Development of Novel Polymeric Architectures for Applications in Drug Delivery
and
Studies Towards the Synthesis of Perfect Polymers by Iterative Exponential Growth "Plus"
(IEG+)
by
Angela X. Gao
B.S., University of Michigan (2012)
Submitted to the Department of Chemistry in Partial Fulfillment of the Requirements for the
Degree of
MACHSTT
NSIT"
Master of Science in Chemistry
MASSACHUSETSI6ltE
OF TECHNOLOGY
at the
JUN 3 0 2014
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
LIBRARIES
May 2014
vt~t4&(J4L
i
C 2014 Massachusetts Institute of Technology. All rights reserved
The author hereby grants to MIT permission to reproduce
and to distribute publicly paper and electronic
copies of this thesis document in whole or in part
in any medium now known or hereafter created.
Signature of Author..
Signature redacted
.......................
Depart ment of Chemistry
May 2 9 th 2014
CerifedbySignature
by .............
Certified
r
redacted
r d a te .............................
Jeremiah A. Johnson
Assistant Professor of Chemistry
Thesis Supervisor
Signature redacted
Accepted by ................
........................................
Robert Warren Field
Robert T. Haslam and Bradley Dewey Professor of Chemistry
Director of Graduate Studies
Development of Novel Polymeric Architectures for Applications in Drug Delivery
and
Studies Towards the Synthesis of Perfect Polymers by Iterative Exponential Growth "Plus"
(IEG+)
by
Angela X. Gao
Submitted to the Department of Chemistry
on May 2 9 1h, 2014 in Partial Fulfillment of the
Requirements for the Degree of
Master of Science in Chemistry
ABSTRACT
Polymeric materials are ubiquitous in numerous facets of everyday life, and their
applications will only become increasingly prevalent as the field of polymer science advances.
The first chapter of this thesis describes the use of polymeric nanoparticles to overcome
challenges in traditional drug delivery. Specifically, a series of novel acid-cleavable bisnorbornene crosslinkers were synthesized and evaluated as building blocks for the formation of
acid-degradable brush-arm star polymers (BASPs) via the brush-first ring-opening metathesis
polymerization (ROMP) method. A bis-norbornene acetal structure was identified that, when
employed in conjunction with a poly(ethylene glycol) (PEG) macromonomer, provided highly
controlled BASP formation reactions. Combination of this new crosslinker with a novel acidlabile doxorubicin (DOX)-branch-PEG macromonomer provided BASPs that simultaneously
degrade and release DOX in cell culture. In vitro cell viability studies using HeLa cells
confirmed that these constructs are cytotoxic.
Even though polymeric materials have found widespread use in current times, polymer
science must overcome certain challenges to contend with the needs of next-generation
technologies. In particular, newer polymer applications require the use of macromolecules with
precisely defined structure and degree of polymerization--challenges that synthetic polymer
chemistry has yet to conquer. The second chapter of this thesis describes a novel synthetic
methodology (IEG+) that gives polymers with defined molar mass and sequence using synthetic
procedures that are precise, scalable, and amenable to diversification. The IEG+ method utilized
monomers equipped with orthogonal protecting groups: epoxides and alkynyl silanes. The
epoxide functionality served both as a protecting group and as a masked synthon for alcohols,
which allowed for side-chain functionalization of the IEG+ scaffold. Combing R- and Smonomers afforded complete stereochemical control of the IEG backbone. Oligomers of
unimolecular molar mass and precise chemical structure were successfully prepared.
Thesis Supervisor: Jeremiah A. Johnson
Title: Assistant Professor of Chemistry
I
TABLE OF CONTENTS
Chapter I. Design and Synthesis of Hydrolytically Labile Brush-Arm Star Polymers
(BASPs) for Controlled Doxorubicin Release
Introduction............................................................................................................................
Results and D iscussion ...................................................................................................
Conclusions........................................................................................................................
Experim ental M ethods.......................................................................................................
Spectral D ata......................................................................................................................
3-4
4-9
10 -10
10 - 27
28 - 42
Chapter II: A Novel Family of Perfect Polymers via Iterative Exponential Growth "Plus"
(IEG+)
Introduction........................................................................................................................
Results and D iscussion .................................................................................................
Conclusions........................................................................................................................
Experim ental
....................................................................................................
Spectral D ata ......................................................................................................................
43 - 45
45 - 49
50 -50
50 -68
-85
Chapter III: References
References..........................................................................................................................
2
86
-
87
Chapter I: Design and Synthesis of Hydrolytically Labile Brush-Arm Star Polymers
(BASPs) for Controlled Doxorubicin Release
Introduction: The development of efficient, modular, multicomponent strategies for
polymer nanoparticle (NP) synthesis will facilitate the formulation of complex NP-based
materials for various applications.1- 5 We have focused on the pursuit of highly convergent
synthetic strategies that build multifunctional NPs directly from densely functionalized
monomers with no extraneous formulation steps.6-8 For example, we recently reported the
synthesis of brush-arm star polymer (BASP) NPs via a versatile "brush-first" method that
involves graft-through ring-opening metathesis polymerization (ROMP) of a norborneneterminated macromonomer (MM) followed by in situ crosslinking with a bis-norbornene
derivative (Figure la).9'"0 The remarkable efficiency and functional group tolerance of ROMP
enables the synthesis of diversely functionalized BASPs on the benchtop within hours. We have
used the brush-first ROMP method to synthesize degradable BASPs with various sizes and
corona compositions including multi-drug-loaded BASPs, two- and three-miktoarm BASPs, and
chloride-functional BASPs for subsequent azide exchange and copper-catalyzed azide-alkyne
(CuAAC) "click" chemistry.6 -8
A.
N
N
0
O
N
H
B. yx
1) 1.0 equiv.
Grubbs 3rd,
H
i"
"Pr
Or
2
1
m equiv
PEGMM (EM
0~0
K
fN,
N QX .
brush-arm
0strplmr(AP
crosslinker
Ph
'Pr 3 Pr
2) N equiv
crossinker
0
Si
Ph' 'Ph
a
'Ph
o -- Oj
star polymer (BASP)5
Figure 1. A. Schematic for the brush-first ROMP process. B. Acid-labile crosslinkers studied in this report.
In our continued effort to translate the brush-first method to chemotherapeutic delivery
applications, we sought to develop a crosslinker that would selectively degrade in the mildly
3
acidic tumor microenvironment, which is 0.5-1.0 pH units lower than normal tissue, or in the
end/lysosome, where the pH can be as low as 4-5.11 The inclusion of acid-cleavable functional
groups into nanostructures and polymers is a common strategy for tumor targeted drug
delivery.1 2 We reasoned that the combination of an acid-cleavable crosslinker with an acid-labile
drug-conjugated MM in the brush-first method would represent a highly convergent strategy for
the synthesis of BASPs for pH-controlled drug delivery applications.
Results and Discussion: Silyl ethers are convenient synthons for acid-sensitive drug
delivery systems.
They can be easily prepared via sequential addition of alcohols to
dichlorosilanes and their rates of hydrolysis can be precisely tuned through choice of Si
substituents.
3
We began our study with the synthesis of a panel of silyl ether-based bis-
norbornene derivatives (1-4, Figure 1b). These compounds were prepared in 35-76% yield via
exposure of an exo-norbomene alcohol derivative to the corresponding dichloro-di(alkyl or
phenyl) silane in the presence of NN-diisopropylethylamine (see experimental methods section
for synthetic details).
We screened each of these silyl ether crosslinkers in the context of brush-first ROMP as
follows. First, a norbomene-poly(ethylene glycol) (PEG) macromonomer 9 (PEG-MM, 10 equiv,
Figure la) was exposed to Grubbs
3rd
generation bis-pyridyl catalyst (1 equiv) for 15 min to
generate living PEG bottlebrush polymers with an average degree of polymerization (m) of ten.
Aliquots of this bottlebrush polymer solution were then transferred to a series of empty vials. A
stock solution of the desired crosslinker was slowly added to each of the vials until the desired N
crosslinker equivalents were achieved.
After 2 h, the reactions were quenched with ethyl vinyl ether and directly analyzed by gel
permeation chromatography (GPC, 0.02 M LiBr in NN-dimethylformamide). The molar mass
4
1.0
-
distributions for these BASPs were
-1
---
N=0
----
N =21
N=10
invariably multimodal (e.g., Figure
~0.8.--N=2
C
0
2) with large fractions of uncoupled
0.
CO 0.6
bottlebrush
0.4-
polymers
and
macromonomer. We hypothesize that
D
=
.crosslinkers
1-4
may
undergo
intramolecular cyclization reactions
Z 0.0.
.4
14
15
16
17
18
19
2k)
,
.
Retention Time / mn
Figure 2. GPC analysis (RI) for BASPs with N
crosslinker 2.
=
that
compete
with
the
desired
21
intermolecular crosslinking reactions.
10. 15, and 20 equiv of
In an effort to identify
a crosslinker that would provide uniform BASPs, we turned to acetal-based crosslinker 5 (Figure
lb).
Acetals are also widely used to impart pH-sensitivity to polymers.
We prepared
compound 5 in 13% yield over three steps starting from cis-5-norbornene-exo-2,3-dicarboxylic
anhydride (see SI for details). Acetal-core BASPs with N = 10, 15, and 20 equivalents of 5 were
prepared following the same procedure described above for crosslinkers 1-4. GPC analysis
(Figure 3)
revealed
monomodal
molar mass distributions and very
C
u 5-
-
-
efficient conversion of brush (N= 0, -4black trace) to BASP for N= 10 and
15; the molar mass distribution
broadened for N = 20. As we have
observed
BASPs
previously
constructed
for
-
(D 3
N M / kDa
0
40.0
10
480
15 1,060
20 2,220
c
2(D
related
using
a
7
Molar mass / Da
Figure 3. GPC analysis (differential weight fraction) for ABASPs with N
10, 15, and 20 equiv of crosslinker 5.
5
photocleavable crosslinker9 0",the weight-average molar mass (M,) for these acetal-core
particles increased geometrically with each addition of five equivalents of crosslinker 5.
Encouraged by these results, we pursued further studies with these materials.
hydrodynamic diameter (DH) of each acetal-core BASP in nanopure water (-
The
mg/mL) was
measured by dynamic light scattering (DLS). DH values ranged from 17 nm to 23 nm as N
increased. The particle size distributions were generally narrow and very little aggregation in
water was observed (Figure 4). Transmission electron microscopy (TEM) of the N= 15 sample
stained with RuO 4 (Figure 3, inset) revealed particles with an average diameter of 114 t 14 nm.
The large size compared to DLS suggests that the particles aggregated during the TEM sample
preparation. In any case, the observation of nanoscopic particles was promising, and led us to
examine the acid-induced degradation of these materials.
N=10
(=Numbr
IIntnsity
10
100
N=15 '
M Nurnbr'
Intensit
'
10
RH n
Figure 4.
100
N=20
Number'
r
- Intensity
10
100
DLS histograms of ABASPs for N = 10, 15, 20 equiv of crosslinker 5.
The N = 15 acetal-core BASP was dissolved in pH 4.0 PBS buffer. Samples of this
solution were taken at various times and subjected to LC/MS analysis.
Figure 5 shows the
LC/MS traces as a function of time. The continuous shift of the initial BASP peak (black trace)
to shorter retention times over the course of 8 days is consistent with particle degradation. After
6
1.0d 0.8
(DO.6--02
0.---
0.
-- N =0 brus
1 week, a drop of concentrated
N = 15 BASP
in pH 4.0 buffer:
0d
1 d
d
3d
-8 d
-- +HCI
HCl (12.1
M)
Complete
acetal
5.5
6.0
6.5
70
~ 10
bottlebrush polymers. In line
with
5.0
cleavage
should regenerate DP
0.0.
4.5
was added.
this
7.5LC/MS
expectation,
trace
Retention time / min
Figure 5. LC-MS traces of the N = 0 bottlebrush polymer and N = 15 acetal core
BASP after exposure to pH 4.0 buffer for up to 8 d.
degradation
of
the
the
HCl
product
overlapped with that of the parent bottlebrush polymer. Furthermore, the DLS histogram of the
degradation product shows some overlap with the parent bottlebrush polymer (Figure 6b).
Finally, the GPC trace of ABASP exposed to trifluoroacetic acid in THF overlapped with that of
the parent bottlebrush (Figure 6a).
A.
1.0
---
0.8
B.
N=15
N=0
N= 15
N=0
WN=15
W
N15+HO inPH
+TFA
k, in THF
0.60.40.20.0.
15
16
17
18
19
20
1
1
Retention ime/ nin
1
102
1o3
RH/nm
Figure 6. A. GPC analysis of degraded N = 15 ABASP compared with parent bottlebrush. B. DLS histogram of degraded N= 15
ABASP compared with parent bottlebrush.
Having shown that 5 can serve as a useful crosslinker for the formation of aciddegradable, uniform BASPs, we turned our attention to the synthesis of drug-conjugated BASPs
that would simultaneously degrade and release a drug molecule in response to acidic conditions.
7
Building off of our previously reported "branched macromonomer" (MM) platform ,22, we
designed a novel doxorubicin (DOX)-based MM (DOX-MM) that possesses an ester linkage that
can be cleaved by esterases present in cell culture2 3 (Figure 7a). 'H NMR and matrix-assisted
laser desorption/ionization (MALDI) analyses confirmed the structure of DOX-MM (see
experimental section for details).
Z 5) A >
HO
A
NN'N
O J
NON-O
0
N N,
0
N
H
0
6
OV
O %
O ~
_
OH
1) 1.0 equiv.
OH
0
HO
0,HOH
DOX-MM
1.0.
Y
MeO
B
0.4-
==0
2) 15 equiv. 5
O0
'
DOX-BASP
C
0.8-
Grubbs 3rd
==
( iminumber7'
hydrolysis
10
08
C.
0.0.
6
10
14
18
Retention time / min
22 1
0
1 2
RH Inn
10'
163
DOX release
Figure 7. A. Schematic for synthesis of DOX-BASPs. B. GPC analysis of N = 15 DOX-BASPs. C. DLS histograms of N= 15
DOX-BASPs.
We prepared a DOX-loaded BASP (Figure 7a) with N = 15 equiv. of 5 following
identical procedures as described above using DOX-MM in place of PEG-MM. GPC analysis
(Figure 7b) of DOX-BASP revealed a lower conversion (-85%) of brush to BASP compared to
the studies described above. This difference may be due to the increased steric hindrance of the
drug-loaded MM compared to PEG-MM.
Nevertheless, we were pleased to find that this
combination of a novel branched MM and crosslinker led to the formation of 22 + 2 nm BASPs
8
(Figure 7c) with an 11.4% DOX loading without
DOX
DOX-BASP
any need for extraneous particle formulation steps.
