A Simple and Versatile Synthetic Strategy to Functional Polypeptides

Aug 21, 2013 - Pesach Perlin , Eric G. Gharakhanian , Timothy J. Deming. Chemical ... David Huesmann , Kristina Klinker , Matthias Barz. Polym. Chem...
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A Simple and Versatile Synthetic Strategy to Functional Polypeptides via Vinyl Sulfone-Substituted L‑Cysteine N‑Carboxyanhydride Jianren Zhou, Peipei Chen, Chao Deng,* Fenghua Meng, Ru Cheng, and Zhiyuan Zhong* Biomedical Polymers Laboratory, and Jiangsu Key Laboratory of Advanced Functional Polymer Design and Application, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou 215123, People’s Republic of China S Supporting Information *

ABSTRACT: Vinyl sulfone-substituted L-cysteine N-carboxyanhydride (VSCys-NCA) monomer was designed and developed to afford a novel and versatile family of vinyl sulfone (VS)-functionalized polypeptides, which further offer a facile access to functional polypeptide-based materials including glycopolypeptides, functional polypeptide coatings, and in situ forming polypeptide hydrogels through Michaeltype addition chemistry under mild conditions. VSCys-NCA was obtained in two straightforward steps with a high overall yield of 76%. The copolymerization of γ-benzyl L-glutamate NCA (BLG-NCA), N-benzyloxycarbonyl-L-lysine NCA (ZLL-NCA), or L-leucine NCA (Leu-NCA) with VSCys-NCA using 1,1,1-trimethyl-N-2-propenylsilanamine (TMPS) as an initiator proceeded smoothly in DMF at 40 °C, yielding P(BLG-coVSCys), P(ZLL-co-VSCys), or P(Leu-co-VSCys) with defined functionalities, controlled molecular weights, and moderate polydispersities (PDI = 1.15−1.50). The acidic deprotection of P(BLG-co-VSCys) and P(ZLL-co-VSCys) furnished water-soluble VS-functionalized poly(L-glutamic acid) (P(Glu-co-VSCys)) and VS-functionalized poly(L-lysine) (P(LL-co-VSCys)), respectively. These VS-functionalized polypeptides were amenable to direct, efficient, and selective postpolymerization modification with varying thiol-containing molecules such as 2-mercaptoethanol, 2-mercaptoethylamine hydrochloride, L-cysteine, and thiolated galactose providing functional polypeptides containing pendant hydroxyl, amine, amino acid, and saccharide, respectively. The contact angle and fluorescence measurements indicated that polymer coatings based on P(Leu-coVSCys) allowed direct functionalization with thiol-containing molecules under aqueous conditions. Moreover, hydrogels formed in situ upon mixing aqueous solutions of P(Glu-co-VSCys) and thiolated glycol chitosan at 37 °C. These vinyl sulfonefunctionalized polypeptides have opened a new avenue to a broad range of advanced polypeptide-based materials.

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polypeptides containing a natural thioether group30 and nonnatural functional groups like propargyl,31−33 allyl/pentenyl,34−37 cinnamyl,38 and vinylbenzyl39 have been designed and prepared without protection and deprotection steps. Moreover, sophisticated functional materials could be obtained by further postpolymerization modification.40,41 For example, allyl-functionalized polypeptides obtained via ROP polymerization of DLallylglycine NCA were modified with different thiol-containing molecules through radical thiol−ene addition chemistry using azobisisobutylonitrile (AIBN) as a radical source at elevated temperature or under strong UV irradiation.34 The degrees of modification for poly(DL-allylglycine) with 2,3,4,6-tetra-O-acetyl1-thio-β-D-glucopyranose were reported to be about 35% and 50% in the presence of AIBN for 1 day at 70 °C and in presence of a photoinitiator Irgacure 819 with mercury medium pressure UV light for 1 day at room temperature, respectively. In this paper, we report on design and development of novel vinyl sulfone (VS)-functionalized polypeptides that provide an

