Engineered C–N Lyases for Stereoselective Synthesis of Tertiary Amines

Abstract Optically pure N ‐functionalized α‐amino acids are valuable chiral building blocks for pharmaceuticals, nutraceuticals, and agrochemicals. Ethylenediamine‐ N,N ‐disuccinic acid lyase from Chelativorans sp. BNC1 catalyzes the addition of a wide range of aliphatic and aromatic primary amines to fumarate, producing the corresponding enantioenriched N ‐substituted L‐ aspartic acids. In this work, the enzyme was subjected to iterative cycles of site‐saturation mutagenesis and screened for increased activity for the addition of 2‐((methylamino)methyl)aniline to fumarate. The final variant displayed an activity of three orders of magnitude higher compared to the wild‐type enzyme. Unexpectedly, the enzyme catalyzed the hydroamination of fumarate with the aliphatic secondary amine of the starting substrate, rather than with the aromatic primary amine, leading to the formation of a tertiary amine. Exploring the substrate scope showed that the enzyme accepts various substituted N ‐methyl‐1‐phenylmethanamines for the hydroamination of fumarate, yielding N,N ‐disubstituted L ‐aspartic acids in high optical purity (up to >99% ee). Furthermore, we showed that the enzyme accepts several ortho ‐substituted anilines that were previously not accepted by the wild‐type enzyme, yielding the corresponding N ‐arylated L ‐aspartic acids in high enantiomeric excess (>99% ee). This serendipitous finding enables a new strategy for the biocatalytic synthesis of tertiary amines, unlocked within the C‐N lyase toolbox.