We next sought to determine if these
DOX-conjugated acetal-core BASPs could deliver
E
therapeutically active free DOX in vitro. First, the
particles were incubated in pH 4.0 PBS buffer for
0
2
8
4
Rtenton
limefI
8
0
4 months.
LC/MS analysis of the resulting
nun
Figure 8. LC/MS trace of DOX-BASPs after incubation in
pH 4.0 buffer.
solution indicated the
presence
of free
DOX
(Figure 8). Next, HeLa cells were exposed to DOX-BASP, non-drug-loaded acetal-core BASP
(N = 15), and free DOX for 72 h. Cell viability (Figure 9) was assessed via MTT assay; halfmaximal inhibitory concentration (IC 50 ) values for each sample were obtained via standard
fitting procedures. The non-drug-loaded acetal-core BASP showed no toxicity over the range of
concentrations studied (blue data, Figure 9). Free DOX and DOX-conjugated BASP displayed
BASP concentration / [tgemLl
IC5o values of 1.3 ± 0.3 mM and
1
8.4 ± 0.5 mM, respectively. As is
--
polymer-drug
--
for
common
higher
than
free
drug.
strongly
suggests
vitro
that
(IC 50
8.4
0. 5 [M)
100.
80
-
in
SP
= 1.3 ± 0.3 RM)
120-
Nevertheless, the observation of
significant
A
50
1000
140-
conjugates, the IC5 0 of the particle
is
free DOX (IC
100
10
60-
toxicity
40
DOX-
0 20
0BASPs
active
release
DOX.
0.1
therapeutically
1.0
"i'i
DOX concentration /
M
6o
Figure 9. Cell viability assay of free DOX, DOX-BASPs. and ABASPs.
9
Conclusion: This work describes the development of novel hydrolytically labile BASP
nanoparticles via the design of acid-cleavable crosslinkers.
We identify an acetal-based bis-
norbornene derivative that enables the brush-first synthesis of uniform BASPs that degrade under
acidic conditions.
We interface this new crosslinker with a novel DOX-based branched
macromonomer for the synthesis of BASPs that degrade and release therapeutic DOX in cell
culture. These two new compounds are important components of our brush-first toolbox for
BASP synthesis.
Experimental Methods
General Considerations: All reagents and solvents were purchased from Aldrich or
VWR and used as supplied unless otherwise noted. Grubbs
3 rd
generation bispyridyl catalyst 2 4 ,
PEG-MM9 and PEG-Alkyne-MM22 were prepared according to literature procedures. Degassed
dichloromethane (DCM) and tetrahydrofuran (THF) were passed through solvent purification
columns prior to use.
Liquid chromatography-mass spectrometry (LC/MS) and preparative HPLC were
performed on an Agilent 1260 LC system equipped with a Zorbax SB-C18 rapid resolution HT
column and a Zorbax SB-C18 semi-preparative column. Solvent gradients consisted of mixtures
of nano-pure water with 0.1% acetic acid (AcOH) and HPLC-grade acetonitrile. Mass spectra
were obtained using an Agilent 6130 single quadrupole mass spectrometer.
Dynamic light scattering (DLS) measurements were made at room temperature using a
Wyatt Technology DynaPro Titan DLS instrument. Samples were dissolved in nanopure water at
a concentration of ~1 mg / mL. A fresh, clean, polystyrene cuvette was washed with compressed
air to remove dust. The sample solution was passed through a 0.4 pm Nylon syringe filter
10
directly into the cuvette; the cuvette was capped and placed in the DLS instrument for particle
sizing. At least 3 measurements were made per sample and average hydrodynamic diameters
were calculated by fitting the DLS correlation function using the CONTIN routine (Dynamics
V6 software package from DynaPro Wyatt Technology)
'H nuclear magnetic resonance ('H-NMR) and
13 C
nuclear magnetic resonance ("C-
NMR) spectra were recorded on Bruker AVANCE-400 NMR spectrometer or INOVA 500 MHz
spectrometer. Chemical shifts are reported in ppm and referenced to the CHCl 3 singlet at 7.24
ppm, DMSO at 2.50 ppm, MeOH at 4.87 ppm, or CH 2Cl 2 at 5.32 ppm.
13 C-NMR
spectra were
referenced to the center peaks of the CDCl 3 triplet at 77.23 ppm, DMSO septet at 39.51 ppm,
MeOH septet at 49.150, or CD 2 Cl 2 quintet at 54.0 ppm. Chemical shifts are expressed in parts
per million (ppm), and splitting patterns are designated as s (singlet), d (doublet), t (triplet), q
(quartet), quin (quintet), m (multiplet) and br (broad). Coupling constants J are reported in Hertz
(Hz). MestReNova NMR 7.0.1 software was used to analyze the NMR spectra.
Gel permeation chromatography (GPC) measurements were performed on an Agilent
1260 LC system with two Shodex KD-806M GPC columns in series at 60 'C and a flow rate of 1
mL / min. NN-Dimethylformamide (DMF) with 0.02 M LiBr was used as the eluent. A T-rEX
refractive index detector (Wyatt) and a DAWN EOS 18-angle laser light scattering (MALLS)
detector (Wyatt) were used for polymer analysis.
High-resolution mass spectrometry (HRMS) was obtained using a Bruker Daltonics
APEXIV 4.7 Tesla Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (FT-ICRMS).
TEM images were obtained at the MIT Center for Materials Science and Engineering on
a JEOL 2011 High Contrast Digital TEM. The samples were prepared as follows: 5.0 p.L of a
11
0.050 mg/mL solution of 20xL (or 15 xL) BASP polymer was deposited via pipet on top of a
carbon film-coated 200-mesh copper grid (purchased from Electron Microscopy Sciences)
placed on a piece of parafilm carbon-coated side up. The sample was allowed to dry at room
temperature. For the stained TEM sample, the particle-coated copper grid prepared as described
above was placed carbon-coated side up in a glass insert for 2-mL vials. The insert was placed
inside a 3-mL scintillation vial, and to the vial (outside the insert) was added 0.50 mL of a 0.5%
RuO 4 (aq).
The vial was capped and allowed to stand for half an hour. The grid was then
removed and was ready for TEM imaging.
Cell Culture Studies: HeLa cells (ATCC) were maintained in RPMI-1640 media
supplemented with 0.01 mg/mL recombinant human insulin (Gibco), 20% fetal bovine serum,
and penicillin/streptomycin in a 5% CO 2 humidified atmosphere (37
'C). Assays were
performed on cells passaged 24 h prior. Dose-response curves were fit using a four-parameter
logistic regression analysis in Origin 8.5.1 software. BASP drug conjugates were reconstituted in
ultrapure water (18 M ) and stored at 4 *C in dark prior to use. Viability was assessed by
CellTiter-Glo assay (Promega) following 72 h total incubation time with ABASPs-spiked OptiMEM reduced serum media.
Synthetic Procedures
Synthesis of Silyl Ether Crosslinkers 1-4
OH
0
DIPEA
0
NH 2
NH2
microwave
toluene, 1700C, 30 m
0
N
OH
1:
C1
12
'
Compound C1. Cis-5-norbomene-exo-2,3-dicarboxylic anhydride (0.625 g, 3.81 mmol), 3aminobenzyl alcohol (0.516 g, 4.18 mmol), NN-diisopropylethylamine (0.730 mL, 4.18 mmol),
and toluene (14 mL) were added to a microwave vial. This reaction mixture was heated and
stirred in a microwave reactor at 170 0 C for 30 min. The crude product was purified via silica
gel chromatography (0%-50% EtOAc/hexanes) to give C1 (0.810 g, 79% yield) as a pure white
solid. 'H NMR (400 MHz, CDCl 3) 6 7.42 (t, J= 7.8 Hz, 1H), 7.34 (d, J = 7.9 Hz, IH), 7.23 (s,
1H), 7.14 (d, J = 7.8 Hz, IH), 6.32 (t, J = 1.8 Hz, 2H), 4.67 (s, 2H), 3.37-3.36 (m, 2H), 2.82 (s,
2H), 2.08 (s, 1H), 1.59 (dt, J = 9.9, 1.4 Hz, lH), 1.47 (d, J = 9.9 Hz, 1H). ' 3 C-NMR (400 MHz,
CDC 3 ) 6 177.31, 142.65, 138.19, 132.17, 129.52, 127.24, 125.68, 124.86, 64.73, 48.09, 46.01,
43.18. HRMS: calcd. for C16 HI5NO 3 [M+NH 4]+, 287.1390; found, 287.1379.
0
DIPEA
-NSOH
-12 H
N
S300
0
microwave
toluene, 175*C, 30 m
N
H
C2
Compound
C2. Cis-5-norbornene-exo-2,3-dicarboxylic
anhydride (1.863
g,
11.4 mmol),
ethanolamine (0.891 mL, 14.8 mmol), NN-diisopropylethylamine (0.198 mL, 1.14 mmol), and
toluene (14 mL) were added to a microwave vial. This reaction mixture was heated and stirred
in a microwave reactor at 175
0C
for 30 min.
The reaction was then diluted with
dichloromethane (100 mL) and washed with water (3 x 100 mL). The organic layer was dried
with anhydrous magnesium sulfate, filtered, and concentrated via rotary evaporation to give C2
(1.54 g, 65% yield) as a pure white solid. 'H NMR (400 MHz, CDCl 3) 6 6.26-6.25 (m, 2H), 3.743.71 (m, 2H), 3.67-3.64 (m, 2H), 3.24 (s, 2H), 2.68 (s, 2H), 2.34 (s, 1H), 1.48 (dt, J = 9.9, 1.4 Hz,
13
1H), 1.31 (d, J
=
9.9 Hz, IH).
13 C-NMR
(400 MHz, CDC13 ) 6 178.92, 138.00, 77.23, 60.47,
48.06, 45.44, 42.95, 41.47. HRMS: calcd. for CIIH 3 NO 3 [M+H]+, 208.0968; found, 208.0958.
0
Pr P r
C Si
DIPEA
0
/
Pr,
,
0
Crosslinker I
DCM, rt, overnight
C2
,Pr
Crosslinker 1. Compound C2 (0.300 g, 1.45 mmol) and NN-diisopropylethylamine (0.419 mL,
2.41
mmol)
were
dissolved
in
3
mL
of
dichloromethane.
To
this
solution,
dichlorodiisopropylsilane (0.065 mL, 0.362 mmol) was added dropwise at room temperature.
The reaction was stirred overnight and then concentrated via rotary evaporation. The crude
product was purified via silica gel chromatography (0%-30% EtOAc/hexanes) to give crosslinker
1 (0.112 g, 59% yield). 'H NMR (400 MHz, CDCL3 ) 6 6.19-6.18 (m, 4H), 3.78 (t, J = 5.7, 4H),
3.56 (t, J = 5.7, 4H), 3.16 (s, 4H), 2.58 (s, 4H), 1.38 (dt, J = 9.8, 1.4 Hz, 2H), 1.29 (d, J = 9.8 Hz,
2H), 0.87-0.83 (m, 14H).
13 C-NMR
(400 MHz, CDC13 ) 6 178.00, 137.84, 59.04, 47.82, 45.27,
42.86, 40.68, 17.10, 11.85. LC/MS m/z: calcd. for C2 8 H3 8N 2 O6 Si [M+Na]+, 549.2; found, 549.3.
o
/
H
N
'OH
0
C2
DIPEA
0
0
Ph% Ph
i'/
CI' ''CI
N
Ph~ Ph
Si
0
O
,
DCM, rt, overnight
N
Crosslinker 2
Crosslinker 2. Compound C2 (0.300 g, 1.45 mmol) and NN-diisopropylethylamine (0.419 mL,
2.41 mmol) were dissolved in 3 mL of dichloromethane. To this solution, dichlorodiphenylsilane
(0.076 mL, 0.362 mmol) was added dropwise at room temperature. The reaction was stirred
14
overnight and then concentrated via rotary evaporation. The crude product was purified via silica
gel chromatography (0%-30% EtOAc/hexanes) to give crosslinker 1 (0.075 g, 35% yield). 'H
NMR (400 MHz, CDCl 3) 6 7.54-7.52 (m, 4H), 7.41-7.37 (m, 2H), 7.34-7.30 (m, 4H), 6.24-6.23
(m, 4H), 3.91 (t, J
13 C-NMR
=
5.5 Hz, 4H), 3.67 (t, J
=
5.5, 4H), 3.21 (s, 4H), 2.60 (s, 4H), 1.33 (s, 4H).
(400 MHz, CDCl 3) 6 178.21, 137.96, 135.02, 131.67, 130.74, 128.06, 59.52, 47.99,
45.40, 42.98, 40.68. LC/MS m/z: calcd. for C34H34N 2O6 Si [M+Na], 617.2; found, 617.2.
*Minor impurity and solvent peaks: 'H NMR (400 MHz, DMSO-d 6 ) 6 4.09 (q, J = 7.1 Hz,
0.08H), 3.77-3.74 (m, 0.21H), 3.26 (s, 0.18H), 2.69 (s, 0.18H), 2.01 (s, 0.08H), 1.23 (t, J = 7.1,
0.14H).
P r , P r/
/'
0
N
OH
0
NN
Si
No
DIPEA
S
Pr "Pr
DCM, rt, overnight
C3
N
Crosslinker 3
Crosslinker 3. Compound C3 (0.122 g, 0.451 mmol) and NN-diisopropylethylamine (0.110 mL,
0.632
mmol)
were
dissolved
in
3
mL
of
dichloromethane.
To
this
solution,
dichlorodiisopropylsilane (0.039 mL, 0.216 mmol) was added dropwise at room temperature.
The reaction was stirred overnight and then concentrated via rotary evaporation. The crude
product was purified via silica gel chromatography (0%-50% EtOAc/hexanes) to give crosslinker
3 (0.106 g, 76% yield). 'H NMR (400 MHz, CDCl3 ) 6 7.38 (d, J = 8.4, 4H), 7.20-7.18 (m, 4H),
6.33-6.32 (m, 4H), 4.82 (s, 4H), 3.38 (s, 4H), 2.83 (s, 4H), 1.59 (dt, J = 9.9, 1.3 Hz, 2H), 1.47 (d,
J
=
9.9 Hz, 2H), 1.11-1.07 (m, 14H).
13 C-NMR
15
(400 MHz, CDCL3) 6 177.30, 141.76, 138.20,
130.71, 126.75, 126.40, 64.26, 48.06, 46.01, 43.17, 17.60, 12.37. LC/MS m/z: calcd. for
C34 H34 N2 0 6 Si [M+Na]+, 617.2; found, 617.2.
0
0
Ph, SPh
N
OH
/
C
Ph, ,Ph
Or"
OCN
N
0
0
DIPEA
0
0
DCM, rt, overnight
Crosslinker 4
C1
Crosslinker 4. Compound C1 (0.120 g, 0.405 mmol) and NN-diisopropylethylamine (0.100 mL,
0.567
mmol)
were
dissolved
in
3
mL
of
dichloromethane.
