a simple and versatile synthetic strategy to functional polypeptides via vinyl sulfone-substituted l...

A Simple and Versatile Synthetic Strategy to Functional Polypeptidesvia Vinyl Sulfone-Substituted L‑Cysteine N‑CarboxyanhydrideJianren 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 ofChemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou 215123, People’s Republic of China

*S Supporting Information

ABSTRACT: Vinyl sulfone-substituted L-cysteine N-carbox-yanhydride (VSCys-NCA) monomer was designed anddeveloped to afford a novel and versatile family of vinylsulfone (VS)-functionalized polypeptides, which further offera facile access to functional polypeptide-based materialsincluding glycopolypeptides, functional polypeptide coatings,and in situ forming polypeptide hydrogels through Michael-type addition chemistry under mild conditions. VSCys-NCAwas obtained in two straightforward steps with a high overallyield of 76%. The copolymerization of γ-benzyl L-glutamateNCA (BLG-NCA), N-benzyloxycarbonyl-L-lysine NCA (ZLL-NCA), or L-leucine NCA (Leu-NCA) with VSCys-NCA using1,1,1-trimethyl-N-2-propenylsilanamine (TMPS) as an initiator proceeded smoothly in DMF at 40 °C, yielding P(BLG-co-VSCys), P(ZLL-co-VSCys), or P(Leu-co-VSCys) with defined functionalities, controlled molecular weights, and moderatepolydispersities (PDI = 1.15−1.50). The acidic deprotection of P(BLG-co-VSCys) and P(ZLL-co-VSCys) furnished water-solubleVS-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 postpolymerizationmodification 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, andsaccharide, respectively. The contact angle and fluorescence measurements indicated that polymer coatings based on P(Leu-co-VSCys) allowed direct functionalization with thiol-containing molecules under aqueous conditions. Moreover, hydrogels formedin situ upon mixing aqueous solutions of P(Glu-co-VSCys) and thiolated glycol chitosan at 37 °C. These vinyl sulfone-functionalized polypeptides have opened a new avenue to a broad range of advanced polypeptide-based materials.

■ INTRODUCTION

Synthetic polypeptides inherit many intriguing properties ofproteins such as excellent biocompatibility, biodegradability,unique hierarchical assembly property, versatile structures andfunctionalities, and biological activity.1−4 They have beenwidely used as biomimetic materials,5−7 drug nanocarriers,8−15

tissue engineering scaffolds,16−18 and potent catalysts.19,20 Thering-opening polymerization (ROP) of N-carboxyanhydride(NCA) is the most viable strategy for the large-scale synthesisof high molecular weight (MW) polypeptides.21 In particular, therecent development of controlled NCA polymerization techniquese.g. using transition metal complexes,22 amine hydrochloride,23

amine under high vacuum,24 amine under low temperature,25 orsilazane derivatives26 renders it possible to prepare polypeptideswith controlled MWs and low polydispersities.The emerging biomedical technology demands advancement

of functional biomaterials.27−29 Functional polypeptides con-taining e.g. carboxylic acid, amine, hydroxyl, and saccharidegroups have been obtained by ROP of side chain protectedNCAs followed by deprotection. This synthetic strategy,however, suffers drawbacks of complex synthesis, low yields,and potential polymer degradation. In recent years, functional

polypeptides containing a natural thioether group30 and non-natural functional groups like propargyl,31−33 allyl/pentenyl,34−37

cinnamyl,38 and vinylbenzyl39 have been designed and preparedwithout protection and deprotection steps. Moreover, sophisti-cated functional materials could be obtained by furtherpostpolymerization modification.40,41 For example, allyl-function-alized polypeptides obtained via ROP polymerization of DL-allylglycine NCA were modified with different thiol-containingmolecules through radical thiol−ene addition chemistry usingazobisisobutylonitrile (AIBN) as a radical source at elevatedtemperature or under strong UV irradiation.34 The degrees ofmodification for poly(DL-allylglycine) with 2,3,4,6-tetra-O-acetyl-1-thio-β-D-glucopyranose were reported to be about 35% and50% in the presence of AIBN for 1 day at 70 °C and in presenceof a photoinitiator Irgacure 819 with mercury medium pressureUV 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