INTRODUCTION Synthetic polypeptides inherit many intriguing properties of proteins such as excellent biocompatibility, biodegradability, unique hierarchical assembly property, versatile structures and functionalities, and biological activity.1−4 They have been widely used as biomimetic materials,5−7 drug nanocarriers,8−15 tissue engineering scaffolds,16−18 and potent catalysts.19,20 The ring-opening polymerization (ROP) of N-carboxyanhydride (NCA) is the most viable strategy for the large-scale synthesis of high molecular weight (MW) polypeptides.21 In particular, the recent development of controlled NCA polymerization techniques e.g. using transition metal complexes,22 amine hydrochloride,23 amine under high vacuum,24 amine under low temperature,25 or silazane derivatives26 renders it possible to prepare polypeptides with controlled MWs and low polydispersities. The emerging biomedical technology demands advancement of functional biomaterials.27−29 Functional polypeptides containing e.g. carboxylic acid, amine, hydroxyl, and saccharide groups have been obtained by ROP of side chain protected NCAs followed by deprotection. This synthetic strategy, however, suffers drawbacks of complex synthesis, low yields, and potential polymer degradation. In recent years, functional © 2013 American Chemical Society

Received: July 12, 2013 Revised: August 5, 2013 Published: August 21, 2013 6723

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Scheme 1. Preparation and Potential Biomedical Applications of VS-Functionalized Polypeptides

lactobionic acid and 2-mercaptoethylamine using EDC/NHS as coupling agents. Ethyl acetate and petroleum ether were dried over CaH2 and distilled prior to use. Tetrahydrofuran (THF) was dried by refluxing over sodium wire under an argon atmosphere followed by distillation. Dimethylformamide (DMF) was distilled under reduced pressure before use. Characterization. 1H NMR spectra were recorded on the Unity Inova 400 operating at 400 MHz. D2O and DMSO-d6 were used as solvents, and the chemical shifts were calibrated against residual solvent signals. The molecular weight and polydispersity of copolymers were determined by a Waters 1515 gel permeation chromatograph (GPC) instrument equipped with two linear PLgel columns (Mixed-C) following a guard column and a differential refractive index detector. The measurements were performed using DMF as the eluent at a flow rate of 1.0 mL/min at 30 °C and a series of narrow polystyrene standards for the calibration of the columns. The static water contact angle measurements were performed on an SL-200C optical contact angle meter (Solon Information Technology Co.) using the sessile drop method. Rheological analysis was performed on RS6000 (ThermoFisher, Germany) with parallel plates (20 mm diameter) configuration at 37 °C in the oscillatory mode. A gap of 0.5 mm, a frequency of 1 Hz, and a strain of 1% were applied to maintain the linear viscoelastic regime. A solvent trap was used to avoid water evaporation. Synthesis of VS-Substituted L-Cysteine N-Carboxyanhydride (VSCys-NCA) Monomer. VSCys-NCA was synthesized in two steps. First, L-cysteine hydrochloride monohydrate (4.45 g, 25 mmol) in methanol (80 mL) was dropwise added to a solution of divinyl sulfone (12.55 mL, 125 mmol) under stirring at 50 °C. After stirring for 100 h, the reaction solution was concentrated under reduced pressure, and the residue was purified by precipitation in ethyl acetate and filtration to yield VS-substituted L-cysteine (VSCys) as a white solid (6.60 g, 95%). 1H NMR (400 MHz, D2O): δ 6.38 and 6.92 (m, 3H, −CH CH2), 4.23 (m, 1H, −CHNH2), 3.57 (t, 2H, −SO2CH2CH2−), 3.18 and 3.23 (m, 2H, −SCH2CH−), 2.98 (t, 2H, −SO2CH2CH2−). Under a nitrogen atmosphere, α-pinene (5 mL, 31.5 mmol) was added to a solution of VSCys (3.75 g, 13.6 mmol) in dry THF (100 mL) at 50 °C. After stirring for 0.5 h, triphosgene (2.02 g, 6.8 mmol) was added, and the reaction solution was stirred at 70 °C for about 2 h. The reaction mixture was then concentrated under reduced pressure, and the residue was precipitated in petroleum ether to give crude VSCys-NCA. The crude product was redissolved in ethyl acetate, washed with cold