To
this
solution,
dichlorodiphenylsilane (0.045 mL, 0.212 mmol) was added dropwise at room temperature. The
reaction was stirred overnight and then concentrated via rotary evaporation. The crude product
was purified via silica gel chromatography (0%-50% EtOAc/hexanes) to give crosslinker 4
(0.112 g, 73% yield). 'H NMR (400 MHz, CDCl 3) 6 7.71-7.68 (m, 4H), 7.45-7.32 (in, 1OH),
7.25-7.22 (in, 2H), 7.17-7.11 (in, 2H), 6.33-6.32 (in, 4H), 4.85 (s, 4H), 3.37 (s, 4H), 2.82 (s, 4H),
1.57-1.49 (m, 2H), 1.44-1.30 (in, 2H). ' 3C-NMR (400 MHz, CDCl 3) 6 177.16, 141.82, 138.16,
135.14, 132.05, 132.01, 130.75, 129.26, 128.19, 126.83, 125.30, 234.58, 64.55, 48.02, 45.98,
43.13. LC/MS m/z: calcd. for C 4 4 H 38N2 0 6Si [M+H]+, 720.3; found, 720.2.
* Minor impurity peaks: 'H NMR (400 MHz, DMSO-d) 6 4.68 (s, 0.24H), 1.97 (s, 0.27H). "CNMR (400 MHz, CDC 3) 6 142.65, 129.48, 127.18, 125.65, 124.83.
Synthesis of Acetal Crosslinker 5
16
0
0
DIPEA
HO
/
NH 2
N
N
microwave
toluene, 140 0C,4h
'
A1
Compound Al. To a 20 mL microwave vial equipped with a magnetic stir bar was added a
solution of cis-5-norbomene-exo-2,3-dicarboxylic
(1.175
anhydride
g,
7.15
mmol),
4-
aminophenol (1.013 g, 9.28 mmol), and NN-diisopropylethylamine (1.62 mL, 9.28 mmol) in
toluene (17 mL). The vial was capped and the solution was placed in the microwave at 140 0C for
4 hours. The reaction was then concentrated via rotary evaporator, re-dissolved in hot methanol
(20 mL), and filtered immediately. The resulting filtrate was cooled to 0*C to give off-white
crystals. The crystals were filtered and recrystallized twice in methanol to give Al as white
crystals (1.133 g, 63% yield). 'H NMR (400 MHz, DMSO-d) 6 9.73 (s, 1H), 7.04-7.00 (m, 2H),
6.84-6.81 (m, 2H), 6.36-6.35 (m, 2H), 3.19-3.18 (m, 2H), 2.80 (s, 2H), 1.47-1.44 (m, 1H), 1.38
(d, J = 9.8 Hz, 1H). '3 C-NMR (400 MHz, DMSO-d 6 ) 6 177.15, 157.34, 137.76, 123.35, 115.39,
47.34, 44.92, 42.62. HRMS: calcd. for C15H12NO 3 [M+H]+, 256.0968; found, 256.0965.
*
Minor solvent peak: 'H NMR (400 MHz, DMSO-d) 6 3.34 (s, 0.13H).
/0
0
C1
N
0OH
N
0I~
PPTS
DCM,
0*C
N
- RT, 12 h
Al
0
K
i
C'
A2
Compound A2. A solution of Al (0.616 g, 2.41 mmol) and pyridinium p-toluenesulfonate
(0.061 g, 0.241 mmol) in DCM (60 mL) was added to a dried, 150 mL round-bottom flask. The
solution was cooled down to 00 C in an ice bath and 2-chloroethyl vinyl ether (1.22 mL, 12.0
17
mmol) was added dropwise. The reaction was stirred for 12 h under nitrogen atmosphere, and
then diluted with DCM (50 mL), transferred to a separatory funnel, and washed with saturated
sodium bicarbonate (100 mL), water (100 mL), and brine (100 mL). The organic solution was
dried over sodium sulfate, filtered, and concentrated via rotary evaporation. The crude product
was purified via silica gel chromatography (0%-30% EtOAc/hexanes) to give A2 (0.804 g, 92%
yield*). 'H NMR (400 MHz, CDCl 3) 6 7.16-7.12 (in, 2H), 7.08-7.04 (in, 2H), 6.30 (t, J =1.8, 2H),
5.45 (q, J= 5.4, 1H), 3.92-3.86 (in, lH), 3.75-3.69 (in, lH), 3.58-3.54 (m, 2H), 3.35 (s, 2H), 2.79
(s, 2H), 1.57 (d, J
=
9.9, IH), 1.42 (d, J
=
9.9, 1H). 13 C-NMR (400 MHz, CDC 3) 6 177.34,
156.64, 138.07, 127.72, 125.81, 117.65, 99.49, 65.20, 47.91, 45.89, 43.06, 43.01, 19.87. LC/MS
m/z: calcd. for CI 9 H 20 C1N0
4
[M+H]+, 362.1; found, 362.1.
* Minor solvent peaks: 'H NMR (400 MHz, CDCl 3 ) 6 5.25 (s, 0.17H), 4.08 (q, J
7.1, 0.29H),
2.00 (s, 0.45H), 1.21 (t, J = 7.2, 0.5 1H).
0
N
S0
N
Al
Al
OH
Na2CO3
0
CI
15-crown-5
DMF, 750 C, 48 h
A2
0
'0N
0 N"
o
0
Acetal-XL
18
N 0
Crosslinker 5. A solution of A2, Al, Na 2 CO 3, and 15-crown-5 in DMF (15 mL) were added to a
dried, 100 mL round-bottom flask. The solution was stirred at 75"C for 48 h under nitrogen. The
reaction was then diluted with ethyl acetate and washed with saturated NaHCO 3, water, and
brine. The organic layer was dried over magnesium sulfate, filtered, and concentrated via rotary
evaporation. The crude product was purified via silica gel chromatography (0%-50%
EtOAc/hexanes) to give crosslinker 5 (0.241 g, 23% yield*). 'H NMR (400 MHz, CDCl 3) 67.167.08 (m, 6H), 6.92-6.90 (m, 2H), 6.32 (m, 4H), 5.51 (q, J = 5.3 Hz, lH), 4.10-4.08 (m, 2H), 4.023.97 (m, lH), 3.88-3.83 (m, lH), 3.38-3.37 (m, 4H), 2.81 (d, J = 5.7, 4H), 1.59 (dqt, J = 9.9, 1.5
Hz, 2H), 1.55 (d, J= 6.8 Hz, 3H), 1.45 (d, J = 9.9 Hz, 2H). "C-NMR (400 MHz, CDCl3 ) 177.51,
177.48, 158.84, 156.90, 138.20, 138.18, 127.81, 127.79, 125.79, 124.93, 117.82, 115.37, 99.49,
67.62, 63.30, 48.04, 48.01, 46.00, 45.99, 43.19, 43.18, 20.04. HRMS: calcd. for C34 H4 2 N2 0 7
[M+H]+, 581.2282; found, 581.2269.
*
Minor solvent peak: 'H NMR (400 MHz, CDC3) 6 1.52 (s, 1.25H).
Synthesis of DOX-MM
0
0
H
~
/
OH
0
Br
HO
EDC-HCI, DMAP
H
DCM, rt, overnight
O0
B1
Compound B1. To a dried 200 mL round bottom flask was added 6-bromohexanoic acid (4.00
g, 20.5 mmol), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide
mmol), and 4-(dimethylamino)pyridine
hydrochloride (4.30 g, 22.5
(0.250 g, 2.05 mmol) in 120 mL of anhydrous
dichloromethane. The solution was stirred for 5 minutes, after which 4-hydroxybenzaldehyde
19
(2.50 g, 20.5 mmol) was added. The reaction was stirred at room temperature overnight. The
reaction was then transferred to a separatory funnel and washed with water (2 x 150 mL) and
brine (150 mL). The organic layer was dried over magnesium sulfate, which was removed by
filtration. The solution was concentrated and run through a silica plug in ethyl acetate. The
fractions were collected and the solvent was removed by rotary evaporation and dried overnight
to give compound BI (4.5 g, 74% yield*). 'H NMR (400 MHz, CDC 3) 9.96 (s, 1H), 7.91-7.88
(in, 2H), 7.27-7.23 (in, 2H), 3.42 (t, J = 6.7, 2H), 2.60 (t, J = 7.4, 2H), 1.94-1.73 (in, 4H), 1.60-
1.52 (in, 2H). 6 ' 3C-NMR (400 MHz, CDCl 3 ) 6 191.11, 171.43, 155.59, 134.19, 131.42, 122.55,
44.89, 34.36, 32.35, 26.49, 24.25. HRMS: calcd. for C13HI 5 BrO 3 [M+NH 4 ]+, 316.0543; found,
316.0532.
* Minor impurity and solvent peaks: 'H NMR (400 MHz, CDCl 3 ) 6 4.09 (q, J= 7.1, 0.13H), 3.55
(t, J = 6.6, 0.46H), 2.14 (s, 0.35H), 2.02 (s, 0.20H), 1.23 (t, J = 7.1, 0.21 H)
0
H
'N
NaBH 4,
0
0Br
MeOH,0 0C, 5 min
HO
00
0Br
B2
B1
Compound B2. Sodium borohydride (0.760 g, 20.1 mmol) was added to compound B1 (4.00 g,
13.4 mmol) in anhydrous methanol (30 mL) at 0 'C under nitrogen. The reaction was stirred for
5 min then quenched with the addition of water. IM HCl was added to neutralize the reaction
mixture until pH = 7. The mixture was extracted with dichloromethane (3 x 50 mL), and the
organic layers were combined and dried with anhydrous magnesium sulfate, which was removed
by filtration. The solution was concentrated and silica gel chromatography was performed using
a gradient of 10% to 80% ethyl acetate in hexanes. The fractions containing product were
20
collected, and the solvent was removed by rotary evaporation and dried overnight to give
compound B2 (2.7 g, yield 67%*). 1H NMR (400 MHz, CDC 3) 6 7.37-7.33 (in, 2H), 7.06-7.03
(m, 2H), 4.65 (s, 2H), 3.42 (t, J = 6.7 Hz, 2H), 2.56 (t, J = 7.4, 2H), 1.94-1.87 (in, 2H), 1.87-1.80
(m, 3H), 1.59-1.52 (in, 2H). "C-NMR (400 MHz, CDCl 3) 6 172.20, 150.25, 138.67, 128.26,
121.84, 64.92, 34.31, 33.64, 32.54, 27.79, 24.24. HRMS: calcd. for C1H
7 BrO 3
[M+NH 4]+,
318.0699; found, 318.0684.
* Minor impurity and solvent peaks: 1H NMR (400 MHz, CDC 3) 6 5.27 (s, 0.04H), 4.10 (q, J
6.5 Hz, 0.19H), 3.55 (t, J
7.1, 0.11H), 3.62 (t, J
=
0.16H), 1.23 (t, J
Hz, 0.19H), 0.05 (s, 0.1OH).
HO
N
= 7.1
=
6.6 Hz, 0.18H), 2.14 (s, 0.04H), 2.02 (s,
NaN 3
Q
Br
HO
0
DMF, 600C, 24h
N3
B3
B2
Compound B3. DMF (20 mL) was added to compound B2 (2.0 g, 6.64 mmol) and sodium azide
(0.650 g, 10 mmol) in a flask, which was heated to 60 'C and stirred for 24 hours. The reaction
was diluted in ethyl acetate (100 mL), and washed with water (2 x 75 mL) and brine (75 mL).
The organic layer was dried with anhydrous magnesium sulfate, which was then filtered. The
solution was concentrated and silica gel chromatography was performed using a gradient of 10%
to 70% ethyl acetate in hexanes. The fractions containing product were collected, and the solvent
was removed by rotary evaporation and dried overnight to give compound B3 (0.716 g, yield
41%).'H NMR (400 MHz, CDCl 3) 6 7.36-7.33 (in, 2H), 7.06-7.02 (m, 2H), 4.65 (s, 2H), 3.28 (t,
J
=
6.5 Hz, 2H), 2.56 (t, J
2H).
1 3C-NMR
=
7.4 Hz, 2H), 1.80-1.73 (in, 3H), 1.68-1.61 (m, 2H), 1.52-1.44 (in,
(400 MHz, CDC 3) 6 172.19, 150.24, 138.68, 128.25, 121.82, 64.90, 51.40,
21
34.32, 28.75, 26.40, 24.59. HRMS: caled. for C13HI 7N 3 0 3 [M+NH 4 ]+, 281.1608; found,
281.1594.
* Minor impurity and solvent peaks: 1H NMR (400 MHz, CDC13) 6 3.61 (t, J
6.5 Hz, 0.16H),
2.14 (s, 0.11 H), 1.39-1.36 (m, 0.28H), 0.05 (s, 0.08H).
o
NO
2
0
HO
0
0
NEt 3
N3
THF, 0*C,l h
B3
N3
0
0
/
B4
N0O
NO 2
Compound B4. A solution of compound B3 (0.179 g, 0.680 mmol) and triethylamine (0.134
mL, 0.954 mmol) in tetrahydrofuran (3 mL) was added dropwise to a flask of 4-nitrophenyl
chloroformate (0.257 g, 1.28 mmol) in tetrahydrofuran (8 mL) at 0 'C under nitrogen. The icebath was removed and the reaction was left to stir for one hour. The mixture was concentrated
via rotary evaporator and purified by silica gel chromatography from 0% to 50% ethyl acetate in
hexanes. The fractions containing product was concentrated on a rotary evaporator and dried on
vacuum overnight to give B4 (0.2138 g, yield 73%*). 'H NMR (400 MHz, CDCl 3) 6 8.27-8.23
(m, 2H), 7.46-7.42 (m, 2H), 7.38-7.34 (m, 2H), 7.13-7.09 (m, 2H), 5.26 (s, 2H), 3.29 (t, J = 6.8
Hz, 2H), 2.58 (t, J = 7.4 Hz, 2H), 1.78 (p, J = 7.8 Hz, 2H), 1.65-1.62 (m, 2H), 1.53-1.46 (m, 2H).
'3 C-NMR (400 MHz, CDC 3 ) 6 171.97, 155.68, 152.62, 151.39, 145.64, 131.96, 130.26, 125.51,
122.20, 121.98, 70.47, 51.40, 34.32, 28.76, 26.41, 24.57. HRMS: calcd. for C2 0H2 0N4 0 7
[M+NH 4]+, 446.1670; found, 446.1661.
22
* Minor impurity and solvent peaks: 'H NMR (400 MHz, CDCl 3) 6 8.03-8.00 (m, 0.16H), 4.494.47 (m, 0.17H), 3.88-3.86 (m, 0.17H), 2.15 (s, 0.10H), 0.05 (s, 0.22H).
