Chapter 3 Synthesis of thermosensitive glycopolymers
containing D-glucose residue: copolymers with N-isopropylacrylamide
162
Chapter 3: Section A: Introduction
3.1.1. Glycopolymers:
The traditional view of carbohydrate polymers as nature’s energy source (starch and
glycogen) and structural materials has expanded. The term glycopolymer is defined as both
natural and artificial carbohydrate-containing polymers, as well as synthetically modified
natural sugar-based polymers. In narrower sense, glycopolymers are defined as synthetic
polymers containing sugar moieties as pendant groups.1 There are different polymerization
techniques which have enabled the synthesis of glycopolymers featuring a wide range of
controlled architectures and functionalities. Methodologies for the synthesis of
glycopolymers can be roughly classified into two main categories: (1) polymerization of
sugar-bearing monomers and (2) chemical modifications of preformed polymers with sugar-
containing reagents. In general, the latter method frequently results in glycopolymers having
less regular structures because of incomplete reactions due to steric hindrance. Therefore, it
is often better to use polymerizations of sugar-carrying monomers for synthesizing linear
glycopolymers of well defined architectures.2 Glycopolymers with different architectures
such as linear polymers, comb polymers, dendrimers, and crosslinked hydrogels have been
reported as shown in Figure 70.
.
Star copolymer Comb copolymer Hyperbranched copolymer Network
Figure 70: Different topologies of glycopolymers
163
Naturally occurring glycoconjugates such as glycoproteins and glycolipids in
animals and plants have been found to play essential roles as recognition sites between cells
and involved in numerous biological functions, adhesion, cell growth regulation, cancer cell
metastasis and inflammation.3 Glycopolymers are increasingly attracting the chemists due to
their role as biomimetic analogues and their potential for investigating glycopolymers-
protein interactions.4 They act as attachment sites for several infectious viruses, toxins and
hormones that result in pathogenesis.5 Therefore, glycopolymers are widely investigated for
pharmaceutical and medical applications.6 Due to their biocompatibility and hydrophilicity,
these polymers are also widely used in biomedical engineering as biocatalysts,7 biosensitive
hydrogels,8 matrices for controlled cell culture,9 stationary phases for separation problems,10
surface modifiers,11 artificial tissues and artificial organ substrates,12 and in drug delivery
systems.13 The recent developments in the field of glycoscience, glycotechnology and the
potential applications of glycopolymers have attracted researchers to develop new synthetic
routes to design a variety of sugar based monomers and polymers.
3.1.2. Stimuli-responsive polymers:
Response to stimulus is a basic process of living systems.14 Based on the lessons
from nature, scientists have been designing useful materials that respond to external stimuli
such as temperature, pH, light, electric field, chemicals and ionic strength. The term
“stimuli-responsive or smart polymers” refers to soluble, surface coated polymers or
crosslinked polymeric gels, which exhibit relatively large and sharp property change in
response to a small physical or chemical stimuli at the phase transition. These responses are
manifested as dramatic changes in one of the properties such as shape, surface
characteristics, solubility and formation of an intricate molecular self-assembly or a sol-to-
gel transition. Temperature and pH are the most widely studied14 stimuli in stimuli
164
responsive polymer systems because they are more convenient and effective controlled-
release systems.
3.1.2.1. Thermosensitive polymers:
Thermosensitive or thermoreversible polymer undergoes phase transition as a
function of temperature. Generally, the solubility of polymer in a solvent increases with an
increase in temperature. However, the thermosensitive polymers exhibit the thermodynamic
lower critical solution temperature (LCST) and show inverse solubility behaviour with an
increasing temperature. The classical example is the solution of poly(N-
isopropylacrylamide) [PNIPAm] in water which shows an LCST in the range of 31-33 °C.15
Below LCST, the polymer is completely soluble in water while it becomes insoluble and
phase-separates above its LCST. This phase separation is accompanied by coil to globule
transition. Figure 71 gives a schematic representation of the phase transition in
thermoresponsive polymers (PNIPAm). The earliest report of PNIPAm was given by Scarpa
et al.16 in 1967. This behaviour has been exploited in biomedical applications varying from
pulsative drug release to control cell adhesion primarily because the LCST is close to
normal body temperature.
The thermosensitive phase transition of these polymers has opened up a multitude of
innovative applications in the areas of sensors or actuators,17 absorbents for solvent
extraction,18 protein-ligand recognition,19 on-off switches for modulated drug delivery,20
artificial organs21 and immobilization of enzyme.22
165
Figure 71: Schematic diagram (a) LCST determination by UV measurement (PNIPAm) (b) Phase transition of thermo-responsive polymer in solution
3.1.2.2. Phase transition in thermosensitive polymers:
The phase transition in thermosensitive polymer is one of the most fascinating and
important phenomena that allow us to explore the principles underlying the molecular
interactions and recognition which exists in synthetic and biological polymers. These
thermosensitive polymers have a balance of hydrophilic and hydrophobic groups. The phase
transition in these polymers is attributed to the fact that, in the solvated state the polymer
chains are extended and are surrounded by water molecules through hydrogen-bonding as
well as structurally layered water molecules around the hydrophobic groups (Figure 72, A).
166
OHN
O NH
HN O
NHO
OHN
NHO
Water molecule=
T < LCST
T > LCST
A B
Figure 72: Temperature induced phase transitions in PNIPAm
However, upon increasing the temperature the hydrogen-bonding between polymer
and water breaks and inter and intra molecular hydrophobic associations dominate leading to
the formation of a network like structure resulting in phase transition (Figure 72, B). Such
transition is an entropy driven process where the release of structured bound water from
hydrophobic groups along the polymer main chain is the major contributing thermodynamic
force.
Badiger et al.23 have demonstrated that the chemical structure of the hydrophobe and
its concentration determines LCST and heat of transition of hydrophobically modified
PNIPAm copolymer gels. In general, the LCST is higher with high hydrophilic content and
decreases with more hydrophobic content. Therefore one can design these polymers with
desired LCSTs by proper balance of hydrophilic/hydrophobic content.
3.1.2.3. Experimental techniques for studying phase transitions in thermosensitive
polymers:
Phase transitions in aq. PNIPAm solutions have been investigated in the literature by
a wide variety of experimental techniques such as IR-spectroscopy, pH measurements,
NMR-spectroscopy, viscometry, light scattering, fluorescence, calorimetry, UV turbidimetry
167
and visual observation of macroscopic separation. IR-spectroscopy provides molecular level
information on possible inter- and intra-molecular interactions between functional groups of
polymer.15,24 NMR is sensitive to the local structural differences of the polymer chains and
has revealed the existence of a discontinuous transition in the relaxation times at the
LCST.25 Viscometry detects the hydrodynamic consequences of aggregation during the
phase transition.26 Light scattering has the ability to monitor concentration fluctuations on a
spatial scale of approximately 1000 Å and has been used to detect the collapse of a single
PNIPAm chain at a temperature lower than that of the macroscopic phase separation.27 An
increased spatial resolution of the measurements has been reported by neutron and X-ray
scattering and fluorescence techniques, although the later requires the use of a probe either
in free solution or covalently bound to PNIPAm in order to ascertain details of polymer
solution behavior.28 Solution calorimetry provides thermodynamic parameters that lead
insight into the forces responsible for the phase separation.29
Amongst these, LCST type transition in PNIPAm and its copolymers is studied in
the present chapter by cloud point determination.
a) Cloud Point Determination:
In 1968, Heskin and Guillet15a reported on the study of solution properties of
PNIPAm, which is more frequently cited by other researchers. They determined the cloud
point by visual observation of the temperature at which first turbidity appeared in a polymer
solution immersed in a water bath as the temperature of bath was raised at 3 °C/h.
