Boosting amidation of Ortho-substituted anilines with esters or acids via hinge and tunnel engineering of lipase

The synthesis of structurally diverse aromatic amides holds significant pharmaceutical importance due to their excellent biological activities. Lipase, which can catalyze the aminolysis of esters to amides without requiring expensive cofactors, are advantageous for enzymatic amide synthesis. However, the weak nucleophilicity of substrates like aniline and the presence of ortho-substituents often hinder their efficacy. Herein, we report the engineering of a previously identified lipase, Ndbn from Rhizorhabdus dicambivorans UGC1, using an integrated approach of hinge and tunnel engineering. The quadruple variant M_4–1 (S37M/L206F/I211G/L212M) exhibited a 165-fold increase in catalytic efficiency for the amidation between o-toluidine and methyl 3-phenylpropanoate compared to the wild-type. Molecular dynamics (MD) simulations revealed that mutations facilitated a more open lid conformation and an optimized amine tunnel, enhancing ligand transport rates and overall catalytic efficiency. Remarkably, M_4–1 also demonstrated superior activity in synthesizing amides from carboxylic acids over esters. The kinetics and potential mechanisms behind this enhanced performance are explored. The efficiency of variant M_4–1 was demonstrated in the chemo-enzymatic synthesis of the local anesthetic prilocaine (28.7 g yield) using acid as substrate. This study offers a blueprint for rationally designing lipases with enhanced catalytic efficiency for sterically hindered substrate.