Synthesis of amino acid based covalently cross-linked polymeric gels using tetrakis(hydroxymethyl) phosphonium chloride as a cross-linker

Accepted Manuscript Synthesis of Amino Acid Based Covalently Cross-Linked Polymeric Gels Using Tetrakis(hydroxymethyl) Phosphonium Chloride as a Cross-Linker Avichal Vaish, Saswati Ghosh Roy, Priyadarsi De PII:

S0032-3861(14)01143-4

DOI:

10.1016/j.polymer.2014.12.043

Reference:

JPOL 17502

To appear in:

Polymer

Received Date: 11 September 2014 Revised Date:

17 December 2014

Accepted Date: 20 December 2014

Please cite this article as: Vaish A, Roy SG, De P, Synthesis of Amino Acid Based Covalently CrossLinked Polymeric Gels Using Tetrakis(hydroxymethyl) Phosphonium Chloride as a Cross-Linker, Polymer (2015), doi: 10.1016/j.polymer.2014.12.043. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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For “Graphical Abstract” Use Only Synthesis of Amino Acid Based Covalently Cross-Linked Polymeric Gels Using Tetrakis(hydroxymethyl) Phosphonium Chloride as a Cross-Linker

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Avichal Vaish, Saswati Ghosh Roy and Priyadarsi De

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Synthesis of Amino Acid Based Covalently Cross-Linked Polymeric Gels Using Tetrakis(hydroxymethyl) Phosphonium Chloride as a Cross-Linker Avichal Vaish, Saswati Ghosh Roy and Priyadarsi De*

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Polymer Research Centre, Department of Chemical Sciences, Indian Institute of Science Education and Research Kolkata, Mohanpur - 741246, Nadia, West Bengal, India. Corresponding author: E-mail: [email protected] (P. De).

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*

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ABSTRACT

This work reports the design and synthesis of side-chain amino acid containing cross-linked polymer gels with high mechanical strength. The Boc-leucine methacryloyloxyethyl ester (BocLeu-HEMA) has been polymerized by reversible addition-fragmentation chain transfer (RAFT) polymerization to obtain the corresponding polymer, P(Boc-Leu-HEMA). After Boc group

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deprotection under acidic condition and pH neutralization amino groups remain in the polymer side chain, which were reacted with tetrakis(hydroxymethyl) phosphonium chloride (THPC) to obtain chemically cross-linked gels. The mechanical properties of gels have been studied by rheological measurements, which reveal that mechanical strength increases as weight % of

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polymer in solution, cross-linking density and polarity of solvents increases, and decreases as molecular weight of polymers increases. A plateau of storage modulus (G′) over a wide range of angular frequency demonstrates that viscoelastic property of gels is independent of angular

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frequency. Field emission scanning electron microscopy (FE-SEM) studies of cross-sectioned gels validate rheological observation that as the G′ values of gel increases, porosity of gel decreases.

Keywords: amino acid; covalent cross-linking; gel.

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1. Introduction Polymer gels, novel materials with three-dimensional network structure, have received appreciable attention for their wide range of applications including drug delivery, cellular

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adhesion,1 nucleic acid purification,2 sensor,3 micro-valves4 and many other engineering fields. Polymer gels may result due to physical interactions (physical gels) or chemical bond formation (chemical gels). Physical gels are formed and stabilized in the solvent mostly through secondary forces such as ionic and hydrophobic interactions, hydrogen bonding, van der Waals forces, helix

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formation, etc.5 Hence physical gels are fragile compared to the chemical gels that are formed as a result of covalent cross-linking of polymer chains.6 Ubiquitous implementation of synthetic polymer gels is often impeded by the narrow operational temperature range, limited performance

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lifetime, and poor toughness.7 For example, many applications including robotics,8 electronic packaging9 and conformance advance may require a much tougher polymer gel. Several strategies have been developed to prepare chemical gels. Matsushita’s group reported nanophase-separated supramolecular assemblies from commercially available carboxylterminated polymers poly(dimethylsiloxane) and branched polyethylenimine via acid-base complexation.10 Thiol-ene chemistry was used to cross-link the internal double bonds in the

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cyclic poly(5-hydroxy-1-cyclooctene) backbone.11 Free radical cross-linking copolymerization (RCC) of a vinyl monomer with a divinyl monomer (cross-linker) is another important and popular strategy to prepare chemical gels.12 However, in case of RCC method intra-molecular

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cross-linking predominates at the early stages polymerization and generates highly heterogeneous gels with irregularly distributed branching points. Whereas, cross-linked gels prepared by controlled radical polymerization (CRP) of a vinyl monomer in the presence of a cross-linker

resulted

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divinyl

well

structured

and

homogeneous

gels.13

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tetrakis(hydroxymethyl) phosphonium chloride (THPC) is employed for the synthesis of protein based covalently cross-linked gels.14 THPC is inexpensive and considered as tetra-functional cross-linker due to presence of four methyl hydroxyl arms which can react with primary and secondary amine groups. Therefore, due to presence of four cross-linking point, it can bind with polymer side chains containing primary and secondary amine functionality to produce covalently cross-linked polymer gels with high cross-linking density and mechanical strength.

