Abstract
The thesis entitled “Design and Synthesis of Carbo-β-peptides from
Monosaccharides” is divided into two chapters.
Chapter I: Novel C-linked Carbo-β-peptides from Mannose derived
Carbo--Amino Acids (-Caas)
This chapter is dealt with the synthesis of new carbo-β-peptides, and their use in the
design and synthesis of super secondary structure helix-turn-helix (HTH), synthesis of
bifunctional amino acids, water soluble carbo-β-peptides and cyclic carbo-β-peptides.
De Novo Design and Synthesis of Helix-Turn-Helix Structure Derived
From C-Linked Carbo--Amino Acids
Peptides and proteins are materials and molecular devices that adopt specific
compact folded and organized structures for performing diverse functions.1 The
formation of such tertiary and quaternary structures, in turn results from the assembly of
stable secondary structures such as helices, sheets and turns. The development of nonnatural peptides2,3 with compact and specific conformation is of considerable interest to
understand the folding and stabilization, besides designing new materials with specific
biological functions. Inspite of rapid studies made in the development and design of
‘foldamers’2,4 from non-natural oligomers, mostly using -amino acids, which exhibit a
variety of helical and other secondary structures, generating tertiary structures has so far
remained a serious challenge.
The helix-turn-helix (HTH) motif,5 a tertiary structure composed of two helices
separated by a turn motif, is one of the simplest functional assembly and has been
implicated in various important functions in DNA binding proteins. However, attempts to
obtain such a structural motif using - or -amino acids have so far not been successful.
Our earlier work on the oligomers derived from carbo-β-amino acids6 (Caas) generated
novel and robust helical structures such as 12/10-helices (right- handed),7 12/10-helices
(left-handed),8 while heterogeneous backbone peptides from L-Ala and -Caa resulted in
hitherto unknown 9/11-helices.9 ‘Alternating chirality’7,10 of the epimeric Caas (S and R)
derived from D-xylose, was efficiently used as the design control to successfully identify
the mixed helical patterns in as short as tri- and tetrapeptides.
Hence, in the present work it was proposed to ‘de novo’ synthesize HTH motif by
the assembly of short peptides with robust helices, derived from -Caa, utilizing D-Pro1
Abstract
Gly as a turn motif.11,12 As first step in this direction, it was envisaged to synthesize the
short peptide helices from Caa monomers. In our earlier studies the Caas used were
having D-xylose side chains in the peptide structures. In the present study, new Caas
from D-mannose, having a D-lyxo-furanoside side chain, which are structurally different
from the D-xylose side chain as shown in Figure 1, are designed.
O
O
R1 R2
H3CO
C
C
O
H3CO
C1 O
C4
R1
C
C
H3CO
O
O
C2
O
1 (S)-Caa(x) R1 = H, R2 = NHBoc
2 (R)-Caa(x) R1 = NHBoc, R2 = H
OCH3
C1
C
C
C3
C2
R2
O
10 (S)-Caa(l) R1 = H, R2 = NHBoc
14 (R)-Caa(l) R1 = NHBoc, R2 = H
Figure 1: Caa(x) and Caa(l) represents epimeric C-linked carbo-β-amino acids of D-xylose
and D-lyxose side chains with respect to amine center S and R.
The main idea behind the preparation of such new Caas was to observe the
impact of the different furanoside side chains, on the synthesis of carbo--peptides and
their helix forming capability and robustness of the thus derived helices. Hence, the new
Caas 10 and 14 were prepared from D-mannose.
Accordingly, commercially available D-mannose was treated with acetone,
methanol and catalytic amount of H2SO4 (Scheme 1) at reflux for 12 h to furnish 3 in
72% yield. Hydrolysis of 3 with 60% aq. AcOH at room temperature for 12 h (Scheme 1)
afforded the diol 4 in 90% yield. Oxidative cleavage of diol in 4 with NaIO4 in the
presence of saturated aqueous NaHCO3 solution in CH2Cl2 at below 20 C for 5 h gave
aldehyde
5
in
82%
yield
which
on
subsequent
Wittig
olefination
with
(methoxycarbonylmethelene)triphenyl phosphorane in benzene at reflux for 5 h gave
α,β-unsaturated ester 6 in 81% yield.