Measuring cloud point is the simplest and most convenient method of determining LCST.
Figure 71(a) illustrates a typical cloud point curve of PNIPAm. Various researchers have
now improved upon these methods by using a standard UV-VIS spectrophotometer. Fixed
168
wavelengths such as 500 nm or 600 nm or computer-averaging turbidity from 400 to 800 nm
have been employed.
3.1.3. Objective:
Among the family of temperature responding polymers, PNIPAm is one of the most
widely studied polymers. Glycopolymers, on the other hand, show a high potential as
biocompatible and bioactive materials for application in tissue engineering and targeted drug
delivery. Therefore, in this work we thought of synthesizing thermosensitive glycopolymers
utilizing new glycomonomer in combination with NIPAm which can be a very promising
approach to improve the biocompatibility of thermoresponsive PNIPAm polymers and even
to induce biological activity due to unique properties of glycopolymers with regard to extra
cellular matrix interactions. This may lead to a targeted control of cell-polymer interactions
with the option of an external control via a temperature stimulus and finally to the
development of optimized intelligent biomaterials.
Also, one can tailormade the LCST of thermosensitive polymers by changing the
hydrophilic/hydrophobic content. Therefore, one of the aims of this study is to clarify the
effect of bioactive hydrophobic/hydrophilic glycomonomer on LCST of PNIPAm by
varying the comonomer composition.
169
Chapter 3: Section B: Synthesis of thermosensitive glycopolymers
Polymerizations of glycomonomers with different functionalities such as alkenyl,30
alkynyl,31 acryloyl,32 methacryloyl,33 acrylamide,34 styryl,35 and vinyl ether36 have been
successfully achieved. Glycopolymers have been prepared by different polymerization
techniques, which include free radical polymerization,37 ionic polymerization,38 coordination
polymerization,39 ring-opening polymerization,40 ring-opening metathesis polymerization,41
reversible addition-fragmentation chain transfer polymerization,42 nitroxide-mediated
polymerization,35a cyanoxyl radical mediated polymerization,43 and atom transfer radical
polymerization.44
3.2.1. Glycopolymer synthesis via free radical polymerization:
Free radical polymerization is a very common synthetic technique in which a
polymer is formed from the successive addition of free radical building blocks. Mainly it
goes through three steps: initiation, propagation and termination. It has the inherent
advantage of robustness; high solvent and monomer purity are not always essential, and it is
tolerant to a wide range of reaction conditions and monomer functionalities. Free radical
polymerization has been widely commercialized and hence initiators are of relatively low
cost. One of the disadvantages associated with the technique is that it is difficult to control
the molecular weight of the resulting polymer without the use of high levels of relatively
toxic initiators and chain transfer agents. Polydispersities of the products tend to be high
(>2.0) and it is nearly impossible to control the terminal functionalities of the polymers with
any degree of precision. Synthesis of polymers of specific architectures is of great
importance. The number of glycopolymers synthesized using free radical conditions is very
170
large. The first glycopolymer synthesis was reported by Horejsi et al.37 in 1978. They
copolymerized acrylamide and allyl glycosides of various sugars 226a-d (Figure 73) in
water using ammonium persulfate as initiator and tetramethylethylenediamine (TMEDA) as
catalyst. The resulting O-glycosyl derivatives of polyacrylamides showed similar activities
to natural polysaccharides towards lectin binding through precipitin assays.
Saccharide O
OHO
HOOH
OH
OHO
HOOH
OH
OHO
HO
HOOH
OHO
HOOH
Saccharide, 226 =
a b c d
Figure 73: Various allyl glycoside monomers reported by Horejsi et al.
Despite the increasing demand for thermosensitive glycopolymers, only a few reports are
available on well-defined glycopolymers. All the methods which are described below
utilized free radical polymerization technique.
3.2.2. Earlier work:
Raku and Tokiwa45 reported copolymers of 6-O-vinyladipoyl-D-glucose with N-
isopropylacrylamide (NIPAm), which resulted in an increase in the LCST accompanied by a
decrease in the heat of transition. Kim and Park46 found that the copolymerization of
hydrophilic acrylamido-2-deoxy-D-glucose with NIPAm produced an upward shift in the
LCST. It was also noted that a copolymer of NIPAm with glucosyloxyethyl methacrylate
shifted the LCST to higher temperatures.47 Zhou et al.48 found an increase in the LCST of a
copolymer gel of NIPAm with acrylamidolactamine. Voit et al.49 synthesized copolymers of
NIPAm with 3′-(1′,2′:5′,6′-di-O-isopropylidene-α-D-glucofuranosyl)-6-methacrylamido
hexanoate and with 3′(1′,2′:5′,6′-di-O-isopropylidene-α-D-glucofuranosyl)-6-
171
methacrylamido undecanoate. After deprotection, it was shown that the LCSTs of the
copolymers were affected by the comonomer content, the spacer chain length of the
glycomonomer, and the chain architecture of the copolymers. Stenzel et al.50 reported the
synthesis of thermosensitive diblock copolymers based on PNIPAm and poly (acryloyl
glucosamine) by reversible addition-fragmentation chain transfer polymerization. Recently,
Pasparakis et al.51 described the reversible aggregation of a bacterial strain, Escherichia coli,
controlled by a thermoresponsive glycopolymer through a combination of a cluster
glycoside effect and polymer conformation.
3.2.3. Present work:
The present work deals with the synthesis of a new glycomonomer namely 3-
acrylamido-3-deoxy-1,2:5,6-di-O-isopropylidene-α-D-glucofuranose (AmIGlc, 228) and its
copolymerization with NIPAm to give hydrophobically modified copolymer by free radical
polymerization using AIBN (2,2′-azobisisobutyronitrile) as an initiator. The isopropylidene
groups of sugar moiety of the copolymer were deprotected by aqueous formic acid to get
water soluble polymer. It was observed that protected and deprotected copolymers show a
downward and upward shift in the LCST with respect to that of PNIPAm. A linear relation
was obtained between the concentration of glycomonomer and the LCSTs of the
copolymers.
3.2.3.1. Glycomonomer synthesis:
To synthesize new glycomonomer 228 (Scheme 43), we have utilized compound
114, which was synthesized from D-glucose and discussed in Chapter 1:section B (1.2.3.1.).
172
This azido compound 114 was subjected to hydrogenation reaction in presence of 10% Pd/C
in methanol to give corresponding amine 227 in almost quantitative yield.
D-glucoseO
OO
O
OO
b) O
OO
TsO
OO
113112
c)
O
OO
N3
OO
114
a)d)
e) f)
O
OO
HO
OO
111
g)O
OO
H2N
OO O
OO
HN
OO
O
AmIGlc, 228
227
Scheme 43: Reagents and conditions: a) dry acetone, anhy. CuSO4, cat. H2SO4, rt, 36 h, 59%; b) PCC, PCC, 4Å mole. sieves, CH2Cl2, rt, 12 h, 93%; c) NaBH4, MeOH:H2O, −10 °C, 2 h, 95%; d) TsCl, pyridine, cat. DMAP, 0 °C-rt, 8 h, 98%; e) NaN3, TBAI, DMF, 110 °C, 72 h, 72%; f) H2, Pd/C, MeOH, 80 psi, rt, 30 min, 99%; g) acryloyl chloride, Et3N, CH2Cl2, 0 °C, 10 min, 85%.