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

Article

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© 2013 American Chemical Society 6723 dx.doi.org/10.1021/ma4014669 | Macromolecules 2013, 46, 6723−6730

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unprecedented access to functional polypeptide materialsincluding glycopolypeptides, functional polypeptide coatings,and in situ forming polypeptide hydrogels through Michael-typeaddition chemistry (Scheme 1). VS-functionalized polypeptideswere readily prepared by controlled copolymerization of a novelmonomer, VS-substituted L-cysteine NCA (VSCys-NCA), withdifferent α-amino acid NCA monomers. VS has a high reactivityand selectivity toward Michael-type conjugate addition that isparticularly appealing for preparation of functional biodegradablematerials and coatings,42 protein immobilization,43 conjugationof targeting ligands to nanoparticles,44 and development of in situforming hydrogels.45−47 Remarkably, VSCys-NCA monomer wasobtained in two straightforward steps with a high overall yield.The resulting VS-functionalized polypeptides are amenable todirect and selective postpolymerization modification with thiol-containing biomolecules, in which no catalyst is required and nobyproduct is generated. More strikingly, these VS-functionalizedpolypeptides allow for the first time direct functionalization ofpolypeptide coatings under aqueous conditions as well as in situformation of robust polypeptide hydrogels. Here, synthesis ofVSCys-NCA monomer, preparation and postpolymerizationmodification of VS-functionalized polypeptides, surface mod-ification of functional polypeptide coatings, and in situ formationof polypeptide hydrogels via Michael-type addition chemistrywere investigated.

■ EXPERIMENTAL SECTIONMaterials. 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%, GLBiochem, Shanghai, China), L-cysteine (>99%, Alfa Aesar), 1,1,1-trimethyl-N-2-propenylsilanamine (TMPS, 96%, Aldrich), 2-mercaptoe-thanol (>99%, Amresco), and fluorescein isothiocyanate (FITC, 98%,Sigma) were used as received. Triphosgene (Shanxi Jiaocheng JinxinChemical Factory, China) was recrystallized from ethyl acetate beforeuse. Thiolated glycol chitosan (GC-SH, Mn = 80 kg/mol, DS 17) wassynthesized according to our previous report.48 Thiolated galactose(galactose-SH, 417.45 Da) was prepared by coupling reaction between

lactobionic acid and 2-mercaptoethylamine using EDC/NHS as couplingagents. Ethyl acetate and petroleum ether were dried over CaH2 anddistilled prior to use. Tetrahydrofuran (THF) was dried by refluxing oversodium wire under an argon atmosphere followed by distillation.Dimethylformamide (DMF) was distilled under reduced pressurebefore use.

Characterization. 1H NMR spectra were recorded on the UnityInova 400 operating at 400 MHz. D2O and DMSO-d6 were used assolvents, and the chemical shifts were calibrated against residual solventsignals. The molecular weight and polydispersity of copolymers weredetermined 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 flowrate of 1.0 mL/min at 30 °C and a series of narrow polystyrenestandards for the calibration of the columns. The static water contactangle measurements were performed on an SL-200C optical contactangle meter (Solon Information Technology Co.) using the sessile dropmethod. Rheological analysis was performed on RS6000 (Thermo-Fisher, Germany) with parallel plates (20 mm diameter) configurationat 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 viscoelasticregime. 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) inmethanol (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, andthe residue was purified by precipitation in ethyl acetate and filtrationto 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, −CHCH2), 4.23 (m, 1H, −CHNH2), 3.57 (t, 2H, −SO2CH2CH2−), 3.18and 3.23 (m, 2H, −SCH2CH−), 2.98 (t, 2H, −SO2CH2CH2−).

