Enzymatic C(sp3)-H Amination: P450-Catalyzed Conversion of Carbonazidates into Oxazolidinones

Abstract

Cytochrome P450 enzymes can effectively promote the activation and cyclization of carbonazidate substrates to yield oxazolidinones via an intramolecular nitrene C-H insertion reaction. Investigation of the substrate scope shows that while benzylic/allylic C-H bonds are most readily aminated by these biocatalysts, stronger, secondary C-H bonds are also accessible to functionalization. Leveraging this "non-native" reactivity and assisted by fingerprint-based predictions, improved active-site variants of the bacterial P450 CYP102A1 could be identified to mediate the aminofunctionalization of two terpene natural products with high regio- and stereoselectivity. Mechanistic studies and KIE experiments show that the C-H activation step in these reactions is rate-limiting and proceeds in a stepwise manner, namely, via hydrogen atom abstraction followed by radical recombination. This study expands the reactivity scope of P450-based catalysts in the context of nitrene transfer transformations and provides first-time insights into the mechanism of P450-catalyzed C-H amination reactions.

Keywords: C−H amination; biocatalysis; carbonazidate; cytochrome P450; oxazolidinones.

Figure 1

P450-catalyzed C–H amination in terpene natural products. The graphs illustrates the relative activity, based on TON, for the FL#62-derived active-site variants selected by fingerprint analysis (SCA). The amino acid mutations in the P450 variants can be found in Table S1. “s.r.c.” = standard reaction conditions, as indicated in Table 2.

Figure 2

Proposed mechanism for P450-catalyzed conversion of carbonazidates to oxazolidinones. The competing pathway leading to the carbamate byproduct is indicated in blue. Y = —OC(O)—.

Figure 3

Rearrangement studies. (a) FL#62-catalyzed cyclization of (E)-12 (or its isomer (Z)-12) leads to formation of trans oxazolidinone (E)-21, as determined by HPLC (Figure S3 in SI) and GC-MS analysis. The Z → E rearrangement in case of (Z)-12 is indicative of the formation radical intermediate via HAA. (b) Rhodium-catalyzed cyclization of carbamate Z-27 is not accompanied by scrambling of the double bond configuration, in agreement with the concerted nitrene C–H insertion mechanism proposed for these catalysts.

Figure 4

Kinetic isotope effect (KIE) experiments. (a) KIE value for FL#62-catalyzed amination of 6 and its deuterated analogue, D-6. The graph reports a plot of initial rate versus substrate concentration for the two reactions. (b) Comparison of KIE_intra and KIE_inter values as determined from intramolecular (D₁-11) and intermolecular (1:1 mixture of 11 and D₂-11) competition experiments, followed by GC-MS analysis (Figure S5).