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Nov 30, 2015 - and the use of organic solvents or toxic phosgene derivatives.2,3. These limitations lead to ... 1), producing an enzyme−substrate (E...
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The Benzyl Ester Group of Amino Acid Monomers Enhances Substrate Affinity and Broadens the Substrate Specificity of the Enzyme Catalyst in Chemoenzymatic Copolymerization Jose Manuel Ageitos, Kenjiro Yazawa, Ayaka Tateishi, Kousuke Tsuchiya, and Keiji Numata* Enzyme Research Team, Biomass Engineering Research Division, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan S Supporting Information *

ABSTRACT: The chemoenzymatic polymerization of amino acid monomers by proteases involves a two-step reaction: the formation of a covalent acyl-intermediate complex between the protease and the carboxyl ester group of the monomer and the subsequent deacylation of the complex by aminolysis to form a peptide bond. Although the initiation with the ester group of the monomer is an important step, the influence of the ester group on the polymerization has not been studied in detail. Herein, we studied the effect of the ester groups (methyl, ethyl, benzyl, and tert-butyl esters) of alanine and glycine on the synthesis of peptides using papain as the catalyst. Alanine and glycine were selected as monomers because of their substantially different affinities toward papain. The efficiency of the polymerization of alanine and glycine benzyl esters was much greater than that of the other esters. The benzyl ester group therefore allowed papain to equally polymerize alanine and glycine, even though the affinity of alanine toward papain is substantially higher. The characterization of the copolymers of alanine and glycine in terms of the secondary structure and thermal properties revealed that the thermal stability of the peptides depends on the amino acid composition and resultant secondary structure. The current results indicate that the nature of the ester group drastically affects the polymerization efficiency and broadens the substrate specificity of the protease.

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reaction.8 The KCS has been optimized through modulation of the reaction conditions. For instance, changes in the enzyme− substrate ratio and the use of an alkaline pH have been demonstrated to enhance the degree of polymerization (DP) of the synthesized polypeptides.9−13 Although multiple studies have been carried out,14−16 KCS still has several limitations; specifically, the DP, amino acid sequence and composition of the synthesized peptides are not adequately controlled.14,15 This lack of control stems from the differing affinity of the protease for different amino acid monomers, which results in the preferential polymerization of amino acids for which the protease has higher affinity. Thus, the feed ratio of the amino acid monomers cannot precisely regulate the composition of the synthesized polypeptide. To regulate the composition of the copolypeptides, we aimed to control the affinity of papain toward amino acid monomers by using different ester groups on the monomers. Papain, a cysteine protease, has been successfully used for the KCS of peptides and is considered a model protease in the chemoenzymatic polymerization of amino acids.12,15,17 The papain

INTRODUCTION Polypeptides are attractive biomacromolecules because of the various biological and chemical functions originating from their hierarchical structures.1 Classical peptide syntheses, such as solid- and liquid-phase syntheses or N-carboxyanhydride ringopening polymerization, require multiple steps of deprotection and the use of organic solvents or toxic phosgene derivatives.2,3 These limitations lead to the high cost of polypeptides and reduce the possibility of bulk-scale polypeptide synthesis.4 By contrast, the chemoenzymatic polymerization of amino acids via aminolysis by proteases is an atom-economical, efficient, and green method.5 The kinetically controlled synthesis (KCS) of polypeptides via proteases and an excess concentration of activated amino acid monomers is the most studied chemoenzymatic polymerization process. In the initiation of KCS, the catalytic residue of the protease (Figure S1) forms a covalent bond with the carboxyl ester group of the monomer (Scheme 1), producing an enzyme−substrate (ES) complex. The amine group of the free monomers competes with water as a nucleophile for the deacylation of the ES, allowing peptide bond formation by aminolysis.6 The efficiency of the synthesis depends on the specific affinity of the enzymatic pocket toward the monomers.7 Thus, the active site of a protease plays the 2fold function of binding the substrate and catalyzing the © XXXX American Chemical Society

Received: October 26, 2015 Revised: November 20, 2015

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DOI: 10.1021/acs.biomac.5b01430 Biomacromolecules XXXX, XXX, XXX−XXX

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Biomacromolecules Scheme 1. Reaction Scheme of the Chemoenzymatic Peptide Synthesis Mediated by Papain

