The use of safe natural catalyst such as enzymes for ring opening polymerization (ROP) of beta-substituted beta-lactones such as benzyl malolactonate (MLABe) is an important objective considering the biomedical applications of the resulting (co)polymers. However, the preparation of well-defined polymeric materials using such systems requires an understanding of enzyme-substrate interactions. In this context, we investigated the mechanism of lipase-catalyzed ROP of MLABe, because it appears that it is probably not the same as the one widely described for other lactones such sigma-caprolactone, propiolactone. and lactide. Enzymatic-catalyzed ROPs of MLABe in the presence of the lipase/acyltransferase CpLip2 and its serine knockout (serine KO) mutant (CpLip2_180A) have led to poly(benzyl malate) (PMLABe) terminated by a monobenzyl fumarate group with monomer conversion higher than 70% and weight-average molar mass of about 3600 g/mol (D = 1.42). On the other hand, only less than 7% of MLABe conversion and no polymer formation were observed when the polymerization reaction was conducted in the presence of inactivated CpLip2 (heated at 100 degreesC). Moreover, the ROP of MLABe in the presence of imidazole, a synthetic mimic of the catalytic histidine, led to a PMLABe terminated by a monobenzyl fumarate group. On the contrary, neither the enzymatic-catalyzed ROP of benzyl dimethylmalolactonate (diMeMLABe), a MLABe with two methyl groups instead of the two "acidic" protons on the lactone's ring, in the presence of CpLip2 and CpLip2_180A nor its chemical ROP in the presence of imidazole were successful. Together, all these results suggested that the lipase-catalyzed polymerization of malolactonates occurred through the abstraction of one of the two "acidic" protons of the lactone's ring by the histidine of the catalytic triad leading to the corresponding monobenzyl fumarate responsible for the polymerization of the remaining monomer. Finally, molecular modeling of the positioning of the monomer into the catalytic site of the CpLip2 and DFT quantum-chemical calculations highlighted an interaction of (R)- and (S)-MLABe with the catalytic histidine of the enzyme preferentially to serine, in the form of a strong hydrogen bond with one of the "acidic" protons of MLABe, thus, supporting the important role of the catalytic histidine in the polymerization of such cyclic lactones.