In IR spectrum, disappearance of strong peak at 2108 cm−1 and appearance of new broad
peak at 3405 cm−1 confirmed the reduction of azide to amine functionality. 1H NMR
spectrum (Figure 74) showed presence of two D2O exchangeable protons at δ 1.92 which
confirmed the formation of amine. This was also supported by 13C NMR spectrum (Figure
75) in which upfield shift of C-3 carbon from δ 66.1 to 57.4 was observed. The amine 227
was treated with acryloyl chloride to give corresponding acrylamide compound 228 in good
yield with mp = 151-151 °C. IR spectrum showed new peaks at 1656 and 1626 cm−1 for
amide carbonyl and olefin respectively. 1H NMR spectrum (Figure 76) showed new signals
at δ, 5.7, 6.1 and 6.3 for three olefinic proton, each with coupling constants 10.2, 16.8 and
17.1 Hz for cis and trans protons of vinyl group, confirmed the successful acrylation.
173
174
175
176
177
This acrylation was also supported by 13C NMR (Figure 77) in which appearance of
signals at δ 127.6 and 130.1 for olefinic carbons and at δ 165.3 for amide carbonyl,
confirmed the amide formation. This glycomonomer was synthesized in 31% overall yield
starting from D-glucose in seven steps.
3.2.3.2. Homopolymers and copolymers with NIPAm (Polymerization):
As shown in scheme 44, the homopolymers and random copolymers of AmIGlc and
NIPAm were prepared with AIBN as an initiator, in 1,4-dioxane at 65 °C by free radical
polymerization as per feed ratio given in Table 6. The 1H NMR spectra of copolymers, S-5,
S-10, S-20, S-25 and their comparison with the spectra of two homopolymers, PNIPAm and
PAmIGlc are given in the Figure 78.
CH2 HC
C
NH
CH2 HCC O
NH
m n
O
OO
OO
NH+NH
CCH3H3C
CHAIBN,
1,4-Dioxane, 65 oC, 24h
R = O
OO
OO
1) HCOOH, rt, 48 h
2) Dialysis, 2d,then freeze drying
OOH
HOOH OH
RH CCH3H3C
H
C O
CH
C O
H2CH2C
C O
NIPAm AmIGlc, 228
Scheme 44: Synthesis of copolymers of PNIPAm and PAmIGlc.
178
Table 6: Summary of copolymers of NIPAm and AmIGlc
Sample code
Feed ratio NIPAm: AmIGlc
Compositiona
NIPAm: AmIGlcMnb
(×10-3) (g/mole)
PDIb Tcc (Pro.) (°C)
Tcd (Depro.)
(°C) PNIPAm 100 : 00 100:00 - - 33.5 -
S-5 95 : 05 97:03 - - 28.6 37.6
S-10 90 : 10 91:09 11.71 1.49 24.3 42.5
S-20 80 : 20 86:14 09.58 2.05 16.1 57.1
S-25 75 : 25 80:20 11.86 1.82 13.1 64.6
PAmIGlc 00 : 100 00:100 03.03 1.52 - - a Determined from 1H NMR integrations. b Determined from GPC (PS calibration). c Cloud temperatures of copolymer containing protected sugar moiety. d Cloud temperatures of copolymer containing deprotected sugar moiety.
3.2.3.3. Deprotection:
The 1,2 and 5,6 di-isopropylidene protection of sugar moiety of the polymer was
removed under mild acidic condition.32a Protected polymer (200 mg) was dissolved in 23
mL of a formic acid solution (85%) and stirred for 48 h at room temperature. The resulting
solution was dialyzed (Sigma-Aldrich, molecular weight cut off: 1000) against double
distilled water for 2 days and freeze-dried. The deprotected polymer was obtained as white
powder (120 mg, yield- 60%).
3.2.3.4. Polymer characterization:
a) Structure determination:
Each polymer was characterized by NMR and IR spectroscopy. In the PAmIGlc
homopolymer and copolymer spectra (Figure 78), the disappearance of olefinic proton peaks
and appearance of signals for the backbone –CH and –CH2 peaks at δ 1.5-2.5 confirmed
formation of polymers. The incorporation of glycomonomer in the copolymer is confirmed
179
by careful analysis of all the spectra. The 1H NMR spectra of homopolymer of PAmIGlc
showed a characteristic peak of H-1 proton at 5.88 ppm of glucofuranose ring which is
absent in the 1H NMR spectrum of PNIPAm homopolymer. However, this peak grows
distinctly in copolymer S-5, S-10, S-20 and S-25 as the glycomonomer content increases.
This clearly indicates the incorporation of glycomonomer in copolymer. The composition of
copolymers was quantitatively estimated from the ratio of integrations of H-1 proton of
glucofuranose ring and -CH proton of isopropyl group of NIPAm. These values were
compared with feed composition. It was observed that there is a fairly good agreement with
the values of the feed, indicating the reaction proceeds to completion.
Figure 78: Comparison of 1H NMR spectra homopolymers and copolymers
180
b) Molecular weight determination:
The average molecular weight of protected homopolymer and copolymers were
determined by Gel Permeation Chromatography (GPC) with RI detector using THF as an
eluent at flow rate of 1.5 mL/min at room temperature. Column of 10 µ SDV gel was used.
For calibration, narrow polydispersity PS standards (Polymer Standards Services) with
molecular weight range of 500-50000 Da were used. As shown in Table 6, molecular
weights (Mn = number average molecular weight) of polymers were found to be 3000 to
11800 g mol-1 with PDI ranging from 1.50 to 2.05. However, determination of molecular
weight of PNIPAm by GPC was difficult as it forms hydrogen bonding and shows
thermosensitive phase transition causing serious problems in GPC analysis. Therefore, we
have not determined the Mn value for PNIPAm homopolymer and S-5 copolymer.
The PAmIGlc homopolymer and its copolymers were converted into water soluble
polymers by treatment with 85% formic acid for 48 hours followed by dialysis against
double distilled water. The 1H NMR spectra of protected homopolymer (PAmIGlc) and
deprotected homopolymer (PAmGlc) are shown in Figure 79. It showed the disappearance
of isopropylidene proton peaks at 1.3-1.5 ppm and upfield shift of H-1 of glucofuranose ring
from 5.88 ppm to 5.14 ppm, confirmed quantitative deprotection of isopropylidene groups
and formation of glucopyranose ring system. Other evidence for quantitative deprotection is
shown by the FTIR spectroscopy. Figure 80 shows the FTIR spectra of both PAmIGlc and
PAmGlc. The PAmGlc showed a broad absorption around 3300 cm−1 due to free hydroxyl
groups of sugar which also confirmed the deprotection of isopropylidene group.
