.3
(7)
C1—C2—H2A
110.5
C8—C9—C10
117.2
C14—C13—C12
122.2
(10)
C3—C2—H2A
110.5
C8—C9—H9
121.4
C15—C14—C13
123.2
(12)
C1—C2—H2B
110.5
C10—C9—H9
121.4
C15—C14—H14
118.4
C3—C2—H2B
110.5
C7—N9—C10
115.3
(7)
C13—C14—H14
118.4
H2A—C2—H2B
108.7
N10—C10—N9
114.8
(7)
C14—C15—C16
119.8
C4—C3—C2
106.9
N10—C10—C9
125.6
(7)
C14—C15—H15
120.1
(2)
(14)
(13)
(8)
219
(2)
(18)
(8)
(2)
(2)
(14)
Appendix B
C4—C3—O1
97.3
(10)
N9—C10—C9
119.6
(7)
C16—C15—H15
120.1
C2—C3—O1
104.3
(6)
C10—N10—C11
125.8
(7)
C15—C16—C17
119.3
C4—C3—H3
115.4
C10—N10—H10N
110
(5)
C15—C16—H16
120.3
C2—C3—H3
115.4
C11—N10—H10N
123
(5)
C17—C16—H16
120.3
O1—C3—H3
115.4
O11—C11—O8
128.3
(8)
C16—C17—C18
120.7
C3—C4—C5
107.6
O11—C11—N10
125.8
(9)
C16—C17—H17
119.6
C3—C4—H4A
110.2
O8—C11—N10
105.9
(8)
C18—C17—H17
119.6
C5—C4—H4A
110.2
C13—C12—O8
109.5
(7)
C13—C18—C17
121.3
C3—C4—H4B
110.2
C13—C12—H12A
109.8
C13—C18—H18
119.3
C5—C4—H4B
110.2
O8—C12—H12A
109.8
C17—C18—H18
119.3
H4A—C4—H4B
108.5
C13—C12—H12B
109.8
O21—C19—O20
123.5
(9)
C4—C5—C1
103.8
O11—C11—N10
125.8
(9)
O21—C19—N1
122.6
(8)
C4—C5—H5A
111.0
O8—C11—N10
105.9
(8)
O20—C19—N1
113.8
(8)
C1—C5—H5A
111.0
C13—C12—O8
109.5
(7)
O20—C20—C21
112.8
(7)
C4—C5—H5B
111.0
C13—C12—H12A
109.8
O20—C20—C22
111.2
(8)
C1—C5—H5B
111.0
O8—C12—H12A
109.8
C21—C20—C22
113.3
(9)
H5A—C5—H5B
109.0
C13—C12—H12B
109.8
O20—C20—C23
101.4
(7)
O62—C6—O61
123.4
(7)
O11—C11—N10
125.8
(9)
C21—C20—C23
109.9
(8)
O62—C6—C1
126.5
(8)
O8—C11—N10
105.9
(8)
C22—C20—C23
107.3
(8)
O61—C6—C1
110.1
(8)
C13—C12—O8
109.5
(7)
C19—O20—C20
120.9
(7)
N8—C7—N9
132.1
(8)
C13—C12—H12A
109.8
C20—C21—H21A
109.5
N8—C7—O1
118.2
(7)
O8—C12—H12A
109.8
C20—C21—H21B
109.5
N9—C7—O1
109.7
(7)
C13—C12—H12B
109.8
H21A—C21—H21B
109.5
C9—C8—N8
126.3
(8)
C13—C12—H12B
109.8
C20—C21—H21C
109.5
C9—C8—H8
116.8
O8—C12—H12B
109.8
H21A—C21—H21C
109.5
N8—C8—H8
116.8
H12A—C12—H12B
108.2
H21B—C21—H21C
109.5
C7—N8—C8
109.2
(7)
C18—C13—C14
115.7
(10)
C20—C22—H22A
109.5
C11—O8—C12
113.1
(7)
C18—C13—C12
122.0
(10)
C20—C22—H22B
109.5
H22A—C22—H22B
109.5
C20—C23—H23A
109.5
H23A—C23—H23C
109.5
C20—C22—H22C
109.5
C20—C23—H23B
109.5
H23B—C23—H23C
109.5
H22A—C22—H22C
109.5
H23A—C23—H23B
109.5
C6—O61—H61
116
H22B—C22—H22C
109.5
C20—C23—H23C
109.5
(8)
(8)
220
(6)
(12)
(12)
(11)
Appendix B
Table 15. Torsion angles [°] for TWIN5..
