Methyl and ethyl esters of monomeric substrates were used in this study because of their relatively higher reactivities in comparison to those of the free amino acids and nylon units [
15,
28]. Because the
pKa of substrate monomer is the main determinant of the reaction efficiency in the chemoenzymatic polymerizatrion, yield and degree of polymerization are largely dependent on the pH of the reaction medium [
14]. Therefore, we set the pH of 8 using phosphate buffer, which is within the range of optimal condition for the chemoenzymatic synthesis by papain [
17], bromelain [
38], proteinase K [
27], and lipase [
39]. Furthermore, we set at 40 °C for the aminolysis reaction, because the aminolysis activity was larger at 40 °C in the polymerization by papain [
17], bromelain [
38], and proteinase K [
27]. In addition, we found that the substrate monomer, namely
l-glutamic acid diethyl ester, tends to be hydrolyzed when the reaction temperature was set over 40 °C [
29]. Therefore, we used pH 8 and 40 °C for the reaction conditions. As the first step, we performed papain-catalyzed chemoenzymatic synthesis using Glu(Et)
2 and nylon 4Et and then obtained a resultant white precipitate at a yield of 53% based on the initial amounts of the monomers used in the reaction. The MALDI-TOF MS spectrum of the resultant product is shown in
Figure 2a. Peaks with constant intervals of 157 g/mol were detected, thus indicating that oligo(GluEt) was successfully synthesized with DP from 7 to 12. In this study, the limiting factor of polymer length is mainly determined by the solubility of the resultant product in the reaction medium. When peptide chains elongate to a length at which they are insoluble, they precipitate from solution, which prevents the increase of the molecular weight. In addition, the other peaks with an interval of 85 g/mol apart from the peaks derived from oligo(GluEt) indicated that oligo(GluEt) and only one nylon 4 unit were copolymerized. We did not detect any peaks derived from peptides containing more than two nylon 4 units. The resultant product was also characterized by
1H-NMR (
Figure 2b), and nylon-derived peaks were detected in addition to the peaks assigned to oligo(GluEt). From the
1H-NMR results, approximately 1 out of 10 oligo(GluEt) had a nylon 4Et unit, according to the integral values of the peaks corresponding to the N-terminal α-proton and C-terminal methylene proton shown as D’ and G, respectively, in
Figure 2b. The formation of γ-glutamate linkage in the oligomer was not detected, based on the
1H–NMR of the resultant product and a previous report on poly(α-methyl γ-
l-glutamate) [
40]. Furthermore, the oligomers formed from diethyl
l-glutamate hydrochloride using papain were exclusively α-linked, which was confirmed by
1H-
1H COSY NMR [
41]. In addition, the papain-catalyzed polymerization of γ-methyl
l-glutamate resulted in no occurrence of the polymerization [
41]. These data indicate that the poly-α-glutamate was exclusively produced in the papain-catalyzed synthesis. The MALDI-TOF MS spectra showed a negligible amount of free pendant carboxylic acid in the resultant product. The major series of signals were derived from the product with ester groups. According to the
1H–NMR spectra, the ratio of the integration value between the peaks derived from the N-terminal α-proton and the C-terminal ester group was nearly identical, which indicates that almost all the ester groups maintained. Thus, our resultant product was mainly composed of the ester moiety at the C-terminus. This is probably because the resultant product was formed as precipitate, which limits accessibility of papain to the resultant product and prevents the hydrolysis of the ester bonds.
To clarify the difference between nylon ethyl ester and nylon methyl ester, we additionally used nylon 4Me as a nylon unit, which resulted in a reaction yield of 45%. The MALDI-TOF MS spectrum of the peptide from Glu(Et)
2 and nylon 4Me showed peaks with constant intervals of 157 g/mol derived from oligo(GluEt) with DP from 7 to 12 in
Figure 2c. Peaks with an interval of 71 g/mol apart from the peaks derived from the oligo(GluEt) were also detected, thus confirming that the nylon 4 unit was introduced into the peptide. The
1H-NMR spectrum indicated that approximately 1 out of 10 oligo(GluEt) contained a nylon 4 unit (
Figure 2d). The comparison between the reactions using nylon 4Et and nylon 4Me demonstrated that the differences in MS peak intervals were 85 g/mol and 71 g/mol apart from the peaks of oligo(GluEt) for nylon 4Et and nylon 4Me, respectively. If the nylon 4 unit were introduced at the N-terminus or the inner part of the resultant peptide, the molecular weight difference between the resultant products using nylon 4Et and nylon 4Me would be the same. Thus, we concluded that the nylon 4 unit was introduced at the C-terminus of oligo(GluEt). In the papain-catalyzed chemoenzymatic synthesis, the catalytic cysteine residue of papain forms a covalent bond with the carboxyl ester group of the monomer, thus resulting in formation of the enzyme-substrate (ES) complex (
Scheme 1) [
21]. The nucleophilic attack by the amine group of monomers allows peptide bond formation by aminolysis. It has been demonstrated that recognition of
d-alanine and β-alanine by papain is approximately 100 times lower than that of
l-alanine [
16]. Although our reaction system contained nine times more nylon monomers than amino acids, introducing the nylon unit into the peptide was still difficult. Therefore, the nylon 4 unit was introduced only at the C-terminus. The results of this study and previous reports suggest that ES complex formation of the nylon 4 unit at the C-terminus with papain is not efficient enough to continue the polymerization (
Scheme 1).