Under a nitrogen atmosphere, α-pinene (5 mL, 31.5 mmol) wasadded 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) wasadded, and the reaction solution was stirred at 70 °C for about 2 h. Thereaction mixture was then concentrated under reduced pressure, and theresidue was precipitated in petroleum ether to give crude VSCys-NCA.The crude product was redissolved in ethyl acetate, washed with cold

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

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dx.doi.org/10.1021/ma4014669 | Macromolecules 2013, 46, 6723−67306724

saturated NaHCO3 aqueous solution and cold water, and dried withanhydrous MgSO4. The evaporation of solvent gave VSCys-NCA as aviscous oil (2.89 g, 80%). 1H NMR (400 MHz, DMSO-d6): δ 6.24and 7.02 (m, 3H, −CHCH2), 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, DMSO-d6): 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 copoly-

merization of VSCys-NCA with γ-benzyl L-glutamate N-carboxyanhy-dride (BLG-NCA), ε-carbobenzyloxy-L-lysine N-carboxyanhydride(ZLL-NCA), or L-leucine N-carboxyanhydride (Leu-NCA) wascarried 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 by1H NMR, as an example. In a glovebox under a nitrogen atmosphere, toa 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 stocksolution of TMPS (6.5 mg, 0.05 mmol) in DMF. The reaction vesselwas sealed and placed in an oil bath thermostated at 40 °C. After 48 hpolymerization, the BLG-NCA was completely consumed (monitoredby FT-IR). The reaction was terminated by two drops of acetic acid.The resulting P(BLG-co-VSCys) copolymers was isolated by precip-itation in diethyl ether, centrifugation, and drying in vacuo. Gravimetricyield: 88.7−96.3%. 1H NMR (400 MHz, DMSO-d6): δ 7.32 (m, 5H,C6H5), 6.23 and 6.94 (m, 3H, −SO2CHCH2), 5.06 and 5.76 (m, 3H,−CHCH2; 2H, −CH2C6H5), 4.29−4.46 (m, 2H, −CHNH−), 3.68(m, 2H, CH2CHCH2−), 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, −SO2CHCH2), 5.73(m, 1H, −CH2CHCH2), 4.95 (m, 2H, −CH2CHCH2; 2H,−CH2C6H5), 4.21−4.44 (m, 2H, −CHNH−), 3.68 (m, 2H,−CH2CHCH2), 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 Leu-

NCA 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, −SO2CHCH2), 5.05 and 5.75 (m, 3H, −CHCH2), 4.32−4.47 (m, 2H, −CHNH−), 3.69 (m, 2H, CH2CHCH2−), 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) ofPVSCys: δ 6.30 and 6.98 (m, 3H, −SO2CHCH2), 5.05 and 5.75 (m,3H, −CHCH2), 4.51 (m, 1H, −CHNH−), 3.69 (m, 2H, CH2CHCH2−), 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. Thefollowing 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

(33%, 0.82 mL, 4.61 mol of HBr, 6 equiv with respect to the benzylgroups). After stirring at room temperature for 2 h, the reactionmixture was precipitated in diethyl ether, filtered, and dried in vacuo.Gravimetric yield: 92.5%. 1H NMR (400 MHz, DMSO-d6) ofP(Glu-co-VSCys): δ 6.28−6.96 (m, 3H, −SO2CHCH2), 5.06−5.76 (m, 3H, −CHCH2), 4.26−4.47 (m, 2H, −CHNH−), 3.68 (m,2H, CH2CHCH2−), 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) ofP(LL-co-VSCys): δ 6.31−7.02 (m, 3H, −SO2CHCH2), 5.06−5.78(m, 3H, −CHCH2), 4.27−4.48 (m, 2H, −CHNH−), 3.93 (m, 2H,CH2CHCH2−), 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 Poly-peptides with Thiol-Containing Molecules. The postpolymeriza-tion modification of VS-functionalized polypeptides was achieved throughMichael-type conjugate addition reaction of VS groups in PVSCyswith thiol groups in functional molecules including 2-mercaptoethanol,2-mercaptoethylamine hydrochloride, L-cysteine, and galactose-SH. TheSH/VS molar ratio was fixed at 2/1, and the reaction proceeded inDMF at room temperature under a nitrogen atmosphere for 1 day. Theresulting modified polypeptides were collected by precipitation in anexcess of cold diethyl ether/ethanol (1/4, v/v), filtration, and drying invacuo at room temperature. The 1H NMR spectra of modified P(Leu-co-VSCys)47% indicated quantitative modification.