181
Figure 79: 1H NMR spectra of (a) PAmIGlc homopolymer (in CDCl3) and (b) the corresponding PAmGlc polymer (in D2O) obtained after removal of isopropylidene groups protection
4 00 0 3 50 0 30 00 25 00 2 00 0 150 0 10 00 50 0
(a )
W a ve nu m b er (cm -1)
(b )
D e p ro te c te d P A m G lc
P ro te c te d P A m IG lc
% T
Figure 80: FTIR spectra of (a) Protected PAmIGlc (b) Deprotected PAmGlc
182
3.2.3.5. Cloud-point temperature determination:
The cloud points of 0.2% solutions of the copolymers in double distilled water were
determined by measuring temperature dependent optical density at 500 nm by using Perkin
Elmer, Lambda 35 UV/Vis Spectrometer equipped with temperature regulated bath. The
temperature scanning rate was 1 °C/min. The cloud point temperatures (Tc) of both,
protected and deprotected polymer were determined by temperature dependent UV/Vis
absorption in water at 500 nm. In Figure 81, PNIPAm homopolymer clearly showed LCST
at 33.5 °C. The protected copolymers showed LCSTs less than that of PNIPAm
homopolymer due to increase in overall hydrophobic content of polymer. It was noticed that
as the amount of glycomonomer increases, the cloud point temperature of copolymer
decreases. It is interesting that the same copolymer after deprotection showed higher LCSTs
than that of protected copolymer and of PNIPAm.
5 10 15 20 25 30 35 40 45 50
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5 S-5 (28.6 oC) S-10 (24.3 oC) S-20 (16.1 oC) S-25 (13.1 oC) PNIPAm (33.5 oC)
Abso
rban
ce
Temperature (oC)
Figure 81: Cloud points of copolymers of PNIPAm with protected sugar moiety
(The value in bracket indicates its LCST)
183
0 10 20 30 40 50 60 70 80
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0 PNIPAm (33.5 oC) Dpr. S-5 (37.6 oC) Dpr. S-10 (42.6 oC) Dpr. S-20 (57.1 oC) Dpr. S-25 (64.6 oC)
Abso
rban
ce
Temperature (oC)
Figure 82: Cloud points of PNIPAm and deprotected copolymers with PAmGlc
(The value in bracket indicates its LCST)
As shown in Figure 82, increase in the LCSTs of deprotected copolymers S-5, S-10, S-20
and S-25, was attributed to the increase in hydrophilicity as after deprotection, the hydroxyl
groups of sugar moiety becomes free and can easily form hydrogen bonding with water even
after LCST of PNIPAm. The above observations also supported the fact that copolymerizing
NIPAm with hydrophobic comonomer resulted in lowering of LCST while copolymerizing
NIPAm with hydrophilic comonomer resulted in higher LCST.
184
Figure 83: Correlation between LCSTs and % mole of sugar moiety in copolymers
#actual molar composition of glycomonomer determined by NMR
The effect of increase in the concentration of the glycomonomer on the LCST is shown in
Figure 83. The linear correlation between LCST and concentration of glycomonomer was
obtained for both protected and deprotected copolymers. These correlations are useful for
designing tailor-made thermosensitive glycopolymers. These polymers can be used for the
study of carbohydrate-protein interactions.
3.2.4. Conclusion:
We have synthesized new glycomonomer (AmIGlc) starting from cheaply available
D-glucose with 31 % overall yield in seven steps. The homopolymerization as well as
copolymerization of glycomonomer with N-isopropylacrylamide (NIPAm) at different
compositions, afforded the thermosensitive glycopolymers. Acid hydrolysis of the protected
glycopolymers gave water soluble polymers. The protected copolymers showed lower LCST
185
while deprotected copolymers showed higher LCST than that of PNIPAm. The increase or
decrease in LCST was found to be proportional to the concentration of glycomonomer. A
linear correlation between the LCST and the concentration of glycomonomer was found to
exist in these copolymers. Such a correlation could be a useful tool in designing
thermosensitive glycopolymers with desired LCST. In our group, work is in progress to
study these polymers in protein-polymer interactions as a function of temperature.
3.2.5. Experimental:
3.2.5.1.: 1,2:5,6-Di-O-isopropylidene-α-D-gluco-1,4-furanose (111):
D-glucose
O
OO
HO
OO
111
OHO
HO OHOH
OH
cat. H2SO4, rt, 36 h, 59%.
Dry acetone, anhy. CuSO4,
D-Glucose (100 g, 555.6 mmol) was added to dry acetone (2 L) at room temperature and
was followed by anhydrous CuSO4 (100 g, 625 mmol). The reaction mixture was cooled to 0
°C, and a catalytic amount of concentrated H2SO4 (4 mL, 16.8 mmol) was added dropwise
over a period of 10 min. The reaction mixture was stirred at room temperature for 30 h. It
was then neutralized with a sat. K2CO3 solution. The solution was filtered, and the filtrate
was evaporated under reduced pressure. The residue thus obtained was extracted with
chloroform (3 × 60 mL). The organic layer was dried over anhydrous sodium sulfate and
concentrated on a rotavapor to afford a yellowish solid, which was recrystallized from
chloroform:hexane (1:9) to give white crystals of 111 (85 g, yield = 59%).
Mp: 108-110 °C. Rf = 0.4 (ethyl acetate/hexane, 3:7). [α]25D: −12.5 (c 1, CHCl3); IR (KBr,
disk): 1024.1 (C-O), 3354.2 (OH) cm−1; 1H NMR (300 MHz, CDCl3): δ 1.31 (s, 3H, CH3),
186
1.36 (s, 3H, CH3), 1.44 (s, 3H, CH3), 1.49 (s, 3H, CH3), 2.97 (bs, 1H -OH, D2O
exchangeable), 3.98-4.03 (m, 2H), 4.14 (m, 1H, H-4), 4.29 (m, 2H), 4.59 (d, J = 4.0 Hz, 1H,
H-2), 5.91 (d, J = 3.8 Hz, 1H, H-1). 13C NMR (75 MHz, CDCl3):δ 25.20, 26.20, 26.84,
26.98 (4 × CH3), 67.56 (C-6), 73.12 (C-3), 74.84 (C-5), 81.15 (C-4), 85.07 (C-2), 105.12 (C-
1), 109.45 (quat. C), 111.63 (quat. C). Elem. Anal. Calcd. for C12H20O6: C, 55.37%; H,
7.74%. Found: C, 55.10%; H, 7.04%.
3.2.5.2.: 1,2:5,6-Di-O-isopropylidine-α-D-gluco-1,4-furan-3-one (112):
O
OO
O
OO
112
O
OO
HO
OO
111 CH2Cl2, rt, 12 h, 93%
PCC, 4Ao mole. sieves,
To a mixture of dry pyridinium chlorochromate (PCC; 150 g) and powdered 4 A° molecular
sieves (150 g) in CH2Cl2 (300 mL) was added a solution of 111 (50 g) in dry CH2Cl2 (300
mL), and the reaction mixture was stirred at room temperature for 12 h. The product 112
was filtered through a silica gel column with ether as an eluent. This filtrate was evaporated
under reduced pressure to obtain the keto compound. This crude, sticky, white solid (27.7 g,
yield = 93%) was used directly for further reaction.