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Naturally occurring amino acid based functional monomers have been largely investigated as important components in synthetic non-biological macromolecules with biomimetic structures and properties for various biomedical applications.15,16 Incorporation of amino acid moieties in the synthetic polymer provides many advantages due to their amphoteric nature, chiral

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recognition,17 stimuli responsiveness,18,19 and their ability to self-assemble into high-order hierarchical structures through intra- and inter-chain associations via non-covalent attraction forces, such as hydrogen bonding.20,21 Amino acid based homopolymers with pendant primary amine groups are fascinating for their potential efficacy in post-polymerization modification

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reactions such as amide and imine formation, ring opening reaction, Michael addition reaction, etc.22 Our group recently reported the synthesis of side-chain amino acid based pH-responsive polymers with free primary –+NH3 group from (meth)acrylate containing side-chain amino acid

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based chiral monomers prepared from L- and D- tryptophan,23 L-alanine, L-phenylalanine,24 Lleucine and L-isoleucine25 via versatile reversible addition-fragmentation chain transfer (RAFT) polymerization technique. Since, THPC reacts readily with primary and secondary amines, free – NH2 of side chain amino acid moieties in the synthetic polymer can be cross-linked effectively. In this work, we have revealed the feasibility of THPC to cross-link synthetic polymers in

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organic solvents by employing RAFT polymerization technique and post polymerization crosslinking reaction. First, poly(Boc-lucine-methacryloyloxyethyl ester) (P(Boc-Leu-HEMA) chiral homopolymers were prepared from their respective monomer Boc-lucine-methacryloyloxyethyl ester (Boc-Leu-HEMA) via RAFT technique.25 Then, Boc group deprotection under acidic

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condition and pH neutralization of amino acid based polymers produced polymers with free NH2 groups which were employed in cross-linking reaction with THPC to form polymer gels.

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The cross-linked polymer gels were characterized by FTIR spectroscopy and the viscoelastic properties of gels prepared with different cross-linking density were studied by rheology.

2. Experimental section 2.1. Materials

Boc-L-Leucine (Boc-L-Leu-OH, 99%) and trifluroacetic acid (TFA, 99.5%) were purchased from

Sisco

Research

Laboratories

Pvt.

Ltd.,

India

and

used

as

received.

4-

Dimethylaminopyridine (DMAP, 99%), dicyclohexylcarbodiimide (DCC, 99%), anhydrous N,Ndimethylformamide (DMF, 99.9%), 2-hydroxyethyl methacrylate (HEMA, 97%) and

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tetrakis(hydroxymethyl) phosphonium chloride solution (THPC, 80% in H2O) were purchased from Sigma-Aldrich and used without any further purification. The 2,2΄-azobisisobutyronitrile (AIBN, Sigma-Aldrich, 98%) was recrystallized twice from methanol and stored in a refrigerator. The NMR solvent CDCl3 (99.8% D) was purchased from Cambridge Isotop

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Laboratories Inc., USA. The RAFT agent, 4-cyano-4-(dodecylsulfanylthiocarbonyl) sulfanyl pentanoic acid (CDP) was synthesized and purified as reported previously.26 The amino acid based vinyl monomer Boc-Leu-HEMA was synthesized by coupling reaction of Boc-L-Leu-OH with HEMA in the presence of DCC and DMAP following the previously reported procedure.27

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Solvents such as hexanes, acetone, diethyl ether, tetrahydrofuran (THF), methanol (MeOH),

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dimethyl sulfoxide (DMSO), etc. were purified by standard procedures.

2.2. Instrumentation

The number average molecular weight (Mn) and molecular weight distribution (dispersity, Ð) values were determined by gel permeation chromatography (GPC) in THF at 30 oC with a flow rate of 1 mL/min. The GPC system contains a Waters Model 515 HPLC pump, Waters Model 2414 refractive index (RI) detector and two columns: Styragel HT4 and Styragel HT3.

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Molecular weights were determined using the conventional calibration curve, which was constructed from poly(methyl methacrylate) (PMMA) standards. The 1H NMR spectroscopic measurements were carried out in a Bruker AVANCEIII 500 MHz spectrometer. Solid-state 13C CP/MAS NMR was also conducted in the same Bruker AvanceIII 500 spectrometer. In a typical

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experiment, a broad band channel was tuned to 125 MHz which is the resonance frequency of 13

C and a 4 mm MAS probe was used for the experiment. A typical 13C value of pulse length was

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used for 4 µs with a relaxation delay of 20 s. The spinning frequency for the sample was synchronized to 8 KHz. FT-IR spectra were recorded using KBr pellets on a Perkin-Elmer Spectrum 100 FT-IR spectrometer. Thermo-gravimetric analysis (TGA) was carried out by using Mettler Toledo TG/SDTA 851e instrument at a heating rate 10 oC/min under N2 atmosphere. Field emission scanning electron microscopy (FE-SEM) was carried out by Carl Ziess Supra SEM instrument.