13 C-NMR
(400 MHz,
CDCl 3) 6 129.87, 128.54.
H
OH
H HO
H2N,-N3
00
0
0*
i'
HCI
+
OH
HO
NO
0
B4
2
0
O
/
DOX-HCI
H
0
N3
0
0- Y
N,,.
0
0
DIPEA
OH
OH
DMF, rt, 12 h
DOX-N 3
H
Me
OH
MeO0
Synthesis of compound DOX-N 3. Doxorubicin hydrochloride (0.304 g, 0.524 mmol) was
dissolved in dimethlyformamide (7 mL). NN-Diisopropylethylamine (0.912 mL, 0.524 mmol)
and B4 (0.2138 g, 0.499 mmol) were added. The solution was stirred at room temperature
overnight. The mixture was then diluted with ethyl acetate (75 mL) and washed with water (2 x
50 mL) and brine (50 mL). The organic layer was dried with anhydrous magnesium sulfate,
23
which was removed by filtration. The solution was concentrated and silica gel chromatography
was performed using a gradient of 0% to 10% methanol in dichloromethane. The fractions
containing product were collected, and the solvent was removed by rotary evaporation and dried
overnight to give DOX-N 3 (0.337 g, yield 81%*). 'H NMR (400 MHz, CDC13) 6 13.95 (s, 1H),
13.21 (s, LH), 8.01 (d, J = 7.7 Hz, 1H), 7.76 (t, J = 8.36 Hz, IH), 7.37 (d, J
(d, J= 8.1 Hz, 2H), 7.00 (d, J= 8.1 Hz, 2H), 5.48 (d, J
=
=
7.8 Hz, 1H), 7.30
3.6 Hz, 1H), 5.26 (bs, 1H), 5.12 (d, J =
8.4, 1H), 4.99 (s, 2H), 4.74 (s, 2H), 4.53 (bs, IH), 4.14-4.09 (m, 1H), 4.06 (s, 3H), 3.83 (bs, 1H),
3.64 (s, 1H), 3.29-3.22 (m, 3H), 3.00-2.86 (m, 2H), 2.53 (t, J = 7.4, 2H), 2.31 (d, J = 14.7 Hz,
lH), 2.15 (dd, J= 7.8, 4.0 Hz, lH), 1.86 (dd, J= 13.4, 5.2 Hz, 1H), 1.79-1.70 (m, 3H), 1.63 (p, J
= 7.3, 2H), 1.50-1.42 (m, 2H), 1.26 (d, J = 6.6 Hz, 3H). "C-NMR (400 MHz, CDCl 3) 6 214.05,
187.28, 186.89, 172.02, 161.26, 156.39, 155.84, 155.61, 150.65, 135.98, 135.69, 135.98, 135.69,
134.17, 133.79, 129.59, 121.82, 121.07, 120.05, 118.67, 111.79, 111.62, 100.93, 77.43, 76.83,
69.88, 69.74, 67.46, 66.29, 65.76, 56.88, 51.40, 47.22, 35.83, 34.31, 34.21, 30.36, 29.48, 28.75,
26.40, 24.57, 17.03. HRMS: calcd. for C4 IH4 4 N4 0 1 5 [M+NH 4]+, 850.3141; found, 850.3150.
*Observed silicone grease: 'H NMR (400 MHz, CDCl 3) 6 0.048 (s, 0.53H)
24
0
/
DOX-N 3
+
O
N(N
oH
67
PEG-Alkyne-MM
N N N
0
0
0
CuOAc
DCM
N
NN"
"'6
O
OH
N
0
N
H
H
0
OH
67
OH
H0O
o
DOX-MM
0
MeO
OH
0
Synthesis of DOX-MM To a solution of DOX-N 3 (32.3 mg, 0.039 mmol) and PEG-AlkyneMM
(120 mg, 0.036 mmol) in 5 ml DCM was added copper(L) acetate (13.2 mg, 0.107 mmol)
under N2 atmosphere. The reaction was allowed to stir at room temperature and monitored by
LC-MS until complete consumption of PEG-Alkyne-MM. Then the solvent was removed under
vacuum and the residue was purified by preparatory HPLC using a linear gradient from 5%
MeCN : 95% H2 0 to 95% MeCN : 5% H 2 0 over 20 minutes. The desired DOX-MM was
obtained as a red powder (35 mg, 23% yield). 'H NMR and MALDI are provided at the end of
the chapter.
General Procedure for BASP Synthesis
Synthesis of Acetal-core BASPs. PEG-MM was added to a 4 mL vial containing a stir bar.
THF was added to the vial with PEG-MM followed with a freshly prepared solution of Gruubs
3rd
generation bis-pyridyl catalyst in THF (0.02 g catalyst / I mL THF, amount added to give
desired PEG-MM:catalyst = 10) such that the total concentration of PEG-MM was 0.1 M.
25
After 10 minutes of stirring at 25 0C, aliquots of the polymerization mixture was transferred to 4 x
4 mL empty vials. A stock solution of crosslinker 5 in THF (0.1 M) was dispensed into the vials
in batches of 5 equivalents until the desired N equivalents were added. The resulting mixtures
were stirred at 25 'C for 1 hr, at which point 1 drop of ethyl vinyl ether was added to quench the
polymerization.
Synthesis of DOX-BASPs. DOX-MM (30 mg) was added to a 4 mL vial containing a stir bar.
THF (45.1 pL) was added to the vial with DOX-MM followed by a freshly prepared solution of
Gruubs
3rd
generation bis-pyridyl catalyst in THF (0.02 g/mL, 25.8 pL) to give desired PEG-
MM:catalyst = 10. Note that the total concentration of MM PEG-MM was 0.1 M. The solution
was allowed to stir for 10 minutes at 25 'C; meanwhile, a stock solution of crosslinker 5 in THF
(0.1M) was made. The stock solution of crosslinker 5 was slowly dispensed into the vial in
batches of 5 equivalents (31.9 pL) until N = 15 equivalents (95.7 piL total) were added. The
resulting mixtures were stirred at 25 'C for 1 hr, at which point I drop of ethyl vinyl ether was
added to quench the polymerization.
26
Spectral Data
0
OH
Cl
--000
95
90
85
190
ISO
170
80
1OD
75
150
I ~IAL
i
I
70
85
40
1
60
130
R hIfp4t
1
40
35
I R
120
B
27
30
SO
70
25
20
60
50
15
40
10
3D
05
2C
0
10
0
N
O
0
C2
_
_I
_
T
95
85
90
1
80
75
70
ON --
J
90o
65
180
170
160
150
140
130
60
flu
T
S
O
T
45
mc
A
40
t(pm)
TI
35
30
25
-113
18
120
8
28
g 4P
0
70
G0
50
~~
20
15
10
4I
3-
2-
40
30
20
05
10
10
0
f
0
Pr,
,Pr
N
N ,,-0Si
0
0
Crosslinker I
p 14 Li
0
95
90
85
80
75
70
65
60
S
~
T
)D
190
180
1w0
10
10
170
160
Th
150
140
140
T130
12
130
120
1&fW
40
45
mc t(pprn)
&M4PA
29
-10I
35
-
-
70
30
25
W0
50
20
15
10
40
3
20
40
30
20
05
-
10
0
-0
0
0
Ph,
N
Ph
N
,-,,Si
0
0
Crosslinker 2
,-
II
iLl
A,
8
82
95
90
85
80
75
70
65
60
190
180
170
160
150
140
130
120
45
30
g
-J
~__
8
2
40
35
30
25
20
15
10
05
880
70
80
50
40
30
20
10
0
0
N
,N
0
Q0 0
,Si
Pr
Pr
Crosslinker 3
95
90
95
a0
190
lao
170
160
75
70
150
140
I7
65
60
130
120
1T
1
45
Vf
-
'e o
-y -
(pp)
35
W0
70
7u
-7
%inm(pI
31
40
30
m
W0
25
20
15
p
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42
m/z
Chapter II: A Novel Family of Perfect Polymers via Iterative Exponential Growth "Plus"
(IEG+)
Introduction: Advances in polymer science have generated functional materials for
numerous applications. Polymers have found widespread use in diverse fields such as medicine,
electronics, and consumer goods. However, many newer polymer applications require the use of
macromolecules with precisely defined structure and degree of polymerization. For example, a
rapidly growing area of research is the application of polymers for drug delivery systems.
Traditional drug delivery methods suffer from many deficiencies such as short half-life, poor
cellular accumulation, and inadequate solubility of the drug. However, use of polymeric drug
delivery systems (PDDS) can overcome these limitations due to their structural stability,
composition, and size.2 5 Furthermore, the side effects resulting from administration of PDDS are
usually reduced relative to the free drug. However, clinical applications of PDDS are limited by
a unique set of challenges. The dispersity and batch-to-batch variation in the size and
morphology of the nanoparticles used in PDDS may result in undesirable variation in the rate of
nanoparticle degradation, the stability of the drug, and the kinetics of drug release.
To this end, there have been several advances towards more uniform polymers.2 1-
Solid-
phase resins and protecting group strategies are routinely used for the synthesis of short
sequence-defined oligomers,3 4 but this approach is inherently limited in its scalability and
generates considerable resin waste. Many modem controlled polymerization techniques rely on
probabilistic monomer addition steps that can result in polymers of low dispersity, but these
techniques still cannot achieve a single uniform mass or precise sequences. 35 Therefore, there is a
demonstrated need for a synthetic methodology that affords unimolecular masses and defined
43
chemical sequences using synthetic procedures that are precise, scalable, and amenable to
diversification.
In order to achieve this, we have developed modifications of the "iterative exponential
growth" (IEG) concept for polymer synthesis; the enhanced version of IEG is herein referred to
as "IEG+". The specific modifications include: (1)
the introduction of functional sidechains in
defined sequences along the backbone of an IEG polymer, and (2) the use of extremely efficient
reactions to build IEG+ polymer scaffolds.
Figure
1. The concept of IEG in which the growing polymer doubles with each iterative coupling step.
To better understand the significance of these modifications, one must be familiar with
traditional IEG.35 This begins with the synthesis of a monomer that possesses orthogonal
protecting groups, PGA and PGB (Figure 1). In separate reaction flasks, PGA and PUB are
removed to generate a batch of PGA-protected monomer and a batch of PUB-protected monomer.
The newly exposed functional groups on each molecule are then coupled together to form a
dimer, which can then undergo subsequent rounds of deprotection and coupling. At each step in
the LEG process, the growing polymer is partitioned in half, each half then undergoes two
separate and complementary deprotection reactions. Those halves are then recombined and
coupled together to give a new protected molecule with twice the molecular weight. In this way,
the number of repeat units of the growing polymer doubles with each iterative coupling step, and
the molecular weight increases exponentially.
44
Although IEG could allow for the synthesis of precise and large polymers, this technique
is not generally recognized as a key component of a polymer chemist's toolbox. Historically, the
lack of enthusiasm for IEG syntheses is due to low yields in coupling and deprotection steps,
which lead to inherently poor yields of polymers.
Our newly proposed synthetic strategy
(IEG+) overcomes these deficiencies in traditional IEG. IEG+ enables the rapid synthesis of
unprecedented diverse macromolecules with unimolecular masses, precise stereochemistries,
defined sequences of sidechain functional groups, and orthogonally addressable end groups.
key monomers
TIPS
0
propargyl alcohol
tetrabutylammonium bisulfate
o
50% w/w aq. NaOH,
84%
CLA,,0
KHMDS
rIpSCI
(R)-TIPS
0
0"C->RT
THF, -78 C
92%
TIPS
0
(S)-TIPS
Figure 2. Synthesis of key monomers (R)-TIPS and (S)-TIPS.
Results and Discussion: We began our study through the synthesis of key monomers
(R)-TIPS and (S)-TIPS (Figure 2). These compounds were prepared in 77% overall yield over
two steps: first, a phase transfer reaction of (R)- or (S)-epichlorohydrin and propargyl alcohol,
followed by protection of the alkyne end with a triisopropylsilyl (TIPS) group. Not only are these
monomers easily synthesized from inexpensive chiral starting materials, but they also fulfill
certain design requirements. First, they are chiral; chirality is a key component found in natural
polymers (e.g., proteins are built from chiral amino acids). Second, they posses orthogonal
epoxide and protected alkyne groups, which can be differentially activated/deprotected.
In a
novel conceptual advance, we use the epoxide as a masked alcohol group, which can serve as a
site for introduction of novel functionality into the polymer backbone. In addition to the
enantiomerically pure monomers, we also synthesized a racemic, t-butyldimethylsilyl- (TBS)
protected version, rac-TBS.
45
Figure 3 depicts one cycle of the IEG+ strategy. First, a stock of (S)-TIPS or (R)-TIPS
was split into two flasks. In one flask, the epoxide was opened with azide anion to generate
azido-alcohol N3-(S)-TIPS or N3 -(R)-TIPS. Unlike traditional IEG wherein a protecting group is
removed on both sides of the molecule, the epoxide served as a site of functionalization and as a
masked alcohol group.
0
OH
NaN3, NH 4CIDMF.60
N3
C 0N
key monomers
o
0'eOH
L-\
(R)-TIPS
o
TIPS
3-(R)-TIPS
TIPS
N3-(S)-TIPS
99%
TIPS
N3
-,O
O
TIPS
(R)-alkyne
!-<-
(S)-TIPS
(R)-(R)-G1
0
N-N
ORS-G
) -G1N
(S)-(S)-G1
o
LA
0
(S)-(R)-G1 LA
from step
ous
OAc
0
N=N
N
OAc
_7,O
TIPS
N N
OAc
TIPS
N
N
N1
-
(S)-alkyne
TIPS
1. CuSO 4, sodium ascorbate
MeOH, RT
then AQ workup
then
N
",
0
OAc
TIPS
2. Ac 2 0, DMAP
THF, RT
AQ workup, column
63-70% for 2 steps
0
Figure 3. The first iteration of IEG+ with (R)-TIPS and (S)-TIPS gives four stereochemical possibilities for dimers.
Typically, the contents of the second flask are treated with a slight excess (1.01 equiv) of
tetrabutylammonium fluoride (TBAF) to remove the TIPS protecting group. However, in the
case of the first IEG+ iteration, the desired alkyne had been previously synthesized as an
intermediate to the monomers. Thus, (R)-alkyne and (S)-alkyne were used directly. The
contents of the two flasks were then recombined in the presence of a copper(I) catalyst to induce
a copper-catalyzed azide-alkyne cycloaddition (CuAAC) "click" reaction. After completion, the
mixture was subjected to a 5 min acylation step to functionalize the newly formed alcohol.