Scheme 1
O
+
D-Mannose
HO
O
O
OCH3
60% aq. acetic acid
acetone, H
MeOH, reflux, 12 h
O
12 h
O
O
O
3
O
OHC
OCH3
NaIO 4
aq. sat. NaHCO3
CH2Cl2, 5 h
O
O
O
HO
Ph3P=CHCOOCH3
O
H3CO
O
6
2
O
4
Benzene, reflux, 5 h
5
OCH3
OCH3
O
Abstract
Reaction of Michael acceptor 6 with benzyl amine (2.5 equivalents)13 at room
temperature for instance gave a separable mixture of epimeric carbo-β-amino acid esters
7 (39%) and 8 (24%) (Scheme 2) respectively with ‘S’ and ‘R’ configuration at the amine
stereocentre.
Scheme 2
O
O
O
H3CO
OCH3
BnNH2
O
NHBn
O
H3CO
OCH3
H3CO
NHBn
O
OCH3
+
O
O
O
6
O
O
O
8
7
The esters 7 and 8 (Schemes 3 and 4) were subjected to hydrogenolysis in the
presence of 10% Pd-C in methanol under hydrogen atmosphere to give 9 and 13
respectively. The amines 9 and 13 were treated with (Boc)2O and Et3N in THF to result
in 10 (92%) and 14 (89%) respectively.
Scheme 3
O
NHBn
O
H3CO
O
O
OCH3
10% Pd-C
NH2
O
H3CO
methanol, 12 h
O
THF, 2 h
O
7
4N NaOH
H3CO
O
9
O
O
(Boc)2O, Et3N
OCH3
10
OCH3
methanol
NHBoc
O
O
HO
NHBoc
O
O
11
OCH3
O
O
H3CO
TFA
O
NH2.CF3COOH
O
OCH
3
CH2Cl 2
12
O
O
Further, 10 and 14 on hydrolysis with NaOH in methanol gave carbo-β-amino
acids 11 (94%) and 15 (92%) respectively. Likewise, compounds 10 and 14 were
exposed to CF3COOH (TFA) in CH2Cl2 to give free amine salts 12 and 16.
3
Abstract
Scheme 4
O
NHBn
O
O
H3CO
O
OCH3
10% Pd-C
NH 2
O
H3CO
methanol, 12 h
O
THF, 2 h
13 O
8
O
4N NaOH
O
H3CO
NHBoc
O
HO
15
OCH3
O
14
H3CO
TFA
O
NHBoc
O
Methanol
O
O
(Boc)2O, Et3N
OCH3
O
OCH3
O
NH2.CF3COOH
O
OCH
3
CH2Cl 2
16
O
O
Having prepared the requisite monomeric Caas, it was next aimed at the synthesis
of peptides by sequential coupling with the design concept of ‘alternating chirality’.
Accordingly, the dipeptide 17, tripeptide 18, tetrapeptide 19 and hexapeptide 20 were
prepared from C-linked carbo-β3-amino acids (Caa) 10 and 14 by adopting segment
condensation method by using standard peptide coupling conditions in the presence of
water soluble 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDCI),
1-hydroxy-1H-benzotriazole (HOBt) and DIPEA as a base in dry CH2Cl2. NMR, CD and
MD studies clearly indicated that, carbo-β-peptides 19 and 20 folded in to right-handed
10/12 mixed helical pattern.
H
N
H
N
H
N
O
O
O
O
O
O O
O
OCH3
O
O
O
O
O
OCH3
O
OCH3
OCH3
18
H
N
H
N
O
O
O
O
O
OCH3
17: n = 1 dipeptide
19: n = 2 tetrapeptide
20: n = 3 hexapeptide
O O
O
O
OCH3 O
OCH3
4
n
Abstract
Likewise, dipeptide 21, tripeptide 22, tetrapeptide 23 and hexapeptide 24 were
prepared with C-linked carbo-β3-amino acid (Caa) 14 and 10 by adopting segment
condensation method by using standard peptide coupling conditions. NMR, CD and MD
studies clearly indicated that carbo-β-peptides 22, 23 and 24 are folding into right-handed
12/10- mixed helical patterns.
12
10
O
H
N
H
N
H
N
O
O
O
O
O O
OCH3
O
O
O
O
O
OCH3
O
OCH3
O
OCH3
22
H
N
H
N
O
O
O
O
O
OCH3
21: n = 1 dipeptide
23: n = 2 tetrapeptide
24: n = 3 hexapeptide
O O
O
O
OCH3 O
OCH3
n
Having successfully utilized the new Caas with D-lyx side chain and observed the
formation of robust helices in as short as a tri-and tetrapeptides, it was next aimed at the
de novo design of peptides with a Helix-Turn-Helix (HTH) structure making use of these
short helical peptides 19 and 22.
