Papain-catalyzed oligomerization of diethyl L-glutamate hydrochloride was conducted in phosphate buffer at 40 °C. Because of rapid oligomerization kinetics, high substrate concentrations were not needed to shift the equilibrium for oligomer synthesis. For example, at 0.03 M diethyl L-glutamate hydrochloride, oligo(γ-ethyl L-glutamate) synthesis and precipitation from solution occurred in 55% yield. MALDI-TOF spectra of precipitated products showed two series of ion peaks separated by 157 m/z units, the mass of oligo(γ-ethyl-L-glutamate) repeat units. The most abundant signals were at DP 8 and 9, in excellent agreement with DP avg values determined by 1 H NMR. Lower intensity peaks with m/z less by 28 correspond to hydrolysis of one ester group either at a chain end or a pendant group along chains. Oligo(γ-ethyl-L-glutamate) synthesis at 40 °C in phosphate buffer (0.9 M, pH 7) occurred rapidly so that by 5, 10, and 20 min the yield reached 70 ( 4%, 78 ( 4% and 81 ( 5%, respectively. High product yields were observed over a broad range of pH values. As long as the pH was maintained from 5.5 to 8.5, the product yield was g60%. Ionic strength had no significant effect on oligopeptide yield. The dominant role of phosphate buffer in reactions was its control of pH. Other influences of phosphate ions on papain, such as nonspecific salt interactions or a "salting out" of product, appear to be of little or no importance. Loss in protein concentration and activity in the supernatant was observed after one reaction. A second reaction cycle performed using recovered supernatants resulted in a decrease in oligo(γ-ethyl-L-glutamate) yield from about 75% to 20%.
Enzymatic synthesis of oligopeptides from l-phenylalanine ethyl ester hydrochloride (l-Phe-Et.HCl) and other l-form hydrophobic amino acid ester hydrochlorides in water miscible organic cosolvents was studied. Different proteases, water miscible cosolvents, and effect of different ratios of water miscible cosolvents for protease-catalyzed oligo-phenylalanine [oligo(l-Phe)] were compared. The importance of the use of water miscible cosolvents in transforming reactions from heterogeneous to homogeneous conditions as a potent medium engineering tool for protease-catalyzed oligopeptide synthesis is highlighted. For example, at 0.125 M l-Phe-Et.HCl, 20% (v/v) methanol, 18.6 mg/mL bromelain, in phosphate buffer (0.25M, pH 8), 40 degrees C, for 3 h, oligo(l-Phe) precipitated from the solution to yield 45 +/- 5%, in contrast, in the absence of cosolvent oligo(l-Phe) yield of 29 +/- 5% was obtained. The following reaction conditions were optimized for bromelain catalyzed oligo(l-Phe) synthesis: pH, temperature, substrate, enzyme, and cosolvent concentrations. DP(avg) and chain length distribution in the product peptides were investigated by (1)H NMR and MALDI-TOF.
One-pot biotransformations gave oligo(γ-l-Et-Glu) decorated with selected amine-functionalized end-groups at C-termini. Motivations for this work were to (i) control the end group structure of peptides synthesized by protease-catalyzed peptide synthesis and (ii) incorporate end-groups that can be used directly or after further modification as polymerizable entities. Papain, bromelain, α-chymotrypsin, Multifect P-3000, and Purafect prime 4000 L were used as catalysts for oligomerization of γ-l-(Et)2-Glu in the presence of monofunctional amines. The series of amine nucleophiles (NH2-R, acyl acceptors) studied mimic phenylalanine in that they possess aromatic rings linked to amine groups by one or more methylenes. Generally, addition of increased quantities of NH2-R from 0 to 30, 50, and 70 mol % with respect to γ-l-(Et)2-Glu results in decreased % yield, but increased mol % of NH2-R end-capped oligo(γ-l-Et-Glu)-NH-R (determined by NMR). Irrespective of the protease used, 2-thiophene methyl amine (TPMA) gave the highest fraction of oligo(γ-l-Et-Glu)-NH-R chains. For example, using Multifect P-3000 and a feed ratio of TPMA-to γ-l-(Et)2-Glu of 7:3, >90 mol % of oligopeptides formed had TPMA C-terminal groups. With all five proteases studied herein, l-phenylalanine and l-histidine did not produce end-capped oligo(γ-l-Et-Glu). In contrast, l-phenylalanine analogs benzylamine (BzA) and l-phenylalaninol (F-OH), both of which lack the α-carboxyl group, gave substantial quantities of oligo(γ-l-Et-Glu)-F-OH or -BzA chains. Hence, the results of this study prove that the promiscuity of proteases used herein can be exploited to create a diverse family of desired end-functionalized oligopeptides. MALDI-TOF spectra recorded of oligo(γ-l-Et-Glu) with amine nucleophiles showed molecular ions that affirmed the formation of corresponding NH2-R functionalized oligo(γ-l-Et-Glu).
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