unprecedented access to functional polypeptide materials including glycopolypeptides, functional polypeptide coatings, and in situ forming polypeptide hydrogels through Michael-type addition chemistry (Scheme 1). VS-functionalized polypeptides were readily prepared by controlled copolymerization of a novel monomer, VS-substituted L-cysteine NCA (VSCys-NCA), with different α-amino acid NCA monomers. VS has a high reactivity and selectivity toward Michael-type conjugate addition that is particularly appealing for preparation of functional biodegradable materials and coatings,42 protein immobilization,43 conjugation of targeting ligands to nanoparticles,44 and development of in situ forming hydrogels.45−47 Remarkably, VSCys-NCA monomer was obtained in two straightforward steps with a high overall yield. The resulting VS-functionalized polypeptides are amenable to direct and selective postpolymerization modification with thiolcontaining biomolecules, in which no catalyst is required and no byproduct is generated. More strikingly, these VS-functionalized polypeptides allow for the first time direct functionalization of polypeptide coatings under aqueous conditions as well as in situ formation of robust polypeptide hydrogels. Here, synthesis of VSCys-NCA monomer, preparation and postpolymerization modification of VS-functionalized polypeptides, surface modification of functional polypeptide coatings, and in situ formation of polypeptide hydrogels via Michael-type addition chemistry were investigated.

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EXPERIMENTAL SECTION

Materials. Divinyl sulfone (95%, Dalian Guanghui, China), L-cysteine hydrochloride monohydrate (99%, Alfa Aesar), α-pinene (98%, Acros), γ-benzyl L-glutamate, ε-carbobenzyloxy-L-lysine, L-leucine (98%, GL Biochem, Shanghai, China), L-cysteine (>99%, Alfa Aesar), 1,1,1trimethyl-N-2-propenylsilanamine (TMPS, 96%, Aldrich), 2-mercaptoethanol (>99%, Amresco), and fluorescein isothiocyanate (FITC, 98%, Sigma) were used as received. Triphosgene (Shanxi Jiaocheng Jinxin Chemical Factory, China) was recrystallized from ethyl acetate before use. Thiolated glycol chitosan (GC-SH, Mn = 80 kg/mol, DS 17) was synthesized according to our previous report.48 Thiolated galactose (galactose-SH, 417.45 Da) was prepared by coupling reaction between 6724

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Scheme 2. Synthetic Pathway to VSCys-NCAa