conditions described in previous reports.25 Briefly, the reactions were performed with stirring (800 rpm) in an EYELA ChemiStation (Tokyo, Japan) at 40 °C in 25 mL glass reaction tubes at a final volume of 5 mL in 1 M phosphate buffer pH 8. The concentrations of monomer and papain were 0.1−1 M and 0.5−10 U/mL, respectively.12,26 The initial pH of the reaction mixture was adjusted with alkali prior to the papain addition. Triplicate results were obtained and averaged. Negative controls without papain or monomer were performed. After the reaction, the products were centrifuged at 8000g for 30 min. The supernatant containing the soluble papain and unreacted monomers was discarded. The product was obtained as a precipitate that was subsequently washed three times with 10 mL of Milli-Q water. The purified products were lyophilized to give a white powdered product. The reaction yields were calculated on the basis of the molar ratio of the resultant products and fed monomers. All the yields in this study were calculated by this method. For the time-course study of the polymerization of Ala, the reaction of 0.6 M of monomer (Ala-OEt, Ala-OMe, Ala-OBzl) with papain (1 U/mL) in 1 M phosphate buffer pH 8 was performed and the reactants (500 μL) were sampled at different time points (0 to 180 min). The reactions were stopped by the addition of 500 μL of 2 M HCl and immediately centrifuged at 16 000g for 5 min at 25 °C. The supernatants were discarded, and the precipitates were washed with Milli-Q water three times. The purified samples were resuspended in 500 μL of Milli-Q water and lyophilized. The reported results are an average of triplicate reactions. For the synthesis of copolypeptides of Ala and Gly [poly(Ala-coGly)], different ratios of Ala-OEt and Gly-OEt (100:0, 80:20, 70:30, 50:50, 30:70, 20:80, and 0:100) with a final concentration of 0.6 M were used. The same ratios of Ala-OBzl and Gly-OBzl were used. For the study of the effects of reaction pH, Ala-OBzl and BrittonRobinson universal buffer27 at different pH (5 to 11) were used as a monomer and reaction buffer, respectively. The pH of the buffers was adjusted using mixtures of sodium hydroxide (NaOH), acetic acid (CH3CO2H), phosphoric acid (H3PO4), boric acid (H3BO3), and potassium chloride (KCl) in different ratios, as previously described.28 Nuclear Magnetic Resonance. 1H NMR, gCOSY (two-dimensional J-correlation spectroscopy with gradient coherence selection) and gHMBCAD (two-dimensional heteronuclear multiple-bond Jcorrelation spectroscopy with adiabatic 180° X-nuclei pulses and gradient coherence selection) spectra were recorded on a Varian NMR System 500 (500 MHz) spectrometer (Varian Medical Systems, Palo Alto, CA, U.S.A.) at 25 °C controlled with the VnmrJ software. The lyophilized samples (10 mg/mL) were suspended in deuterated dimethyl sulfoxide (DMSO-d6) containing 10% deuterated trifluoroacetic acid (TFA-d) or pure TFA-d. One hundred twenty-eight scans were recorded during each 1H NMR experiment. 13C NMR spectra were acquired at 126 MHz with the samples dissolved in pure TFA-d. Tetramethylsilane (TMS) was used as an internal reference at 0.00 ppm. Data were processed and analyzed by ACD/NMR Processor Academic Edition, version 12.01 (Advanced Chemistry Development, Inc., Toronto, ON, Canada, 2010). The average DP (DPavg) of the peptides was calculated on the basis of the relative peak area of the amine terminal α carbon [CHαi for Ala (1 H) and CH2αi for Gly (2 H)] in relation to the relative area of the peaks of the chain [CHαn for Ala (n H) and CH2αn for Gly (n 2H)] and the carboxyl terminal alpha

immobilized on silica particles demonstrates recyclability in chemoenzymatic polymerization.17 Four types of ester group (methyl, ethyl, benzyl and tert-butyl esters), as well as L-alanine (Ala) and glycine (Gly), were chosen as activated monomers in the present study. Ala and Gly are the simplest model amino acids, and the affinity of Ala toward papain is markedly higher than that of Gly,18 thus allowing a comparison of the effect of the ester group on the synthesis using favorable and unfavorable substrates for papain.19 Herein, we verify that the KCS efficiency is influenced by the affinity of papain toward the amino acids as well as by the ester group of the monomers. Particularly, a benzyl ester group (OBzl) enhances the KCS efficiency, even when the enzyme concentration is relatively lower or when an amino acid with a low affinity toward papain is used. As a consequence, an ideal copolymerization can be performed in which the feed ratio of monomers and the composition of the polypeptides are identical. This work represents the first report in which the KCS using monomers with OBzl enables the chemoenzymatic polymerization and copolymerization of Gly, which exhibits a low affinity toward papain. Furthermore, copolypeptides of Ala and Gly [poly(Ala-co-Gly)] are attractive because they are the main components of structural proteins such as collagen,20 elastin21 and fibroin.22−24 We also show the first result related to the secondary structure and thermal properties of poly(Alaco-Gly).