Rf = 0.5 (ethyl acetate/n-hexane, 3:7); [α]25D: +44.0 (c 1, CHCl3); IR (KBr, disk): 1081 (C-
O), 1773 (C=O) cm−1; 1H NMR (300 MHz, CDCl3): δ 1.31 (s, 6H, 2 × CH3), 1.45 (s, 3H,
CH3), 1.47 (s, 3H, CH3), 4.02 (m, 2H), 4.33-4.39 (m, 3H), 6.12 (d, J = 4.5 Hz, 1H, H-1).
13C NMR (75 MHz, CDCl3): δ 25.44, 26.12, 27.32, 27.71 (4 × CH3), 64.43 (C-6), 76.52 (C-
4), 77.40 (C-5), 79.10 (C-2), 103.75 (C-1), 109.84 (quat. C), 112.63 (quat. C), 209.05
(C=O). Elem. Anal. Calcd. for C12H18O6: C, 55.82%, H, 7.02%. Found: C, 55.21%; H,
7.47%.
187
3.2.5.3.: 1,2:5,6-Di-O-isopropylidene-α-D-allo-1,4-furanose (A):
O
OO
O
OO O
OO
HO
OO
112
−10 oC, 2 h, 95%
NaBH4, MeOH:H2O,
A
To a solution of ketone 112 (18.4 g, 71.4 mmol) in MeOH (100 mL) and water (10 mL),
sodium borohydride (NaBH4; 3.2 g, 85.6 mmol) was added in portions (0.2 g each with 7.5
min intervals over 2 h) at −10 °C, and the mixture was stirred continuously. After
completion of the reaction, 10% aqueous HCl was added until pH = 7 to quench the excess
of NaBH4. It was then extracted with CH2Cl2 and washed with water. The organic layer was
dried over anhydrous sodium sulfate and evaporated on a rotavapor to get a white solid, A;
(17.6 g, yield = 95%).
mp: 75-76 °C. Rf = 0.3 (ethyl acetate/n-hexane, 3:7); [α]25D: +37.6 (c 1, CHCl3); IR (KBr,
disk): 1026.1 (C-O), 3359.5 (OH) cm−1; 1H NMR (300 MHz, CDCl3): δ 1.35 (s, 3H, CH3),
1.36 (s, 3H, CH3), 1.45 (s, 3H, CH3), 1.56 (s, 3H, CH3), 2.55 (d, J = 8.4 Hz 1H -OH, D2O
exchangeable), 3.80 (dd, J = 4.8, 8.5 Hz, 1H, H-6b), 3.97-4.08 (m, 3H, H-3, H-4, H-6a),
4.29 (ddd, J = 4.9, 6.6, 6.6 Hz, 1H, H-5), 4.59 (dd, J = 4.0, 5.1 Hz, 1H, H-2), 5.80 (d, J = 3.8
Hz, 1H, H-1). 13C NMR (75 MHz, CDCl3): δ 25.20, 26.24, 26.41, 26.59 (4 × CH3), 65.72
(C-6), 72.44 (C-3), 75.51 (C-5), 78.81 (C-4), 79.69 (C-2), 103.75 (C-1), 109.84 (quat. C),
112.63 (quat. C). Elem. Anal. Calcd. for C12H20O6: C, 55.37%, H, 7.74%. Found: C,
55.97%; H, 7.47%.
188
3.2.5.4.: 1,2:5,6-Di-O-isopropylidene-3-O-tosyl-α-D-allo-1,4-furanose (113):
O
OO
HO
OO O
OO
TsO
OO
113
cat. DMAP, 0 °C-rt, 8 h, 98%
TsCl, Pyridine,
A
To a solution of above allose derivative A (17.3 g, 66.5 mmol) in dry pyridine (125 mL),
tosyl chloride (13.9 g, 73.2 mmol) followed by a catalytic amount of 4-N,N-
dimethylaminopyridine (DMAP; 0.02 g, 0.16 mmol) was added at 0 °C under a nitrogen
atmosphere. The reaction mixture was stirred for 8 h at room temperature. After completion
of the reaction, the mixture was neutralized (pH = 7) with 10% aqueous HCl. Then, it was
extracted with ethyl acetate. The organic layer was dried with anhydrous sodium sulfate and
evaporated under reduced pressure. Column purification of the product afforded a white,
crystalline solid 113; (27.2 g, yield = 98%).
Mp: 107-109 °C; Rf = 0.6 (ethyl acetate/n-hexane, 2:8); [α]25D: +64.00 (c 0.084, CHCl3); IR
(KBr, disk): 1026.1 (C-O), 1371.3 (S=O), 2987 (C-H) cm−1; 1H NMR (300 MHz, CDCl3): δ
1.32 (s, 3H, CH3), 1.38 (s, 3H, CH3), 1.53 (s, 3H, CH3), 1.58 (s, 3H, CH3), 2.45 (s, CH3-Ar),
3.78 (t, J = 8.4 Hz, 1H, H-4), 3.92 (dd, J = 6.6, 8.1 Hz, 1H, H-3), 4.13-4.22 (m, 2H, H-5 and
H-6a), 4.67 (m, 2H, H-2 and H-6b), 5.75 (d, J = 3.0 Hz, 1H, H-1), 7.33 (d, J = 7.8 Hz, 2H,
ArH), 7.85 (d, J = 7.8 Hz, 2H, ArH). 13C NMR (75 MHz, CDCl3): δ 21.75 (Ar-CH3), 25.15,
26.14, 26.67, 26.73 (4 × CH3), 65.20 (C-6), 74.66 (C-5), 76.61 (C-4), 77.01 (C-3), 77.96 (C-
2), 103.75 (C-1), 109.84 (quat. C), 113.52 (quat. C), 128.24 (2 × Ar-C), 129.56 (2 × Ar-C),
133.04 (Ar-C-CH3), 145.05 (Ar-C-SO3). Elem. Anal. Calcd. for C19H26O8S: C, 55.06%; H,
6.32%. Found: C, 55.91%; H, 6.71%.
189
3.2.5.5.: 3-Azido-3-deoxy-1,2:5,6-di-O-isopropylidene-α-D-gluco-furanose (114):
O
OO
TsO
OO
113
O
OO
N3
OO
114 DMF, 110 oC,
72 h, 72%
NaN3, TBAI,
To a stirred solution of 113 (11.0 g, 26.6 mmol) in anhydrous dimethylformamide (DMF;
120 mL), sodium azide (4.3 g, 66.5 mmol) and tetrabutylammonium iodide (TBAI; 4.9 g,
13.3 mmol) were added under a nitrogen atmosphere. The solution was heated at 110 °C in
an oil bath for 72 h. After completion of the reaction, the solvent was removed under
reduced pressure. The reaction mixture was extracted with ethyl acetate. The organic layer
was washed with water, dried over anhydrous sodium sulfate, and concentrated on a
rotavapor. Silica gel column purification afforded a thick, yellowish liquid 114; (5.5 g, yield
= 72%).
