Directed Evolution of Nonheme Fe Enzymes for Enantioselective and Regiodivergent 1,3- and 1,4-Nitrogen Migration: Biocatalytic Asymmetric Synthesis of Noncanonical α- and β-Amino Acids.

July 27, 2026| Source: Journal of the American Chemical Society

Biocatalyst-controlled regioselective C(sp)-H functionalization of carboxylic acid derivatives provides a powerful method for the synthesis of noncanonical α- and β-amino acids. Herein, we report a nonheme Fe enzyme-catalyzed, regiodivergent, and enantioselective nitrogen migration, enabled by directed evolution of 1-aminocyclopropane-1-carboxylic acid oxidase from Petunia hybrida (PhyACCO). Through systematic evaluation of azanyl ester N-protecting groups and nonheme Fe enzymes, as well as iterative rounds of protein engineering, we developed two complementary nitrogen migratases, ACCO Nimα and ACCO Nimβ , that enabled biocatalyst-controlled 1,3- and 1,4-nitrogen migration with excellent regioselectivity. ACCO Nimα catalyzed the efficient enantioselective synthesis of α-amino acids via amidation of unactivated (nonbenzylic) secondary C(sp)-H bonds with up to 1900 total turnover numbers (TTN) and a k cat of 1560 min -1 , affording diverse noncanonical α-amino acids. ACCO Nimα further allowed the enantioconvergent synthesis of challenging α,α-disubstituted amino acids from racemic substrates via tertiary C(sp)-H bond amidation. In contrast, ACCO Nimβ enabled the regio- and enantioselective synthesis of β-amino acids via a catalyst-controlled 1,6-hydrogen atom transfer pathway that remains largely underexplored. Kinetic and intramolecular hydrogen-deuterium competition studies indicated rate-determining HAT, with the more exergonic 1,6-HAT showing a smaller KIE than 1,5-HAT. ACCO Nimα and ACCO Nimβ exhibited similar kinetic isotope effects for both 1,5- and 1,6-HAT pathways, suggesting that enzyme engineering controls regio- and enantioselectivity but does not alter the intrinsic transition-state properties of the HAT event. Together, these results further established engineered nonheme Fe enzymes as an excellent platform for the development of stereoselective, synthetically useful non-native biocatalytic transformations.

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Directed Evolution of Nonheme Fe Enzymes for Enantioselective and Regiodivergent 1,3- and 1,4-Nitrogen Migration: Biocatalytic Asymmetric Synthesis of Noncanonical &#x3b1;- and &#x3b2;-Amino Acids.