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MATERIALS AND METHODS

Materials. L-Alanine ethyl ester hydrochloride (Ala-OEt), L-alanine methyl ester hydrochloride (Ala-OMe), L-alanine benzyl ester hydrochloride (Ala-OBzl HCl), L-alanine benzyl ester p-toluenesulfonate salt (Ala-OBzl), L-alanine tert-butyl ester hydrochloride (AlaOtBu), glycine methyl ester hydrochloride (Gly-OMe), glycine ethyl ester hydrochloride (Gly-OEt), poly(Gly) [500−5000 g/mol], trifluoroacetic acid (TFA), and deuterated trifluoroacetic acid (TFAd) were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Glycine benzyl ester hydrochloride (Gly-OBzl) and glycine tert-butyl ester hydrochloride (Gly-OtBu) were purchased from Wako Pure Chemical Industries (Osaka, Japan). Poly(Ala) [1000−5000 g/mol] was purchased from MP Biomedicals, LLC (Santa Ana, CA, U.S.A.). Deuterated dimethyl sulfoxide (DMSO-d6) was purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). Standard chemicals were purchased from Wako Chemical Co. (Kanagawa, Japan). All the chemicals were used without any purification. Papain (EC No. 3.4.22.2), derived from the unripe fruit of the papaya tree (Carica papaya), was purchased from Wako Pure Chemical Industries. The papain was purified and activated according to a previous report.12 The activity was approximately 0.097 ± 0.011 U/mg, where one unit is defined to hydrolyze and release 1 mmol of fluorescein isothiocyanate (FITC) from FITC-labeled casein per minute at pH 7.5, 25 °C (protease activity assay kit, Abcam, U.K.). Chemoenzymatic Polymerization of Ala and Gly. The reaction conditions for the peptide synthesis were based on the B

DOI: 10.1021/acs.biomac.5b01430 Biomacromolecules XXXX, XXX, XXX−XXX

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Figure 1. 1H NMR (a,c,e) and MALDI-TOF spectra (b,d,f) of poly(Ala) synthesized from Ala-OMe (a and b), Ala-OEt (c and d), and Ala-OBzl (e and f) using papain as a catalyst. carbons [CHαC for Ala (1 H) and CH2αC for Gly (2 H)]. The percent hydrolysis was calculated on the basis of the relative peak area of the N-terminal alpha protons [CHαi for Ala (1 H) and CH2αi for Gly (2 H)] in relation to the theoretical relative area of the ester signal [3 H for CH3OMe, 2 H for CH2OEti and 2H for CH2OBzli]. Identification of Ala αCH (αi, αn and αC) signals in copolymers was conducted by the analysis of the gCOSY spectra by the correlation between the αCH and CH3 signals (βi, βn and βC). The relative composition of the copolymers was calculated on the basis of the relation between the integration value of the peak area of the CHα of the Ala residues and the CH2α peaks of Gly. Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-TOF). MALDI-TOF mass spectral data were acquired using an Autoflex speed L MALDI-TOF-MS system (Bruker, Bremen, Germany).12 α-Cyano-4-hydroxycinnamic acid (Sigma) was dissolved in a mixture containing 0.1% TFA, 50% acetonitrile, and 50% water (TA solution) and used as the matrix. Lyophilized peptide was dissolved in TA solution and mixed with the matrix at a 1:1 ratio. The prepared sample (2 μL) was spotted onto the target plate and allowed to air-dry at room temperature. The acquired data were processed by Flex Analysis 3.4, Polytools 1.18 (Bruker Daltonik GmbH, Bremen, Germany), and Polymerix software 2.0.0 (Sierra Analytics Inc., Modesto, CA) to analyze the molecular weight, degree of

oligomerization, and copolymer composition.25 The maximum DP (DPmax) of the peptides was determined on the basis of the MALDITOF mass spectral data. Fourier Transform Infrared Spectroscopy (FTIR). FTIR absorption spectra from 700 to 4000 cm−1 were collected using a Shimadzu IR-Prestige-21 FTIR spectrometer (Shimadzu, Kyoto, Japan) equipped with a multiple-reflection, horizontal MIRacle ATR attachment (using a Ge crystal, from Pike Tech, Madison, WI) at 2 cm−1 resolution, using 252 scans. Thermal Analysis. Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) measurements were carried out using a Mettler Toledo TGA/DSC 2 Star system (Greifensee, Switzerland). Samples (3 mg) were encapsulated in aluminum pans and heated under a nitrogen atmosphere from 30 to 500 °C at a heating rate of 20 °C min−1.29 After the water removal phase (100 °C), the degradation temperature (Td) was determined as the peak of the derivative thermogravimetry (DTG) plots.