46
a
(S)R)-G1
(SHS)-Gi
(RHRJ-G1
9-22%
for
4 tp 9"
(RH-RHR)-R)-G2
0
NN
OAC
(SH-S)-SH-S)-G2
o
N=N
OAc
N=N
OAc
(SH-R)-(S)-R)-G2
(
b
0o
,,,
N=N
QAc
N=N
OAc
TIPS
N=N
OAc
N=N
OAc
TIPS
N=N
OAc
N=N
OAc
7-,,
N
C, DO EA,B H J
F
0-
TIPS
0
OAcH H N=N
OAc
N=N
OAc
J F H H J F GH
TIPS
I
11
S-SR
SSSS
RRRR
H
F
B
G
C
A
5.3 5.2 5.1
5.0
4.9 4.8 4.7 4.6 4.5 4.4 4.3 4.2 4.1 4.0 3.9 3.8 3.7 3.6
fl (ppm)
0
C
C, D
E
F
0JF
H H J
3.53.4
H
FGH
3.2 3.1 3.0 2.9 2.8 2.7 2.6 2.5
OAc
N=N
OAc
N=N
OAc
N=N
BH
A,B H J
D
E
H
JF
H J FI H
TIPS
I
SRSR
SSSS
RRRR
i
CDCI3
A-
A7.70
a
JkA
7.65
7.60
7.55
7.50
fl
7.45
(ppm)
7.40
7.35
7.30
7.25
7.20
Figure 4. a) Tetramers with defined stereochemistry; b-c) Overlay of H NMR spectra of (R)-(R)-(R)-(R)-G2, (S)-(S)-(S)-(S)G2, and (S)-(R)-(S)-(R)-G2.
47
Stereochemical Diversity in IEG+: Simple combinations of (S)-TIPS and (R)-TIPS
generated four dimers with distinct stereochemistry (Figure 3), each of which could be used as
building blocks for further iterations of IEG+. From these dimers, it is possible, in principle, to
generate IEG+ oligomers and polymers with any desired stereochemistry at any point along the
backbone--a feat unprecedented for synthetic macromolecules. In our preliminary work, we have
synthesized three examples of tetramers with defined stereochemistry (Figure 4a). As excpected,
the 'H NMR spectra of the enantiomers, (R)-(R)-(R)-(R)-G2 and (S)-(S)-(S)-(S)-G2, overlap and
the spectrum of the diastereomer, (S)-(R)-(S)-(R)-G2 does not. These tetramers were obtained in
four steps from the dimers in a manner analogous to the synthesis of dimers from monomers. In
obtaining tetramers from dimers, all of the intermediate steps required only aqueous workups.
No chromatographic separation was required until after acylation of the tetramer. Future work
will involve optimizing these reactions to improve overall yields of the tetramer.
This initial study will hopefully lead to building a library of higher oligomers and
polymers that possess all of the possible stereochemical permutations. The thermal properties of
these polymers can be studied by differential scanning calorimetry (DSC), and it will be possible
to precisely correlate the glass transition and/or melting temperatures of these materials with
their backbone stereochemistry.
Functional Diversity in IEG+: Sidechain Sequence Control: The above studies have
focused on stereo- and regio- chemistry in IEG+ molecules that posses exclusively -OAc groups.
However, numerous other hydroxyl group modifications can be used to achieve a wide range of
functionalities. As a proof of concept, three functionalized dimers were synthesized with
sidechains comprised of alkyl chains, oligoethylene glycol, and isopropylidene ketals (Figure
5a). These functional dimers were synthesized via EDC coupling to free alcohol of the hydroxyl
48
dimer. With these building blocks, it will be possible to generate alternating, palindromic, block,
and other defined
sequences. Figure 5b represents one possibility--a poly(alkyl)-co-
poly(ethylene glycol) A-B diblock copolymer. Additionally, the isopropylidene ketal allows for
branching off of the IEG+ scaffold. The solution- and solid-state self-assembly of perfect block
copolymers, such as in Figure 5b, could give rise to very uniform assemblies. As part of this
preliminary study, a simple homo-tetramer (Figure 5c) was synthesized from (S)-(S)-hexyl-G1.
This tetramer, (S)-(S)-(S)-(S)-hexyl-G2, was synthesized from (S)-(S)-hexyl-G1
in four steps
with 28% overall yield.
a)
(R)-alkyne
(RHR)-PEG-G1
N3-(R)-TBS
O
(S)-alkyne
4 steps
+
42-61%
N3-(S)-TBS
N=N
O
O
~zN
O
overall
N
TBS
4
BS (SHS)-hexyl-G1
0
0
rac-alkyne
O
N3.rac-TBS
N=N
0
rac-acetonide-Gi
TBS
0
0
ON
b)
0
2
0
N=N
0
0 0
4
TIPS
0
TIPS
0
N=N
0
2n-1
2m14
0
O'
0
O
N=N
OH
N=N
0
N=N
O2n-1
'_\
0
C)
0
4
0
N=N
NA
N-0
TIPS
0
4
0
N=N
N~N
0
-0
0
N=N
4
TBS
0
N
0'-1O
28% yield for 4 steps (from dimer), MW: 986.4
Figure 5. a) Synthesis of functional dimers. b) Proposed synthesis of block copolymer. c) Synthesis of tetramer with alkyl
sidechains.
49
Conclusion: This chapter described a novel synthetic methodology (IEG+) that gives
polymers with defined molar mass and sequence using synthetic procedures that are precise,
scalable, and amenable to diversification. The IEG+ method required monomers equipped with
orthogonal protecting groups: epoxides and alkynyl silanes. The epoxide functionality served
both as a protecting group and as a masked synthon for alcohols, which allowed for side-chain
functionalization of the IEG+ scaffold. Combing R- and S- monomers afforded complete
stereochemical control of the IEG backbone. Oligomers of unimolecular molar mass and precise
chemical structure were successfully prepared.
Experimental Methods
General Considerations: All reagents and solvents were purchased from Aldrich or
VWR and used as supplied unless otherwise noted. Degassed dichloromethane (DCM) and
tetrahydrofuran (THF) were passed through solvent purification columns prior to use.
'H nuclear magnetic resonance (IH-NMR) and
13 C
nuclear magnetic resonance ("C-
NMR) spectra were recorded on Bruker AVANCE-400 NMR spectrometer or INOVA 500 MHz
spectrometer. Chemical shifts are reported in ppm and referenced to the CHCl 3 singlet at 7.24
ppm, DMSO at 2.50 ppm, MeOH at 4.87 ppm, or CH 2 Cl 2 at 5.32 ppm.
13 C-NMR
spectra were
referenced to the center peaks of the CDC13 triplet at 77.23 ppm, DMSO septet at 39.51 ppm,
MeOH septet at 49.150, or CD 2CI 2 quintet at 54.0 ppm. Chemical shifts are expressed in parts
per million (ppm), and splitting patterns are designated as s (singlet), d (doublet), t (triplet), q
(quartet), quin (quintet), m (multiplet) and br (broad). Coupling constants J are reported in Hertz
(Hz). MestReNova NMR 7.0.1 software was used to analyze the NMR spectra.
50
Analytical high-performance liquid chromatography mass spectrometry (HPLC-MS or
LC-MS) data were obtained using an Agilent 1260 series HPLC system equipped with a variable
wavelength ultraviolet-visible (UV-Vis) detector and an Agilent 6130 single quadrupole mass
spectrometer. Solvent gradients consisted of mixtures of nano-pure water with 0.1% acetic acid
(AcOH) and HPLC-grade acetonitrile. High-resolution mass spectrometry data were obtained on
a Bruker Daltonics APEXIV 4.7 Tesla Fourier Transform Ion Cyclotron Resonance Mass
Spectrometer (FT-ICR-MS).
Synthetic Procedures:
propargyl alcohol
tetrabutylammonium bisulfate
50% w/w aq. NaOH,
0*C->RT
O
rac-alkyne
8 To a
Compound rac-alkyne. The following procedure was modified fom a previous report."
cooled (0*C) solution of 50% w/w aqueous sodium hydroxide (62 mL), pentanes (100 mL),
epichlorohydrin (30 mL, 0.383 mol), and tetrabutylammonium hydrogen sulfate (6.40 g, 18.8
mmol), was added prop-2-yn- 1 -ol (11 mL, 0.189 mol). The solution was then allowed to warm to
room temperature. After 3 h, the solution was diluted with H2 0 (100 mL), and the aqueous layer
was extracted with Et2 0 (3 x 100 mL). The combined organic layers were washed with brine (2 x
100 mL), dried over MgSO 4 , filtered, and concentrated under vacuum. The resulting oil was
purified by flash chromatography (0-30% Et 20/pentanes) to give the product as a colorless oil
(18.0 g, 85%). 'H NMR (400 MHz, CDCl 3) 6 4.16 (m, 2H), 3.77 (m, 1H), 3.43 (m, IH), 3.11 (m,
1H), 2.75 (m, 1H), 2.57 (m, IH), 2.41 (t, J= 2.4 Hz, 1H);
13 C
NMR (100 MHz, CDC13) 6 79.4,
75.0, 70.4, 58.5, 50.6, 44.3; HRMS (ESI) m / z calcd for C6H8 0 2 (M + H)+ 113.0597, found
113.0603.
51
propargyl alcohol
tetrabutylammonium bisulfate
0
50% w/w aq. NaOH,
0
0 C->RT
(R)-1 kyn
(R)-alkyne
Compound (R)-alkyne. The following procedure was modifiedfrom a previous report." To a
cooled (0"C) solution of 50% w/w aqueous sodium hydroxide (64 mL), pentanes (100 mL), (R)(-)-epichlorohydrin (42 mL, 0.537 mol), and tetrabutylammonium hydrogen sulfate (9.17 g, 27.0
mmol), was added prop-2-yn-1-ol (15.7 mL, 0.270 mol). The solution was then allowed to warm
to room temperature. After 3 h, the solution was diluted with H20 (100 mL), and the aqueous
layer was extracted with Et 20 (3 x 100 mL). The combined organic layers were washed with
brine (2 x 100 mL), dried over MgSO 4 , filtered, and concentrated under vacuum. The resulting
oil was purified by flash chromatography (0-30% Et 2 0/pentanes) to give the product as a
colorless oil (16.3 g, 54%). 'H NMR (400 MHz, CDCl 3) 64.18 (m, 2H), 3.79 (dd, J= 11.3, 3.0
Hz, 1H), 3.44 (dd, J= 11.3, 5.8 Hz, 1H), 3.13 (m, lH), 2.77 (dd, J= 5.0, 4.2 Hz, 1H), 2.60 (dd, J
= 5.0, 2.7, 1H), 2.42 (t, J= 2.4 Hz, I H);
3
C NMR (100 MHz, CDCl 3) 6 79.4, 75.0, 70.5, 58.6,
50.6, 44.4; HRMS (ESI) m /z calcd for C6H80 2 (M + H)+ 113.0597, found 113.0604.
Cl
propargyl alcohol
tetrabutylammonium bisulfate
50% w/w aq. NaOH,
0C->RT
0
(S)-alkyne
8 To a
Compound (S)-alkyne. The following procedure was modified from a previous report."
cooled (0"C) solution of 50% w/w aqueous sodium hydroxide (50 mL), pentanes (100 mL), (S)(+)-epichlorohydrin (33 mL, 0.422 mol), and tetrabutylammonium hydrogen sulfate (7.15 g, 21.1
mmol), was added prop-2-yn-l-ol (12.3 mL, 0.211 mol). The solution was then allowed to warm
to room temperature. After 3 h, the solution was diluted with H20 (100 mL), and the aqueous
layer was extracted with Et 20 (3 x 100 mL). The combined organic layers were washed with
52
brine (100 mL), dried over MgSO 4, filtered, and concentrated under vacuum. The resulting oil
was purified by flash chromatography (0-30% Et 20/pentanes) to give the product as a colorless
oil (13.2 g, 56%). 'H NMR (400 MHz, CDCl 3) 64.17 (in, 2H), 3.77 (dd, J= 11.3, 3.0 Hz, 1H),
3.43 (dd, J= 11.3, 5.8 Hz, 1H), 3.12 (in, 1H), 2.75 (dd, J= 5.0, 4.2 Hz, IH), 2.58 (dd, J= 5.0,
2.7 Hz, 1H), 2.41 (t, J= 2.4 Hz, IH); '3 C NMR (100 MHz, CDCl 3 ) 6 79.4, 75.0, 70.5, 58.5, 50.6,
44.4; HRMS (ESI) m /z calcd for C6H80 2 (M + H)+ 113.0597, found 113.0606.
0
L
O"
KHMDS
TIPSCI
/
THF, -78 0C
(R)-alkyne
o
TIPS
L\
(R)-TIPS
Compound (R)-TIPS. A solution of (R)-alkyne (4.80 g, 42.8 mmol) in THF (150 mL) was
cooled to -78*C, and a IM THF solution of KHMDS (50 mL, 50 mmol) was added dropwise.
After addition, the reaction was allowed to stir for 15 minutes, and then TIPSCl (12.0 mL, 56.0
mmol) was added. The reaction was allowed to warm up to room temperature and stirred
overnight. The solution was diluted with saturated NH 4 Cl (150 mL) and extracted with ether (3 x
75 mL). The organic layers were combined and washed with brine (200 mL), dried over MgSO 4 ,
filtered, and concentrated under vacuum. The crude product was run through a silica plug (30%
ether/pentanes) and carried on to the next step without further purification.
0
OH
TIPS
NaN, NH 4CIv
(R)-TIPS
DMF, 600C
TIPS
N 3 kO
<
N3-(R)-TIPS
Compound N3-(R)-TIPS. A solution of (R)-TIPS (9.08 g, 33.9 mmol), NaN3 (6.60 g, 0.101
mol), and NH 4 Cl (5.40 g, 0.101 mol) in DMF (50 mL) was heated to 60 0 C. The mixture was
stirred for 24 h, and then diluted with saturated sodium bicarbonate (100 mL) and extracted with
ether (3 x 50 mL). The combined organic layers were washed with brine (100 mL), dried over
53
MgSO 4 , filtered, and concentrated under vacuum. The resulting oil was purified by flash
chromatography (0-30% Et2 O/pentanes) to give the product as a colorless oil (4.50 g, 34%
overall yield for two steps). 'H NMR (400 MHz, CDCl 3) 6 4.22 (s, 2H), 3.96 (quin, J = 4.7 Hz,
IH), 3.59 (dq, J= 9.6, 4.2 Hz, 2H), 3.36 (m, 2H), 2.36 (bs, IH), 1.06 (s, 21 H);
3
1
C NMR (100
MHz, CDC 3 ) 8 102.7, 88.8, 70.9, 69.8, 59.7, 53.7, 18.8, 11.3; HRMS (ESI) m / z calcd for
C15H2 9N 30 2 Si (M + NH 4)+329.2367, found 329.2361.