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2.3. General RAFT polymerization procedure A typical RAFT polymerization procedure of Boc-Leu-HEMA is as follows: Boc-LeuHEMA (1.0 g, 2.91 mmol), CDP (23.5 mg, 0.058 mmol), AIBN (0.82 mg, 5.0 µmol; 0.5 mL solution of 16.4 mg AIBN in 10 mL DMF), DMF (3.5 g) and a magnetic stir-bar were taken in a

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20 mL septa-sealed glass vial. The vial was purged with dry N2 for 20 min and polymerization reaction was carried out in a preheated reactor at 70 oC. The polymerization reaction was quenched at predetermined time by cooling the vial in an ice-water bath and exposing the solution to atmosphere. Approximately 0.1 mL solution was analyzed by 1H NMR spectroscopy

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to determine monomer conversion by comparing the integration of the monomer vinyl protons with the DMF protons at 8.02 ppm at the begining and end of polymerization. The remaining

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reaction mixture was diluted with acetone and precipitated from a large volume of hexanes. The polymer, P(Boc-Leu-HEMA), was solubilized in acetone and reprecipitated four times from hexanes. The resulting polymer was dried under high vacuum at room temperature for 6 h to obtain a yellow powder.

2.4. Deprotection of Boc groups from polymers

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Boc-protecting groups of side chains amino acid units of the homopolymers were removed by using TFA in DCM to obtain trifluoroacetate polymers salts P(TFA-H3N+-Leu-HEMA) as shown in Scheme 1. In a typical example, 0.5 g of P(Boc-Leu-HEMA) was dissolved in 5.0 mL DCM in a 20 mL glass vial and stirred for 10 min to ensure that the mixture was homogeneous.

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Then, 5 mL of TFA was added drop-wise under ice-water bath condition and was allowed to stir for 2 h at room temperature. The resulting polymer was precipitated from diethyl ether and separated by centrifugation. The precipitate was washed with diethyl ether twice and dried under

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high vacuum for 8 h to get a pale yellow powder. Free –NH2 pendants in the side chains of the homopolymers were obtained by adjusting the pH of the polymer solution to 7-8. Typically, 0.3 g of P(TFA¯H3N+-Leu-HEMA) was dissolved in 3 mL de-ionised (DI) water in a 20 mL vial. The solution was allowed to stir for 10 min to get a homogeneous solution. Then, 1N NaOH solution was added drop-wise under stirring until precipitate appeared. The precipitate, P(H2N-Leu-HEMA), was isolated by centrifugation, washed with DI water twice and finally dried under high vacuum for 24 h to get a pale yellow powder.

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2.5. Gel synthesis Gels were prepared by the reaction between P(H2N-Leu-HEMA) and THPC at room temperature. Briefly, 0.1 g of P(H2N-Leu-HEMA) was dissolved in 0.5 mL THF in a 20 mL vial. Then, 0.5 mL DI water was added to the vial and stirred for 5 min to get a uniform solution.

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Calculated amount of THPC was added to the vial and mixed quickly at room temperature to get gel. 2.6. Rheological studies

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Rheological measurements were performed with an AR-G2 rheometer (TA Instruments) to determine storage modulus (G΄) and loss modulus (G΄΄) of synthesized cross-linked polymer gels. All measurements were performed at 25 oC in oscillatory mode using 40 mm parallel plate

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geometry with gap settings of about 1 mm. The shear strain of 2.0 % was selected, which was determined to be within the linear viscoelastic regime. The angular frequency was varied from 0.1 to 100 rad/s. 2.7 FE-SEM analysis

The interior morphology and porous structures of synthesized cross-linked polymer gel matrixes

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were studied by FE-SEM. First, gels were prepared and aged overnight. Then gels were cross sectioned, loaded on the silicon wafer, frozen in liquid nitrogen, and freeze dried under high vacuum at -50 oC for 32 h using a lyophilizer (Orleon instrument). Dry gel samples were coated

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with a thin layer of gold-palladium alloy for 1 min under high vacuum and studied by FE-SEM

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(Carl Ziess Supra SEM instrument).

3. Results and discussions

3.1. Synthesis and characterization of gels First, the monomer Boc-Leu-HEMA was polymerized by RAFT method using DMF as solvent, CDP as RAFT agent and AIBN as a radical initiator at 70 oC to prepare corresponding polymer, P(Boc-Leu-HEMA) (Scheme 1).25 During the polymerization [Boc-Leu-HEMA]/CDP ratios were altered at constant [CDP]/[AIBN] = 1/0.1 ratio to obtain P(Boc-Leu-HEMA) with different molecular weights. The GPC refractive index (RI) traces for all the homopolymers are symmetric and unimodel (Fig. S1). From the GPC analysis, number average molecular weight

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(Mn,GPC) and Ð values were determined for homopolymers and results are shown Table 1. Theoretical molecular weights (Mn,theo) predicted from stoichiometry and monomer conversion match well with the Mn,GPC values and narrow Ð values were observed for all the polymers.