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RESULTS AND DISCUSSION Effects of the Ester Group of the Ala Monomer on Its Polymerization. Ala-OMe, Ala-OEt, and Ala-OBzl were successfully polymerized by papain, giving white precipitates. The synthesis of poly(Ala) was confirmed by 1H NMR, C

DOI: 10.1021/acs.biomac.5b01430 Biomacromolecules XXXX, XXX, XXX−XXX

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Biomacromolecules gCOSY, gHMBCAD, and MALDI-TOF (Figure 1 and Figures S2 and S3). The negative control reactions without enzyme or monomers did not show any precipitation. By contrast, the reaction with Ala-OtBu did not yield a precipitate, indicating that papain does not recognize the tert-butyl ester groups of Ala-OtBu. This lack of recognition is attributed to the tert-butyl group being too bulky to be recognized by papain in the current reactions. The result relating to the tert-butyl ester monomer agrees with the results of a study of the papainmediated synthesis of dipeptides using tert-butyl ester as a carboxyl-protecting group.30 Poly(Ala) synthesized with different ester groups presented homologous NMR spectra, which in turn were similar to the spectrum of the commercial poly(Ala) reference sample (Figure S2). In the gCOSY spectra in DMSOd6:TFA-d (9:1) (Figure S3), an interconnection between the Nterminal CHαi (3.90 ppm) and NH2i (8.12 ppm) and CH3i (1.38 ppm) was detected. The second N-terminal CHαii (4.40 ppm) was connected to NHii (8.56 ppm) and CH3ii (1.39 ppm). The repetition unit CHαn (4.36−4.39 ppm) was connected to NHn (8.0−8.20 ppm) and CH3n (1.33−1.22 ppm). In the spectrum of poly(Ala)OBzl, signals of the CH2 and benzyl ring appeared at 5.13 ppm and 7.40−7.30, respectively. The 13C and 1H NMR assignments of poly(Ala)OBzl were confirmed by gHMBCAD (Figure S4). The counterion of the Ala-OBzl used in this study differed from those of the other employed monomers (p-toluenesulfonate and hydrochloride). To clarify the effect of ptoluenesulfonate, L-Ala benzyl esters with hydrochloride and p-toluenesulfonate salt were compared in terms of yield and DP. As a result, both the monomers yielded similar DPs and yields (0.3 M Ala-OBzl HCl: yield =62.6 ± 1.1%, DPavg = 10.7 ± 1.6: DPmax: 23.0 ± 0.7; 0.3 M Ala-OBl: yield =62.3 ± 3.0%, DPavg= 11.0 ± 1.3: DPmax: 23.7 ± 0.3). Both DPavg and DPmax were evaluated to imply the distribution of the molecular weight of the polypeptides, because of the poor solubility of the synthesized polypeptides in this study. Thus, the p-toluenesulfonate salt does not differentiate the results of chemoenzymatic polymerization by papain. The p-toluenesulfonate salt was detected by 1H NMR (Figure S2). The absence of the signals therefore confirmed that the purification process completely removed the p-toluenesulfonate salt from the products. To compare the kinetics of the KCS with different ester groups of the monomers, a time-course study was performed using Ala-OMe, Ala-OEt and Ala-OBzl with 1 U/mL of papain (Figure 2). Because only insoluble products were purified in this study, low-molecular-weight products that did not precipitate were not collected.25 The yield of poly(Ala) in the reaction involving Ala-OMe increased as a function of time up to 30 min and then decreased at 120 min. The yield with AlaOEt increased to approximately 60% with increasing reaction time. The polymerization of Ala-OBzl showed an exponential cumulative curve as a function of time and a plateau after 120 min. However, in contrast to previous reports in which other ester groups were used,11,26,31 the yield from Ala-OBzl did not decrease within 180 min. This result indicates that Ala-OBzl significantly enhances the polymerization efficiency and inhibits exotype hydrolysis compared with other ester groups.32 The reactivity of the ester groups of the Ala monomer was investigated using various monomer/enzyme ratios; specifically, the concentration effects of the monomer using a fixed concentration of enzyme and of the enzyme using a fixed concentration of monomer were studied. The highest yield of KCS using Ala-OMe was achieved at a monomeric concen-

Figure 2. Time-course study of the synthesis of poly(Ala) using AlaOMe (open triangle), Ala-OEt (gray triangle), and Ala-OBzl (closed triangle). Reactions were carried out at 40 °C using 0.6 M of the monomer in 1 M phosphate buffer at pH 8 containing 1 U/mL of papain. Error bars represent the standard deviations of the replicates (n = 3).

tration of 0.3 M (Figure 3a). The DPavg was similar among the concentrations (avg. 7.5 ± 0.3), whereas the DPmax slightly increased with increasing concentration. In the case of the KCS

Figure 3. Effect of the polymerization conditions on the yield (bars) and degree of polymerization (average: circles, maximum: squares) in the synthesis of poly( L -Ala) using different monomers. (a) Concentration effects of Ala-OMe, Ala-OEt, and Ala-OBzl. The reactions were carried out in 1 M phosphate buffer at pH 8 containing 1 U/mL of papain. (b) Concentration effects of papain with Ala-OMe, Ala-OEt, and Ala-OBzl at concentrations of 0.7 M. (c) Effect of initial reaction pH on the polymerization of Ala-OBzl. Reactions were carried out using 0.3 M of Ala-OBzl and 1 U/mL of papain. Error bars represent the standard deviations of the replicates (n = 3). D