*Observed diethyl ether and acetone 'H NMR (400 MHz, CDCl3) 6 3.46 (q, J = 7.0 Hz, 0.56H),
2.15 (s, 0.61H), 1.19 (t, J= 7.0 Hz, 0.83H);
0
13
C NMR (100 MHz, CDCl 3 ) 6 100.2, 66.1, 15.5.
KHMDS
TIPSCI
(S)-alkyne
TIPS
0
THF, -78*C
(S)-TIPS
Compound (S)-TIPS. A solution of (S)-alkyne (7.20 g, 64.2 mmol) in THF (225 mL) was
cooled to -78'C, and a IM THF solution of KHMDS (75 mL, 75 mmol) was added dropwise.
After addition, the reaction was allowed to stir for 15 minutes, and then TIPSCl (18.0 mL, 84.0
mmol) was added. The reaction was allowed to warm up to room temperature and stirred
overnight. The solution was diluted with saturated NH 4 Cl (225 mL) and extracted with ether (3 x
100 mL). The organic layers were combined and washed with brine (300 mL), dried over
MgSO 4 , filtered, and concentrated under vacuum. The crude product was run through a silica
plug (30% ether/pentanes) and carried on to the next step without further purification.
TIPS
(S)-TIPS
NaN 3 , NH 4CI ,
0
DMF, 60 C
3
HHN
TIPS
N3 -(S)-TIPS
Compound N 3 -(S)-TIPS. A solution of (S)-TIPS (14.6 g, 54.5 mmol), NaN3 (10.6 g, 0.163
mol), NH4 Cl (8.75 g, 0.163 mol) in DMF (100 mL) was heated to 60'C. The mixture was stirred
54
for 24 h, and then diluted with saturated sodium bicarbonate (100 mL) and extracted with ether
(3 x 100 mL). The combined organic layers were washed with brine (200 mL), dried over
MgSO 4 , filtered, and concentrated under vacuum. The resulting oil was purified by flash
chromatography (0-30% Et 2 O/pentanes) to give the product as a colorless oil (8.44 g, 42%
overall yield for two steps). 'H NMR (400 MHz, CDCl 3 ) 6 4.22 (s, 2H), 3.96 (quin, J= 4.7 Hz,
IH), 3.59 (dq, J= 9.6, 4.2 Hz, 2H), 3.36 (in, 2H), 2.37 (bs, lH), 1.06 (s, 21 H);
1C
NMR (100
MHz, CDC 3) 6 102.7, 88.8, 70.9, 69.8, 59.7, 53.7, 18.8, 11.3; HRMS (ESI) m / z calcd for
C1 5H2 9N 30 2 Si (M + NH4 ) 329.2367, found 329.2344.
*Observed diethyl ether, acetone, and triisopropylsilanol 'H NMR (400 MHz, CDCl 3) 6 3.46 (q,
J= 7.0 Hz, 0.70H), 2.15 (s, 0.61H), 1.19 (t, J= 7.0 Hz, 1.04H), 1.03 (s, 4.89H); '3 C NMR (100
MHz, CDCl 3) 6 66.1, 17.9, 15.5, 12.5.
0
L-\-,o /
rac-alkyne
KHMDS
TIPSCI
THF, -78-C
o
,
TBS
o
rac-TBS
Compound rac-TBS. A solution of 1-rac-alkyne (2.60 g, 23.2 mmol) in THF (80 mL) was
cooled to -78"C, and a 1 M THF solution of KHMDS (25 mL, 25 mmol) was added dropwise.
After addition, the reaction was allowed to stir for 15 minutes, and then TBDMSC (4.20 g, 27.9
mmol) was added. The reaction was allowed to warm up to room temperature and stirred
overnight. The solution was diluted with saturated NH 4 CI (100 mL) and extracted with ether (3 x
50 mL). The organic layers were combined and washed with brine (100 mL), dried over MgSO 4,
filtered, and concentrated under vacuum. The crude product was run through a silica plug (30%
ether/pentanes) and carried on to the next step without further purification.
55
o
o
rac-TBS
TBS
NaN 3 , NH
4 CI ,
N3
OH
TBS
O
N3-rac-TBS
DMF, 600C
Compound N3-rac-TBS. A solution of rac-TBS (4.14 g, 18.3 mmol), NaN3 (3.57 g, 54.8
mmol), NH 4 C1 (2.93 g, 54.8 mmol) in DMF (50 mL) was heated to 60'C. The mixture was
stirred for 24 h, and then diluted with saturated sodium bicarbonate (50 mL) and extracted with
ether (3 x 75 mL). The combined organic layers were washed with brine (150 mL), dried over
MgSO 4 , filtered, and concentrated under vacuum. The resulting oil was purified by flash
chromatography (0-30% Et 20/pentanes) to give the product as a colorless oil (3.95 g, 57%
overall yield for two steps). 'H NMR (400 MHz, CDCl3 ) 6 4.19 (s, 2H), 3.96 (quin, J = 4.7 Hz,
IH), 3.57 (dq, J= 9.6, 4.2 Hz, 2H), 3.37 (m, 2H), 2.38 (bs, IH), 0.92 (s, 9H), 0.10 (s, 6H);
3
C
NMR (100 MHz, CDCl 3) 6 101.6, 90.8, 71.1, 69.8, 59.7, 53.6, 26.2, 16.7, 4.51; HRMS (ESI) m/
z calcd for C 12 H2 3N 3O 2 Si (M + NH 4)+ 287.1898, found 287.1880.
*Observed diethyl ether, acetone, water, and hexanes 'H NMR (400 MHz, CDC13 ) 6 3.46 (q, J
7.0 Hz, 0.83H), 2.15 (s, 0.15H), 1.56 (bs, 0.73H), 1.27 (m, 2.5 H), 1.19 (t, J= 7.0 Hz, 1.37H),
0.86 (t, J= 7.3, 2.59H);
13 C
NMR (100 MHz, CDCl 3) 6 66.1, 34.3, 22.6, 15.5, 14.3.
Synthesis of dimers
0
L
,,
O1.
(S)-alkyne
(S)a
OH
CuSO 4, sodium ascorbate
knethen
TIPS
MeOH, RT
AQ workup
2. Ac20, DMAP
TH, RT
then AQ workup, column
,
0
21.,O
OAc
N=N
TIPS
N
(S)-(S)-G1
N3-(S)-TIPS
Compound (S)-(S)-G1. To a vial equipped with a stirbar, (S)-alkyne (0.180 g, 1.61 mmol), N3 (S)-TIPS (0.500 g, 1.61 rnmol), and MeOH (5 mL) were added. Sodium ascorbate (0.642 mL;
1.0 M solution in H 2 0; 0.642 mmol) and copper sulfate (0.321 mL; 0.5 M solution in H 2 0; 0.161
56
mmol) were added and the solution was stirred at room temperature for 15 min. The reaction was
monitored via LC-MS and upon completion, 4 drops of H2 0 2 were added. The reaction was then
diluted with EtOAc (50 mL) and washed with saturated EDTA (50 mL). The aqueous layer was
then extracted with EtOAc (2 x 30 mL). The combined organic layers were then washed with
brine (100 mL), dried over MgSO 4, filtered, and concentrated under vacuum. The intermediate
was carried on to the next step without further purification.
The intermediate was dissolved in THF (10 mL) and to this was added acetic anhydride (0.455
mL, 4.81 mmol) and DMAP (9.80 mg, 0.080 mmol). The reaction was stirred for 5 min while the
reaction was monitored by LC-MS. Upon completion, the reaction was diluted with EtOAc (50
mL) and washed with 0.1 M HCl (50 mL), saturated sodium bicarbonate (50 mL), and brine (50
mL). The organic layer was dried over MgSO 4 , filtered, and concentrated under vacuum. The
crude product was purified by flash chromatography (0-10% MeOH/DCM) to give (S)-(S)-GJ
(0.507 g obtained, 68% overall yield for 2 steps). 'H NMR (400 MHz, CDCl 3) 6 7.58 (s, 1H),
5.30 (m, IH), 4.68 (in, 2H), 4.61 (m, 2H), 4.22 (s, 2H), 3.81 (dd, J = 11.4, 2.8 Hz, IH), 3.65 (dq,
J= 10.5, 4.8 Hz, 2H), 3.42 (dd, J= 11.4, 6.0 Hz, 1H), 3.14 (in, 1H), 2.77 (t, J= 5.0 Hz, IH),
2.59 (dd, J= 5.0, 2.7 Hz. IH), 2.03 (s, 3H), 1.04 (s, 21H); '3 C NMR (100 MHz, CDCl 3) 6 170.0,
145.2, 123.7, 102.3, 89.1, 71.2, 70.9, 67.7, 64.8, 59.7, 50.9, 50.3, 44.4, 21.0, 18.8, 11.3; HRMS
(ESI) m /z calcd for C23 H39N 30 5Si (M + H)+ 466.2738, found 466.2625.
*Observed acetone and dichloromethane 'H NMR (400 MHz, CDCl 3) 6 5.27 (s, 0.28H), 2.14 (s,
1.05H);
13 C
NMR (100 MHz, CDC13 ) 6 31.2.
57
-. 1O
'
1. CuSO 4, sodium ascorbate
-
MeOH, RT
then AQ workup
(R)-alkyne
OH
TIPS
3
0
N=N
OAc
TIPS
2. Ac 2O, DMAP
then AQ workup, column
N3-(R)-TIPS
Compound (R)-(R)-GJ. This compound was synthesized using the same procedure as (S)-(S)G1, starting with IR-alkyne and N3 -1R-TIPS. 0.5196 g, 70% overall yield for 2 steps. 'H NMR
(400 MHz, CDCl 3) 6 7.58 (s, 1H), 5.30 (m, 1H), 4.68 (m, 2H), 4.61 (m, 2H), 4.22 (s, 2H), 3.81
(dd, J= 11.4, 2.8 Hz, IH), 3.65 (dq, J= 10.5, 4.8 Hz, 2H), 3.42 (dd, J= 11.4, 6.0 Hz, 1H), 3.14
(m, IH), 2.77 (t, J= 5.0 Hz, IH), 2.59 (dd, J= 5.0, 2.7 Hz, 1H), 2.03 (s, 3H), 1.04 (s, 21H);
3
1
C
NMR (100 MHz, CDC 3) 6 170.0, 145.2, 123.7, 102.3, 89.1, 71.2, 70.9, 67.7, 64.8, 59.7, 50.9,
50.3, 44.4, 21.0, 18.8, 11.3; LC-MS m /z caled for C2 3H39N 30 5 Si (M + H)+ 466.3, found 466.3.
*Observed acetone and dichloromethane (400 MHz, CDC 3 ) 6 5.27 (s, 0.60H), 2.14 (s, 0.98H).
0
O
L
'
1. CuSO 4, sodium ascorbate
MeOH, RT
(R)-alkyne
OH
N3
then AQ workup
TIPS
N0THERT
0
L-\
N=N
.
N
OAc
TIPS
O
,
2. Ac2 ODMAP
then AQ workup, column
(R)-(S)-G1
N3-(S)-TIPS
Compound (R)-(S)-GJ. This compound was synthesized using the same procedure as (S)-(S)G1, starting with IR-alkyne and N3-1S-TIPS. 0.4719 g obtained, 63% overall yield for 2 steps.
'H NMR (400 MHz, CDC13) 6 7.58 (s, 1H), 5.30 (m, 1H), 4.68 (m, 2H), 4.61 (m, 2H), 4.22 (s,
2H), 3.81 (dd, J= 11.4, 2.8 Hz, 1H), 3.65 (dq, J= 10.5, 4.8 Hz, 2H), 3.42 (dd, J= 11.4, 6.0 Hz,
1H), 3.14 (m, 1H), 2.77 (t, J= 5.0 Hz, 1H), 2.59 (dd, J= 5.0, 2.7 Hz, IH), 2.03 (s, 3H), 1.04 (s,
21H); '3 C NMR (100 MHz, CDC13 ) 6 170.0, 145.2, 123.7, 102.3, 89.1, 71.2, 70.9, 67.7, 64.8,
59.7, 50.9, 50.3, 44.4, 21.0, 18.8, 11.3; HRMS (ESI) m / z calcd for C2 3 H3 9N 3O 5Si (M + H)
466.2738, found 466.2631.
58
*Observed ethyl acetate and acetone 'H NMR (400 MHz, CDCl 3) 6 4.09 (q, J= 7.1 Hz, 1.28H),
2.14 (s, 1.01H), 2.02 (s, 2.03H), 1.23 (t, J= 7.1 Hz, 2.04H);
13 C
NMR (100 MHz, CDC 3) 6 60.6,
21.3, 14.4.
0
0
(S-lyeMeOH,
(S)-alkyne
OH
TIPS
N'O
3
<-
1.CuSO 4, sodium ascorbate
RT
then AQ workup
,
~THFRT
OcTIPS
0NN
N
2. Ac 2O, DMAP
O
N
(S)-(R)-G1
then AQ workup, column
N3-(R)-TIPS
Compound (S)-(R)-GJ. This compound was synthesized using the same procedure as (S)-(S)G1, starting with 1S-alkyne and N3 -1R-TIPS. 0.5076 g obtained, 66% overall yield for 2 steps.
'H NMR (400 MHz, CDCl 3) 6 7.58 (s, 1H), 5.30 (m, 1H), 4.68 (m, 2H), 4.61 (m, 2H), 4.22 (s,
2H), 3.81 (dd, J= 11.4, 2.8 Hz, 1H), 3.65 (dq, J= 10.5, 4.8 Hz, 2H), 3.42 (dd, J= 11.4, 6.0 Hz,
1H), 3.14 (m, iH), 2.77 (t, J= 5.0 Hz, 1H), 2.59 (dd, J= 5.0, 2.7 Hz, 1H), 2.03 (s, 3H), 1.04 (s,
21H);
13 C
NMR (100 MHz, CDCl 3) 6 170.0, 145.2, 123.7, 102.3, 89.1, 71.2, 70.9, 67.7, 64.8,
59.7, 50.9, 50.3, 44.4, 21.0, 18.8, 11.3; LC-MS m /z calcd for C23H39N30 5Si (M + H)+ 466.3,
found 466.3.
0
O
1.CuSO 4, sodium ascorbate
MeOH, rt
(R)-alkyne
then AQ workup
OH
N3
OHO
TBS
3
N3-(R)-TBS
2.
O-[2-(2-methoxyethoxy)ethyl]glycolic
EDC-HCI, DMAP
~~DOMV,
rt
then AQ workup, column
0
0
acid
N
0
=N
0
0
TBS
NN
"""
(R)-(R)-PEG-G1
Compound (R)-(R)-PEG-GJ. To a vial equipped with a stirbar, IR-alkyne (0.180 g, 1.61
mmol), N3 -1R-TBS (0.432 g, 1.61 mmol), and MeOH (5 mL) were added. Sodium ascorbate
(0.642 mL; 1.0 M solution in H2 0; 0.642 mmol) and copper sulfate (0.321 mL; 0.5 M solution in
H2 0; 0.161 mmol) were added and the solution was stirred at room temperature for 15 min. The
59
reaction was monitored via LC-MS and upon completion, 4 drops of H2 0 2 were added. The
reaction was then diluted with EtOAc (50 mL) and washed with saturated EDTA (50 mL). The
aqueous layer was then extracted with EtOAc (2 x 30 mL). The combined organic layers were
then washed with brine (100 mL), dried over MgSO 4 , filtered, and concentrated under vacuum.