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Homopolymers were characterized by 1H NMR spectroscopy and the typical resonance signals for the different protons from the polymer are assigned on the spectrum (Fig. S2). Comparison of the integration areas from the terminal -CH2-CH2- protons (from the HOOC-CH2CH2-C(CN)(CH3)- chain end) at 2.4-2.6 ppm and the repeating unit protons at 4.1-4.5 ppm (from

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-O-CH2-CH2-O- and chiral proton) allowed determination of the number average degrees of polymerization (DPn). The number average molecular weights of the homopolymers were determined from NMR (Mn,NMR) spectra by using the DPn, molecular weight of monomer and the

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molecular weight of CDP (Table 1). Excellent agreement between the theoretical, NMR, and GPC molecular weights were observe for the RAFT polymerization of Boc-Leu-HEMA in DMF at 70 oC.

HOOC O O

S

HOOC

O

S

C12H 25

S

AIBN, DMF, 70oC

HN O O

S S C 12H 25 n O S

HOOC NC O

TFA

O

O

O

O

HN

-

OOCF3CH3N

O

O

P(H 3N +-Leu-HEMA)

P(Boc-Leu-HEMA)

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Boc-Leu-HEMA

C 12H 25

O

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NC

O

S S n O S

NC

NaOH S S C 12H 25 n O S

HOOC NC HN

NH

HN

NH

O THPC

O O H 2N

Cross-linked gel

P(H2N-Leu-HEMA)

Scheme 1. Synthesis of P(Boc-Leu-HEMA) by RAFT polymerization, deprotection of Boc groups from the side chains and gel formation between deprotected polymer and THPC.

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Table 1. RAFT polymerization of Boc-Leu-HEMA at different [Boc-Leu-HEMA] (M)/[CDP] ratios in DMF at 70 oC. Time

Conv.a

Mn,GPCb

(min)

(%)

(g/mol)

Ðb

P9

10/1/0.1

210

70

3,400

1.12

P15

20/1/0.1

210

65

5,600

1.16

P22

25/1/0.1

240

82

8,000

1.14

P37

50/1/0.1

270

70

13,200

1.20

P45

100/1/0.1

240

43

16,000

1.31

Mn,theoc

Mn,NMRd

DPne

(g/mol)

(g/mol)

2,800

3,200

9

4,800

5,100

15

7,450

7,200

22

12,420

12,600

37

15,170

15,400

45

Determined by 1H NMR spectroscopy. bMeasured by gel permeation chromatography (GPC)

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a

[M]/[CDP]/[AIBN]

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Polymer

using conventional calibration in THF. cTheoretical number average molecular weight (Mn,theo) =

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(([Boc-Leu-HEMA]/[CDP] × molecular weight (MW) of Boc-Leu-HEMA × conversion) + (MW of CDP)). dDetermined by 1H NMR study. eDegrees of polymerization (DPn) = (Mn,GPC – MW of CDP)/(MW of Boc-Leu-HEMA).

c

a b

DMSO-d 6

n O

d O

* b,i,a

e

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f

O O g h H2 N

*

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d,g

200

a b

f

k

150

h HN k O l

f,h

*

j

j

e

i

n

c

(B)

c

d O

O

d,g

n

n O e

O g

n

j

b,i

i

100

n,j,c

e f,h

O m

m a

l

(A) 50

0

ppm 13

Fig. 1. C NMR spectra of (A) P(Boc-Leu-HEMA) homopolymer (solid state) and (B) P(H2NLeu-HEMA) with free –NH2 in DMSO-d6.

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In the next stage, Boc groups were removed by treatment with TFA at room temperature and thus obtained Boc-deprotected water soluble polymer salts, P(TFA¯H3N+-Leu-HEMA) (Scheme 1). Successful Boc-deprotection was confirmed by 1H NMR spectroscopy, where the – C(CH3)3 protons for the Boc group at 1.44 ppm was completely disappeared in deprotected

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P(TFA¯H3N+-Leu-HEMA) homopolymers (Fig. S2).25 Then, the –NH3+ groups in the side chain were transformed to free –NH2 groups by dissolving them into water and precipitation at basic condition (pH 7.0 to 8.0). Boc deprotection of P(Boc-Leu-HEMA) was also studied by 13C NMR spectroscopy (Fig. 1). The C(CH3)3 and C(CH3)3 carbons for Boc group resonate respectively at

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30.3 and 157.3 ppm in Boc protected homopolymers. These carbon signals disappeared in the P(H2N-Leu-HEMA) homopolymers. These P(H2N-Leu-HEMA) homopolymers with different

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molecular weights were used for the polymeric gel synthesis.

Table 2. Gel formation from the cross-linking reaction between P(H2N-Leu-HEMA) with THPC at room temperature.