a

Conditions: (i) divinyl sulfone, 50 °C, methanol, 100 h; (ii) triphosgene, α-pinene, reflux, 2 h, THF. (33%, 0.82 mL, 4.61 mol of HBr, 6 equiv with respect to the benzyl groups). After stirring at room temperature for 2 h, the reaction mixture was precipitated in diethyl ether, filtered, and dried in vacuo. Gravimetric yield: 92.5%. 1H NMR (400 MHz, DMSO-d6) of P(Glu-co-VSCys): δ 6.28−6.96 (m, 3H, −SO2CHCH2), 5.06− 5.76 (m, 3H, −CHCH2), 4.26−4.47 (m, 2H, −CHNH−), 3.68 (m, 2H, CH2CHCH2−), 2.68−2.88 (m, 4H, −CH2SCH2−), 2.23 (t, 2H, −CH2CH2COOH), 1.73−1.88 (m, 2H, −CH2CH2COOH), 7.94−8.45 (m, 3H, −NH−). 1H NMR (400 MHz, DMSO-d6) of P(LL-co-VSCys): δ 6.31−7.02 (m, 3H, −SO2CHCH2), 5.06−5.78 (m, 3H, −CHCH2), 4.27−4.48 (m, 2H, −CHNH−), 3.93 (m, 2H, CH2CHCH2−), 2.79−2.89 (m, 4H, −CH2SCH2−; m, 2H, −CH2NH2), 1.34−1.56 (m, 6H, −CH2CH2CH2CH2NH2), 7.87− 8.06 (m, 3H, −NH−; 2H, −NH2). Postpolymerization Modification of VS-Functionalized Polypeptides with Thiol-Containing Molecules. The postpolymerization modification of VS-functionalized polypeptides was achieved through Michael-type conjugate addition reaction of VS groups in PVSCys with thiol groups in functional molecules including 2-mercaptoethanol, 2-mercaptoethylamine hydrochloride, L-cysteine, and galactose-SH. The SH/VS molar ratio was fixed at 2/1, and the reaction proceeded in DMF at room temperature under a nitrogen atmosphere for 1 day. The resulting modified polypeptides were collected by precipitation in an excess of cold diethyl ether/ethanol (1/4, v/v), filtration, and drying in vacuo at room temperature. The 1H NMR spectra of modified P(Leu-coVSCys)47% indicated quantitative modification. Preparation of VS-Functionalized Polypeptide Films and Direct Modification with Thiol-Containing Molecules. Thin films were prepared by dip-coating a solution of P(Leu-co-VSCys) copolypeptides in DMF (5 mg/mL) on the microscope slides and dried in vacuo. The films were incubated in aqueous solution of a thiolcontaining molecule (e.g., 2-mercaptoethanol, cysteamine, L-cysteine hydrochloride) at a concentration of 1 mg/mL for 24 h. The resulting modified films, after exhaustively rinsed with deionized water, were dried over phosphorus pentoxide under reduced pressure. The water contact angles of both modified and unmodified polypeptide films were examined on an SL-200C optical contact angle meter (Solon Information Technology Co.) using the sessile drop method. The experiments were conducted in triplicate, and the results presented were the average data with standard deviation. The successful immobilization of cysteamine on P(Leu-co-VSCys) films and chemical reactivity of amino groups on surface were further studied by fluorescence microscopy. Typically, P(Leu-co-VSCys) films following modification with cysteamine was treated with 0.5 mg/mL FITC in phosphate buffered saline (PBS, 20 mM, pH 9.0) at 37 °C for 24 h in the dark. The FITC-modified films were thoroughly rinsed with deionized water and then observed using fluorescence microscope (Leica DM4000M). P(Leu-co-VSCys) films directly treated with FITC in aqueous condition were used as a control. Rapidly in Situ Forming Polypeptide Hydrogels through Michael-Type Addition Chemistry. Hydrogels were fabricated in vials by thoroughly mixing solutions of P(Glu-co-VSCys) in 2-(Nmorpholino)ethanesulfonic acid (MES, pH 5.3, 10 mM) and thiolated glycol chitosan (GC-SH) in (4-(2 hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES, pH 8.0, 1 mM). Rheological analysis was performed by quick mixing P(Glu-co-VSCys) solution and GC-SH solution and then operating on the test platform of RS6000 at 37 °C in the oscillatory mode. The evolution of storage modulus (G′) and loss modulus (G″) was recorded as a function of time. The gelation time was defined as the time point where G′ = G″. The storage modulus of