Thus, in the present study on the do novo design and synthesis of HTH, the carbo
-peptides 19 and 22 would be utilized as helical counterparts, while D-Pro-Gly would
participate in ‘turn motif’.12,13 Further, it was proposed to flank the D-Pro-Gly dipeptide
with two units of -hGly (hG) on both the sides (C-and N-termini) to form a tetrapeptide
-hGly-D-Pro-Gly--hGly
(hGPGhG),
wherein,
the
-hGly
provides
required
conformational flexibility to the mixed helices that would be interlinked with the turn
motif, eventually to form HTH tertiary structure.
Accordingly,
tetrapeptide
25
-hGly-D-Pro-Gly--hGly
(hGPGhG)
and
heptapeptide 26 was synthesized from known dipeptide D-Pro-Gly, -hGly and tripeptide
22 by using standard peptide coupling conditions. NMR, and CD studies suggesting it to
be a type II' -turn around Pro-Gly in tetrapeptide hGPGhG 25 and heptapeptide 26 is
folding into super secondary structure helix-turn.
5
Abstract
H
O
H
N
N
O
N
H
O
O
O
HN
25
10
OCH3
O
(A)
(B)
Figure 2: A) Circular dichroism spectrum of 26 in CH3OH at concentration of 0.2 mM. B)
Superimposed 15 minimum energy structures of 26 (sugars are replaced with methyl groups and
protons have been removed for clarity)
The presence of a super secondary structure of the helix-turn family in
heptapeptide 26 is noteworthy and provides credence to our desire of generating a helixturn-helix tertiary structure involving a minimal number of amino acid residues, by
piecing the three structural motifs together by amide bonds.
Further, decapeptide 27 was prepared with heptapeptide 26 and tripeptide 22 by
standard peptide coupling conditions. The 1H NMR spectrum of decapeptide 27 showed
presence of cis- and trans isomers (1:9).
12
O
10
H
N
H
N
O
O O
O
O
O
H
N
H
N
O
OCH3 O
O
O
O
12
O
OCH3
O
O
11
OCH3
H3CO
O
O
H 12
N
H
N
O
O
H3CO
H
O
HN
15
10
N
O
O
O
H
N
O
O
O
O
O H CO O H3CO
3
27
6
NH
O
10
Abstract
From the NMR studies it was amply evident that the 10-membered turn that was
observed in 26 is disrupted in the decapeptide 27. Instead of the expected HTH structure,
the turn motif has shown coupling constants and long rang nOe’s of 11- and 15membered H-bondings, whereby there was a distraction of 10-membered turn. It was felt
that presence of (R)-Caa unit of tripeptide at the C-terminus of decapeptide, is causing the
damage to the turn.
To avoid such fallout and to protect the turn structure, in the new design it was
envisioned to have a -peptide with an N-terminus (S)-Caa monomer, coupled with the
C-terminus -hGly in the heptapeptide.
Accordingly, coupling of 26 and 19 under the standard coupling conditions gave
undecapeptide 28 (Scheme 24) in 58% yield. 1H NMR spectrum of 28 showed the
presence of cis- and trans-isomers in 1:3 ratio. NMR, CD and MD studies clearly
indicated that undecapeptide 28 is folding into super secondary structure helix-turn-helix
with 12/10- and 12/10/12/10-helices and a type II' -turn.
(A)
(B)
Figure 3: A) Circular dichroism spectrum of 28 in CH3OH at concentration of 0.2 mM. B)
Superimposed 15 minimum energy structures of 28 (sugars are replaced with methyl groups and
protons have been removed for clarity)
7
Abstract
The helix-turn-helix motif has been thus generated by a de novo synthesis with as
small as 11 amino acids residues. The robustness of the oligomers obtained from Caa and
the classical D-Pro-Gly turn motif retain structure of the individual secondary structure to
generate a very well defined tertiary structure with the helices orthogonal to each other.
Synthesis of Bifunctional C-Linked Carbo--Amino Acids: Design of
Water soluble Carbo--peptides for Biological applications
The availability of conformationally structured, water soluble -peptide
sequences, together with their known stability and resistance to enzymatic degradation,14
has led to some early observations regarding the biological activity of this oligomer class.