The intermediate was carried on to the next step without further purification.
The intermediate was dissolved in DCM (10
mL) and to this was added O-[2-(2-
methoxyethoxy)ethyl]glycolic acid (0.369 mL, 2.40 mmol), N-(3-dimethylaminopropyl)-N'ethylcarbodiimide hydrochloride (0.492 g, 2.57 mmol), and DMAP (0.196 g, 1.60 mmol). The
reaction was stirred for 1 hour and then quenched with brine (50 mL) and extracted with DCM (3
x 30 mL). The organic layer was dried over MgSO 4 , filtered, and concentrated under vacuum.
The crude product was purified by flash chromatography (0-10% MeOH/DCM) to give (R)-(R)PEG-GJ (0.3674 g, 42% overall yield over 2 steps). 'H NMR (400 MHz, CDCl 3) 6 7.55 (s, 1H),
5.26 (in, IH), 4.58-4.47 (in, 4H), 4.07 (d, J= 2.8 Hz, 2H), 4.00 (d, J= 2.8 Hz, 2H), 3.69 (dd, J=
11.5, 2.8 Hz, 1H), 3.53 (s, 2H), 3.51-3.48 (in, 3H), 3.41-3.39 (in, 3H), 3.28 (dd, J= 11.4, 6.1 Hz,
IH), 3.23 (s, 2H), 3.22 (s, 3H), 3.01 (m, IH), 2.64 (t, J= 5 Hz, IH), 2.46 (dd, J= 5.0, 2.7 Hz,
lH), 0.79 (s, 9H), -0.02 (s, 6H);
13
C NMR (100 MHz, CDCl3 ) 6 169.4, 144.8, 123.7, 101.0, 90.6,
71.7, 70.9, 70.8, 70.7, 70.4, 70.3, 68.2, 67.4, 64.4, 59.3, 58.8, 53.5, 50.5, 49.9, 44.0, 25.9, 16.3, 4.84; HRMS (ESI) m /z calcd for C2 5H4 3N30 8Si (M + H)+ 542.2892, found 542.2909.
*Observed DCM and impurity 'H NMR (400 MHz, CDCl 3) 6 5.19 (s, 1.75H), 4.17-4.15 (m,
0.51H), 4.04 (s, 0.45H);
13 C
NMR (100 MHz, CDCl 3) 6 170.3, 68.8, 68.4, 63.6, 53.5.
60
1. CuSO 4, sodium ascorbate
MeOH, rt
0
0
then AQ workup
/
rac-alkyne
o
2.
0
O
OH
N3
TBS
OH
0
N=N
0
TBS
N
7o
O
rac-acetonide-G1
EDC-HCI, DMAP
DCM, rt
then AQ workup, column
N3-rac-TBS
Compound rac-acetonide-GJ. To a vial equipped with a stirbar, rac-alkyne (0.174 g, 1.55
mmol), N3-rac-TBS (0.500 g, 1.55 mmol), and MeOH (5 mL) were added. Sodium ascorbate
(0.620 mL; 1.0 M solution in H2 0; 0.620 mmol) and copper sulfate (0.3 10 mL; 0.5 M solution in
H2 0; 0.155 mmol) were added and the solution was stirred at room temperature for 15 min. The
reaction was monitored via LC-MS and upon completion, 4 drops of H20 2 were added. The
reaction was then diluted with EtOAc (50 mL) and washed with saturated EDTA (50 mL). The
aqueous layer was then extracted with EtOAc (2 x 30 mL). The combined organic layers were
then washed with brine (100 mL), dried over MgSO 4, filtered, and concentrated under vacuum.
The intermediate was carried on to the next step without further purification.
The intermediate was dissolved in DCM (10 mL) and to this was added 2,2,5-trimethyl-1,3dioxane-5-carboxylic
acid
(0.405
g,
2.32
mmol),
N-(3-dimethylaminopropyl)-N'-
ethylcarbodiimide hydrochloride (0.475 g, 2.48 mmol), and DMAP (0.190 g, 1.55 mmol). The
reaction was stirred for 1 hour and then quenched with brine (50 mL) and extracted with DCM (3
x 30 mL). The organic layer was dried over MgSO 4 , filtered, and concentrated under vacuum.
The crude product was purified by flash chromatography (0-10% MeOH/DCM) to give racacetonide-GJ (0.4546 g, 54% overall yield over 2 steps). 'H NMR (400 MHz, CDCl3 ) 6 7.69 (s,
0.83H), 5.32 (quin, J= 5.6 Hz, IH), 4.65-4.55 (m, 4H), 4.11 (d, J= 7.1 Hz, 2H), 4.02 (d, J= 7.1
Hz, 2H), 3.72 (dd, J= 11.4, 2.9 Hz, 1H), 3.55-3.51 (m, 4H), 3.35 (dd, J= 11.4, 5.9 Hz, lH), 3.06
61
(m, 1H), 2.69 (t, J= 4.9 Hz, IH), 2.51 (dd, J= 5.0, 2.6 Hz, IH), 1.34 (s, 3H), 1.27 (s, 3H), 0.98
(s, 3H), 0.83 (s, 9H), 0.02 (s, 6H); "C NMR (100 MHz, CDC13) 6 173.3, 145.0, 123.9, 101.2,
98.2, 90.7, 71.0, 70.9, 67.6, 66.0, 65.9, 64.5, 59.5, 50.7, 50.2, 44.2, 42.2, 26.1, 21.0, 18.2, 16.4,
14.2, 4.69; HRMS (ESI) m /z caled for C2 6H4 3N30 7 Si (M + H)+ 538.2943, found 538.2951.
*Observed ethyl acetate and impurity 'H NMR (400 MHz, CDCl 3) 6 9.28 (d, J= 7.9 Hz, 0.07H),
7.86 (s, 0.07H), 7.36 (d, J = 12.7 Hz, 0.06H), 5.63 (dd, J= 12.7, 7.9 Hz, 0.09H), 4.06-4.03 (m,
1.50H), 1.94 (s, 2.17H), 1.16 (t, J= 7.1 Hz, 2.22H);
13 C
NMR (100 MHz, CDC 3) 6 191.0, 171.1,
169.5, 142.0, 124.8, 111.2, 60.4, 21.3.
0
sodium ascorbate
MeOH, RT
then AQ workup
0
1.CuSO 4 ,
1,O
(S)-alkyne
OH
3
1
TBS
O
''
0
2. hexanoic anhydride, DMAP
THF, RT
then AQ workup, column
N
,,-O
=N
4
0
TBS
NN
(S)-(S)-hexyl-G1
N3-(S)-TBS
Compound (S)-(S)-hexyl-GJ. To a vial equipped with a stirbar, IS-alkyne (0.180 g, 1.61 mmol),
N3-IS-TBS (0.432 g, 1.61 mmol), and MeOH (5 mL) were added. Sodium ascorbate (0.642 mL;
1.0 M solution in H 2 0; 0.642 mmol) and copper sulfate (0.321 mL; 0.5 M solution in H 2 0; 0.161
mmol) were added and the solution was stirred at room temperature for 15 min. The reaction was
monitored via LC-MS and upon completion, 4 drops of H20 2 were added. The reaction was then
diluted with EtOAc (50 mL) and washed with saturated EDTA (50 mL). The aqueous layer was
then extracted with EtOAc (2 x 30 mL). The combined organic layers were then washed with
brine (100 mL), dried over MgSO 4, filtered, and concentrated under vacuum. The intermediate
was carried on to the next step without further purification.
62
The intermediate was dissolved in THF (10 mL) and to this was added hexanoic anhydride
(0.741 mL, 3.21 mmol) and DMAP (9.80 mg, 0.080 mmol). The reaction was stirred for 20 min
while the reaction was monitored by LC-MS. Upon completion, the reaction was diluted with
EtOAc (50 mL) and washed with 0.1 M HCl (50 mL), saturated sodium bicarbonate (50 mL),
and brine (50 mL). The organic layer was dried over MgSO 4 , filtered, and concentrated under
vacuum. The crude product was purified by flash chromatography (25-75% EtOAc/hexanes) to
give (S)-(S)-hexyl-G1 (0.4676 g, 61% overall yield for 2 steps). 'H NMR (400 MHz, CDCl 3 ) 6
7.58 (s, 1H), 5.31 (m, IH), 4.71-4.56 (m, 4H), 4.18 (d, J= 2.5 Hz, 2H), 3.80 (dd, J= 11.4, 2.9
Hz, 1H), 3.61 (d, J= 4.9 Hz, 2H), 3.42 (dd, J= 11.4, 6.0Hz, lH), 3.14 (m, IH), 2.77 (t, J= 4.9
Hz, IH), 2.59 (quin, J= 2.5 Hz, 1H), 2.27 (t, J= 7.5 Hz, 2H), 1.54 (quin, J= 7.4 Hz, 2H), 1.271.18 (m, 4H), 0.90 (s, 9H), 0.86 (t, J= 6.8 Hz, 3H); "C NMR (100 MHz, CDCl 3) 6 172.9, 145.2,
123.7, 101.2, 91.0, 71.3, 70.5, 67.8, 64.8, 59.7, 50.9, 50.4, 44.4, 34.3, 31.3, 26.2, 24.6, 22.4, 16.6,
14.1, -4.53; LC-MS m /z calcd for C2 4 H4 1N3 0 5 Si (M + H)+ 480.3, found 480.3.
*Observed DCM and impurity 'H NMR (400 MHz, CDCl 3) 6 5.27 (s, 0.63H); '3 C NMR (100
MHz, CDCL3) 6 100.2.
63
Synthesis of Tetramers
NaN3 , NH 4CI
DMF, 60C
N3
OH
kO
OAc
N=N
TIPS
O
NNiJ
>
N3-(R)-(R)-G1
N=N
0
OAc
TIPS
0
(R)-(R)-G1
TBAFN
L
TBAF
THF, rt
0
N=N
OAc
N=N
OAc
N=N
.~
O
N=N
OAc
k(R)-(R)-G1-alkyne
OAc
TIPS
1. CuSO 4, sodium ascorbate
MeOH, RT
then AQ workup
2. Ac20, DMAP
THF, RT
(R)-(R)-(R)-(R)-G2
then AQ workup, column
Compound (R)-(R)-(R)-(R)-G2. (R)-(R)-GI was divided into two batches. The first batch of (R)(R)-G1 (0.230 g, 0.494 mmol) was added to a 20 mL microwave vessel equipped with a stirbar.
To this was added NaN, (0.096 g, 1.48 mmol), NH4 Cl (0.079 g, 1.48 mmol) and DMF (15 mL).
The vessel was then sealed and placed in a microwave reactor for 25 min at 125"C. The reaction
was then diluted with EtOAc (60 mL) and washed with saturated sodium bicarbonate (2 x 60
mL) and brine (1 x 60 mL). The organic layer was then dried over MgSO 4, filtered, and
concentrated under vacuum to give crude N3-(R)-(R)-GI, which was used without further
purification.
The second batch of (R)-(R)-G1 (0.258 g, 0.553 mmol) was added to a 20 mL scintillation vial
equipped with a stirbar. The dimer was dissolved in THF (10 mL) and TBAF (0.558 mL; IM in
THF; 0.558 mmol) was added dropwise. The reaction was shown to be complete after 15
minutes by LC-MS. The excess TBAF was quenched by adding CaCO 3 (0.116 mg, 1.16 mmol),
DOWEX 50WX8-400 (347 mg), and MeOH (2 mL), and the mixture was stirred for I h. 3 9 All
64
insoluble materials were removed by filtration through a pad of Celite, and the filter cake was
washed with MeOH. The filtrate was then concentrated under vacuum to give the crude
compound (R)-(R)-GJ-alkyne, which was used without further purification.
The crude compounds N3-(R)-(R)-Gt
and (R)-(R)-G1-alkyne were combined in a 20 mL
scintillation vial equipped with a stirbar. The compounds were dissolved in MeOH (15 mL), and
to this was added sodium ascorbate (0.221 mL; 1.0 M solution in H2 0; 0.221 mmol) and copper
sulfate (0.111 mL; 0.5 M solution in H2 0; 0.056 mmol). The reaction was stirred at room
temperature for 25 minutes and monitored via LC-MS. Upon completion, 4 drops of H20 2 were
added, and the reaction was diluted with EtOAc (60 mL). The organic solution was then washed
with saturated EDTA (60 mL), and the aqueous layer was back-extracted with EtOAc (2 x 30
mL). The combined organic layers were then washed with brine (100 mE), dried over MgSO 4 ,
filtered, and concentrated under vacuum to give the crude cycloaddition adduct. The crude
intermediate was carried on to the next step without further purification.
The crude intermediate was dissolved in THF (10 mL) and to this was added acetic anhydride
(0.140 mL, 1.48 mmol) and DMAP (6.0 mg, 0.049 mmol). The reaction was stirred for 5 min
while the reaction was monitored by LC-MS. Upon completion, the reaction was diluted with
EtOAc (50 mL) and washed with 0.1 M HCl (50 mL), saturated sodium bicarbonate (50 mL),
and brine (50 mL). The organic layer was dried over MgSO 4 , filtered, and concentrated under
vacuum. The crude product was purified by flash chromatography (0-10% MeOH/DCM) to give
(R)-(R)-(R)-(R)-G2 (46.1 mg, 11% for four steps). 'H NMR (400 MHz, CDCl 3) 6 7.61 (s, IH),
7.59 (s, 1H), 7.58 (s, 1H), 5.32-5.21 (m, 3H), 4.68-4.55 (m, 12H), 4.21 (s, 2H), 3.79 (dd, J= 8.7,
65
2.8 Hz, 1H), 3.68-3.60 (m, 2H), 3.58-3.49 (in, 4H), 3.38 (dd, J= 11.4, 6.1 Hz, 1H), 3.11 (m, IH),
2.76 (t, J= 4.2 Hz, 1H), 2.57 (dd, J= 5.0, 2.6 Hz, IH), 2.00 (in, 9H), 1.02 (s, 21H); '3 C NMR
(100 MHz, CDC13) 6 170.01, 169.96, 145.09, 144.48, 144.45, 124.02, 123.85, 123.81, 102.29,
89.02, 71.21, 70.78, 70.66, 70.63, 68.23, 68.16, 67.69, 64.79, 64.75, 64.71, 59.62, 50.83, 50.40,
50.13, 50.07, 44.35, 21.02, 20.97, 18.71, 11.23; HRMS (ESI) mr/z calcd for C39H 6lN
9 0,,Si
(M +
H)+ 860.4333, found 860.4311.
