Modular Synthesis of Amphiphilic Ionic Polyacrylamides for Noncovalent Modulation of Esterase Activity

Abstract

Engineering the local environment of enzymes provides a complementary strategy to protein engineering for improving biocatalyst performance under process‐relevant conditions. Here, a modular post‐polymerization modification platform based on poly(pentafluorophenyl acrylate) (poly(PFPA)) is used to prepare a focused library of amphiphilic ionic polyacrylamide‐based copolymers with systematic variation in degree of polymerization, ionic/hydrophilic composition, and hydrophobic side‐chain length. Limiting first‐stage aminolysis installs trimethylammonium, oligo(ethylene glycol), and alkyl side chains, whereas residual activated esters are subsequently capped as propylamides or hydrolyzed to carboxylates. In a parallel microplate fluorogenic esterase assay, the polymers produce distinct concentration‐dependent changes in apparent enzyme activity. High polymer concentrations generally inhibit product formation, whereas selected C6‐ and C10‐containing polymers, especially at DP 60 and 100, increase apparent esterase activity at low concentrations by approximately 20% relative to the enzyme‐only control. C16 analog are predominantly inhibitory, consistent with overly strong hydrophobic association, self‐assembly, or substrate sequestration, whereas PEG incorporation attenuates both activation and inhibition, and zwitterionic charge balance partially mitigates hydrophobe‐induced inhibition. These results identify hydrophobicity, multivalency, and charge balance as coupled design variables in noncovalent polymer‐enzyme complexation and establish a practical framework for screening synthetic polymer microenvironments for hydrolase catalysis.