Seebach and co-workers designed cyclic‚ -peptides containing only four residues that
bind to the somatostatin receptor with micromolar affinity.15 Hexa-, hepta-, and
nonameric‚ -peptides carrying one to seven water-solubilizing groups have also been
shown to be inhibitors of small intestinal cholesterol and fat adsorption.16 DeGrado17 and
Gellman18 used similar designs in mimicking natural membrane-active peptide toxins and
antibiotics. A strong correlation exists among the sequences studied between the helical
content and antibacterial activity. The design principal used by Gellman for water soluble
-peptides with helical structure, involved having charged hydrophilic residues on one
face of the helix and hydrophobic residue on its other face, providing amphiphilicity to
the helix.
The preceding section described the synthesis of a new class of carbo-β-peptides
from C-linked carbo-β-amino acids 10 and 14. It has also been demonstrated the use of
the concept of ‘alternating chirality’ for successful synthesis of carbo-β-peptides with
robust mixed helical structures such as 10/12- and 12/10-helical patterns in organic
solvents. These peptides, however, are not soluble in water. In the present design, to
probe 10/12- and 12/10-helical structures in water, we required water soluble carbo animo acid residues with a proper conformational constraint, as a compliment to -amino
acids Caa 10 and Caa 14. Therefore new bifunctional amino acids such as 40 and 43 were
designed to help in the water solubility through the additional amine group. We have
therefore incorporated the thus prepared a bifunctional Caa residue after every two Caa
8
Abstract
residues, yet preserving the concept of alternating chirality, in conformity with the design
for the mixed helices.
Accordingly, known 2, 3; 5, 6 di-O-isopropyledine D-mannose 29, obtained from
commercially available D-mannose, was treated with trimethylsulfoxonuim iodide in the
presence of potassium tert-butoxide in dry DMSO at room temperature for 3 h to afford
mannose methanol 30 in 86% yield (Scheme 25). Conventional tosylation of primary
alcohol 30 with p-TsCl, Et3N and catalytic DMAP in CH2Cl2 at room temperature for 3 h
gave 31 in 83% yield. Tosylate 31 was further treated with NaN3 in DMF at 80 C for 8 h
to afford azide 32 in 65% yield (Scheme 3). Reduction of 32 with triphenylphosphene in
methanol followed by treatment with (Boc)2O afforded 33 in 76% yield.
Scheme 5
O
D-Mannose
O
O
O
cat. H2SO4, Dry CuSO4
O
Acetone, RT, 24 h
O
OH
O
TMSOI, t-BuOK
DMSO, RT, 3 h
O
OH TsCl, Et N
3
O
29
O
O
O
OTs
O
NaN3,DMF
O
O
O
80 0C, 8 h
O
O
O
NHBoc Ph P=CHCOOCH
3
3
NaIO4
aq. sat. NaHCO3
CH2Cl2, 5 h
O
Benzene, reflux, 5h
O
35
34
PTSA
CH3OH, H2O, 8 h
O
33
O
OHC
NHBoc
O
CH3OH, 6 h
NHBoc
32
O
HO
O
O
N3 Ph3P, (Boc)2O
O
31
HO
30
O
O
CH2Cl2, 3 h
O
O
O
H3CO
NHBoc
O
O
36
Further, hydrolysis of 5, 6-acetonide in 33, on reaction with PTSA in MeOH and
water at room temperature for 8 h (Scheme 26) afforded the required diol 33 in 87%
yield. Oxidative cleavage of diol in 33 with NaIO4 in the presence of saturated aqueous
NaHCO3 solution in CH2Cl2 at below 20 C for 5 h gave aldehyde 35 in 82% yield, which
on
subsequent
Wittig
olefination
with
(methoxycarbonylmethylene)triphenyl
phosphorane in benzene at reflux for 5 h gave α,β-unsaturated ester 36 in 81% yield.
Michael addition of α, β-unsaturated ester 36 with benzyl amine (2.5 equivalents)
at room temperature gave a mixture of carbo-β-amino acid esters 37 (41%) and 38 (25%)
(Scheme 6).