C6—C1—N1—C19
68.9
(10)
O1—C7—N9—C10
−174.6
(8)
C2—C1—N1—C19
−61.3
(10)
C7—N9—C10—N10
−178.6
(8)
C5—C1—N1—C19
−170.9
(8)
C7—N9—C10—C9
−0.6
(13)
N1—C1—C2—C3
−90.3
(8)
C8—C9—C10—N10
176.5
(9)
C6—C1—C2—C3
141.3
(8)
C8—C9—C10—N9
−1.2
(14)
C5—C1—C2—C3
21.5
(9)
N9—C10—N10—C11
178.1
(8)
C1—C2—C3—C4
0.2
(11)
C9—C10—N10—C11
0.3
C1—C2—C3—O1
−102.2
C12—O8—C11—O11
−4.4
(13)
C7—O1—C3—C4
90.5
C12—O8—C11—N10
175.5
(7)
C7—O1—C3—C2
−160.0
(9)
C10—N10—C11—O11
0.3
C2—C3—C4—C5
−22.7
(13)
C10—N10—C11—O8
−179.7
(8)
O1—C3—C4—C5
84.7
(9)
C11—O8—C12—C13
−167.2
(8)
C3—C4—C5—C1
37.0
(12)
O8—C12—C13—C18
168.8
(9)
N1—C1—C5—C4
82.1
(9)
O8—C12—C13—C14
−14.3
(13)
C6—C1—C5—C4
−157.2
(8)
C18—C13—C14—C15
−0.5
C2—C1—C5—C4
−34.5
(10)
C12—C13—C14—C15
−177.5
N1—C1—C6—O62
−3.2
C13—C14—C15—C16
−0.2
C2—C1—C6—O62
125.2
C14—C15—C16—C17
0.3
(19)
C5—C1—C6—O62
−120.1
C15—C16—C17—C18
0.4
(18)
N1—C1—C6—O61
179.2
(7)
C14—C13—C18—C17
1.2
(15)
C2—C1—C6—O61
−52.4
(10)
C12—C13—C18—C17
178.3
C5—C1—C6—O61
62.2
(9)
C16—C17—C18—C13
−1.2
C3—O1—C7—N8
−8.5
(14)
C1—N1—C19—O21
−178.8
C3—O1—C7—N9
171.4
(8)
C1—N1—C19—O20
4.4
N9—C7—N8—C8
−6.3
(16)
O21—C19—O20—C20
−10.8
(11)
O1—C7—N8—C8
173.4
(9)
N1—C19—O20—C20
166.0
(7)
C9—C8—N8—C7
3.6
C21—C20—O20—C19
−62.3
(10)
N8—C8—C9—C10
−0.4
C22—C20—O20—C19
66.3
N8—C7—N9—C10
5.1
C23—C20—O20—C19
−179.8
(8)
(9)
(13)
(10)
(10)
(15)
(16)
(17)
221
(14)
(14)
(16)
(10)
(19)
(10)
(16)
(7)
(11)
(9)
(7)
Appendix B
Table 16. Hydrogen bonds for TWIN5 [Å and °].
______________________________________________________________________
D-H...A
d(D-H)
d(H...A)
d(D...A)
<(DHA)
______________________________________________________________________
N(1)-H(1N)...O(21)#1
0.88(2)
2.08(2)
2.956(8)
175(7)
N(10)-H(10N)...O(62)#2 0.89(2)
2.01(3)
2.879(8)
163(8)
O(61)-H(61)...N(9)#2
0.89(2)
1.83(3)
2.716(8)
170(9)
O(61)-H(61)...O(1)#2
0.89(2)
2.61(7)
3.192(9)
124(7)
______________________________________________________________________
Symmetry transformations used to generate equivalent atoms:
#1 -x+1,-y+2,-z+1
#2 -x+1,-y+1,-z
222
Appendix B
major component
OH disorder – also on C3 (18%) as well as C4 (82%)
223
Appendix B
Table 17. Crystal data and structure refinement for x81315.
Identification code
x81315
Empirical formula
C12 H21 N O5
Formula weight
259.30
Temperature
100(2) K
Wavelength
0.71073 Å
Crystal system
Monoclinic
Space group
P2(1)/n
Unit cell dimensions
a = 6.4326(6) Å
= 90°.
b = 20.7240(18) Å
= 94.603(5)°.
c = 10.0612(9) Å
 = 90°.