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peptides.35 Papain has also been reported to display a 2-fold higher turnover rate for N-protected Ala esters in comparison to N-protected Gly esters.18 In the aminolysis of glycine, the deacylation of the papain-acyl complex by glycine, the formation of a peptide bond,19,34 and the polymerization of Ala-Gly-OEt by papain have been reported.13 Thus, the KCS of poly(Gly) has not been previously described, mainly because of its low affinity toward proteases.18,19,34 To polymerize Gly monomers, we incubated a fixed concentration (0.6 M) of Gly-OMe, Gly-OEt, Gly-OBzl, and Gly-OtBu with several concentrations of papain (0.5 to 10 U/ mL) (Figure 4). The synthesis of poly(Gly) with the different

with Ala-OEt, the maximum yield was achieved using an AlaOEt concentration of 0.5 M. The DPavg of poly(Ala)OEt was maintained at the concentration assayed (avg. 7.6 ± 0.8). The DPmax detected by MALDI-TOF was 17 ± 1 in the 0.3 and 0.5 M reactions. These results are comparable to those reported by Baker and Numata for the KCS of poly(Ala)OEt under alkaline conditions.12 The KCS using Ala-OBzl as a monomer exhibited the highest yields and DPs of poly(Ala) at different monomer concentrations compared with the other types of esters. The DPavg of poly(Ala)OBzl was 11.1 ± 1.3, whereas the DPmax was highest at each concentration compared with the DPmax of the other monomers studied (Figure 3a). In particular, at Ala-OBzl concentrations of 0.7 and 1 M, more than 30 repetition units were polymerized (Figures 1 and 3a). These results for AlaOBzl indicate that the yields from Ala-OBzl were constant at monomer concentrations ranging from 0.3 to 1.0 M and that the DP of poly(Ala)OBzl also increased with increasing concentration of the monomer. Similar to the concentrations of the monomers, the concentration of papain primarily affected the reaction yield (Figure 3b). The use of Ala-OBzl as a monomer resulted in the efficient synthesis of poly(Ala) at lower concentrations of papain compared with the concentrations used in the polymerizations of Ala-OMe and Ala-OEt. In the cases of Ala-OEt and Ala-OMe, 2- to 5-fold concentrations of papain were required to achieve a yield comparable to that achieved in the case of Ala-OBzl. When the concentration of papain was greater than 2.5 U/mL, the reaction yield from all the monomers decreased because of hydrolysis reactions. On the basis of the 1H NMR results, the hydrolysis products for 1 U/ mL of papain were 40 ± 6% for Ala-OMe, 20 ± 2% for Ala-OEt and 11 ± 4% for Ala-OBzl, respectively. The DPavg of poly(Ala) was not drastically affected by the concentration of papain. The DPmax of poly(Ala)OBzl increased with increasing concentration of Ala-OBzl, which was the most reactive Ala monomer in this study. Furthermore, papain is one of the most studied proteases for chemoenzymatic polymerizations, because of higher polymerization activity than the other proteases such as proteinase K, bromelain, and alpha-chymotrypsin.17,26 The enhancement in reactivity with amino acid benzyl ester can broaden choices of enzyme catalyst, which demonstrates a lower activity but different substrate specificity, for chemoenzymatic polymerization of amino acids. The pH of the reaction has been reported to be an important factor in the KCS of peptides,9,12,26 and it is known to affect the yield and length of the poly(Ala)OEt syntheses.12 However, chemoenzymatic polymerization using a high concentration of the acidic monomer leads to more acidic reaction conditions, which induces difficulty in controlling the pH. In the polymerization of Ala-OBzl mediated by papain (1 U/mL) at different pH levels (Figure 3c), the yield increased with increasing initial pH of the reaction and reached a maximum value at pH 9. No polymerization was detected at pH 5, whereas the yield at pH 6 was approximately 20%. Although the DPmax and DPavg increased with increasing pH of the reaction, the yield achieved with Ala-OBzl was not drastically affected by alkaline pH levels because the stability of the benzyl esters against alkaline saponification is relatively greater than that of ethyl esters.33 Effects of the Ester Group of the Gly Monomers on the Synthesis. According to the literature, papain can neither recognize unprotected Gly19 nor hydrolyze the dipeptide glycylglycine34 but can hydrolyze the Gly-Gly bond of

Figure 4. Effect of papain concentration on the yield (bars) and degree of polymerization (average: circles, maximum: squares) in poly(Gly) synthesis using Gly-OMe, Gly-OEt, and Gly-OBzl. The reactions were carried out at 40 °C using 0.6 M of monomer in 1 M phosphate buffer, pH 8. Error bars represent the standard deviations of the replicates.