9
Abstract
Scheme 6
O
O
H3CO
NHBoc
O
H3CO
BnNH 2
H3CO
NHBoc
NHBn
O
NHBoc
+
12 h, RT
O
O
NHBn
O
O
O
36
O
O
O
38
37
The esters 37 and 38 were subjected to hydrogenolysis in the presence of 10% PdC in methanol under hydrogen atmosphere to give 39 and 42 (Scheme 7) respectively.
The amines 39 and 42 were treated with Cbz-Cl and DIPEA in CH2Cl2 to result in 40
(82%) and 43 (86%) respectively. Further, 40 and 43 on hydrolysis with NaOH in
methanol gave carbo-β-amino acids 41 (86%) and 44 (89%).
Scheme 7
O
O
NHBn
O
H3CO
NHBoc
O
NH2
O
H3CO
10% Pd-C
NHBoc Cbz-Cl, DIPEA
methanol, 12 h
O
O
39
37
O
O
NHCbz
O
H3CO
NHBoc
O
HO
4N NaOH
NHCbz
O
NHBoc
O
methanol
O
O
NHBn
O
H3CO
NHBoc
O
10% Pd-C
O
NHBoc Cbz-Cl, DIPEA
methanol, 12 h
O
H3CO
O
CH2Cl, 2 h
O
42
O
NHCbz
O
NHBoc
O
NH2
H3CO
38
O
O
41
40
O
CH2Cl, 2 h
O
O
4N NaOH
methanol
HO
NHCbz
O
NHBoc
O
O
44
43
Having successfully prepared the bifunctional C-linked carbo--amino acids 40
and 43, converted them into requisite acids 41 and 44; and amines 39 and 42 required
peptide coupling, the work for the synthesis of water soluble peptides was initiated.
10
Abstract
O
H3CO
O
NHCbz
O
H3CO
NHBoc
O
NHCbz
O
NHBoc
O
O
40
O
43
Figure 5: Bifunctional carbo--amino acids
Accordingly, the hexapeptides 45 and 46 were prepared from bifunctional Clinked carbo--amino acids 40 and 43, and dipeptides 17 and 21 by adopting segment
condensation method by using standard peptide coupling conditions. NMR, CD and MD
studies clearly indicated that, carbo-β-peptide 45 folded in a right-handed 10/12- mixed
helical pattern and 46 folded in a right-handed 12/10- mixed helical pattern.
12
10
H
N
O
O
H
N
O
O
O
H
N
O
O
45
12
O
H
N
O
O
O
O
BocHN
O
O
OCH3
O
O
O
O
H
N
O
O
O
O
OCH3
O O
O
H
N
O O
O
H
N
O
OCH3
OCH3
12
H
N
O
O
OCH3 O
BocHN
10
H
N
O
O
O
OCH3
BocHN
O
O
O
O
10
H
N
H
N
O O
O
12
10
OCH3 O
BocHN
O
10
H
N
O O
O
OCH3
O
O
O
O
OCH3
O
OCH3
46
Removal of the acid and amine protecting groups (-OMe, Cbz, Boc) using basic
hydrolysis with NaOH in methanol, 10% Pd/C in methanol and trifluoracetic acid
resulted in water-soluble peptides, which were further confirmed by mass spectral
analysis.
The CD spectra of all the peptides in methanol display distinct signatures of a
right handed mixed helix with a maxima at about 203 nM, with very little excursion in
the negative molar ellipticity, as expected in mixed 10/12-helical structure. The intensity
of this absorption is drastically reduced in aqueous buffer solution (Figure 4). This
suggests that the 12/10-helical conformation of these peptides is far less stable in water
than methanol.
11
Abstract
Although the CD-spectroscopic analysis shows that these peptides are only
partially folded in aqueous media, still it showed antibacterial activity against a variety of
bacterial strains. Studies on both - and -peptides have established that a preorganized
amphiphilic structure is not a prerequisite for antimicrobial activity, as no straightforward
relation between helical stability and antimicrobial activity exists.
A
B
C
Figure 4: Circular dichroism spectrum of 45. The peptides were measured as its di-and tris-TFA
salt, in methanol (A and B) and aqueous media (C) at concentration of 0.2 mM.
