Rapid Regioselective Oligomerization of l-Glutamic Acid Diethyl Ester Catalyzed by Papain

Abstract

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%.

Chemoenzymatic Synthesis of Polypeptides for Use as Functional and Structural Materials

“…Methyl, [49] ethyl [45] Proteinase I, [49] proteinase K (5-9) [45] l-Glutamic acid Ethyl [45,46,49,59,62,[71][72][73] Proteinase I, [49] papain (6-9.5), [59,62,[71][72][73] α-chymotrypsin (5-12), [59] proteinase K (5-9), [45] bromelain (5-9), [46,59] protease SG (6-11) [59] l-Aspartic acid Ethyl [49] Proteinase I [49] l-Tryptophan…”

“…[59,71,72] Although l-glutamic acid is a hydrophilic amino acid that contains a carboxylic acid residue in its side chain, the conversion of the carboxylic acid to an ester causes the resulting poly(l-glutamate) to be hydrophobic, leading to its precipitation from aqueous buffer solutions. In the reported studies, diethyl l-glutamate was selected as a mono mer, and the polymerization was conducted using papain to yield polyGlu with a DP of 9.5 as estimated by 1 H nuclear magnetic resonance (NMR) spectroscopy.…”

Polymerization of Peptide Polymers for Biomaterial Applications

“…[47][48][49][50][51][52][53][54] This reaction is initiated by the formation of an acyl-enzyme intermediate between the Cys or Ser residue in the active site and the ester group on modified carboxylic acid. In presence of high concentration of the substrate, the free amine group of the L-amino acids acts as the nucleophile resulting in propagation.…”

Synthesis of peptides with narrow molecular weight distributions via exopeptidase-catalyzed aminolysis of hydrophobic amino-acid alkyl esters

“…14,15 Although these methods have both drawbacks and beneficial aspects, the protease-catalyzed chemoenzymatic synthesis of peptides is an alternative to these conventional methods. 10,[16][17][18][19][20] Importantly, this method offers advantages over other processes because it requires limited or no side-chain protection of amino acids. 21 In addition, this alternative uses mild conditions and retains stereospecificity.…”

“…A kinetic model for serine protease-catalyzed peptide synthesis has been proposed. 19,[22][23][24][25][26] This model suggests that the protease-catalyzed synthesis of peptides proceeds via kinetically controlled synthesis. The activated amino acid and the enzyme first form an acyl-enzyme intermediate (ES complex) (Scheme 1), which is then competitively deacylated by a nucleophile or water.…”