DPn of P(H2NLeu-HEMA) 37 37 37 37 37 37 37 37 9 15 22 45 37 37 37

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Weight % of polymer 10 10 10 10 5 15 20 25 10 10 10 10 10 10 10

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G11 G12 G13 G14 G21 G22 G23 G24 G31 G32 G33 G34 G41 G42 G43

[P(H2N-LeuHEMA)]/[THPC] 0.5/1.0 0.75/1.0 1.0/1.0 1.0/0.5 0.5/1.0 0.5/1.0 0.5/1.0 0.5/1.0 0.5/1.0 0.5/1.0 0.5/1.0 0.5/1.0 0.5/1.0 0.5/1.0 0.5/1.0

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Gel

Solvent THF THF THF THF THF THF THF THF THF THF THF THF MeOH DMSO DMF

Gelation time (min) Immediately Immediately 2 4 5 Immediately Immediately Immediately Immediately Immediately 3 No gelation 2 Immediately 3

According to the previous report by Chung et al.,14 THPC should react with free -NH2 pendants of P(H2N-Leu-HEMA) through a Mannich-type reaction by the generation of formaldehyde (HCHO) in the presence of H2O as catalyst. Then, HCHO reacts with the -NH2

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group to form an immonium ion, which reacts with the THPC derivative to finish the hydroxymethyl arm replacement, resulting in the amine coupling. Reaction with other free NH2 pendants of P(H2N-Leu-HEMA) occur in a similar manner with the remaining unreacted hydroxymethyl arms to yield gel (Fig. 2). Therefore, to prepare 3-dimentional amino acid based

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polymeric gels; THPC was used as a cross-linker. Various stoichiometric ratios of THPC were added to the P(H2N-Leu-HEMA) solutions in THF/water (1/1, v/v) at room temperature. During the gel formation [P(H2N-Leu-HEMA)]/[THPC] ratio (G11 to G14 in Table 2), wt % of P(H2NLeu-HEMA) (G11 and G21 to G24 in Table 2) and molecular weight of P(H2N-Leu-HEMA)

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(G11 and G31 to G34 in Table 2) were varied. Also, THF was replaced with other solvents such as MeOH, DMSO and DMF (G41 to G43 in Table 2) to study the gelation reactions. Gelation

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times for these different gels were monitored visually and shown in Table 2. As expected, gelation time increases with the increase in [P(H2N-Leu-HEMA)]/[THPC] ratio and concentration of polymer (wt % of P(H2N-Leu- HEMA)]) in solution, because gelation depends on availability of free –NH2 groups and cross-linker. But gelation time increases as the molecular weight of polymer increases because high molecular weight polymer is less soluble in the THF/water (1:1, v/v) mixed solvent. Hence, there would be less interaction between the cross-

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linker and polymer side chains. We observed that as the polarity of the solvent decreases, gelation time increases. Highly polar solvent helps the polymer to solubilise in aqueous based

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mixed solvents to give better interaction between the cross-linker and free –NH2 pendants.

Fig. 2. Formation of gel after addition of THPC to the P(H2N-Leu-HEMA) solution in THF/water (1/1, v/v) at room temperature.

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(A)

80 (B)

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70

C-P Peak Appeared

Amine Peak Disappeared

60 50 40 2000

1750

1500

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%Transmittance (a.u.)

90

1250

1000

750

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-1

Wavenumber (cm )

Fig. 3. FT-IR spectra of (A) P(H2N-Leu-HEMA) homopolymer with free –NH2 pendants and (B) dried polymer gel prepared by using 20 wt % polymer and 1:2 polymer to THPC stoichiometric ratio (G23 in Table 2).

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To reveal the interaction between P(H2N-Leu-HEMA) and THPC in the gels, FT-IR measurements were carried out for P(H2N-Leu-HEMA) homopolymer and dried polymer gel (Fig. 3). Characteristic absorption peak of amine group at 1580 cm-1 is vanished in the dried gel and a new absorption peak was observed at 1050 cm-1 which corresponds to the carbon-

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phosphorous bond.28 Thermal stability of P(H2N-Leu-HEMA) and gels was investigated by TGA study (Fig. S3). The P(H2N-Leu-HEMA) homopolymers showed two-step degradation, where

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the first stage (160–370 oC) primarily originates from the side group decomposition of repeating units (amine and ester groups). The residual chain was further decomposed in the last step beyond 370 oC. The gel showed higher thermal stability than the P(H2N-Leu-HEMA) which is provided by the cross-linked network structure. However, gels also degraded in two stages, where the first stage (210-375 oC) was originated from the breaking of coupling between the cross-linker and the homopolymer as well as ester group decomposition from repeating units. In the second stage, beyond 375 oC, residual chains were decomposed.

11

10

5

10

4

10

3

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G' G"

10

1

% Strain

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G ', G " (Pa)

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10

2

10

3

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Fig. 4. Storage modulus G′ and loss modulus G′′ on strain sweep with the gel prepared from 10 wt % of P(H2N-Leu-HEMA) (DPn = 37) in THF at room temperature at [P(H2N-LeuHEMA)/[THPC] = 1:2 ratio (G23 in Table 2).

