Stereoselective Enzymatic Synthesis of Heteroatom-Substituted Cyclopropanes

Abstract

The repurposing of hemoproteins for non-natural carbene transfer activities has generated enzymes for functions previously accessible only to chemical catalysts. With activities constrained to specific substrate classes, however, the synthetic utility of these new biocatalysts has been limited. To expand the capabilities of non-natural carbene transfer biocatalysis, we engineered variants of Cytochrome P450 BM3 that catalyze the cyclopropanation of heteroatom-bearing alkenes, providing valuable nitrogen-, oxygen-, and sulfur-substituted cyclopropanes. Four or five active-site mutations converted a single parent enzyme into selective catalysts for the synthesis of both cis and trans heteroatomsubstituted cyclopropanes, with high diastereoselectivities and enantioselectivities and up to 40 000 total turnovers. This work highlights the ease of tuning hemoproteins by directed evolution for efficient cyclopropanation of new substrate classes and expands the catalytic functions of iron heme proteins.

Highly Diastereo‐ and Enantioselective Synthesis of Nitrile‐Substituted Cyclopropanes by Myoglobin‐Mediated Carbene Transfer Catalysis

“…These results thus showed that diazoacetonitrile can be effectively utilized as carbene donor by the Mb-based carbene transferase. In addition, the chiral induction imposed by Mb(H64V,V68A) in the cyclopropanation reaction with diazoacetonitrile mirrors that observed in styrene cyclopropanation with EDA [9c] and DTE [9g] , highlighting the conserved stereoselectivity of this biocatalyst across these acceptor-only carbene donors. Further experiments showed that other hemoproteins can catalyze this reaction, but only with lower activity and/or stereoselectivity compared to Mb(H64V,V68A) (Table S1).…”

“…Heme-containing proteins [9] as well as engineered/artificial metalloenzymes [10] have been recently identified as viable biocatalysts for promoting olefin cyclopropanations in the presence of diazo reagents. In particular, we previously reported the ability of engineered variants of myoglobin (Mb) to catalyze the cyclopropanation of vinylstyrenes with ethyl α-diazoacetate (EDA) with a high degree of diastereo- and enantioselectivity.…”

“…In particular, we previously reported the ability of engineered variants of myoglobin (Mb) to catalyze the cyclopropanation of vinylstyrenes with ethyl α-diazoacetate (EDA) with a high degree of diastereo- and enantioselectivity. [9c, 9d] Despite this progress, the scope of biocatalytic cyclopropanations has been largely restricted to α-diazoacetates, limiting the types of functionalized cyclopropanes accessible using these systems. To overcome these limitations, our attention was drawn to diazoacetonitrile, a largely underutilized reagent in organic chemistry.…”

“…[12] On the other hand, protocols for in situ generation of diazoacetonitrile through diazotization of 2-amino-acetonitrile [11, 13] —a shelf-stable, readily available, and inexpensive reagent—, are incompatible with protein stability and function. Our recent success in engaging gaseous 2-diazo-trifluoroethane (DTE) in myoglobin catalysis [9g] prompted us to apply an analogous compartmentalized reaction system for testing the ability of myoglobin to accept diazoacetonitrile as a carbene donor for olefin cyclopropanation. Accordingly, we tested a reaction system in which diazoacetonitrile ( 1a ) generated in situ via diazotization of 2-amino-acetonitrile ( 1b ) in a ‘reagent generation chamber’, is carried through a ‘reaction chamber’ containing the biocatalyst and a model olefin substrate ( p -chloro-styrene, 2a ) via a continuous flow of inert gas (Ar) (Table 1 and Figure S1).…”

“…The myoglobin variant Mb(H64V,V68A) was selected as the catalyst in reason of its high stereoselectivity in olefin cyclopropanations with EDA. [9c, 9d] Gratifyingly, a reaction system involving Mb(H64V,V68A)-containing whole cells (OD 600 : 80) and ex situ generated diazoacetonitrile resulted in the accumulation of the desired nitrile-substituted cyclopropane product, 3a , in good conversion (44%; Table 1, Entry 2). A similar reaction with purified protein did not yield any product (Table 1, Entry 1), indicating that the whole cell system protects the biocatalyst from inactivation by the gas flow.…”