Preparation of VS-Functionalized Polypeptide Films andDirect Modification with Thiol-Containing Molecules. Thinfilms were prepared by dip-coating a solution of P(Leu-co-VSCys)copolypeptides in DMF (5 mg/mL) on the microscope slides anddried in vacuo. The films were incubated in aqueous solution of a thiol-containing molecule (e.g., 2-mercaptoethanol, cysteamine, L-cysteinehydrochloride) at a concentration of 1 mg/mL for 24 h. The resultingmodified films, after exhaustively rinsed with deionized water, weredried over phosphorus pentoxide under reduced pressure. The watercontact angles of both modified and unmodified polypeptide filmswere examined on an SL-200C optical contact angle meter (SolonInformation Technology Co.) using the sessile drop method. Theexperiments were conducted in triplicate, and the results presentedwere 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 furtherstudied by fluorescence microscopy. Typically, P(Leu-co-VSCys) filmsfollowing modification with cysteamine was treated with 0.5 mg/mLFITC in phosphate buffered saline (PBS, 20 mM, pH 9.0) at 37 °C for24 h in the dark. The FITC-modified films were thoroughly rinsedwith deionized water and then observed using fluorescence microscope(Leica DM4000M). P(Leu-co-VSCys) films directly treated with FITCin aqueous condition were used as a control.

Rapidly in Situ Forming Polypeptide Hydrogels throughMichael-Type Addition Chemistry. Hydrogels were fabricated invials by thoroughly mixing solutions of P(Glu-co-VSCys) in 2-(N-morpholino)ethanesulfonic acid (MES, pH 5.3, 10 mM) and thiolatedglycol chitosan (GC-SH) in (4-(2 hydroxyethyl)-1-piperazineethane-sulfonic acid) (HEPES, pH 8.0, 1 mM). Rheological analysis wasperformed by quick mixing P(Glu-co-VSCys) solution and GC-SHsolution and then operating on the test platform of RS6000 at 37 °C inthe oscillatory mode. The evolution of storage modulus (G′) and lossmodulus (G″) was recorded as a function of time. The gelation timewas defined as the time point where G′ = G″. The storage modulus of

Scheme 2. Synthetic Pathway to VSCys-NCAa

aConditions: (i) divinyl sulfone, 50 °C, methanol, 100 h; (ii) triphosgene, α-pinene, reflux, 2 h, THF.



polypeptide hydrogels was determined in triplicate, and the resultspresented were the average data with standard deviation.

■ RESULTS AND DISCUSSIONSynthesis of VS-Substituted L-Cysteine N-Carboxyan-

hydride (VSCys-NCA) Monomer. VSCys-NCA was synthe-sized by treating L-cysteine hydrochloride with excess of divinylsulfone in methanol at 50 °C followed by cyclization using

triphosgene in the presence of α-pinene (Scheme 2). 1H NMRof VSCys showed signals of L-cysteine moieties at δ 4.23 and3.18, signals attributable to vinyl protons at δ 6.92 and 6.38, andtwo 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 closeto 2:1:2, confirming equivalent coupling of L-cysteine hydro-chloride and divinyl sulfone. VSCys-NCA was isolated as aviscous oil in a high overall yield of 76%. 1H NMR displayedclearly signals at δ 6.24 and 7.02 owing to vinyl protons, δ 4.77and 9.15 to methine and amide protons, and δ 2.81, 3.00, and3.42 to the three methylene protons neighboring to the thioetheror sulfone group (Figure 1A). The signals at δ 7.02 (methineproton of VS group) and 4.77 (methine proton of NCA ring)had an intensity ratio close to 1:1, indicating that VSCys-NCAwas obtained with quantitative functionality. 13C NMR detectedbesides four alkane carbons at δ 26.32−59.44 also two vinylcarbons at δ 131.96 and 137.89 as well as two carbonyl carbonsat δ 153.34 and 171.77 (Figure 1B). The elemental analysisrevealed a composition close to that calculated for VSCys-NCAand furthermore ESI-MS showed an exact mass of 265.01. Theseresults point out that VSCys-NCA monomer is readily obtainedwith a defined structure and high yield.

