Biocatalytic Asymmetric Cyclopropanations via Enzyme-Bound Iminium Ion Intermediates

Abstract

Cyclopropane rings are an important structural motif frequently found in many natural products and pharmaceuticals. Commonly, biocatalytic methodologies for the asymmetric synthesis of cyclopropanes rely on repurposed or artificial heme enzymes. Here, we engineered an unusual cofactor-independent cyclopropanation enzyme based on a promiscuous tautomerase for the enantioselective synthesis of various cyclopropanes via the nucleophilic addition of diethyl 2-chloromalonate to α,β-unsaturated aldehydes. The engineered enzyme promotes formation of the two new carbon-carbon bonds with excellent stereocontrol over both stereocenters, affording the desired cyclopropanes with high diastereo- and enantiopurity (d.r. up to 25:1; e.r. up to 99:1). Our results highlight the usefulness of promiscuous enzymes for expanding the biocatalytic repertoire for non-natural reactions.

Keywords: biocatalysis; catalytic promiscuity; cyclopropanation; enzyme engineering.

Scheme 1

Iminium ion biocatalysis, highlighting the key catalytic role of the N‐terminal proline residue (Pro‐1) of the 4‐OT enzyme. A) Proposed mechanism of the 4‐OT catalyzed Michael addition of nitromethane to cinnamaldehydes. B) Proposed mechanism of the 4‐OT catalyzed epoxidation reaction between hydroperoxides and cinnamaldehydes. C) Proposed mechanism of the 4‐OT catalyzed cyclopropanation reaction between diethyl 2‐halomalonates and cinnamaldehydes.

Figure 1

Engineering of 4‐OT for cyclopropanations. A) Reaction of the 4‐OT catalyzed cyclopropanation of diethyl 2‐chloromalonate and cinnamaldehyde to afford 1 a. B) Comparison of the specific activity of wild type 4‐OT and engineered 4‐OT variants for the cyclopropanation reaction between diethyl 2‐chloromalonate and cinnamaldehyde to yield 1 a. The specific activities are as follows: wt (3.0 mU mg⁻¹), F50V (16.7 mU mg⁻¹), M45C/F50A (36.0 mU mg⁻¹), M45T/F50A (40.8 mU mg⁻¹), M45L/F50V (43.7 mU mg⁻¹), M45I/F50A (67.9 mU mg⁻¹), M45V/F50A (89.4 mU mg⁻¹). Reaction conditions: 1 mM cinnamaldehyde, 5 mM diethyl 2‐chloromalonate, 0.1 mg mL⁻¹ 4‐OT variant, 50 mM HEPES pH 6.5. Error bars represent the standard deviation of two measurements using the same enzyme batch (n=2).