saturated NaHCO3 aqueous solution and cold water, and dried with anhydrous MgSO4. The evaporation of solvent gave VSCys-NCA as a viscous oil (2.89 g, 80%). 1H NMR (400 MHz, DMSO-d6): δ 6.24 and 7.02 (m, 3H, −CHCH2), 4.77 (t, 1H, −CHNH−), 3.42 (t, 2H, −SO2CH2CH2−), 3.00 (t, 2H, −SO2CH2CH2−), 2.81 (d, 2H, −SCH2CH−), 9.15 (s, 1H, −NH−). 13C NMR (400 MHz, DMSOd6): δ 171.77, 153.34, 137.89, 131.96, 59.44, 54.54, 33.77, 26.32. Anal. Calcd for C8H11O5S2N: C, 36.22; H, 4.18; N, 5.28. Found: C, 37.11; H, 4.68; N, 5.09. Electrospray ionization mass spectrometry (ESI-MS, m/z): calcd for C8H11O5S2N 265.01; found 265.01. Synthesis of VS-Functionalized Polypeptides. The copolymerization of VSCys-NCA with γ-benzyl L-glutamate N-carboxyanhydride (BLG-NCA), ε-carbobenzyloxy-L-lysine N-carboxyanhydride (ZLL-NCA), or L-leucine N-carboxyanhydride (Leu-NCA) was carried out in DMF at 40 °C for 48 h using TMPS as an initiator. Take synthesis of P(BLG-co-VSCys)17% copolymer, wherein xx% means molar fraction of VSCys units in copolypeptides determined by 1 H NMR, as an example. In a glovebox under a nitrogen atmosphere, to a solution of BLG-NCA (0.43 g, 1.62 mmol) and VSCys-NCA (0.11 g, 0.41 mmol) in DMF (10 mL) under stirring was quickly added a stock solution of TMPS (6.5 mg, 0.05 mmol) in DMF. The reaction vessel was sealed and placed in an oil bath thermostated at 40 °C. After 48 h polymerization, the BLG-NCA was completely consumed (monitored by FT-IR). The reaction was terminated by two drops of acetic acid. The resulting P(BLG-co-VSCys) copolymers was isolated by precipitation in diethyl ether, centrifugation, and drying in vacuo. Gravimetric yield: 88.7−96.3%. 1H NMR (400 MHz, DMSO-d6): δ 7.32 (m, 5H, C6H5), 6.23 and 6.94 (m, 3H, −SO2CHCH2), 5.06 and 5.76 (m, 3H, −CHCH2; 2H, −CH2C6H5), 4.29−4.46 (m, 2H, −CHNH−), 3.68 (m, 2H, CH2CHCH2−), 2.76−2.87 (m, 4H, −CH2SCH2−), 2.37 (t, 2H, −COCH2CH2−), 1.80−1.93 (m, 2H, −COCH2CH2−), 8.10−8.48 (m, 3H, −NH−). In a similar manner, P(ZLL-co-VSCys)19% was synthesized by copolymerization of ZLL-NCA and VSCys-NCA. Gravimetric yield: 94.8%. 1H NMR (400 MHz, DMSO-d6) of P(ZLL-co-VSCys) 19%: δ 7.28 (m, 5H, C6H5), 6.23 and 6.93 (m, 3H, −SO2CHCH2), 5.73 (m, 1H, −CH2CHCH2), 4.95 (m, 2H, −CH2CHCH2; 2H, −CH2C6H5), 4.21−4.44 (m, 2H, −CHNH−), 3.68 (m, 2H, −CH2CHCH2), 2.76−2.93 (m, 4H, −CH2SCH2−; m, 2H, −CH2NH−), 1.21−1.61 (m, 6H, −CHCH2CH2CH2CH2NH−), 7.89−8.13 (m, 3H, −NH−). P(Leu-co-VSCys) was prepared by direct copolymerization of LeuNCA with VSCys-NCA in DMF. Gravimetric yield: 57.6%. 1H NMR (400 MHz, DMSO-d6) of P(Leu-co-VSCys)47%: δ 6.30 and 6.98 (m, 3H, −SO2CHCH2), 5.05 and 5.75 (m, 3H, −CHCH2), 4.32− 4.47 (m, 2H, −CHNH−), 3.69 (m, 2H, CH2CHCH2−), 2.65−2.88 (m, 4H, −CH2SCH2−), 1.56 (m, 1H, −CH2CH(CH3)2), 1.43 (m, 2H, −CH2CH(CH3)2), 0.86 (d, 6H, −CH2CH(CH3)2), 7.91−8.57 (m, 3H, −NH−). PVSCys was prepared by direct polymerization of VSCys-NCA in DMF at 40 °C for 48 h. 1H NMR (400 MHz, DMSO-d6) of PVSCys: δ 6.30 and 6.98 (m, 3H, −SO2CHCH2), 5.05 and 5.75 (m, 3H, −CHCH2), 4.51 (m, 1H, −CHNH−), 3.69 (m, 2H, CH2 CHCH2−), 2.66−2.90 (m, 4H, −CH2SCH2−). The deprotection of P(BLG-co-VSCys) and P(ZLL-co-VSCys) was carried out by acidolysis using a 33% solution of HBr in AcOH. The following is a typical example on deprotection of P(BLG-co-VSCys) copolypeptides. To a solution of P(BLG-co-VSCys)17% (0.2 g, 0.024 mmol) in CF3COOH was added a solution of HBr in AcOH 6725

