Enzymatic construction of highly strained carbocycles

Abstract

Small carbocycles are structurally rigid and possess high intrinsic energy due to their significant ring strain. These unique features lead to broad applications, but also create challenges for their construction. We report the discovery and engineering of hemeproteins that catalyze the formation of chiral bicyclobutanes, one of the most strained four-membered systems, via successive carbene addition to unsaturated carbon–carbon bonds. Enzymes that produce cyclopropenes, putative intermediates to the bicyclobutanes, were also identified. These genetically-encoded proteins are readily optimized by directed evolution, function in Escherichia coli, and act on structurally diverse substrates with high efficiency and selectivity, providing an effective route to many chiral strained structures. This biotransformation is easily performed on preparative scale and the resulting strained carbocycles can be derivatized, opening myriad potential applications.

Computational Investigations of Heme Carbenes and Heme Carbene Transfer Reactions

“…[2] However,o nly recently,t he involvement of heme carbenes was reported in biocatalytic reactions, which have been rapidly expanded to ab road range of carbene transfer reactions, including cyclopropanations, [3] NÀHi nsertion, [3h,p, 4] SÀH insertion, [3q, 5] CÀHf unctionalization, [3h, 6] SiÀHi nsertion, [3p, 7] BÀH insertion, [8] aldehyde olefinations, [9] sigmatropic rearrangements, [10] cyclopropenation, [11] and bicyclobutanation. [11] These biocatalysts are based on heme proteins, such as cytochrome P450, horseradish peroxidase,c ytochromec,a nd myoglobin (Mb), but are all engineered with mutations, compared with native heme proteins,t op ossess good to superb biocatalytic properties. These studies are inspired by the excellent catalytic performance of native cytochrome P450s for numerous biochemicalr eactions, [12] which has also stimulated the development of biomimetic iron porphyrin complexes for av arietyo f catalytic carbene transfer reactions.…”

Unleashing the Synthetic Power of Plant Oxygenases: From Mechanism to Application

“…However, successful fusion of plant P450s to partner reductases has enabled an increase in their catalytic efficiency (Didierjean et al, 2002;Schückel et al, 2012;Sadeghi and Gilardi, 2013). Directed evolution of P450s has also yielded novel activities and even some previously unobserved for any natural catalyst, such as carbene/nitrene insertions to generate cyclopropyl/butyl groups, aziridination, and sulfimidation, greatly expanding the utility of this enzyme scaffold (Coelho et al, 2013;Wang et al, 2014;Brandenberg et al, 2017;Chen et al, 2018a). Such exhaustive evolution of any plant P450 has yet to be explored, owing to their difficulty in heterologous expression, but their native promiscuity could yield improved access to a plethora of plant secondary metabolites.…”

A Continuing Career in Biocatalysis: Frances H. Arnold

“…122 In a recent study, the Arnold group described the directed evolution of a P411 variant, called P411 BM3 -E10-FA, capable of accepting phenylacetylene and ethyl diazoacetate as substrates to make bicyclobutane product as a single stereoisomer (Scheme 3). 123 Key to this success is the enzyme's ability to stabilize the putative reactive cyclopropene intermediate and exert precise stereocontrol over two carbene transfer events. P411 BM3 variants that yield cyclopropenes as the final products were also identified, such that each enantiomer of a 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 18 cyclopropene can be made enzymatically with thousands of turnovers and high enantioselectivity (Scheme 3).…”

“…126 These efforts are complemented by the mechanistic work of other groups, 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 19 further our understanding of how iron porphyrins and heme proteins catalyze carbene transfer to olefins, N-H, and C-H bonds. [127][128][129][130][131][132][133] Genetically programmable chiral organoborane synthesis was also realized by the laboratory evolution of Rma cyt c. 123 This platform successfully yields structurally distinct organoboranes through divergent directed evolution (Scheme 4B), providing bacterial borylation catalysts that are suitable for gram-scale biosynthesis, and offering up to 15,300 turnovers and excellent selectivity (99:1 e.r., 100% chemoselectivity). Moreover, these enzymes are readily tunable to accommodate the synthesis of lactonebased organoboranes 134 and a broad range of chiral α-trifluoromethylated organoboranes (Scheme 4C).…”