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

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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