Synthesis of Self-assembying Cyclic Carbo-β-peptides from Carbo-amino acids:
The cyclic peptides that stack on top of one another forming nanotube-like
structures have been the subject of many investigations due to their utility in chemical,
biological and materials science.19 These cyclic oligomers, composed of carbohydrate
units, have been used in the areas of drug delivery, asymmetric synthesis and also as
enzymemimetics.20 Vancomycin, a representative of the glycopeptide class of
antibiotics,21 was isolated in the 1950s and used for over four decades as a weapon of last
resort to combat bacterial disease. The cyclic peptides adopt antiproliferative activity by
inhibiting the growth of several cancer cell lines22 and cyclic peptides form tublar ionconducting channels in phospholipid layers.23 Kessler et al.24 prepared cyclic peptides
using constrained sugar amino acid and β-hGly alternatively, which showed symmetry in
acetonitrile. Kessler at al.25 reported sugar amino acid (SAA) somatostatin analogues,
which possess antiproliferative and apoptotic activity against both multidrug-resistant and
drug-sensitive hepatoma carcinoma cells. Recently several groups26 reported cyclic
homooligomers from furanoid and pyranoid sugar amino acids.
12
Abstract
After successful preparation of linear C-linked carbo-β3-peptides with secondary
structures, synthesis of cyclic oligomers from linear peptides is achieved. Accordingly,
dipeptide 47, tripeptide 48 and tetrapeptide 49 were prepared with epimeric C-linked
carbo-β3-amino acid (Caa) 10, by adopting segment condensation method.
H
N
O
OCH3
47: n = 2 dipeptide
48: n = 3 tripeptide
49: n = 4 tetrapeptide
O O
O
O
O
OCH3
n
For the preparation of cyclic peptides, pentafluorophenyl active ester method was
used for cyclisation.27 Cyclic carbo-β3-peptides 50 and 51 were prepared from their open
chain oligomers and cyclic mixed β-peptide 52 derived from bifunctional C-linked carboβ3-amino acid 40 with dipeptide 47.
The analysis of cyclic peptides 50-52 by NMR, IR and TEM indicated that these
cyclic carbo-β3-peptides stack on top of one another forming nanotube-like structures.
H3CO
H3CO
O
O
O
O
O HN
NH
OCH 3
HN
O
H3CO
NH
O
O
HN
O
O
O
O
O
O
O
O
O HN
O
O
O
O
H3CO
50
NHBoc
O
52
O
O
OCH 3
O
O
O
H3CO
O
H
N
O
O
O
HN
O
O
NH
O
O
H3CO
O
N
H
O
O
OCH 3
O
O
51
Figure 5: Transmission Electron Microscopy (TEM) images of 51 nanotubes (scale bar, 250 nm)
13
Abstract
O
O
O
O
R
N N
H H
N
H
N
H
R
O
O
O
O
N
H
OCH 3
R=
N N
H H
O
O
R
O
O
O
N
H
R
O
O
R
N
H
N N
H H
N
H
R
Figure 6: Cyclic carbo-β3-peptide 51 subunits are represented in a self-assembled tubular
configuration and extensive inter-subunit hydrogen bonding (For clarity most side chains are
omitted.)
Formation of nanotubes in the present work is concentration and time dependent.
Except cyclic carbo-β3-peptide 52 all the peptides have shown symmetry. The TEM study
indicated the formation of sharp and long nanotubes for 50, while TEM study on 52 in
methanol solution, clearly indicated the absence of nanotube formation. Eventhough FTIR studies strongly indicated intermolecular hydrogen bonding, TEM study in methanol
indicated no nanotubes in 52.
Thus, in the present study, a) a new class of cyclic carbo-β-peptides are prepared
from C-linked carbo-β-amino acids (Caa) derived from D-mannose, b) formation of
nanotubes in solution is time dependent, c) nanotubes observed in tripeptide 50 are thin
and longer as compared to others.
Chapter II: Split behaviour of -hGly and -Caa (R): Control on the
Conformational behaviour in Carbo-β-peptides
This chapter is dealt with the synthesis of mixed peptides from -hGly and -Caas and
the role of split behaviour of -hGly and R-Caa in robust helix formation
Earlier it was observed that, the carbo-β-peptides derived by the concept of
‘alternating chirality’7 resulted in the peptides with robust helical patterns while under the
influence of (R)- and (S)-Caas, the mixed peptides derived from alternating -Caa, hGly gave robust structures,8 wherein, -hGly participated in a 10-membered H-bonding.
However, the conformation in homo-oligomeric carbo-β-peptides from (R)-Caa(x) could
not be well defined by NMR due to overlapping of the chemical signals, eventhough the
CD spectrum indicated the presence of secondary structure.