3.2. Rheological studies

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In order to evaluate the cross-linking effect on the rheological properties, four series of gels have been prepared at varying [P(H2N-Leu-HEMA)/[THPC] ratios, weight percentage of P(H2NLeu-HEMA) in the solution, molecular weight of the P(H2N-Leu-HEMA) and solvent as listed in Table 2, were analyzed by rheometer. In order, First, strain sweep experiment was carried out to

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determine the linear viscoelastic regime by measuring the storage modulus (G′) and loss modulus (G′′) as a function of % strain (Fig. 4) for the gel synthesized from P(H2N-Leu-HEMA) with Mn

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= 13,000 g/mol at [P(H2N-Leu-HEMA)/[THPC] = 1:2 at 10 wt % in THF/H2O (1:1, v/v) (G11 in Table 1). For this gel, the shear strain up to almost 40 % at the angular frequency of 0.1 rad/s was found to be in the viscoelastic range. Hence all other frequency sweep experiments were carried out at a constant strain of 2.0 %, which is well below the deformation range in the viscoelastic range of P(H2N-Leu-HEMA) gel. Within the viscoelastic range, higher value of G′ than G′′ confirmed that the rheological behaviour is dominated by an elastic property rather than a viscous property.

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5

G ' (Pa)

G ' (Pa)

10

10

4

10

5

(B)

4

RI PT

(A)

5 wt% 10 wt% 15 wt% 20 wt% 25 wt%

NH2 : THPC = 1:2 NH2 : THPC = 1:1.33 NH2 : THPC = 1:1

10

NH2 : THPC = 1:0.5 0

1

2

10 10 Angular Frequency (rad/s)

10

DPn = 37 DPn = 22 DPn = 15

0

10

1

10

2

Angular Frequency (rad/s)

(D) 5

10

G ' (Pa)

G ' (Pa)

10

(C)

DPn = 9

5

4

0

10

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10

10 -1 10

10

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6

3

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10

1

10

Angular Frequency (rad/s)

2

10

DMSO MeOH DMF THF

4

10

0

10

1

10

2

10

Angular Frequency (rad/s)

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Fig. 5. Storage modulus (G′) versus angular frequency (ω) at a shear strain of 2.0 % of gels prepared (A) at 10 wt % having [P37]/[THPC] ratios of 1:2, 1:1.33, 1:1 and 1:0.5 in THF/H2O

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(1:1, v/v), (B) at [P37]/[THPC] = 1:2 at 5, 10, 15, 20 and 25 wt % in THF/H2O (1:1, v/v), (C) at [P(H2N-Leu-HEMA)]/[THPC] = 1:2 using 10 wt % solution in THF/H2O (1:1, v/v), having DPn of P(H2N-Leu-HEMA) 9, 15, 22 and 37, and (D) at 10 wt % having [P37]/[THPC] ratio of 1:2 in THF/H2O (1:1, v/v), DMF/H2O (1:1, v/v), MeOH/H2O (1:1, v/v) and DMSO/H2O (1:1, v/v).

To understand the effect of cross-linking on the rheological properties, gels were synthesized (10 wt % polymer solution in THF/H2O, 1:1 v/v) by tuning the stoichiometry of P(H2N-Leu-HEMA) (DPn = 37) to THPC. The frequency dependence of the G′ for the polymer

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gels was determined in the angular frequency range of 0.1 to 100 rad/s using a constant strain of 2.0 %. A plateau G′of ~10000 Pa was found for the gel prepared at 1:0.5 polymers to THPC stoichiometry (Fig. 5A). By increasing the cross-linking density, the G′ values increases and it could be increased to ~59000 Pa for gels obtained using 1:2 polymer to THPC stoichiometry

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(Fig. 5A), because the elastic free energy of a polymer network depends upon the number of active polymer chains between the cross-links in a network.29 For the same reason, the plateau G′ for the polymer gels with 1:2 polymer to THPC stoichiometry ratio could be decreased from ~50000 to ~2200 Pa, when the cross-linking density was decreased by changing the wt % of

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polymer from 20 to 5 % as shown in Fig. 5B. Further increase in polymer concentration limits their solubility and hence did not increase G′ value for the gel prepared at 25 % polymer

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concentration (Fig. 5B).

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Fig. 6. Loss modulus (Gʹ′) versus angular frequency (ω) at a shear strain of 2.0 % of gels prepared (A) at 10 wt % having [P37]/[THPC] ratios of 1:2, 1:1.33, 1:1 and 1:0.5 in THF/H2O (1:1, v/v), (B) at [P37]/[THPC] = 1:2 at 5, 10, 15, 20 and 25 wt % in THF/H2O (1:1, v/v), (C) at [P(H2N-Leu-HEMA)]/[THPC] = 1:2 using 10 wt % solution in THF/H2O (1:1, v/v), having DPn

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of P(H2N-Leu-HEMA) 9, 15, 22 and 37, and (D) at 10 wt % having [P37]/[THPC] ratio of 1:2 in THF/H2O (1:1, v/v), DMF/H2O (1:1, v/v), MeOH/H2O (1:1, v/v) and DMSO/H2O (1:1, v/v).

