Enantioselective oxidation of unactivated C–H bonds in cyclic amines by iterative docking-guided mutagenesis of P450BM3 (CYP102A1)

Abstract

Selective oxidation of ring C-H bonds is an attractive route to functionalised cyclic amines which are versatile intermediates in drug synthesis and important fragment molecules in drug discovery. Here we report a combined substrate and enzyme engineering approach to achieve enantioselective functionalisation of all unactivated C-H bonds of azepane, azocane, 7azabicyclo[2.2.1]heptane, and 8-azaspiro[4.5]decane by cytochrome P450BM3 (CYP102A1).Different N-modifying groups provide product diversity at high enantioselectivity (up to 99% e.e.) from a panel of just 48 variants of P450BM3. Substrate docking into molecular dynamics simulation structures of enzyme variants is shown to be useful for designing mutations to increase enantioselectivity by disfavouring binding poses leading to the unwanted enantiomer, and to increase enzymatic activity by disfavouring non-productive poses from 10 or so variants per generation. The synthetic application of remote C-H activation within cyclic amines is exemplified by the synthesis of anisodamine via enantioselective hydroxylation of N-Bocnortropinone.

Choose Your Own Adventure: A Comprehensive Database of Reactions Catalyzed by Cytochrome P450 BM3 Variants

“…All positions of the P450 BM3 heme domain that have been mutated and show improvement toward large alkyl hydroxylation are shown (light green spheres). Mutated positions in the heme domain (residues 1–471) and in the reductase domain (472–1049) are listed, with refs: 1 (), 21 (), 25 (), 26 ( , , , , , ), 27 (), 28 (), 30 (), 47 ( , , , , , , , , , , , , , , , − ), 51 ( , , , , , , , ), 52 ( , , , , ), 55 (), 58 ( , , , ), 62 (), 67 (), 69 (), 70 (), 72 ( , , ), 73 ( , ), 74 ( , , , , , , , , , , , , , , , , ), 75 ( , , , , , , , , − ), 78 (…”

“…It has been the topic of extensive research over the past three decades and numerous laboratories have worked on evolving this enzyme to generate novel enzymatic properties and features. As a result, P450 BM3 and its variants have been shown to catalyze a wide variety of reactions, including hydroxylation, − epoxidation, − carbene transfer, − and nitrene transfer, − with a diverse range of substrates. The advances reported by the group of Frances Arnold on the evolution of P450 BM3 toward novel biocatalytic reactions, which were prominently featured in her 2018 Nobel-winning research, are among the most transformative.…”

“…All positions of the P450 BM3 heme domain that have been mutated and show improvement toward large alkyl hydroxylation are shown (light green spheres). Mutated positions in the heme domain (residues 1−471) and in the reductase domain (472−1049) are listed, with refs: 1 (320), 21 (325), 25 (446), 26(2,287,320,429,446,447), 27 (325), 28 (320), 30 (325), 47(2, 195, 217, 238, 241, 244, 245, 287, 288, 293, 348, 426, 429, 433, 438, 446−454), 51(2,238,241,245,270,325,446,451), 52(217,244,290,348,433), 55 (325), 58(290,359,455,456), 62(381), 67(325), 69(24), 70 (325), 72 (2, 24, 296), 73(195,313), 74(2,157,238,241,244,287,288,306,429,431,441,442,446,447,453,457,458), 75 (2, 12, 24, 166, 219, 309, 319, 452, 458−461), 78 (2, 12, 24, 217, 220, 244, 245, 296, 309, 319, 348,...…”

Selective P450BM3 Hydroxylation of Cyclobutylamine and Bicyclo[1.1.1]pentylamine Derivatives: Underpinning Synthetic Chemistry for Drug Discovery

“…The enzyme library evolved from four parent mutants of the P450 BM3 wild-type, through studies on the oxidation of a variety of substrate classes as described in earlier publications . Substrates were screened against the same 48-member subset of the wider library (with minor variations, see Supporting Information section S2), selected on the basis of reactivity profiles established in previous work with substrates of similar molecular weights and structural motifs, including anilides, cyclic amines, and cycloalkanes …”

Z-Schematic Artificial Leaf Structure for Biosolar Oxyfunctionalization of Hydrocarbons

“…The unbiased PEC reaction is further combined with enantioselective oxyfunctionalization of C–H bonds using unspecific peroxygenase − from Agrocybe aegerita, which was expressed recombinantly in Pichia pastoris (r Aae UPO). Selective oxyfunctionalization of (inert) C–H bonds is the long-standing goal in chemistry because the reaction offers novel synthetic strategies applicable to ubiquitous C–H bonds , in organic compounds (e.g., alkane feedstocks, pharmaceuticals). Compared with (in)­organic catalysts and P450 monooxygenases, heme-dependent UPOs accomplish the challenging reactions with several advantages , (e.g., balancing high reactivity and selectivity, using H 2 O 2 as the sole oxidant, requiring mild conditions).…”