monomers was confirmed by MALDI-TOF, 1H NMR, and gHMBCAD (Figure 5 and Figure S5) using purchased poly(Gly) as a reference sample. Similar to the Ala monomers, no Gly-OtBu was polymerized. Gly-OMe and Gly-OEt were polymerized at concentrations greater than 2.5 U/mL, whereas Gly-OBzl was polymerized to poly(Gly) at all investigated concentrations. On the basis of the yield and the required concentration of papain, the poly(Gly) synthesis efficiency with OBzl was 10 and 5 times higher than those with OMe and OEt, respectively (Figure 4). Compared with Ala, the polymerization of Gly required a higher concentration of papain to achieve a similar yield because of the lower affinity of papain toward Gly than toward Ala.18 The NMR signals of poly(Gly) synthesized with different esters differed only in the signals of the terminal groups (Figure 5). The N-terminal CH2αi (3.66 ppm) connected with NH2i (8.10 ppm), the second N-terminal CH2αii (3.90 ppm) connected with NHii (8.67 ppm), and the chain repetition unit CH2αn (3.75−3.85 ppm) connected with NHn (8.13−8.22 ppm). However, in the case of poly(Gly), separate signals were observed for the terminal amino acid CH2αC and NHc: poly(Gly)OMe 3.88 and 8.31, poly(Gly)OEt 3.86 and 8.26, and poly(Gly)OBzl 3.94 and 8.33. The 13C and 1H NMR assignments of poly(Gly)OBzl were confirmed on the basis of the gHMBCAD analysis (Figure S5). The relatively high reactivity of Gly-OBzl and Ala-OBzl might enhance the affinity of the monomers in the enzymatic groove, suggesting that there is both an interaction with the active site and the stabilization of the oxyanion hole.32 The interaction by the OBzl group is explained by the interaction with the residues that surround the active site (Figure S1), especially those that have been reported to interact with aromatic moieties, such as Gly 65,32 Pro 68,36 Val 133,36,37 and E

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Figure 5. 1H NMR (a,c,e) and MALDI-TOF (b,d,f) spectra of poly(Gly) produced with Gly-OMe (a and b), Gly-OEt (c and d), and Gly-OBzl (e and f) using papain as a catalyst.

Ala 160.36 The reaction of amino acids with an OBzl group was reported to be 4 times faster with less hydrolysis in comparison to similar reactions with an OMe group.32 The polymerization of Gly-OBzl at a relatively low papain concentration is also supported by the claim that the OBzl group enhances the affinity of the substrate to papain by the ester group, improving the initiation of the KCS (Scheme 1). Effect of the Ester Group of the Monomers on the Copolymerization. The affinity of papain toward Gly is lower compared with that toward Ala,18 which hinders the simultaneous copolymerization of Gly via chemoenzymatic aminolysis.14,15,25 For the copolymerization, Ala-OEt/Gly-OEt and Ala-OBzl/Gly-OBzl at different monomer ratios were tested using 1 U/mL of papain. The monomers with the OMe group were not used because of their low reactivity (Figure 4). In the KCS using the ethyl ester monomers, the Gly content in the copolymer was lower than the feed ratio. At the same time, the yield decreased with an increase in the feed content of GlyOEt. The copolymerization of the 50/50 monomer ratio of AlaOEt/Gly-OEt gave only 2% yield (Figure 6A), which did not increase even when higher concentrations (up to 10 U/mL) of papain were used, thereby enabling the poly(Gly)OEt synthesis. In addition, copolymerization did not occur when the concentration of Gly-OEt was higher than that of Ala-OEt in

the feed. These results confirm the difference in the affinity of papain toward Ala-OEt and that toward Gly-OEt, as was observed in their polymerization (Figures 3 and 4). By contrast, the copolymerization of Ala-OBzl and Gly-OBzl yielded copolymer poly(Ala-co-Gly) in all feed ratios, as confirmed by 1H and 13C NMR (Figures S6 and S7) and MALDI-TOF analyses (Figure 6B). The MALDI-TOF spectra of the copolymers contained repeating series of mass peaks spaced 57 m/z and 71 m/z units apart, corresponding to Gly and Ala, respectively (Figure 6B). The highest maximum (MWmax) and average (MWavg) molecular weights were obtained in poly(80 mol % Ala-co-20 mol % Gly) (Ala/Gly feeding ratio =20/80), whereas the reaction with the Ala/Gly feed ratio =80/20 gave poly(82 mol % Ala-co-18 mol % Gly) with the maximum yield. The MWavg and MWmax of poly(53 mol % Ala-co-47 mol % Gly) were 1059 ± 114 g/mol and 2186 ± 203 g/mol, respectively. The Gly and Ala contents were determined by the integration of the 1H NMR signals of the copolymers and were in accordance with the feed ratio employed (Figure 6c), demonstrating that the reactivities of both monomers with OBzl were similar. Poly(53 mol % Ala-co-47 mol % Gly) exhibited low solubility in DMSO/TFA (9/1) and was therefore dissolved into TFA as a solvent for NMR measurements.13 In the 1H NMR spectra, F