Rf = 0.7 (ethyl acetate/n-hexane, 1.5:8.5). [α]25D: −24.37 (c 0.087, CHCl3). IR (thin film):
1072 (C-O), 2108 (-N3), 2985 (C-H) cm−1; 1H NMR (300 MHz, CDCl3): δ 1.33 (s, 3H,
CH3), 1.37 (s, 3H, CH3), 1.44 (s, 3H, CH3), 1.52 (s, 3H, CH3), 3.98 (dd, 1H, J = 4.8 and 8.7
Hz, H-4), 4.07-4.16 (m, 3H, H-3, H-6a, and H-6b), 4.23 (m, 1H, H-5), 4.61 (d, 1H, J = 3.6
Hz, H-2), 5.9 (d, 1H, J = 3.6 Hz, H-1). 13C NMR (75 MHz, CDCl3): δ 24.92, 24.95, 26.41,
26.63 (4 × CH3), 66.08 (C-3), 67.32 (C-6), 72.74 (C-5), 80.19 (C-4), 83.10 (C-2), 105.08 (C-
1), 109.01 (quat. C), 111.83 (quat. C). Elem. Anal. Calcd. for C12H19N3O5: C, 50.52%; H,
6.71%; Found: C, 51.01%; H, 6.81%.
190
3.2.5.6.: 3-Amino-3-deoxy-1,2:5,6-di-O-isopropylidene-α-D-glucofuranose (227):
O
OO
N3
OO
114
O
OO
H2N
OO
227
H2, Pd/C, MeOH
80 psi, rt, 30 min, 99%
A solution of 114 (1.6 g, 5.6 mmol) in dry MeOH (20 mL) and 5% Pd/C (0.1 g) was
hydrogenated under 2.76 × 106 Pa for 30 min. After completion of the reaction, it was
filtered through a Celite bed, washed with MeOH, and concentrated on a rotavapor to give
amine 227 as a sticky, white solid (1.4 g, yield = 98.5%).
Rf: 0.2 (ethyl acetate). [α]25D: −19.82 (c, 0.032, CHCl3). IR (KBr, disk, cm−1): 3405 and
1590 (N-H), 1110 (C-N). 1H NMR (300 MHz, CDCl3): δ 1.31 (s, 3H, CH3), 1.36 (s, 3H,
CH3), 1.42 (s, 3H, CH3), 1.51 (s, 3H, CH3), 1.92 (bs, 2H, -NH2, D2O exchangeable), 3.57 (d,
1H, J = 2.7 Hz, H-3), 3.98 (dd, 1H, J = 4.5 and 8.4 Hz, H-6a), 4.03 (dd, 1H, J = 2.7 and 8.7
Hz, H-4), 4.15 (dd, 1H, J = 6 and 8.4 Hz, H-6b), 4.21 (m, 1H, H-5), 4.43 (d, 1H, J = 3.3 Hz,
H-2), 5.90 (d, 1H, J = 3.5 Hz, H-1). 13C NMR (75 MHz, CDCl3): δ 25.32, 26.25, 26.81,
26.93 (4 × CH3), 57.41 (C-3), 68.12 (C-6), 72.84 (C-5), 81.19 (C-4), 86.10 (C-2), 104.88 (C-
1), 109.41 (quat. C), 111.63 (quat. C). Elem. Anal. Calcd. for C12H21NO5: C, 55.58%; H,
8.16%. Found: C, 55.31%; H, 8.99%.
3.2.5.7.: 3-Acrylamido-3-deoxy-1,2:5,6-di-O-isopropylidene-α-D-glucofuranose (228):
O
OO
HN
OO
O
O
OO
H2N
OO
227
Acryloyl Chloride, Et3N
CH2Cl2, 0 oC, 10 min, 85%
228
To a stirred solution of 227 (1.5 g, 5.8 mmol) in CH2Cl2 (20 mL) was added triethyl amine
(NEt3, 0.8 mL, 6.9 mmol), which was followed by the dropwise addition of acryloyl chloride
191
(0.5 mL, 6.3 mmol) through a syringe at 0 °C. The mixture was stirred at the same
temperature (10 min) and quenched by the addition of water (10 mL). The mixture was
extracted with CH2Cl2 (15 mL × 3) and concentrated under reduced pressure. The residue
was purified by silica gel column chromatography to afford compound 228 as a white,
crystalline solid; (1.54 g, yield = 85%).
Mp: 151-152 °C. Rf: 0.7 (ethyl acetate). [α]25D: −58.21 (c 0.070, CHCl3). IR (KBr, disk):
3319 (N-H), 1656 (C=O), 1626 (C=C) cm−1. 1H NMR (300 MHz, CDCl3): δ 1.31 (s, 3H,
CH3), 1.37 (s, 3H, CH3), 1.44 (s, 3H, CH3), 1.53 (s, 3H, CH3), 3.82 (app. t, 1H, J = 7.2 and
8.1 Hz, H-3), 4.13 (dd, 1H, J = 6.3 and 8.1 Hz, H-4), 4.23 (m, 1H, H-5), 4.42 (m, 2H, H-
6a,6b), 4.69 (d, 1H, J = 3.3 Hz, H-2), 5.68 (d, 1H, J = 10.2 Hz, H-9), 5.88 (d, 1H, J = 3.3
Hz, H-1), 6.06 (dd, 1H, J = 10.2 and 16.8 Hz, H-8), 6.32 (d, 1H, J = 17.1 Hz, H-10), 6.57
(bs, 1H, -NH, D2O exchangeable). 13C NMR (75 MHz, CDCl3): δ 25.02, 26.05, 26.48, 30.96
(4 × CH3), 56.43 (C-3), 69.35 (C-6), 73.16 (C-5), 79.06 (C-4), 83.87 (C-2), 104.22 (C-1),
109.69 (C-10), 111.84 (C-13), 127.63, 130.11 (C-8, C-9), 165.31 (C-7). Elem. Anal. Calcd.
for C15H23NO6: C, 57.50%; H, 7.40%; Found: C, 57.31%; H, 7.09%.
3.2.5.8. Polymerization:
In a typical procedure, a mixture of monomer 228 (0.064 g, 0.20 mmol), NIPAm
(0.43 g, 3.85 mmol) and AIBN (7.0 mg) in 1,4-dioxane (5 mL), was taken in glass tube and
nitrogen gas was bubbled through the tube for 20 min. The reaction was maintained at 65 °C
for 24 h. The content was precipitated in n-hexane and again dissolved in acetone and
reprecipitated in n-hexane. Finally, the product was dried under reduced pressure at 50 °C
for 2 days. All polymers were obtained as white powders in quantitative yield.
192
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196
Ph. D. Thesis Abstract
Abstract of the thesis entitled “Synthesis and Bioevaluation of 3-Hydroxypiperidines, α-
Hydroxylactones and Synthesis of Thermosensitive Glycopolymers from D-Glucose” to
be submitted to the University of Pune for the Degree of Doctor of Philosophy in
Chemistry by Mr. Vishwas U. Pawar under the guidance of Dr. (Mrs.) Vaishali S. Shinde,
Department of Chemistry, University of Pune, Pune, India.
The present thesis is divided into 3 chapters.
Chapter 1: Synthesis and bioevaluation of hydroxypiperidines.
This chapter includes introduction towards hydroxylated piperidines such as azasugars,
pipecolic acids and alkaloids from Cassia and Prosopis species and further divided into
three sections.
Section A: Introduction of hydroxypiperidines
This section deals with introduction, biological importance and earlier methods for the
synthesis of alkyl substituted pipecolic acid derivatives.
