Protease-catalyzed co-oligomerizations of L-leucine ethyl ester with L-glutamic acid diethyl ester: Sequence and chain length distributions

Four proteases (papain, bromelain, (x-chymotrypsin and protease SG) were used to catalyze cooligomerizations Of L-leucine ethyl ester (L-Et-Leu) with diethyl-L-glutamate (L-(Et)(2)-Glu). Protease added to reaction was normalized based on their relative activities for casein hydrolysis. With the exception of papain that shows a very broad pH optimum for oligo(gamma-Et-L-Glu) synthesis, the pH optimum for peptide synthesis was above that for peptide hydrolysis. For L-(Et)(2)-Glu/L-Et-Leu (1:1 mol/mol) co-oligomerizations, the relative order of activities of the four proteases is papain approximate to bromelain > alpha-chymotrypsin > protease SG. H-1 NMR analyses showed that co-oligopeptide compositions were close in value to the monomer feed ratio. Reactivity ratios, r(Leu) and r(Glu), are nearly identical and have values close to one, suggesting propagation during oligomer synthesis tends to occur randomly giving random sequences along oligopeptides. MALDI-TOF spectra information was obtained on both chain length distribution and chemical composition distribution for oligo(gamma-Et-L-Glu-co-similar to 50mol%-L-Leu) prepared using papain, bromelain, alpha-chymotrypsin or protease SG as catalysts. From MALDI-TOF generated total abundance intensities, DPavg values of these samples are 7.5, 6.1, 8.5 and 8.7 respectively, which agrees well with values determined by H-1 NMR (7.3, 6.5, 8.2 and 8.5 respectively). Oligo(gamma-Et-L-Glu-co-similar to 50mol%-L-Leu), synthesized using alpha-chymotrypsin and protease SG, consists of the broadest chain length distributions (5-12 and 6-11, respectively). In contrast, oligopeptides from bromelain and papain catalysis have narrower chain length distributions (5-8 and 6-9, respectively). Also, MALDI-TOF shows a large heterogeneity of leucine contents for oligo(gamma-Et-L-Glu-co-similar to 50mol%-L-Leu) synthesized by all four protease catalysts, consistent with co-oligomerizations occurring by random propagation events. Eight tripeptides representing all possible sequences were prepared by standard solid-state Fmoc-based peptide chemistry. Oligo(gamma-Et-L-Glu-co-similar to 50mol%-L-Leu) chains prepared using all four proteases were degraded to oligomers where the primary constituent in the population was trimers. Using LC-MS to analyze the relative content of tripeptide sequences, and calculating the abundance of triad sequences for a statistically random process, it was found for all four proteases that experimental and theoretical values are in excellent agreement. Hence, in agreement with MALDI-TOF and reactivity ratio results, LC-MS analysis showed that random co-oligopeptides were formed. In other words, for this set of monomers and proteases, no preference or selectivity was observed for addition of L-(Et)(2)-Glu or L-Et-Leu to propagating oligomer chains.