Protease-catalyzed routes to oligo (alpha-glutamate) and corresponding co-oligopeptides: Synthesis and structural analysis

Papain-catalyzed oligomerization of diethyl L-glutamate hydrochloride was conducted in phosphate buffer at 40°C. Due to rapid oligomerization kinetics, high substrate concentrations were not needed to shift the equilibrium for oligomer synthesis. Oligo(gamma-ethyl- L-glutamate) synthesis at 40°C in phosphate buffer (0.9M, 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. Ionic strength had no significant effect on oligopeptide yield. The dominant role of phosphate buffer in reactions was its control of pH. Four proteases (papain, bromelain, alpha-chymotrypsin and protease SG) were used to catalyze co-oligomerizations of L-leucine ethyl ester ( L-Et-Leu) with diethyl-L-glutamate ( L-(Et)2-Glu). 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 &ap bromelain > alpha-chymotrypsin > protease SG. MALDI-TOF spectra information was obtained on both chain length distribution and chemical composition distribution for oligo (gamma-Et-L-Glu-co-&sim50mol% L-Leu) prepared using papain, bromelain, alpha-chymotrypsin or protease SG as catalysts. Using LC-MS to analyze the relative content of tripeptide sequences, 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.Oligopeptide-based lipopeptides were prepared via conjugating proteases catalyzed peptide products with fatty acids. Synthesized lipopeptides include fatty acids with chain length ranging from 8 to 18, coupled to oligo(Glu), oligo(Leu-co-Glu). C12/Oligo(Glu) is found to be very surface active with a surface tension lowering from 72 nMm-1 to 30 mNm-1 and a CMC of 4&sim5 mM. Incorporation of hydrophobic amino acids within the oligo (Glu) segment further modified lipopeptide colloidal properties.