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triphosgene in the presence of α-pinene (Scheme 2). 1H NMR of VSCys showed signals of L-cysteine moieties at δ 4.23 and 3.18, signals attributable to vinyl protons at δ 6.92 and 6.38, and two linking ethylene protons at δ 3.57 and 2.98 (Figure S1). The signals at δ 6.38, 4.23, and 3.57 had an intensity ratio close to 2:1:2, confirming equivalent coupling of L-cysteine hydrochloride and divinyl sulfone. VSCys-NCA was isolated as a viscous oil in a high overall yield of 76%. 1H NMR displayed clearly signals at δ 6.24 and 7.02 owing to vinyl protons, δ 4.77 and 9.15 to methine and amide protons, and δ 2.81, 3.00, and 3.42 to the three methylene protons neighboring to the thioether or sulfone group (Figure 1A). The signals at δ 7.02 (methine proton of VS group) and 4.77 (methine proton of NCA ring) had an intensity ratio close to 1:1, indicating that VSCys-NCA was obtained with quantitative functionality. 13C NMR detected besides four alkane carbons at δ 26.32−59.44 also two vinyl carbons at δ 131.96 and 137.89 as well as two carbonyl carbons at δ 153.34 and 171.77 (Figure 1B). The elemental analysis revealed a composition close to that calculated for VSCys-NCA and furthermore ESI-MS showed an exact mass of 265.01. These results point out that VSCys-NCA monomer is readily obtained with a defined structure and high yield. Synthesis of VS-Functionalized Polypeptides. VSCysNCA was readily copolymerized with different α-amino acid NCA monomers such as γ-benzyl L-glutamate NCA (BLGNCA), N-benzyloxycarbonyl-L-lysine NCA (ZLL-NCA), and L-leucine NCA (Leu-NCA) using TMPS as an initiator in DMF at 40 °C (Scheme 3). The results of copolymerization are summarized in Table 1. 1H NMR of P(BLG-co-VSCys) showed characteristic vinyl sulfone protons at δ 6.23 and 6.94 (Figure 2A), indicating that vinyl sulfone was intact during polymerization and following work-up procedures. VSCys fractions (FVSCys) of P(BLG-co-VSCys) determined by comparing the intensities of signals at δ 6.23 owing to VS protons of VSCys and 7.32 assignable to the benzene ring of BLG increased from 8.5 to 28 mol % at increasing VSCys-NCA monomer feed ratios

Figure 1. 1H NMR (400 MHz) (A) and 13C NMR (100 MHz) (B) of VSCys-NCA in DMSO-d6. polypeptide hydrogels was determined in triplicate, and the results presented were the average data with standard deviation.

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RESULTS AND DISCUSSION Synthesis of VS-Substituted L-Cysteine N-Carboxyanhydride (VSCys-NCA) Monomer. VSCys-NCA was synthesized by treating L-cysteine hydrochloride with excess of divinyl sulfone in methanol at 50 °C followed by cyclization using Scheme 3. Synthesis of VS-Functionalized Polypeptides

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Table 1. Synthesis of VS-Functionalized Polypeptidesa Mn (kg/mol) entry

copolymer

M/Ib

f VSc (%)

FVSd (%)

NMRe

GPCf

PDI GPCf

yield (%)

1 2 3 4 5 6

P(BLG-co-VSCys) P(BLG-co-VSCys) P(BLG-co-VSCys) P(BLG-co-VSCys) P(ZLL-co-VSCys) P(Leu-co-VSCys)

40 40 40 60 40 57

10 20 30 20 20 30

8.5 17 28 21 19 47

10.5 8.50 8.00 12.5 9.94 5.30

10.7 7.05 7.60 10.9 17.7 4.34

1.25 1.50 1.45 1.28 1.15 1.23

93.2 88.7 91.7 96.3 94.8 57.6

a

VS-functionalized polypeptides were prepared through ring-opening copolymerization of BLG-NCA, ZLL-NCA, or Leu-NCA with VSCys-NCA in DMF at 40 °C for 2 days using TMPS as an initiator. bTotal monomer-to-initiator molar ratio. cMolar fraction of VSCys-NCA monomer in the feed. d Molar fraction of VSCys units in resulting copolymer determined by 1H NMR. eEstimated by 1H NMR end-group analysis. fDetermined by GPC (eluent: DMF, flow rate: 1.0 mL/min, standards: polystyrene).