DOI: 10.1021/acs.biomac.5b01430 Biomacromolecules XXXX, XXX, XXX−XXX

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Biomacromolecules

employed in the feed (Figures 6c and 7a). The spectra of poly(53 mol % Ala-co-47 mol % Gly) in Figure S7 were assigned according to previous reports on an alternative copolymer of Ala and Gly, poly(Ala) and poly(Gly).13 In comparison with the homopolymers, the CHα signals of Ala slightly shifted downfield as the Gly content increased, whereas the CH2α signal of Gly shifted upfield with an increase in the Ala content. It was therefore possible to distinguish the residues between Ala-Gly (AG) and Gly-Ala (GA) connections (Figure 7a). At Ala:Gly ratios ranging from 8:2 to 5:5, the presence of the Ala CHαi signal implies that the polymerization was initiated preferentially with Ala rather than Gly. According to the carbonyl region in the 13C NMR spectra (Figure S7), the chemical shift of the Ala-Gly (A-G) carbonyl connection moved downfield from the Ala-Ala (An) carbonyl signal, and the chemical shift of the Gly-Ala (G-A) carbonyl connection moved upfield from the Gly-Gly (Gn) carbonyl signal (Figure 7b). On the basis of the connections between Ala and Gly, we confirmed that the copolymers of Ala and Gly were successfully synthesized using the OBzl ester group, as we designed. Characterization of Poly(Ala-co-Gly). This work represents the first report of the chemoenzymatic synthesis of poly(Ala-co-Gly), which is considered as a model peptide of Bombyx mori silk.40 The structure−function relationship of poly(Ala-co-Gly) can reveal the roles of Ala and Gly in B. mori and spider dragline silks. The poly(Ala-co-Gly) synthesized via chemoenzymatic polymerization in this study was characterized in its secondary structure by FTIR (Figure 8 and Figure S8). Because TFA induces changes in the structure to random coil and helical,39,41 the NMR studies in this solvent are only useful to confirm the assignment and observe the connections. The FTIR spectra of homopolymers with different ester groups were used as controls (Figure S8). According to previous assignments,42−45 the poly(Ala)OMe, poly(Ala)OEt, and poly(Ala)OBzl spectra showed bands corresponding to the βform, whereas the secondary structure of poly(Gly)OMe, poly(Gly)OEt, and poly(Gly)OBzl were assigned as polyglycine II. Polyglycine I has been described as having a 3-fold helical conformation,45 whereas polyglycine II has a planar zigzag conformation similar to a β-sheet.44 The results indicate that the ester group of the monomer did not affect the structure. The FTIR spectra of poly(18 mol % Ala-co-82 mol % Gly)OBzl were identical to those of poly(Gly)OBzl; hence, the conformation of poly(18 mol % Ala-co-82 mol % Gly)OBzl was polyglycine II. Interestingly, according to the bands at 1516 and 1015 cm−1, the conformation of poly(27 mol % Ala-co-73 mol % Gly)OBzl was assignable to polyglycine I.44,46 The spectra of poly(Ala-co-Gly) were similar to those of poly(Ala)OBzl in the amide I and amide II regions (1630 and 1550 cm−1). The band at 1630 cm−1 and the shoulder at approximately 1690 cm−1 originate from antiparallel β-sheets.47−49 These results relating to the conformational change based on the Ala/Gly composition suggest that approximately 30 mol % of either Ala or Gly are sufficient to induce a sheet conformation in the copolymers, whereas the copolymer containing greater than 80 mol % Gly tends to form polyglycine II, similar to poly(Gly). The monomeric composition of poly(Ala-co-Gly) is therefore one of the major factors, in addition to the spinning condition, that determine the secondary structure. The effect of the Ala/Gly composition and secondary structures on the thermal stability was also studied by TGA/ DSC (Figure 9). DTG traces were calculated from the TGA profiles, and commercial poly(Gly) and poly(Ala) samples were

Figure 6. Studies of the copolymerization of Ala and Gly monomers. (a) Effects of Ala/Gly feed ratio on yield and molecular weight. Bars: percent yields of reactions. Circles: average molecular weights calculated by 1H NMR. Squares: maximum molecular weights detected by MALDI-TOF. The total concentration of monomers was 0.6 M. The reaction was performed in 1 M phosphate buffer at pH 8 containing 1 U/mL of papain. Error bars represent the standard deviations of the replicates (n = 3). (b) MALDI-TOF analysis of poly(53 mol % Ala-co-47 mol % Gly). The inset denotes each assignment of the copolymers of Ala and Gly. (c) Ala and Gly compositions of the poly(Ala-co-Gly) synthesized at different feed ratios of Ala-OBzl to Gly-OBzl. Each error bar represents the standard deviation of the replicates (n = 3).

the CHα and CH2α in TFA shifted downfield relative to those obtained with the sample dissolved in a DMSO/TFA (9/1) mixture. The carbonyl signals in the 13C NMR spectra shifted to the values reported for a helical structure, as previously described.38,39 By contrast, the amine signals decreased in intensity and shifted upfield. As previously mentioned, the 1H NMR spectra of the copolymers presented signals of Ala and Gly with integration values in accordance with the ratio G