Volume
1336.9(2) Å3
Z
4
Density (calculated)
1.288 Mg/m3
Absorption coefficient
0.100 mm-1
F(000)
560
Crystal size
0.40 x 0.24 x 0.16 mm3
Theta range for data collection
2.26 to 34.33°.
Index ranges
-10<=h<=10, -32<=k<=32, -15<=l<=15
Reflections collected
36326
Independent reflections
5549 [R(int) = 0.0306]
Completeness to theta = 25.00°
99.9%
Absorption correction
Semi-empirical from equivalents
Max. and min. transmission
0.986 and 0.908
Refinement method
Full-matrix least-squares on F2
Data / restraints / parameters
5549 / 6 / 182
Goodness-of-fit on F2
1.054
Final R indices [I>2sigma(I)]
R1 = 0.0499, wR2 = 0.1359
R indices (all data)
R1 = 0.0648, wR2 = 0.1454
Largest diff. peak and hole
1.185 and -0.338 e.Å-3
224
Appendix B
Table 18. Bond lengths [Å] and angles [°] for x81315.
C1—N1
1.4609
(12)
C7—H7A
0.9800
C3B—C2—H2B
110.9
C1—C6
1.5315
(14)
C7—H7B
0.9800
C3A—C2—H2B
110.9
C1—C5
1.5520
(14)
C7—H7C
0.9800
C1—C2—H2B
110.9
C1—C2
1.5575
(14)
C9—C11
1.5181
(15)
H2A—C2—H2B
108.9
N1—C8
1.3465
(12)
C9—C12
1.5201
(17)
C4A—C3A—C2
102.23
N1—H1N
0.903
C9—C10
1.5231
(15)
C4A—C3A—H3A
111.3
C2—C3B
1.5222
(15)
C10—H10A
0.9800
C2—C3A—H3A
111.3
C2—C3A
1.5222
(15)
C10—H10B
0.9800
C4A—C3A—H3B
111.3
C2—H2A
0.9900
C10—H10C
0.9800
C2—C3A—H3B
111.3
C2—H2B
0.9900
C11—H11A
0.9800
H3A—C3A—H3B
109.2
C3A—C4A
1.5195
C11—H11B
0.9800
O1A—C3B—C4B
113.0
(3)
C3A—H3A
0.9900
C11—H11C
C4A—H4
O1A—C3B—C2
115.9
(3)
C3A—H3B
0.9900
C12—H12A
C4B—C5
C4B—C3B—C2
102.22
C3B—O1A
1.388
C12—H12B
C4B—H4B1
O1A—C3B—H3C
108.5
C3B—C4B
1.5196
C12—H12C
C4B—H4B2
C4B—C3B—H3C
108.5
C3B—H3C
1.0000
O1—H1
0.8400
C2—C3B—H3C
108.5
O1A—H1A
0.8400
N1—C1—C6
110.84
(8)
C3B—O1A—H1A
109.5
C4A—O1
1.4184
(16)
N1—C1—C5
112.55
(8)
O1—C4A—C3A
105.88
(10)
C4A—C5
1.5448
(16)
C6—C1—C5
112.74
(8)
O1—C4A—C5
112.77
(10)
C4A—H4
1.0000
N1—C1—C2
106.98
(8)
C3A—C4A—C5
104.80
(9)
C4B—C5
1.5448
C6—C1—C2
109.09
(8)
O1—C4A—H4
111.0
C4B—H4B1
0.9900
C5—C1—C2
104.23
(8)
C4A—C5—C1
106.15
C4B—H4B2
0.9900
C8—N1—C1
125.41
(8)
C4B—C5—H5A
110.5
C5—H5A
0.9900
C8—N1—H1N
115.8
(10)
C4A—C5—H5A
110.5
C5—H5B
0.9900
C1—N1—H1N
118.3
(10)
C1—C5—H5A
110.5
O2—C8
1.2286
(11)
C3B—C2—C3A
0.0
C4B—C5—H5B
110.5
O3—C8
1.3409
(11)
C3B—C2—C1
104.14
(8)
O3—C9
1.4749
(12)
C3A—C2—C1
104.13
(8)
C4A—C5—H5B
C1—C5—H5B
110.5
110.5
O4—C6
1.2052
(12)
C3B—C2—H2A
110.9
H5A—C5—H5B
108.7
O5—C6
1.3450
(13)
C3A—C2—H2A
110.9
C8—O3—C9
120.87
O5—C7
1.4440
(13)
C1—C2—H2A
110.9
C1—C5—H5B
110.5
(15)
(16)
(5)
(16)
(16)
225
(9)
(9)
(8)
(8)
Appendix B
Table 19. Torsion angles [°] for x813
C6—C1—N1—C8
51.87
(13)
O1—C4A—C5—C1
−93.08
(11)
C5—C1—N1—C8
−75.41
(12)
C3A—C4A—C5—C1
21.62
(11)
C2—C1—N1—C8
170.70
(9)
N1—C1—C5—C4B
−111.07
(9)
N1—C1—C2—C3B