Synthesis of VS-Functionalized Polypeptides. VSCys-NCA was readily copolymerized with different α-amino acidNCA monomers such as γ-benzyl L-glutamate NCA (BLG-NCA), N-benzyloxycarbonyl-L-lysine NCA (ZLL-NCA), andL-leucine NCA (Leu-NCA) using TMPS as an initiator in DMFat 40 °C (Scheme 3). The results of copolymerization aresummarized in Table 1. 1H NMR of P(BLG-co-VSCys) showedcharacteristic vinyl sulfone protons at δ 6.23 and 6.94 (Figure 2A),indicating that vinyl sulfone was intact during polymerizationand following work-up procedures. VSCys fractions (FVSCys) ofP(BLG-co-VSCys) determined by comparing the intensitiesof signals at δ 6.23 owing to VS protons of VSCys and 7.32assignable to the benzene ring of BLG increased from 8.5 to28 mol % at increasing VSCys-NCA monomer feed ratios

Scheme 3. Synthesis of VS-Functionalized Polypeptides

Figure 1. 1H NMR (400 MHz) (A) and 13C NMR (100 MHz) (B) ofVSCys-NCA in DMSO-d6.



( f VSCys) from 10 to 30 mol % (Table 1, entries 1−3). Moreover,the number-average molecular weight (Mn) of P(BLG-co-VSCys) estimated from the intensity ratios of signals at δ 6.23(VSCys) and 7.32 (BLG) to those at δ 5.76 (methine proton ofTMPS moiety) were close to the theoretical values and increasedin proportion to monomer-to-initiator (M/I) ratios (Table 1,entries 1−4). Notably, GPC measurements showed that allP(BLG-co-VSCys) copolymers had moderate polydispersities(PDI = 1.25−1.50) and Mn values close to those determined by1H NMR end-group analyses (Table 1, entries 1−4). It is clearthat copolymerization of VSCy-NCA and BLG-NCA proceeds ina controlled manner to give P(BLG-co-VSCys) copolymers withtailored molecular weights and functionalities.In a similar way, copolymerization of VSCy-NCA and

ZLL-NCA yielded well-defined P(ZLL-co-VSCys) copolymer.1H NMR analysis showed that P(ZLL-co-VSCys) was obtainedwith a VSCys content of 19 mol % and an Mn of 9.94 kg/mol atan 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 17.7kg/mol and a low PDI of 1.15 (Table 1, entry 5). The deviationof Mn value determined by GPC from that calculated by1H NMR is most likely due to use of polystyrene standards formolecular weight calibration in our GPC measurements. Incomparison, copolymerization of VSCy-NCA and Leu-NCAresulted in only partial monomer conversion under otherwisethe same conditions, probably due to low polymerizability ofLeu-NCA. 1H NMR indicated that P(Leu-co-VSCys) preparedat an f VSCys of 30 mol % had an elevated VSCys content of47 mol % (Figure S2). In accordance, both 1H NMR end-groupanalysis and GPC showed that P(Leu-co-VSCys) had a com-parably low Mn though PDI remained low (1.23) (Table 1,entry 6). It should further be noted that VSCys-NCA could alsoundergo homopolymerization to yield PVSCys with an Mn of16.0 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) furnishedVS-functionalized poly(L-glutamic acid) (P(Glu-VSCys)) andVS-functionalized poly(L-lysine) (P(LL-VSCys)), respectively(Scheme 3). The deprotection of P(BLG-co-VSCys) was carriedout in 33% HBr/AcOH (6 equiv of HBr with respect to thebenzyl groups) at room temperature for 2 h, which has shownto successfully remove the protecting benzyl groups of PBLGwithout obvious main chain cleavage.49−51 Both P(Glu-VSCys)and P(LL-VSCys) were freely water-soluble. 1H NMR inDMSO-d6 showed that signals at around δ 7.30 and 5.00attributable to the benzyl protons completely disappeared whileno change was observed for signals assignable to VS protons(δ 6.93 and 6.23) (Figure 3), indicating quantitative removal of

