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

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.