90.46
(9)
C6—C1—C5—C4B
122.66
(9)
C6—C1—C2—C3B
−149.59
(8)
C2—C1—C5—C4B
4.49
C5—C1—C2—C3B
−28.95
(10)
N1—C1—C5—C4A
−111.07
(9)
N1—C1—C2—C3A
90.45
(9)
C6—C1—C5—C4A
122.66
(9)
C6—C1—C2—C3A
−149.59
(8)
C2—C1—C5—C4A
4.49
(10)
C5—C1—C2—C3A
−28.95
(10)
C7—O5—C6—O4
2.94
(14)
C3B—C2—C3A—C4A
−118.4
(4)
C7—O5—C6—C1
179.73
(8)
C1—C2—C3A—C4A
42.55
(10)
N1—C1—C6—O4
−143.87
(10)
C3A—C2—C3B—O1A
−175.1
(5)
C5—C1—C6—O4
−16.69
(14)
C1—C2—C3B—O1A
165.9
(3)
C2—C1—C6—O4
98.59
(11)
C3A—C2—C3B—C4B
61.6
(4)
N1—C1—C6—O5
39.40
(11)
C1—C2—C3B—C4B
42.54
(10)
C5—C1—C6—O5
166.58
(8)
C2—C3A—C4A—O1
79.80
(11)
C2—C1—C6—O5
−78.14
(10)
C2—C3A—C4A—C5
−39.62
(11)
C9—O3—C8—O2
6.37
O1A—C3B—C4B—C5
−164.9
(3)
C9—O3—C8—N1
−173.31
(9)
C2—C3B—C4B—C5
−39.61
(11)
C1—N1—C8—O2
177.63
(10)
C1—N1—C8—O3
−2.69
(14)
C8—O3—C9—C11
−67.33
(12)
C3B—C4B—C5—C4A
C3B—C4B—C5—C1
0(22)
21.61
(11)
(10)
(16)
O1—C4A—C5—C4B
0(76)
C8—O3—C9—C12
57.59
(12)
C3A—C4A—C5—C4B
0(22)
C8—O3—C9—C10
175.15
(9)
Table 20. Hydrogen bonds for x81315 [Å and °].
______________________________________________________________________
D-H...A
d(D-H)
d(H...A)
d(D...A)
<(DHA)
______________________________________________________________________
N(1)-H(1N)...O(2)#1
0.903(15)
1.970(16)
2.8720(11)
177.3(14)
O(1A)-H(1A)...O(4)#2
0.84
2.05
2.855(6)
159.4
O(1)-H(1)...O(5)#3
0.84
2.02
2.8498(13)
171.7
______________________________________________________________________
Symmetry transformations used to generate equivalent atoms:
#1 -x+1,-y,-z+2
#2 -x,-y,-z+1
#3 x-1,y,z
226
Appendix B
Table 21. Crystal data and structure refinement for X81218.
Identification code
x81218
Empirical formula
C13 H23 N O5
Formula weight
273.32
Temperature
100(2) K
Wavelength
0.71073 Å
Crystal system
Monoclinic
Space group
P2(1)/c
Unit cell dimensions
a = 9.6941(16) Å
= 90°.
b = 14.156(2) Å
= 111.391(6)°.
c = 11.191(2) Å
 = 90°.
227
Appendix B
Volume
1430.0(4) Å3
Z
4
Density (calculated)
1.270 Mg/m3
Absorption coefficient
0.097 mm-1
F(000)
592
Crystal size
0.25 x 0.22 x 0.06 mm3
Theta range for data collection
2.26 to 23.30°.
Index ranges
-10<=h<=10, -15<=k<=13, -12<=l<=11
Reflections collected
8817
Independent reflections
2056 [R(int) = 0.0433]
Completeness to theta = 23.30°
99.5%
Absorption correction
Semi-empirical from equivalents
Max. and min. transmission
0.994 and 0.797
Refinement method
Full-matrix least-squares on F2
Data / restraints / parameters
2056 / 2 / 182
Goodness-of-fit on F2
1.235
Final R indices [I>2sigma(I)]
R1 = 0.0950, wR2 = 0.1951
R indices (all data)
R1 = 0.1190, wR2 = 0.2044
Largest diff. peak and hole
0.718 and -0.409 e.Å-3
228
Appendix B
Table 22. Bond lengths [Å] and angles [°] for X81218.