14
Abstract
Based on the above observations, a new design for the synthesis of homooligomeric peptides was conceived, wherein, two (R)-Caa monomers derived both from
D-xylose and D-mannose, which differ in the spatial arrangement in the side chains, were
envisaged to avoid the overlapping of the side chains in the helix formation.
Thus in the new design, it was planned to synthesize the carbo-β-peptides using
the R-Caa(x) 2 and R-Caa(l) 14 alternatingly. The monomers 2 and 14 (Figure 6), with
homochiral geometry at the amine center (‘R’ amine centers respectively), were
successfully utilized for the synthesis of new carbo-β-peptides.
O
O
NHBoc
O
O
H3CO
Ca
Cb
C4
C1
H3CO
O
C3
C2
H3CO
NHBoc
O
Ca
Cb
C4
C3
O
(R)-Caa(x) 2
OCH3
C1
C2
O
(R)-Caa (l) 14
Figure 6 : R-Caa(x) and R-Caa(l) represents epimeric C-linked carbo-ß-amino acids
of D-xylose and D-lyxose
Accordingly, dipeptide 53, tetrapeptide 54 and hexapeptide 55 were prepared with
C-linked carbo-β3-amino acids (Caa) 2 and 14 ‘alternatingly’ by adopting segment
condensation method by using standard peptide coupling conditions.
H
N
H
N
O
O
H3CO
O
O
O
OCH3
O
53: n = 1 dipeptide
54: n = 2 tetrapeptide
55: n = 3 hexapeptide
O
O
O
O
OCH3
n
In the 1H NMR of tetrapeptide 54 and hexapeptide 55 in CDCl3, due to severe
overlap in the CH and CH region, most of the specific assignments could not be made.
Several of the amide resonances appeared at low field ( > 7 .5 ppm) and solvent titration
studies indicated that many of the amide protons are participating in hydrogen bonding.
Eventhough the CD spectrum of hexapeptide 55 indicated the presence of a secondary
structure, it could not be defined properly. Hence it was planed to provide conformational
flexibility by use of β-hGly alternatingly with ‘RR’-dipeptide repeat.
15
Abstract
Accordingly, the peptides 56-60 were prepared with 53, 2 and β-hGly, using
standard peptide coupling conditions. NMR, CD and MD studies clearly indicated that
carbo-β-peptides 57, 58 and 59 fold into right-handed 10/12-mixed helical pattern, and 60
was folding into right-handed 12/10-mixed helical pattern.
H
N
H
N
O
O
O
O
O
O O
O
O
H
N
O
OCH3
H
N
O
O
H
N
O
O
O
58: n = 1 tetrapeptide
59: n = 2 hexapeptide
H3CO
O
O
OCH3
O
O
H3CO
O
56: n = 1 tripeptide
57: n = 2 hexapeptide
n
H
N
O
O
OCH3
O
H3CO
O
H
N
OCH3
O
O
n
10
12
H
N
O
H
N
O
O
O
O
O
H
N
O
OCH3
10
H
N
O
O
H3CO
O
H3CO
12
O
O
H
N
O
OCH3
O
O
H3CO
O
O
O
O
60
It is rather interesting that in the mixed peptides, placement of a monomer like hGly, which does not have any substituent on either of the carbons, made a significant
change and a secondary structure was observed in the oligomers having (R)-Caas.
Further, it was also observed that in peptides 58 and 59, the N-terminus (R)-Caa and in 57
first and fourth (R)-Caa residue participated in H-bonding like (S)-Caa with their amide
participating in 10-membered H-bonding. In the peptide 60 second (R)-Caa participated
in 10-membered H-bonding like (S)-Caa. Thus, in the above peptides (R)-Caa has shown
‘split behaviour’ in its H-bonding participation.
This study on homo-oligomers, along with -hGly, and (R)-Caa was observed to
show ‘split behaviour’ and proved as an important tool to design the mixed helical
structures, even in the homo-oligomers. Based on the above results from peptide 57,
having R, R-homo-dipeptide repeat with alternating -hGly giving a robust helix, it was
felt to observe the impact of -hGly with RS-and SR-hetero-dipeptide repeats.
16
Abstract
Accordingly, the peptides 61-66 were prepared with C-linked carbo-β3-amino
acids 1, 2 and β-hGly, using standard peptide coupling conditions.
