An enzymatic platform for the asymmetric amination of primary, secondary and tertiary C(sp3)-H bonds

Abstract

The ability to selectively functionalize ubiquitous C-H bonds streamlines the construction of complex molecular architectures from easily available precursors. Here we report enzyme catalysts derived from a cytochrome P450 that use a nitrene transfer mechanism for the enantioselective amination of primary, secondary and tertiary C(sp³)-H bonds. These fully genetically encoded enzymes are produced and function in bacteria, where they can be optimized by directed evolution for a broad spectrum of enantioselective C(sp³)-H amination reactions. These catalysts can aminate a variety of benzylic, allylic and aliphatic C-H bonds in excellent enantioselectivity with access to either antipode of product. Enantioselective amination of primary C(sp³)-H bonds in substrates that bear geminal dimethyl substituents furnished chiral amines that feature a quaternary stereocentre. Moreover, these enzymes enabled the enantioconvergent transformation of racemic substrates that possess a tertiary C(sp³)-H bond to afford products that bear a tetrasubstituted stereocentre, a process that has eluded small-molecule catalysts. Further engineering allowed for the enantioselective construction of methyl-ethyl stereocentres, which is notoriously challenging in asymmetric catalysis.

Fig. 1.. Three major types of asymmetric C(sp³)–H functionalization and biocatalytic C(sp³)–H amination.

a, Enantioselective functionalization of secondary C(sp³)–H bonds. b, Enantioselective functionalization of primary C(sp³)–H bonds (i.e., desymmetrization of gem-dimethyl substituents). c, Enantioconvergent functionalization of tertiary C(sp³)–H bonds. d, Proposed enzymatic synthesis of chiral diamines using C(sp³)–H amination. FG = functional group.

Fig. 2.. Enantioselective amination of secondary C(sp³)–H bonds.

a, Directed evolution of P411ΔFAD for the enantioselective synthesis of 1,2-diamines. Crystal structure of a variant closely related to P411_Diane1 is shown (Protein Data Bank ID: 5UCW); b, Substrate scope of 1,2-diamine and 1,3-diamine synthesis. c, Enantiodivergent amination of unactivated secondary C(sp³)–H bonds to access either antipode of the diamine product. Experiments were performed using E. coli expressing cytochrome P41l_Diane2 or P41l_Diane1 (OD₆₀₀ = 30) with 10 mM substrate at room temperature under anaerobic conditions for 12–24 h. †High TTN experiments were performed using E. coli expressing cytochrome P411_Diane2 or P411_Diane1 (OD₆₀₀ = 1.9) with 20 mM substrate at room temperature under anaerobic conditions for 24 h.

Fig. 3.. Enantioselective amination of primary and tertiary C(sp³)–H bonds.

a. asymmetric amination of primary C(sp³)–H bonds (i.e., desymmetrization of geminal dimethyl substituents); b. Enantioconvergent amination of tertiary C(sp³)–H bonds; c. Enantioconvergent construction of ‘methyl-ethyl’ stereocentre using tertiary C(sp³)–H amination. Experiments were performed using E. coli expressing cytochrome P41l_Diane1 I327P, P41l_Diane1 L78A A87G I327V Q437G, and or P411_Diane1 L82C L181V I327T A330M Q437G (OD600 = 30–40) with 10 mM substrate at room temperature under anaerobic conditions for 12–24 h.

Fig. 4.. Mechanistic and computational studies.

a. Scrambling of olefin stereochemistry during the allylic C(sp³)–H amination of (E)- and (Z)-1d using P411 biocatalyst (P411_Diane1 I327P). b. Mechanistic proposal to account for the enantioconvergent amination of tertiary C(sp³)–H bonds. c. Free energy profile of the iron porphyrin-catalysed C(sp³)–H amination. Density functional theory (DFT) calculations were performed at the B3LYP-D3(BJ)/6–311+G(d,p)-LANL2TZ(f)/SMD(chlorobenzene)//B3LYP-D3(BJ)/6–31+G(d)-LANL2DZ level of theory. Triplet structures are shown in black and quintet structures are shown in blue. Mulliken spin densities of Fe and N of the key iron nitrenoid 7 are shown in italic (blue).