C(1)-N(1)
1.444(7)
C(13)-C(16)
1.520(8)
O(3)-C(3)-H(3A)
112.2
C(1)-C(6)
1.531(8)
C(13)-C(15)
1.526(10)
C(2)-C(3)-H(3A)
112.2
C(1)-C(2)
1.540(8)
C(14)-H(14A)
0.9800
C(3)-O(3)-H(3)
108(4)
C(1)-C(5)
1.557(8)
C(14)-H(14B)
0.9800
C(5)-C(4)-C(3)
103.3(6)
N(1)-C(11)
1.349(7)
C(14)-H(14C)
0.9800
C(5)-C(4)-H(4A)
111.1
N(1)-H(1)
0.89(2)
C(15)-H(15A)
0.9800
C(3)-C(4)-H(4A)
111.1
C(2)-C(3)
1.577(9)
C(15)-H(15B)
0.9800
C(5)-C(4)-H(4B)
111.1
C(2)-H(2A)
0.9900
C(15)-H(15C)
0.9800
C(3)-C(4)-H(4B)
111.1
C(2)-H(2B)
0.9900
C(16)-H(16A)
0.9800
H(4A)-C(4)-H(4B)
109.1
C(3)-C(4)
1.477(10)
C(16)-H(16B)
0.9800
C(4)-C(5)-C(1)
106.8(5)
C(3)-O(3)
1.511(9)
C(16)-H(16C)
0.9800
C(4)-C(5)-H(5A)
110.4
C(3)-H(3A)
1.0000
C(1)-C(5)-H(5A)
110.4
O(3)-H(3)
0.89(2)
N(1)-C(1)-C(6)
111.3(5)
C(4)-C(5)-H(5B)
110.4
C(4)-C(5)
1.462(9)
N(1)-C(1)-C(2)
113.6(5)
C(1)-C(5)-H(5B)
110.4
C(4)-H(4A)
0.9900
C(6)-C(1)-C(2)
111.1(5)
H(5A)-C(5)-H(5B)
108.6
C(4)-H(4B)
0.9900
N(1)-C(1)-C(5)
109.3(5)
O(6)-C(6)-O(7)
123.7(5)
C(5)-H(5A)
0.9900
C(6)-C(1)-C(5)
106.0(5)
O(6)-C(6)-C(1)
123.7(5)
C(5)-H(5B)
0.9900
C(2)-C(1)-C(5)
105.0(5)
O(7)-C(6)-C(1)
112.4(5)
C(6)-O(6)
1.201(7)
C(11)-N(1)-C(1)
120.4(5)
C(6)-O(7)-C(8)
115.7(5)
C(6)-O(7)
1.334(7)
C(11)-N(1)-H(1)
115(4)
O(7)-C(8)-C(9)
107.8(5)
O(7)-C(8)
1.451(7)
C(1)-N(1)-H(1)
121(4)
O(7)-C(8)-H(8A)
110.1
C(8)-C(9)
1.498(8)
C(1)-C(2)-C(3)
102.7(5)
C(9)-C(8)-H(8A)
110.1
C(8)-H(8A)
0.9900
C(1)-C(2)-H(2A)
111.2
O(7)-C(8)-H(8B)
110.1
C(8)-H(8B)
0.9900
C(3)-C(2)-H(2A)
111.2
C(9)-C(8)-H(8B)
110.1
C(9)-H(9A)
0.9800
C(1)-C(2)-H(2B)
111.2
H(8A)-C(8)-H(8B)
108.5
C(9)-H(9B)
0.9800
C(3)-C(2)-H(2B)
111.2
C(8)-C(9)-H(9A)
109.5
C(9)-H(9C)
0.9800
H(2A)-C(2)-H(2B)
109.1
C(8)-C(9)-H(9B)
109.5
C(11)-O(11)
1.222(7)
C(4)-C(3)-O(3)
104.6(7)
H(9A)-C(9)-H(9B)
109.5
C(11)-O(12)
1.347(7)
C(4)-C(3)-C(2)
104.5(5)
C(8)-C(9)-H(9C)
109.5
O(12)-C(13)
1.472(7)
O(3)-C(3)-C(2)
110.5(5)
H(9A)-C(9)-H(9C)
109.5
C(13)-C(14)
1.512(9)
C(4)-C(3)-H(3A)
112.2
H(9B)-C(9)-H(9C)
109.5
229
Appendix B
O(11)-C(11)-O(12)
126.0(5)
H(14A)-C(14)-H(14C)
109.5
O(11)-C(11)-N(1)
123.3(6)
H(14B)-C(14)-H(14C)
109.5
O(12)-C(11)-N(1)
110.7(5)