benzyl groups and VS group unspoiled during acidic treatment.It is evident, therefore, that VSCys-NCA can copolymerize withdifferent NCA monomers, which provides a facile access to arange of VS-functionalized polypeptides with distinct hydro-philicity, charge, and functionalities.

Postpolymerization Modification of P(Leu-co-VSCys)with Thiol-Containing Molecules. Interestingly, VS-func-tionalized polypeptides were amenable to versatile and sel-ective postpolymerization modification with thiol-containingmolecules such as 2-mercaptoethanol, 2-mercaptoethylamine

Table 1. Synthesis of VS-Functionalized Polypeptidesa

Mn (kg/mol)

entry copolymer M/Ib f VSc (%) FVS

d (%) NMRe GPCf PDI GPCf yield (%)

1 P(BLG-co-VSCys) 40 10 8.5 10.5 10.7 1.25 93.22 P(BLG-co-VSCys) 40 20 17 8.50 7.05 1.50 88.73 P(BLG-co-VSCys) 40 30 28 8.00 7.60 1.45 91.74 P(BLG-co-VSCys) 60 20 21 12.5 10.9 1.28 96.35 P(ZLL-co-VSCys) 40 20 19 9.94 17.7 1.15 94.86 P(Leu-co-VSCys) 57 30 47 5.30 4.34 1.23 57.6

aVS-functionalized polypeptides were prepared through ring-opening copolymerization of BLG-NCA, ZLL-NCA, or Leu-NCA with VSCys-NCA inDMF 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.dMolar 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).

Figure 2. 1H NMR spectra (400 MHz) of copolypeptides in DMSO-d6: (A) P(BLG-co-VSCys)17% (Table 1, entry 2) and (B) P(ZLL-co-VSCys)19% (Table 1, entry 5).



hydrochloride, L-cysteine, and galactose-SH at a ligand-SH/VSmolar ratio of 2/1 in the absence of any catalyst under mildconditions (Scheme 4). Typical 1H NMR spectra of P(Leu-co-VSCys)47% copolymer following treatment with 2-mercaptoe-thanol and galactose-SH showed complete vanishing of signalsat δ 6.93 and 6.23 owing to VS protons and emergence ofnew signals characteristic of 2-mercaptoethanol (δ 3.56) orgalactose-SH (δ 4.27, 3.92, and 3.39) moieties (Figure S3),signifying quantitative functionalization. These results corrob-orate that Michael-type conjugate addition between VS andthiol groups is highly selective and tolerant to various functionalgroups including amines and carboxylic acids. It is of particularinterest to note that through VS-functionalized polypeptidesglycopolypeptides can be readily obtained with controlledsaccharide contents without tedious protection/deprotectionprocess, which might find tremendous applications in fields ofcontrolled drug release and regenerative medicine.52−55

Surface Modification of P(Leu-co-VSCys) Films withThiol-Containing Molecules. More strikingly, films prepared