Fig. 5C shows that molecular weight of P(H2N-Leu-HEMA) plays a crucial role to decide

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the cross-linking density of the polymer network. It was accepted that as the molecular weight of the polymer would increase, the value of G′ should increase.30 However, in contrast, we

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observed a loss in the storage modulus of cross-linked gels as degree of polymerization of the P(H2N-Leu-HEMA) increases from 9 to 37 as shown in Fig. 5C. This could be due to the decreased solubility of P(H2N-Leu-HEMA) in THF/H2O (1:1, v/v) mixed solvent with increasing molecular weigh, which affects the cross-linking density. Note that we previously mentioned that the P(H2N-Leu-HEMA) with DPn = 45 did not give gel.

To study the effect of solvent polarity during gel formation, cross-linking reactions were

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carried out at different solvents; THF (dielectric constant (ε) = 7.5), DMF (ε = 38), MeOH (ε = 33) and DMSO (ε = 47) with water in 1:1 (v/v) mixture. Gels were prepared from P(H2N-LeuHEMA) with DPn = 37 at 10 wt % at [P(H2N-Leu-HEMA)]/[THPC] = 1:2 in the above four

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different mixed solvent systems. Fig. 5D shows that the G′ values of gels could be increased from ~15000 to ~71000 Pa, as the solvent was changed from THF/H2O to DMSO/H2O. Water is one of the most important components of this cross-linking reaction since HCHO is generated

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from THPC in presence of water, which reacts with –NH2 groups in polymer. Thus, polymer dissolved in more polar solvent will more efficiently react with the cross-linker to give much more stable polymer gel. However, there is irregularity in the G′ values of gels prepared in MeOH/H2O and DMF/H2O (Fig. 5D). Although MeOH is less polar than DMF, the G′ = 41000 Pa of gel from MeOH as solvent is higher than the G′ = 30000 Pa of gel prepared in DMF. This could be explained by the hydrogen bonding interaction between the MeOH and water, which overcome the dipole-dipole interaction between DMF and water and as a result stronger gel is obtained in MeOH solvent.

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Strong polymer gel with covalent cross-linked network has two characteristic properties: G′ exceeds G′′ by about 2 orders of magnitude and G′ exhibit a plateau in wide frequency region, independent of angular frequency.31 Previously, Deng et al. reported a strong covalently crosslinked polymer gel with average G′ of 6.4×103 Pa which exceeds G′′ (average 13 Pa) by about 2

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orders of magnitude.32 In this work, we have prepared many chemically cross-linked polymer gels using THPC as a cross-linker which have G′ value greater than 104 Pa and also exceed the value of G′′ by 2 orders of magnitude (Fig. 6). From Fig. 5 and Fig. 6 we can say that all gels have shown more elasticity than viscosity and in all cases tan delta (tan δ = G′′/ G′) are < 1.0.

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Hence, these polymer gels also could be considered as mechanically strong polymer network.

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3.3. Gel morphology

To understand the morphological behaviour of the gels at nanoscopic scale, FE-SEM measurements were carried out. Homogeneity of cross-linked network depends on the parameters which affect the cross-linking density, such as concentration of cross-linker in the solution, molecular weight of polymer, concentration of polymer, etc.33 First, gels were prepared from the reaction of 10 wt % P(H2N-Leu-HEMA) (DPn = 37) in THF/water (1/1, v/v) with

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THPC at [P(H2N-Leu-HEMA)]/[THPC] = 1:2 (Fig. 7A) and [P(H2N-Leu-HEMA)]/[THPC] = 1:0.5 (Fig. 7B). Figure 7A shows more heterogeneous and less porous network than Fig. 7B because less amount of cross-linker was used to prepare the gel in Fig. 7B and less amount of cross-linkers tend to give homogeneous cross-linking with low cross-link density. Because of

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this low cross-link density, the gel in Fig. 7B is more porous than the gel in Fig. 7A. Also, DPn of polymers were varied to prepare gels keeping all other conditions constant.

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For example, gels were prepared from the reaction of 10 wt % polymer solution in THF/water (1/1, v/v) with THPC at [P(H2N-Leu-HEMA)]/[THPC] = 1:2 with two different molecular weights; DPn = 37 (Fig. 7A) and 22 (Fig. 7C). Since lower molecular weight is more soluble in THF, it helps to form more homogeneous network with high cross-link density. Due to this high cross-link density, the gel in Fig. 7C is less porous than the gel in Fig. 7A, which supports the rheological data (vide supra). Furthermore, the wt % of P(H2N-Leu-HEMA) in the solution was varied from 5 (Fig. 7D), 10 (Fig. 7A) and 25% (Fig. 7E) for the gel synthesis with polymer having DPn = 37 at [P(H2N-Leu-HEMA)]/[THPC] = 1:2. As the wt % of P(H2N-Leu-HEMA) in the solution increases, both porosity and homogeneity decreases as expected. For the less

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concentrated solution, the THPC cross-linker can uniformly attach to the side chains of the polymer which gives homogeneity to the gel. On the other hand, molecular packing becomes tighter with increasing P(H2N-Leu-HEMA) concentration in solution which provides less

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porosity to the gel.