C(13)-C(15)-H(15A)
109.5
C(11)-O(12)-C(13)
121.8(5)
C(13)-C(15)-H(15B)
109.5
O(12)-C(13)-C(14)
109.3(5)
H(15A)-C(15)-H(15B)
109.5
O(12)-C(13)-C(16)
110.8(5)
C(13)-C(15)-H(15C)
109.5
C(14)-C(13)-C(16)
112.8(6)
H(15A)-C(15)-H(15C)
109.5
O(12)-C(13)-C(15)
102.2(5)
H(15B)-C(15)-H(15C)
109.5
C(14)-C(13)-C(15)
110.4(6)
C(13)-C(16)-H(16A)
109.5
C(16)-C(13)-C(15)
110.8(5)
C(13)-C(16)-H(16B)
109.5
C(13)-C(14)-H(14A)
109.5
H(16A)-C(16)-H(16B)
109.5
C(13)-C(14)-H(14B)
109.5
C(13)-C(16)-H(16C)
109.5
H(14A)-C(14)-H(14B)
109.5
H(16A)-C(16)-H(16C)
109.5
C(13)-C(14)-H(14C)
109.5
H(16B)-C(16)-H(16C)
109.5
Table 23. Torsion angles [°] for X81218.
C(6)-C(1)-N(1)-C(11)
-58.7(7)
C(2)-C(1)-C(6)-O(6)
31.5(8)
C(2)-C(1)-N(1)-C(11)
67.6(7)
C(5)-C(1)-C(6)-O(6)
-82.0(7)
C(5)-C(1)-N(1)-C(11)
-175.5(5)
N(1)-C(1)-C(6)-O(7)
-25.1(7)
N(1)-C(1)-C(2)-C(3)
110.1(5)
C(2)-C(1)-C(6)-O(7)
-152.8(5)
C(6)-C(1)-C(2)-C(3)
-123.4(5)
C(5)-C(1)-C(6)-O(7)
93.7(6)
C(5)-C(1)-C(2)-C(3)
-9.3(6)
O(6)-C(6)-O(7)-C(8)
-1.3(8)
C(1)-C(2)-C(3)-C(4)
31.9(7)
C(1)-C(6)-O(7)-C(8)
-177.0(5)
C(1)-C(2)-C(3)-O(3)
-80.1(6)
C(6)-O(7)-C(8)-C(9)
-167.8(5)
O(3)-C(3)-C(4)-C(5)
73.4(6)
C(1)-N(1)-C(11)-O(11)
-10.9(8)
C(2)-C(3)-C(4)-C(5)
-42.8(7)
C(1)-N(1)-C(11)-O(12)
170.2(5)
C(3)-C(4)-C(5)-C(1)
37.0(7)
O(11)-C(11)-O(12)-C(13)
-0.9(9)
N(1)-C(1)-C(5)-C(4)
-138.6(5)
N(1)-C(11)-O(12)-C(13)
178.0(5)
C(6)-C(1)-C(5)-C(4)
101.3(6)
C(11)-O(12)-C(13)-C(14)
-68.1(7)
C(2)-C(1)-C(5)-C(4)
-16.4(6)
C(11)-O(12)-C(13)-C(16)
56.9(7)
N(1)-C(1)-C(6)-O(6)
159.2(6)
C(11)-O(12)-C(13)-C(15)
174.9(5)
230
Appendix B
Table 24. Hydrogen bonds for X81218 [Å and °].
______________________________________________________________________
D-H...A
d(D-H)
d(H...A)
d(D...A)
<(DHA)
______________________________________________________________________
O(3)-H(3)...O(11)#1
0.89(2)
1.93(2)
2.821(6)
172(6)
N(1)-H(1)...O(3)#2
0.89(2)
2.07(2)
2.960(6)
170(6)
______________________________________________________________________
Symmetry transformations used to generate equivalent atoms:
#1 x,-y+1/2,z+1/2
#2 -x+1,-y,-z+2
231
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