H
N
H
N
O
O
O
H3CO
O
H3CO
O
O
O
O
O
O
H
N
O
63: n = 1 tripeptide
64: n = 2 hexapeptide
O
O
n
12
10
H
N
O
H
N
O
O
H3CO
H3CO
O
O
10
H
N
O
O
O
O
O
O
H
N
O
O
H3CO
O
OCH3
12
H
N
H3CO
H
N
O
O
10
O
n
O
O
H3CO
H3CO
61: n = 1 tripeptide
62: n = 2 hexapeptide
O
H
N
O
OCH3
O
H
N
O
H
N
O
O
O
O
O
OCH3
O
65
10
12
H
N
O
O
H3CO
O
H
N
O
O
H3CO
O
O
12
H
N
O
O
O
10
H
N
O
H3CO
O
66
H
N
O
O
H3CO
O
O
H
N
O
O
OCH3
O
O
NMR, CD and MD studies clearly indicated that the peptides 62 and 64 have not
shown any secondary structures, while 65 and 66 with altered design formed well-defined
12/10- and 10/12-mixed helices, due to the ‘split’ behaviour of -hGly. In peptide 65, hGly participated in 10-membered H-bonding, while in 66 it preferred to have 12membered H-bonding. In the peptides 62 and 64, -hGly was between one (S)- and one
(R)-monomer, where -hGly was in ‘confused’ situation, while in the other design, where
-hGly was between two (S) residues and participated in 12-membered H-bonding while,
between two (R)-residues it participated in 10-membered H-bonding.
17
Abstract
Based on the above results from peptides 65 and 66, having (R,S)- and (S,R)hetero-dipeptide repeats with alternating -hGly giving a robust helix due to the split
behaviour of -hGly, it was felt to observe the impact of -hGly with (R,S,R)- and
(S,R,S)-hetero-tripeptide repeats.
Accordingly, the heptapeptides 67 and 68 were prepared using the tripeptides 18
and 22 and β-hGly. NMR, CD and MD studies clearly indicated that carbo-β-peptides 67
and 68 folding to right-handed 12/10- and 10/12-mixed helical patterns respectively.
O
H
N
O
O
O
O
O
OCH3 O
10
H
N
O
O
O O
O
OCH3 O
O
O
O
OCH3 O
O
H
N
OCH3 O
O O
O
67
O
O
O
OCH3
O O
OCH3
O
OCH3
10
H
N
H
N
O
O O
O
H
N
O
OCH3
O
OCH3
O O
O
O
O
OCH3
O
10
O
O
OCH3
12
H
N
H
N
O
O
OCH3
10
O O
O
H
N
H
N
H
N
12
O
O
H
N
H
N
O
O
12
10
H
N
O
12
10
12
O
OCH3 O
OCH3
68
The split behaviour of -hGly was also observed in heptapeptides 67 and 68 with
altered design formed well-defined 12/10- and 10/12-mixed helices. In peptide 67, hGly participated in 10-membered H-bonding, while in 68 it preferred to have 12membered H-bonding.
Thus, in the present study a) -hGly and (R)-Caa have shown ‘split behaviour’
which was responsible for the formation of robust mixed helical patterns, b) a
homologous R,R-dipeptide repeat with -hGly gave peptide 57, which a well defined
10/12-helix, wherein, the N-terminal (R)-Caa participated in 10-membered H-bonding.
The ‘spilt behaviour’ of (R)-Caa thus was responsible in the peptides 57, 58 and 59 to
result in the mixed helices, c) ‘split behaviour’ of -hGly was observed in peptides made
from di- and tripeptide repeats with alternating chirality. In peptides 65 and 67, -hGly
participated in 10-membered H-bonding, while in 66 and 68 it preferred to have 12membered H-bonding. Thus, -hGly, with no substitution and high flexibility behaved
very well under the influence of Caa and formed robust mixed helices due to ‘split
18
Abstract
behaviour’. Further it was observed that, -hGly in between two different Caa monomers
such as (R)- and (S)-Caa, resulted in ‘mismatching’ peptides with no helical structures,
while, in between two similar monomers, such as
(S)-Caa--hGly-(S)-Caa, -hGly
participated in 12-membered H-bonding, while in (R)-Caa--hGly-(R)-Caa has
participated in a 10-membered H-bonding. Thus, this study has very well established the
importance of -hGly in robust helix formation.
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