Protease-Catalyzed l-Aspartate Oligomerization: Substrate Selectivity and Computational Modeling

Abstract

Poly(aspartic acid) (PAA) is a biodegradable water-soluble anionic polymer that can potentially replace poly(acrylic acid) for industrial applications and has shown promise for regenerative medicine and drug delivery. This paper describes an efficient and sustainable route that uses protease catalysis to convert l-aspartate diethyl ester (Et₂-Asp) to oligo(β-ethyl-α-aspartate), oligo(β-Et-α-Asp). Comparative studies of protease activity for oligo(β-Et-α-Asp) synthesis revealed α-chymotrypsin to be the most efficient. Papain, which is highly active for l-glutamic acid diethyl ester (Et₂-Glu) oligomerization, is inactive for Et₂-Asp oligomerization. The assignment of α-linkages between aspartate repeat units formed by α-chymotrypsin catalysis is based on nuclear magnetic resonance (NMR) trifluoacetic acid titration, circular dichroism, and NMR structural analysis. The influence of reaction conditions (pH, temperature, reaction time, and buffer/monomer/α-chymotrypsin concentrations) on oligopeptide yield and average degree of polymerization (DP_avg) was determined. Under preferred reaction conditions (pH 8.5, 40 °C, 0.5 M Et₂-Asp, 3 mg/mL α-chymotrypsin), Et₂-Asp oligomerizations reached maximum oligo(β-Et-α-Asp) yields of ∼60% with a DP_avg of ∼12 (M _n 1762) in just 5 min. Computational modeling using Rosetta software gave relative energies of substrate docking to papain and α-chymotrypsin active sites. The substrate preference calculated by Rosetta modeling of α-chymotrypsin and papain for Et₂-Asp and Et₂-Glu oligomerizations, respectively, is consistent with experimental results.