Section B: Synthesis and enzyme inhibitory study of methyl substituted pipecolic acid
derivatives
This section deals with the present work on the synthesis of 6-methyl-3-hydroxy derivative
of pipecolic acid 1 and its acid reduced analogue 2. Wittig reaction on D-glucose derived
azido aldehyde 3 underwent smoothly to afford the α,β-unsaturated methyl ketone 4 in good
yields. A stereoselective intramolecular reductive amination strategy was used as a key step
to build the required piperidine skeleton as a single diastereomer. (Scheme 1)
D-glucose
NCbz
O
O
OH3C
N3
O
O
OH3C
OO
N3
O
O
O
single diastereomer
a)
NH3C
OO
O
H
HH H
b)
H
3 4
6
5 Scheme 1: Reagents and Conditions: a) Ph3P=CHCOCH3, dry THF, 0 °C, 2 h, 87%. b) H2, Pd/C, MeOH, rt, 260 psi, 12 h, 93%. c) CbzCl, NaHCO3, aq. MeOH, 0 °C - rt, 6 h, 94%.
197
Stereochemistry at newly generated centre was confirmed by decoupling and 2D-NOESY
studies after converting piperidine into its N-Cbz protected derivative 6. The piperidine
intermediate 6 was successfully transformed in to target molecules 1 and 2 by simple
organic transformations (Scheme 2). Glycosidase inhibitory activity of these two molecules
was tested and compound 1 showed strong competitive inhibition of commercially available
α-glucosidase (Sigma Aldrich) with Ki = 71 µM and IC50 = 1150 µM and non competitive
inhibition of α-galactosidase with Ki = 26 µM and IC50 = 180 µM isolated from the bacteria
Geobacillus toebii BK1. On the other hand no inhibition was observed for compound 2.
NCbz
O
O
OH3C N
Cbz
O
OH
OHH3C N
Cbz
OCHO
OH3C N
Cbz
OH
H3COH
(S)NH
(R)
(R) OH
H3COHN
Cbz
OH
H3COH
O
(S)NH
(S)
(R) OH
H3COH
O
a) b) c)
d)e)
d)
1 2
H
6
7
8
Scheme 2: Reagents and Conditions: a) TFA:H2O (3:2), 0 °C, 1 h. b) NaIO4, acetone:H2O (8:2), 0 °C - rt, 2 h. c) NaBH4, MeOH, 0 °C, 1 h, 89%. d) H2, Pd/C, MeOH, rt, 80 psi, 12 h, 97% for 1, 93% for 2. e) NaClO2, NaH2PO4, 30% H2O2, aq. MeCN, 0 °C - rt, 10 h, 87%.
Section C: Introduction and studies towards the synthesis of (−)-deoxoprosophylline.
This section contains introduction, reported methods for the synthesis of (−)-
deoxoprosophylline and our attempts for the synthesis of target molecule. As shown in
scheme 3, D-glucose was transformed into triflate 10 in two steps which was then displaced
by NaN3 to afford azide 11. Selective deprotection of 5,6-acetonide, conversion of diol to
dimesylate and treatment with NaI under reflux condition yielded azido olefin 14 in good
yield. Acetonide deprotection, oxidative cleavage and hydride reduction gave the azido diol,
which in turn transformed into required homo allyl amine 19. This chiral homo allyl amine
was then subjected to aza-Prins cyclization (Scheme 4) to get the required tri-substituted
piperidine. Unfortunately all our attempts failed to get required cyclized product. Changing
the amine protection from -Cbz to -Ts did not work for the aza-Prins cyclization. Presence
of strong Lewis acid labile ether functionality was attributed to the failure of key reaction in
our strategy. Work is in progress to explore the compound 17 and 19 in the synthesis of
jaspine B and its isomers.
198
D-glucose
O
O
ON3
O
O
ON3
OHHOO
O
OTfO
OOO
O
OHO
OO
O
O
ON3
OO
O
O
ON3
OMsMsO
a) b) c) d)
e) f) g)
OH
N3OH
O
OH
OHN3
OCHO
ON3
OBn
N3OBn
OBn
P-NHOBn
h)
i) j) k)
11
13 14
17 19
9
15
18
1210
16
Scheme 3: Reagents and Conditions: a) Acetone, Anhy. CuSO4, cat. H2SO4, rt, 36 h, 59%. b) (TfO)2O, Pyridine, CH2Cl2, −10 °C, 2 h. 92%. c) NaN3, Bu4NI, DMF, 50 °C, 5 h, 70%. d) 10% H2SO4, MeOH-H2O, rt, 3 h, 88%. e) MsCl, NEt3, CH2Cl2, 0-25 °C, 2.5 h, 98%. f) NaI, Me2CO, reflux, 12 h, 87%. g) TFA:H2O (3:2), 0-25 °C, 3 h. h) NaIO4, (CH3)2CO-H2O (9:1), 0-25 °C, 2.5 h. i) NaBH4, MeOH, 0-25 °C, 1 h. j) NaH, BnBr, DMF, 0-25 °C, 2 h, 85%. k) i) Ph3P, THF:H2O (10:1), rt, 2 h. ii) Cbz-Cl, NaHCO3, MeOH:H2O, (4:1), 0-25 °C, 6 h, (78 % over two steps); or TsCl, TEA, CH2Cl2, 5 h, (72 % over two steps).
NR
OBn
OBn
(-)-Deoxoprosophylline, 20
10
OBn
PNHOBn
i)
P = Ts or Cbz
Nu
NH
OH
OH10
C12H25CHO,CH2Cl2
19
Scheme 4: Reagent and conditions: i) a) I2, rt, 24 h. b) I2, reflux, 12 h. c) FeCl3, rt, 24 h. d) FeCl3, reflux, 24 h. e) BiCl3, rt, 24 h. f) BiCl3, reflux, 24 h.
Chapter 2: Synthesis and bioevaluation of new α-hydroxy-γ-lactones as anticancer
compounds.
This chapter is further divided into three sections.
199
Section A: Introduction.
This section deals with introduction and earlier methods for the synthesis of α-hydroxy
lactones and their biological importance.
Section B: Synthesis and bioevaluation of new α-hydroxy-γ-lactones.
This section includes present work for the synthesis of homologues of Harzialactone A.
O
O
OTfO
OO O
O
O
OO
b) c)
e)
O
O
O
OO
d)
O
O
O
OHHO
O
O
OH
Og)
Z:E = 85:15
f) O
O
O
O
OH
OH
a)
21
23
2524
10 22
O
O
O
Scheme 5: Reagents and conditions: a) DBU, MeCN, rt, 12 h, 94%. b) H2, Pd/C, ethyl Acetate, rt, 2 h, 93%. c) 30% HClO4, THF, 0 °C, <5 min, 88%. d) NaIO4, sat. NaHCO3, CH2Cl2, 0 °C to rt, 1.5 h, 87%. e) Ph3P+CH2PhBr−, n-BuLi, THF, 0 °C to rt, 12 h, (Z:E = 85:15), 71%. f) H2, Pd/C, ethyl acetate, rt, 2 h, 96%. g) cat. H2SO4, THF:H2O (4:1), 65 °C, 2.5 h, 85%.
3-Deoxy-gulose derivative 21 was synthesized by using reported method (scheme 5),
which was then converted to aldehyde and to the corresponding Wittig product 23. Olefin
reduction and acetonide deprotection afforded mixture of hemiacetal 25 in good yield.
Selective anomeric oxidation was carried out by using celite supported Ag2CO3 to afford the
target molecule 26 in good yield.