(f VSCys) from 10 to 30 mol % (Table 1, entries 1−3). Moreover, the number-average molecular weight (Mn) of P(BLG-coVSCys) estimated from the intensity ratios of signals at δ 6.23 (VSCys) and 7.32 (BLG) to those at δ 5.76 (methine proton of TMPS moiety) were close to the theoretical values and increased in proportion to monomer-to-initiator (M/I) ratios (Table 1, entries 1−4). Notably, GPC measurements showed that all P(BLG-co-VSCys) copolymers had moderate polydispersities (PDI = 1.25−1.50) and Mn values close to those determined by 1 H NMR end-group analyses (Table 1, entries 1−4). It is clear that copolymerization of VSCy-NCA and BLG-NCA proceeds in a controlled manner to give P(BLG-co-VSCys) copolymers with tailored molecular weights and functionalities. In a similar way, copolymerization of VSCy-NCA and ZLL-NCA yielded well-defined P(ZLL-co-VSCys) copolymer. 1 H NMR analysis showed that P(ZLL-co-VSCys) was obtained with a VSCys content of 19 mol % and an Mn of 9.94 kg/mol at an f VSCys of 20 mol % and an M/I ratio of 40/1 (Figure 2B). GPC displayed that P(ZLL-co-VSCys)19% had an Mn of 17.7 kg/mol and a low PDI of 1.15 (Table 1, entry 5). The deviation of Mn value determined by GPC from that calculated by 1 H NMR is most likely due to use of polystyrene standards for molecular weight calibration in our GPC measurements. In comparison, copolymerization of VSCy-NCA and Leu-NCA resulted in only partial monomer conversion under otherwise the same conditions, probably due to low polymerizability of Leu-NCA. 1H NMR indicated that P(Leu-co-VSCys) prepared at an f VSCys of 30 mol % had an elevated VSCys content of 47 mol % (Figure S2). In accordance, both 1H NMR end-group analysis and GPC showed that P(Leu-co-VSCys) had a comparably low Mn though PDI remained low (1.23) (Table 1, entry 6). It should further be noted that VSCys-NCA could also undergo homopolymerization to yield PVSCys with an Mn of 16.0 kg/mol and a moderate PDI of 1.58. The acidic deprotection of the benzyl and carbobenzyloxy groups in P(BLG-co-VSCys) and P(ZLL-co-VSCys) furnished VS-functionalized poly(L-glutamic acid) (P(Glu-VSCys)) and VS-functionalized poly(L-lysine) (P(LL-VSCys)), respectively (Scheme 3). The deprotection of P(BLG-co-VSCys) was carried out in 33% HBr/AcOH (6 equiv of HBr with respect to the benzyl groups) at room temperature for 2 h, which has shown to successfully remove the protecting benzyl groups of PBLG without obvious main chain cleavage.49−51 Both P(Glu-VSCys) and P(LL-VSCys) were freely water-soluble. 1H NMR in DMSO-d6 showed that signals at around δ 7.30 and 5.00 attributable to the benzyl protons completely disappeared while no change was observed for signals assignable to VS protons (δ 6.93 and 6.23) (Figure 3), indicating quantitative removal of

Figure 2. 1H NMR spectra (400 MHz) of copolypeptides in DMSOd6: (A) P(BLG-co-VSCys)17% (Table 1, entry 2) and (B) P(ZLL-coVSCys)19% (Table 1, entry 5).

benzyl groups and VS group unspoiled during acidic treatment. It is evident, therefore, that VSCys-NCA can copolymerize with different NCA monomers, which provides a facile access to a range of VS-functionalized polypeptides with distinct hydrophilicity, charge, and functionalities. Postpolymerization Modification of P(Leu-co-VSCys) with Thiol-Containing Molecules. Interestingly, VS-functionalized polypeptides were amenable to versatile and selective postpolymerization modification with thiol-containing molecules such as 2-mercaptoethanol, 2-mercaptoethylamine 6727