*Observed grease and impurity 'H NMR (400 MHz, CDC13 ) 6 8.05 (s, 0.16H), 1.21 (s, 4.45H);
13 C
NMR (100 MHz, CDC13 ) 6 129.63, 29.83.
NaN 3 , NH04CI
DMF, 60 C
OH
N3
N=N
0
OAc
~ ~~
TIPS
0.
N3-(S)-(S)-G1
TIPS
OAc
N=N
0
N=N
0.
(S)-(S)-Gi
L.~
TBAF
THF, rt
0
N=N
,A
OAc
0
N=N
NN
OAc
N=N
O
0TBA
N=N
OO
O
(S)-(S)-G1-alkyne
OAc
N
TIPS
1- CuSO 4 , sodium ascorbate
MeOH, RT
then AQ workup
2. Ac2 0, DMAP
THF, RT
then AQ workup, column
(S)-S)(S)(S)-G2
Compound (S)-(S)-(S)-(S)-G2. Tetramer synthesized in an analogous manner to (R)-(R)-(R)(R)-G2. 26.7 mg obtained, 9% for four steps from (S)-(S)-G1. 'H NMR (400 MHz, CDC 3) 6
7.62 (s, 1H), 7.59 (s, 1H), 7.58 (s, 1H), 5.33-5.21 (m, 3H), 4.68-4.28 (in, 12H), 4.21 (s, 2H), 3.79
(dd, J= 11.4, 2.8 Hz, 1H), 3.65 (in, 2H), 3.54 (in, 4H), 3.39 (dd, J= 11.4, 6.1 Hz, 1H), 3.12 (in,
1H), 2.75 (t., J= 4.8 Hz, 1H), 2.57 (dd, J= 5.0, 2.6 Hz, 1H), 2.01 (in, 9H), 1.03 (s, 21H);
13
C
NMR (100 MHz, CDC13) 6 170.02, 169.97, 145.11, 144.49, 144.46, 124.02, 123.85, 123.81,
102.29, 89.04, 71.22, 70.79, 70.68, 70.65, 68.24, 68.17, 67.70, 64.80, 64.76, 64.73, 59.63, 50.84,
66
50.41, 50.15, 50.08, 44.37, 21.03, 20.98, 18.72, 11.24; HRMS (ESI) m / z calcd for
C39H6 1N90 ,Si (M + H)+ 860.4333, found 860.4348.
*Observed grease and impurity 'H NMR (400 MHz, CDCl 3) 8 8.05 (s, 0.23H), 1.21 (s, 1.34H);
'3 C NMR (100 MHz, CDC 3 ) 6 129.63, 39.07, 29.84, 24.14, 23.12.
NaN 3 , NH 4CI
DMF, 600 C
N3
OH
O
OAc
N=N
N
TIPS
O
N3-(S)-(R)-G
N=N
0
OAc
TIPS
(S)-(R)-G1
TBAFN'QOc
N=N
0
OAc
K-Yo
TBAF
rt
THF,
K
(S)-(R)-G1-alkyne
1. CuSO 4, sodium ascorbate
O
N=N
OAc
N=N
OAc
N=N
OAc
TIPS
MeOH, RT
then AQ workup
2. Ac 2O, DMAP
THF, RT
then AQ workup, column
(S)-(R)-(S)-(R)-G2
Compound (S)-(R)-(S)-(R)-G2. Tetramer synthesized in an analogous manner to (R)-(R)-(R)(R)-G2. 92.7 mg obtained, 22% for four steps from (S)-(R)-GJ. 'H NMR (400 MHz, CDCl 3) 6
7.60, (s, 1H), 7.58 (s, IH), 7.56 (s, 1H), 5.30-5.20 (m, 3H), 4.65-4.50 (m, 12H), 4.19 (s, 2H),
3.77 (dd, J= 11.5, 2.8 Hz, 1H), 3.62 (m, 2H), 3.51 (m, 4H), 3.35 (dd, J= 11.5, 6.1 Hz, 1H), 3.10
(m, 1H), 2.72 (t, J= 4.8 Hz, 1H), 2.54 (dd, J= 5.0, 2.6 Hz, 1H), 1.98 (m, 9H), 1.00 (s, 21H); '3 C
NMR (100 MHz, CDC13) 6 169.95, 169.90, 144.99, 144.39, 144.37, 124.00, 123.84, 123.79,
102.25, 88.94, 71.15, 70.72, 70.60, 70.56, 68.18, 68.13, 67.63, 64.70, 64.66, 64.63, 59.55, 50.76,
50.34, 50.08, 50.02, 44.28, 20.94, 20.89, 18.64, 11.16; HRMS (ESI) m / z calcd for
C39H 6 N 90, Si (M + H)+ 860.4333, found 860.4354.
67
*Observed grease and impurity 'H NMR (400 MHz, CDC13) 6 8.05 (s, 0.14H), 1.19 (s, 0.97H);
13 C
NMR (100 MHz, CDCl3 ) 6 129.56.
0
NaN 3, NH 4CI
0
DMF,60 C
0
-. .N
N=N
TBS
N3-(S)-(S)-hexyl-G1
TBS
0
KO
N=N
OH
4
.J
0
(S)-(S)-hexyl-G1
0
TBAF
THF, rt
N=N
*
.,
0
O
(S)-(S)-hexyl-G1-alkyne
N=N
0
O
0
O
4
N=N
0
O
N
0,
,,
(S)-(S)-(S)-(S)-hexyl-G2
Compound (S)-(S)-(S)-(S)-hexyl-G2.
TBS
0
N=N
0
1. CuSO 4, sodium ascorbate
4
MeOH, RT
then AQ workup
2. hexanoic anhydride, DMAP
THF, RT
then AQ workup, column
Tetramer synthesized in an analogous manner to (R)-(R)-
(R)-(R)-G2. 0.109 mg obtained, 28% for four steps from (S)-(S)-hexyl-G1. 'H NMR (400 MHz,
CDCI3 ) 6 7.59 (s, IH), 7.57 (s, 1H), 7.55 (s, IH), 5.31-5.21 (m 3H), 4.65-4.51 (m, 12H), 4.15 (s,
2H), 3.76 (dd, J= 11.4, 2.8 Hz, 1H), 3.58 (d, J= 4.7 Hz, 2H), 3.55-3.46 (m, 4H), 3.36 (dd, J=
11.4, 6.1 Hz, 1H), 3.09 (m, 1H), 2.72 (t, J= 4.2 Hz, I H), 2.54 (dd, J= 5.0, 2.6 Hz, 1H) 2.22 (t, J
=
7.5 Hz, 6H), 1.49 (quin, J= 7.4 Hz, 6H), 1.26-1.16 (m, 12H), 0.87 (s, 9H), 0.81 (t, J= 6.7 Hz,
9H), -0.05 (s, 6H);
13 C
NMR (100 MHz, CDC13) 6 172.78, 172.75, 144.96, 144.39, 144.37,
123.91, 123.78, 123.73, 101.15, 90.90, 71.13, 70.40, 70.32, 70.30, 68.26, 68.19, 67.77, 64.73,
64.71, 64.63, 59.55, 50.75, 50.33, 50.13, 50.09, 44.28, 34.13, 31.21, 31.20, 26.10, 24.51, 24.49,
22.32, 16.50, 13.96, -4.63; LC-MS m /z caled for C48H79N90 1 S i (M + H)+ 986.6, found 986.4.
68
NMR Spectra
0
rac-alkyne
Lt
,A%-
C
to
95
170
90
180
85
80
150
7T
140
65
70
130
120
J
-
60
i10
4
cha PPMP
40
70
1
69
35
25
30
80
50
20
40
15
30
10
20
05
10
0
(R)-alkyne
c
95
90
I?, _.
10
170
850
85
-
60
I
75
65
70
-..- 1 _d-, .A. . KF
160
150
140
130
120
60
45
40
110
25
3C
35
AhI IE E
L
-
-I f It
70
l
70
60
0
25
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-3
.I
50
20
.
-
40
15
10
05
AU AL7i1
.
I
30
20
10
0
II
0
(S)-alkyne
a8
c
95
90
85
80
75
95
70
Ti
l0
170
10
150
140
130
120
5
80
45
hppm)
1C&
40
35
30
25
20
15
10
05
-I
I~~iim~inj
I
110
8
8
809
0
71
70
60
50
-
40
30
20
10
0
OH
TIPS
N3
o
N3-(R)-TIPS
I
J
L
C
95
90
80
85
75
85
70
-11
170
160
150
140
130
120
110
r
W4 5
~t45*
oM)
0
a0
L.i
18
-
I
1
r
40
20
25
15
60
005
10
hI.
70
72
y
30
35
L
50
40
30
20
10
OH
TIPS
N3
O
,
N3-(S)-TIPS
o
9s
90
65
80
15
65
10
60
40
lqi&dl
I
35
30
110
160
150
140
130
120
20
15
10
110
10
m
73
05
II
I
r
10
25
-
1
SO
-
T
40
- - "
30
20
10
3
OH
TBS
N3
0
N3-rac-TBS
______
C
9_
9
s
95
90
SS5
7
_
0
d0
05
70
7S
50
0A
5
~1L~
__________
NOR*
to
170
150
ISO
140
130
120
40
J~
Ito
flu-1
70
74
35
2S
30
-
60
_
A
Al
20
15
I
,
SC
I
I
40
30
05
10
III
20
IQ
01
TIPS
OAc
N=N
N
0
O
(R)-(R)-G1
7 95
oo
85
so
is
70
85
60
Iii ~I~
I
35
40
1 11 1
-***mow
1 1
I
r
10
170
160
150
140
130
120
110
V&M
AW
75
70
OC
T
50
25
30
40
30
20
10
I
i
20
15
1
0
0
0
TIPS
OAc
N=N
(S)-(S)-G1
.
95
90
s0
I
10
170
-C
-r-rC
a5
7
I
10
15D
65
70
-,
I
I
140
130
120
-0+1
60
110
T
T
s
11
30
30
25
25
60
15
'
20
20
10
05
I
II I
70
76
35
35
I
1~
-
I-
40
40
a
s0
40
30
20
10
0
0
N-N
QAc
TIPS
0
N.
(R)-(S)-G1
97g55
'o
o
i
70
55
6
"
50
40
4
-I
170
180
150
1
40
1 30
120
Ito
I~
77
35
I
30
25
20
15
1 0
05
0
11. 11. - -I
0 0Rw ,%
Cl
70
80
50
40
30
20
10
0
N=N
TIPS
OAc
(s)-(R)-G1
LI
Ja.
9
08 0i
95
as 5 o
9s
r0
14
rI -pill
-W
as
I
I
Oil
o
75
l
60
I
Iw
ISO
140
i3o
120
110
%
78
a1
35
3C
25
20
it
I0 a
III I
i
0"
at
4D
4
I
1;0
I
l&kAit
VPMIO
70
I
I
60
50
I
40
3D
20
1(1
05
O
TBS
O
N=N
N
0
(R)-(R)-PEG-G1
9
95
-1
10
170
90
0
s
75
150
iv
5
40
I 1
140
130
120
110
120
90
Chia
m ams a
'o
35
a 2
25
30
20
15
10
0
05
1.jIL~ I]
I1
-160
t
70
5h10
79
80
(pm
70
s0
50
40
30
20
'0
0
TBS
0
N=N
rac-acetonide-G1
90
5
85
75
83
~li
70
T
65
T
60
TT
4
C
35
00101)
30
V
25
20
15
1c
~
05
CO
C
11
L. i
-~
i
55
I
190
180
170
1
0
1o0
140
130
120
110
80
1a
.
80
70
60
50
40
30
20
10
0
O
0
4
N=N
0--.,,
TBS
0
N
o
(S)-(S)-hexyl-G1
__
5
90
85
80
I
75
65
70
1
to
17D
140
150
AA 10
JA±
140
60
-1
30
120
4
S5
35
I
10D
90
80
ShtO
Clmnia
81
25
05
20
I!II .II
Ii
110
30
70
{pn
s0
50
I
40
30
20
10
co
C
QAc
0N=N
0
0~-
-
TIPS
QAc
N=N
QAc
N=N
,
,,z,,
(R)-(R)-(R)-(R)-G2
951
0
85
go
80
75
72
85
i I1
0o
1 70
G
so1
5 0
1
130
~
60 c
5
11 to
40
35
30
~I
l&fWW&
82
75
20
15
1
0
e5
IK
I
I
120
45
7C
60
5C
A0
30
20
10
0
N=N
OAc
N=N
0
N
OAc
N
TIPS
OAc
(S)-(S)-(S)-(S)-G2
-At
L
-
-U
8
,c
95
90
as
75s
oo
I
10
170
160
150
70
I,
140
130
"5
60
I
120
45
I
I
110
22
40
35
30
II I I
70
83
1l
60
25
20
i
50
15
ii
40
30
10
05
II
20
10
3
o
N=N
OAc
N=N
oR(-, (,(o
OAc
N=N
TIPS
OAc
(S)-(R)-(S)-(R)-G2
L
I
A
8
0
95
90
85
8
10
75
s0
I
10
110
1SO
150
140
130
A
AA~KA
65
60
110
e
40
a
35
30
25
20
1.11
i
I
I
120
4
I
A
I
70
84
60
50
A-.
40
10
15
30
05
1
20
10
0
oj'.
4
oN=N
O"W4
0
N=N
~.NK0
LA0
O"W4
0
N=N
0
K0
N.
NN,-0
TBS
(S)-(S)-(S)-(S)-hexyl-G2
5
90
8t,
80
15
10
65
i
10
170
140)
150
1 40
1 30
55
s0
5g,4
1 20
110
85
25
20
Is
10
05
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10
0
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30
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.1
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4 0
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80
50
40
30
20
C
Chapter I1. References
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
(11)
(12)
(13)
(14)
(15)
(16)
(17)
(18)
(19)
(20)
(21)
(22)
(23)
(24)
(25)
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Cohen, J. A.; Beaudette, T. T.; Cohen, J. L.; Broaders, K. E.; Bachelder, E. M.; Frdchet,
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Gillies, E. R.; Goodwin, A. P.; Frdchet, J. M. J. Bioconjugate Chem. 2004, 15, 12541263.
Gillies, E. R.; Frechet, J. M. J. Bioconjugate Chem. 2005, 16, 361-368.
Murthy, N.; Thng, Y. X.; Schuck, S.; Xu, M. C.; Frdchet, J. M. J. J Am. Chem. Soc.
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Chan, Y.; Bulmus, V.; Zareie, M. H.; Byrne, F. L.; Barer, L.; Kavallaris, M. J.Control.
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