from VS-functionalized polypeptides could be directly modifiedwith thiol-containing biomolecules under aqueous conditions.Water contact angle analysis showed that VS-functionalizedP(Leu-co-VSCys) films following treatment with 2-mercaptoe-thanol and cysteine became much more hydrophilic, as evidencedby a significant reduction of water contact angle (8°−20°) forP(Leu-co-VSCys)47% (Figure 4A). To further confirm occur-rence of surface modification, P(Leu-co-VSCys)47% filmfollowing treatment with cysteamine was reacted with fluoresceinisothiocyanate (FITC) in phosphate buffered saline (PBS,20 mM, pH 9.0). Fluorescence microscopy showed thatstrong fluorescence was observed throughout the whole film(Figure 4B), indicating homogeneous surface modification withcysteamine and furthermore amine groups at the surfacemaintaining high reactivity. In contrast, no fluorescence wasdetected for P(Leu-co-VSCys) film directly treated with FITCunder otherwise the same conditions (Figure 4B). This representsa first report on direct surface modification of polypeptide coatingsunder aqueous conditions, which might have great applica-tions in medical implants as well as bioactive tissue engineeringscaffolds.

Rapidly in Situ Forming Polypeptide Hydrogelsthrough Michael-Type Addition Chemistry. Notably, weare also able to prepare in situ forming robust polypeptidehydrogels based on water-soluble VS-functionalized polypeptides using polythiols as a cross-linking agent. For example,hydrogels formed rapidly upon mixing aqueous solutions ofP(Glu-co-VSCys) and thiolated glycol chitosan (GC-SH) at37 °C without any catalyst (Figure 5A). Rheology analysisshowed 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-co-VSCys)17% and (B) P(LL-co-VSCys)19%.

Scheme 4. Postpolymerization Modification of VS-Functionalized Polypeptides

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



pH 5.3) at a VS/SH molar ratio of 1/1 led to almost instanta-neous gelation (gelation time <10 s) and storage modulus (G′)reached a plateau within 15 min (Figure 5B), indicating a fastcross-linking process. G′ increased from 2.34 to 6.12 kPa withincreasing P(Glu-co-VSCys) concentrations from 1.2 to 2.0 wt %(Figure 5C). A control experiment performed using unmodifiedGC and P(Glu-co-VSCys) showed that no gel was formed in 2 hunder otherwise the same conditions, supporting that hydrogelsof GC-SH and P(Glu-co-VSCys) were formed by Michaeladdition of VS with the thiol groups instead of amine groups inGC-SH. The high selectivity of VS toward thiol group ascompared to amino group has previously been reported.56 Tothe best of our knowledge, this is a first report on in situ formingpolypeptide hydrogels via Michael-type addition chemistry.These in situ forming polypeptide hydrogels are interesting forcell encapsulation and controlled release of drugs andproteins.57−60

■ CONCLUSIONS

We have demonstrated for the first time that vinyl sulfone-functionalized polypeptides offer unprecedented access tofunctional polypeptide-based biomaterials that can be tailoredto meet the specific requirements of distinct biomedical applica-tions. These novel vinyl sulfone-functionalized polypeptideshave several unique advantages: (i) They are readily preparedwith controlled structures, molecular weights, and compositions.Importantly, no protection and deprotection steps are required.(ii) They are amenable to direct, efficient, and selective post-polymerization modification with thiol-containing biomoleculesvia Michael-type conjugate addition chemistry under mildconditions. (iii) Their films can be directly modified with thiol-containing biomolecules under aqueous conditions, providinga versatile platform to obtain functional polypeptide surfaces.(iv) In situ forming robust polypeptide hydrogels can be facilely

designed and prepared based on water-soluble vinyl sulfone-functionalized polypeptides.

■ ASSOCIATED CONTENT*S Supporting Information1H NMR spectra of VSCys, PVSCys, P(Leu-co-VSCys), andP(Leu-co-VSCys) modified with 2-mercaptoethanol or gal-actose-SH. This material is available free of charge via theInternet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*Tel +86-512-65880098; e-mail [email protected] (C.D.),[email protected] (Z.Z.).NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis work was supported by the National Natural ScienceFoundation of China (NSFC 51003070, 51103093, 51173126,51273137 and 51273139), the National Science Fund forDistinguished Young Scholars (51225302), and a ProjectFunded by the Priority Academic Program Development ofJiangsu Higher Education Institutions.

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



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mailto:[email protected]

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a simple and versatile synthetic strategy to functional polypeptides via vinyl sulfone-substituted l...

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