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Article

Figure 4. Surface modification of P(Leu-co-VSCys)47% polypeptide films. (A) Water contact angles of polypeptide films modified with mercaptoethanol and cysteine; data are presented as mean ± SD (n = 3). (B) fluorescence images of polypeptide films treated with cysteamine and FITC successively in aqueous condition or treated with FITC directly (control).

from VS-functionalized polypeptides could be directly modified with thiol-containing biomolecules under aqueous conditions. Water contact angle analysis showed that VS-functionalized P(Leu-co-VSCys) films following treatment with 2-mercaptoethanol and cysteine became much more hydrophilic, as evidenced by a significant reduction of water contact angle (8°−20°) for P(Leu-co-VSCys)47% (Figure 4A). To further confirm occurrence of surface modification, P(Leu-co-VSCys)47% film following treatment with cysteamine was reacted with fluorescein isothiocyanate (FITC) in phosphate buffered saline (PBS, 20 mM, pH 9.0). Fluorescence microscopy showed that strong fluorescence was observed throughout the whole film (Figure 4B), indicating homogeneous surface modification with cysteamine and furthermore amine groups at the surface maintaining high reactivity. In contrast, no fluorescence was detected for P(Leu-co-VSCys) film directly treated with FITC under otherwise the same conditions (Figure 4B). This represents a first report on direct surface modification of polypeptide coatings under aqueous conditions, which might have great applications in medical implants as well as bioactive tissue engineering scaffolds. Rapidly in Situ Forming Polypeptide Hydrogels through Michael-Type Addition Chemistry. Notably, we are also able to prepare in situ forming robust polypeptide hydrogels based on water-soluble VS-functionalized poly peptides using polythiols as a cross-linking agent. For example, hydrogels formed rapidly upon mixing aqueous solutions of P(Glu-co-VSCys) and thiolated glycol chitosan (GC-SH) at 37 °C without any catalyst (Figure 5A). Rheology analysis showed that mixing 2.0 wt % P(Glu-co-VSCys) in HEPES (1 mM, pH 8.0) and 2.0 wt % GC-SH (DS 17) in MES (10 mM,

Figure 3. 1H NMR spectra (400 MHz, DMSO-d6) of (A) P(Glu-coVSCys)17% and (B) P(LL-co-VSCys)19%.

hydrochloride, L-cysteine, and galactose-SH at a ligand-SH/VS molar ratio of 2/1 in the absence of any catalyst under mild conditions (Scheme 4). Typical 1H NMR spectra of P(Leu-coVSCys)47% copolymer following treatment with 2-mercaptoethanol and galactose-SH showed complete vanishing of signals at δ 6.93 and 6.23 owing to VS protons and emergence of new signals characteristic of 2-mercaptoethanol (δ 3.56) or galactose-SH (δ 4.27, 3.92, and 3.39) moieties (Figure S3), signifying quantitative functionalization. These results corroborate that Michael-type conjugate addition between VS and thiol groups is highly selective and tolerant to various functional groups including amines and carboxylic acids. It is of particular interest to note that through VS-functionalized polypeptides glycopolypeptides can be readily obtained with controlled saccharide contents without tedious protection/deprotection process, which might find tremendous applications in fields of controlled drug release and regenerative medicine.52−55 Surface Modification of P(Leu-co-VSCys) Films with Thiol-Containing Molecules. More strikingly, films prepared

Scheme 4. Postpolymerization Modification of VS-Functionalized Polypeptides

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dx.doi.org/10.1021/ma4014669 | Macromolecules 2013, 46, 6723−6730

Macromolecules

Article

Figure 5. In situ forming polypeptide hydrogels prepared from VS-functionalized polypeptides. (A) Images of polypeptide hydrogels prepared from the solutions of P(Glu-co-VSCys) in HEPES and GC-SH in MES. (B) Evolution of storage modulus and loss modulus upon mixing P(Glu-coVSCys) (FVSCys = 17%, 2 wt %) and GC-SH (DS 17) at 37 °C. (C) Storage moduli of hydrogels formed at different concentrations of P(Glu-coVSCys) (FVSCys = 17%); data are presented as mean ± SD (n = 3). Molar ratio of SH/VS was fixed at 1/1.

pH 5.3) at a VS/SH molar ratio of 1/1 led to almost instantaneous gelation (gelation time