Fig. 7. FE-SEM images of gel prepared from P(H2N-Leu-HEMA) in THF/water (1/1, v/v) with

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(A) [P37]/[THPC] = 1:2, 10 wt % polymer, (B) [P37]/[THPC] = 1:0.5, 10 wt % polymer, (C) [P22]/[THPC] = 1:2, 10 wt % polymer, (D) [P37]/[THPC] = 1:2, 5 wt % polymer, and (E)

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[P37]/[THPC] = 1:2, 25 wt % polymer. Scale bar indicates 2 µm.

4. Conclusions

Covalently cross-linked polymeric gels with high mechanical strength have been successfully prepared from amino acid based polymers containing pendant leucine units with free -NH2 moieties and THPC as cross-linker using Mannich type condensation reaction. Chemical gelation reaction was confirmed from FT-IR spectroscopy, where characteristic absorption peak of amine group at 1580 cm-1 is vanished in the dried gel and a new absorption

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peak appeared at 1050 cm-1 due to the carbon-phosphorous bond. Rheological studies demonstrated that viscoelastic property of gels is independent of angular frequency, whereas, mechanical strengths increased with the wt % of polymer in solution, cross-linking density and polarity of the reaction medium. However, G′ values decreased with the increasing molecular

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weight of P(H2N-Leu-HEMA) due the decreased solubility of P(H2N-Leu-HEMA) in THF/H2O (1:1, v/v) gelation medium. This concept evidently can be elaborated to other polymers having primary –NH2 groups in the side chains to prepare novel and interesting covalently cross-linked polymeric gel materials. These covalently cross-linked chemical gels are expected to show high

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biocompatibility because of the natural origin of amino acids. Due to the high mechanical

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strength, these polymer gels can be used in many engineering applications.

Supporting Information

The GPC and 1H NMR of homopolymers. TGA curves for the homopolymer and polymer gel.

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Acknowledgements

We would like to thank The Department of Science and Technology (DST), India [Project:

References

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SR/S1/OC-51/2010] for financial support.

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[8] Otake M, Kagami Y, Kuniyoshi Y, Inaba MA, Inoue HA. IEEE Int. Conf. Robot Autom. 2002;3:3224-3229. [9] Lenhart JL, Cole PJ, Unal B, Hedden R. Appl. Phys. Lett. 2007;91:061929-061933.

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[25] Bauri K, Roy SG, Pant S, De P. Langmuir 2013;29:2764-2774. [26] Moad G, Chong YK, Postma A, Rizzardo E, Thang SH, Advances in RAFT Polymerization 2005;46:8458–8468. [27] Bauri K, Pant S, Roy SG, De P. Polym. Chem. 2013;4:4052-4060. [28] Daasch L, Smith D. Anal. Chem. 1951;23:853–868. [29] Lowman, AM.; Peppas, NA. Hydrogels. In Encyclopedia of Controlled Drug Delivery; Wiley: New York;1999:397−418. [30] Cooper-White JJ, Mackay ME,. J. Poly. Sc. Part B: Polym. Phys. 1999;37:1803-1814.

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[31]. Kavanagh GM, Ross-Murphy SB. Prog. Polym. Sci. 1998;23:533–562. [32] Deng G, Tang C, Li F, Jiang H, Chen Y. Macromolecule 2010;43:1191-1194.

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Highlights of the present investigation


Cross-linked polymer gels have been synthesized from side-chain amino acid containing polymer with free amine groups and four-armed cross-linker.

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The mechanical properties of gels have been studied by rheological measurements.
Mechanical strength of gels increases as weight % of polymer in solution, cross-linker density and polarity of solvents increases.


All gels show high mechanical strength and may find potential applications in

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engineering fields.

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Supporting Information for Synthesis of Amino Acid Based Covalently Cross-Linked Polymeric Gels Using Tetrakis(hydroxymethyl) Phosphonium Chloride as a Cross-Linker

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Avichal Vaish, Saswati Ghosh Roy, and Priyadarsi De*

Polymer Research Centre, Department of Chemical Sciences, Indian Institute of Science

Corresponding Author: E-mail: [email protected] (P.D.).

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*

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Education and Research Kolkata, Mohanpur - 741246, Nadia, West Bengal, India

P22 P37

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Relative RI Signal

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Elution Volume (mL)

Fig. S1. The GPC RI traces of P22, P37 and P45 obtained by varying [Boc-LeuHEMA]/[CDP] ratios.

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Fig. S2. The 1H NMR spectra of P(Boc-Leu-HEMA) in CDCl3 (black) and P(H2N-LeuHEMA) in D2O (red).

G23 P(H2N-Leu-HEMA)

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Temperature (oC)

Fig. S3. TGA thermograms of P(H2N-Leu-HEMA) (blue line) and dried gel (G23 in Table 2 in the main manuscript, red line).

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