O
OH
OH(S)
O
(R)OH
Oa)
(S)O
(R)OH
O
b)Br c)
25 26
27
Scheme 6: Reagents and Conditions: a) Ag2CO3-Celite, toluene, reflux, 3 h, 79%. b) Br2, BaCO3, dioxane-H2O, (2:1), (in dark), rt, 1 h, 94%. c) H2, Pd/C, ethyl acetate, 260 psi, rt, 24 h, 96%.
200
In case of Br2-BaCO3 mediated oxidation, we observed the ring bromination at para
position in phenyl ring with required oxidation to afford 27. This brominated lactone could
also be debrominated to give lactone 26. Anticancer activity of these target molecules was
assessed against five cancer cell lines, P388D1, HL60, COLO-205, Zr-75-1 and HeLa. Both
compound 26 and 27, showed significant activity against colon cancer (COLO-205) and
cervical cancer (HeLa) and moderate with others unlike the harzialactone A which is
reported as a cytotoxic against P388 cell line. To the best of our knowledge, this is the first
report of harzialactone analogues as potent inhibitors of human colon and cervical cancer.
Section C: Introduction to diarylheptanoids and synthesis of (−)-Yashabushidiol B,
(3S,5S)-1-(4'-hydroxyphenyl)-7-phenyl-3,5-heptanediol and its 4'-methoxy derivative.
This section deals with earlier details of isolation, biological importance, earlier synthesis of
some linear diarylheptanoids and present work.
a) b) c)
d)
O
OH
OMeO
OBn
OMe
(S)O
(R)OBn
OH(S) (R)
OH OBn
e)
Z:E = 64:36 (3S,5S)-Yashabushidiol B
24 28
29 3130
O
O
O
(S) (S)
OH OH
Scheme 7: Reagents and Conditions: a) Dowex H+ form, MeOH, rt, 12 h. quant. b) NaH, BnBr, THF, 0 °C - rt, 2 h. 93%. c) Dowex H+ form, cat. H2SO4, reflux, 12 h, 71%. d) Ph3P+CH2PhBr−, n-BuLi, THF, reflux, 4 h; (Z:E = 64:36) 76%. e) H2, Pd/C, ethyl acetate, 80 psi, rt, 12 h, 96%.
Herein we describe the synthesis of antipode of natural products from
diarylheptanoid family. Required Wittig reagents were used with suitably protected
hemiacetals 29 to give olefins which were subsequently reduced to give antipode of
yashabushidiol B 31 and its 4'-methoxy analogue 33. O-demethylation of compound 33 was
carried out to afford corresponding hydroxy compound 34 which is also antipode of
naturally occurring heptanediol.
201
a)
OH OBn
(S) (S)
OH OH
b)
Z:E = 69:31 OMe
(S) (S)
OH OH
OH
c)
(3S,5S)-1-(4'-hydroxyphenyl)-7-phenyl-3,5-heptane diol
(3S,5S)-1-(4'-methoxyphenyl)-7-phenyl-3,5-heptane diol
29
32
33
34
OMe
Scheme 8: Reagents and Conditions: a) Ph3P+CH2Ph-OMeBr−, n-BuLi, THF, reflux, 4 h; 81%, (Z:E = 61:39). b) H2, Pd/C, ethyl acetate, 80 psi, rt, 12 h, 96%. c) AlCl3, EtSH, CH2Cl2, −20 °C - rt, 87%.
Chapter 3: Synthesis of thermosensitive glycopolymers containing D-glucose residue:
copolymers with N-isopropylacrylamide.
This chapter is further divided into 3 sections.
Section A: Introduction
This section deals with introduction of glycopolymer and more specifically thermosensitive
glycopolymers and earlier reports for the synthesis of thermosensitive glycopolymers.
Section B: Synthesis and characterization of thermosensitive glycopolymers
This section includes present work for the synthesis of new glycomonomer from D-glucose
and its copolymerization with NIPAm to produce thermosensitive glycopolymers. As shown
in scheme 9, we have synthesized the glycomonomer (AmIGlc, 40) with 31% overall yield
in 7 steps from D-glucose. This glycomonomer was then copolymerized with NIPAm in
presence of AIBN as an initiator. We have synthesized these polymers with different molar
feed ratio and their actual composition was determined by 1H NMR analysis. Acetonide
protection was then removed by acid hydrolysis to obtain hydrophilic glycopolymers. The
cloud-point temperatures (LCST) of both the protected and deprotected polymers were
determined by temperature-dependent UV–visible absorption in water at 500 nm.
202
O
O
OO
OOO
O
OHO
OO
b) c)
e)d) f)
O
O
OHO
OO O
O
OTsO
OO
O
O
ON3
OO O
O
OH2N
OO O
O
OHN
OO
OAmIGlc, 40
a)
9 35 36 37
38 39
Scheme 9: Reagents and Conditions: a) PCC, 4Å mole. sieves, CH2Cl2, rt, 12h, 93%. b) NaBH4, aq. MeOH, −10 °C, 2 h, 95%. c) TsCl, Pyridine, cat. DMAP, 0 °C - rt, 12 h, 98%. d) NaN3, TBAI, DMF, 110 °C, 72 h; 72%. e) H2, Pd-C, MeOH, 80 psi, rt, 30 min, 99%; f) Acryloyl Chloride, TEA, CH2Cl2, 0 °C, 10 min, 85%.
The protected copolymers showed LCSTs lower than that of the PNIPAm homopolymer
because of an increase in the overall hydrophobic content of the polymer. As the amount of
the glycomonomer increased, the cloud-point temperature of the copolymer decreased. It is
interesting that the same copolymer after deprotection showed higher LCSTs than those of
the protected copolymer and PNIPAm.
CH2 HC
C
NH
CH2 HCC O
NH
m n
O
OO
OO
NH+NH
CCH3H3C
CHAIBN,
1,4-Dioxane, 65 oC, 24h
R = O
OO
OO
1) HCOOH, rt, 48 h
2) Dialysis, 2d,then freeze drying
O
OH
HO
OH OH
RH CCH3H3C
H
C O
CH
C O
H2CH2C
C O
NIPAm AmIGlc, 40
Scheme 10: Synthesis of copolymers of PNIPAm and PAmIGlc.
A linear correlation between the LCST and concentration of the glycomonomer was
obtained for both protected and deprotected copolymers. These correlations are useful for
203
designing tailor-made thermosensitive glycopolymers. These polymers can be used for the
study of carbohydrate-protein interactions.
Table 1: Summary of the copolymers of PNIPAm and PAmIGlc.
Sample code
Feed ratio NIPAm: AmIGlc
Compositiona
NIPAm: AmIGlcMnb
(×10-3) (g/mole)
PDIb Tcc (Pro.) (oC)
Tcd (Depro.)
(oC) PNIPAm 100 : 00 100:00 - - 33.5 -
S-5 95 : 05 97:03 - - 28.6 37.6 S-10 90 : 10 91:09 11.71 1.49 24.3 42.5 S-20 80 : 20 86:14 09.58 2.05 16.1 57.1 S-25 75 : 25 80:20 11.86 1.82 13.1 64.6
PAmIGlc 00 : 100 00:100 03.03 1.52 - - a Determined from 1H NMR integrations b Determined from GPC (PS calibration) c Cloud temperatures of copolymer containing protected sugar moiety
d Cloud temperatures of copolymer containing deprotected sugar moiety