DOI: 10.1021/acs.biomac.5b01430 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules

Figure 7. 1H and 13C NMR spectra of poly(Ala), poly(Gly) and poly(Ala-co-Gly) at different ratios produced with Ala-OBzl and Gly-OBzl. (a) 1H NMR spectra of the alpha-carbon region. (b) 13C NMR spectra of the carbonyl region for clear assignment of Ala and Gly connections.

used as reference materials. The molecular weight of the commercial poly(Gly) and poly(Ala) were 500−5000 and 1000−5000 g/mol, respectively. Additionally, the chemical structures of both the commercial polypeptides were confirmed by 1H NMR (See Supporting Information). In the TGA and DSC profiles of all the samples, an initial weight loss that originated from the removal of water molecules was detected at temperatures under 100 °C.50 Transition and melting temperatures were not detected in all the samples. In addition, the thermal degradation temperature, Td, was determined (Table S1). Similar to the commercial reference samples poly(Ala)OH and poly(Gly)OH, poly(Ala)OBzl exhibited a higher Td than poly(Gly)OBzl. Only poly(Ala)OH was decomposed almost completely. This might be because of the impurities detected by 1H NMR (Figure S2D). The Td of the copolymers increased when the Ala content exceeded 18 mol % because the greater amount of Ala induced more hydrophobic interactions as well as the formation of sheet-like structures (Figure 10) according to the results of structural characterization by FTIR. Poly(Gly) formed a polyglycine II sheet structure and exhibited a higher Td comparison to that of poly(18 mol % Ala-co-82 mol % Gly)OBzl with a polyglycine I helical structure. Thus, the thermal degradation behavior and stability of the copolymers of Ala and Gly depend on the amino acid composition and resultant secondary structure. Furthermore, these thermal properties of poly(Ala-co-Gly) were similar to and reproduced the reported thermal properties of spider dragline silk and silkworm silk.51,52

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CONCLUSIONS

In summary, we have studied the effect of the ester groups of amino acid monomers on the KCS mediated by papain. The polymerization results of Ala and Gly monomers indicate that the efficiency of the ester group in KCS with papain can be classified as OBzl > OEt > OMe, whereas OtBu is not sufficiently reactive for the polymerization. The Ala-OBlz and Gly-OBlz monomers exhibited higher reactivity and enhanced the yield and peptide length in a shorter polymerization time and with a lower concentration of papain. This work represents

Figure 8. FTIR spectra of poly(Ala), poly(Gly), and poly(Ala-co-Gly) synthesized in this study. Assignments are as follows: Bend: b. Rock: r. Stretch: s. Twist: tw.

H

DOI: 10.1021/acs.biomac.5b01430 Biomacromolecules XXXX, XXX, XXX−XXX

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Biomacromolecules

Figure 9. Thermogravimetric analysis (TGA), differential scanning calorimetry (DSC) and derivative thermogravimetry (DTG) curves of poly(Ala)OH (a), poly(Ala)OBzl (b), poly(80 mol % Ala-co-20 mol % Gly)OBzl (c), poly(74 mol % Ala-co-26 mol % Gly)OBzl (d), poly(53 mol % Ala-co-47 mol % Gly)OBzl (e), poly(27 mol % Ala-co-73 mol % Gly)OBzl (f), poly(18 mol % Ala-co-82 mol % Gly)OBzl (g), poly(Gly)OBzl (h) and poly(Gly)OH (i).

group drastically affects the KCS efficiency; therefore, these factors should be considered during the design of chemoenzymatic peptide syntheses.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.5b01430. Figures S1−S10, Table S1, and the details of NMR measurements (PDF)

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AUTHOR INFORMATION

Corresponding Author

Figure 10. Thermal degradation temperature (Td) as a function of the Ala composition of poly(Ala-co-Gly).

*E-mail: [email protected]. Author Contributions

the first report of the chemoenzymatic synthesis of poly(Gly) by papain using the active monomer Gly-OBlz; the success of this approach indicates that the OBzl group broadens the substrate specificity of papain and enhances the reactivity of the monomeric substrates, specifically, Gly and Ala. This higher reactivity enabled the controlled copolymerization of Ala and Gly, which exhibit dramatically different reactivities and affinities toward papain. The resultant copolymers of Ala and Gly were appropriate model copolymers of silkworm and spider dragline silks in terms of their secondary structures and thermal properties. Our findings indicate that the nature of the ester

The manuscript was written through the contributions of all authors. All authors have approved the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was financially supported by the RIKEN Biomass Engineering Program and the Impulsing Paradigm Change through Disruptive Technologies Program (ImPACT). I

DOI: 10.1021/acs.biomac.5b01430 Biomacromolecules XXXX, XXX, XXX−XXX

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Biomacromolecules

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DOI: 10.1021/acs.biomac.5b01430 Biomacromolecules XXXX, XXX, XXX−XXX