Enzymatic Assembly of Diverse Lactone Structures: An Intramolecular C-H Functionalization Strategy.

Lactones are cyclic esters with extensive applications in materials science, medicinal chemistry, and the food and perfume industries. Nature's strategy for the synthesis of many lactones found in natural products always relies on a single type of retrosynthetic strategy, a C-O bond disconnection. Here, we describe a set of laboratory-engineered enzymes that use a new-to-nature C-C bond-forming strategy to assemble diverse lactone structures. These engineered "carbene transferases" catalyze intramolecular carbene insertions into benzylic or allylic C-H bonds, which allow for the synthesis of lactones with different ring sizes and ring scaffolds from simple starting materials. Starting from a serine-ligated cytochrome P450 variant previously engineered for other carbene-transfer activities, directed evolution generated a variant P411-LAS-5247, which exhibits a high activity for constructing a five-membered ε-lactone, lactam, and cyclic ketone products (up to 5600 total turnovers (TTN) and >99% enantiomeric excess (ee)). Further engineering led to variants P411-LAS-5249 and P411-LAS-5264, which deliver six-membered δ-lactones and seven-membered ε-lactones, respectively, overcoming the thermodynamically unfavorable ring strain associated with these products compared to the γ-lactones. This new carbene-transfer activity was further extended to the synthesis of complex lactone scaffolds based on fused, bridged, and spiro rings. The enzymatic platform developed here complements natural biosynthetic strategies for lactone assembly and expands the structural diversity of lactones accessible through C-H functionalization.

Enantio- and Diastereoenriched Enzymatic Synthesis of 1,2,3-Polysubstituted Cyclopropanes from (Z/E)-Trisubstituted Enol Acetates.

In nature and synthetic chemistry, stereoselective [2 + 1] cyclopropanation is the most prevalent strategy for the synthesis of chiral cyclopropanes, a class of key pharmacophores in pharmaceuticals and bioactive natural products. One of the most extensively studied reactions in the organic chemist's arsenal, stereoselective [2 + 1] cyclopropanation, largely relies on the use of stereodefined olefins, which can require elaborate laboratory synthesis or tedious separation to ensure high stereoselectivity. Here, we report engineered hemoproteins derived from a bacterial cytochrome P450 that catalyze the synthesis of chiral 1,2,3-polysubstituted cyclopropanes, regardless of the stereopurity of the olefin substrates used. Cytochrome P450BM3 variant P411-INC-5185 exclusively converts (Z)-enol acetates to enantio- and diastereoenriched cyclopropanes and in the model reaction delivers a leftover (E)-enol acetate with 98% stereopurity, using whole Escherichia coli cells. P411-INC-5185 was further engineered with a single mutation to enable the biotransformation of (E)-enol acetates to α-branched ketones with high levels of enantioselectivity while simultaneously catalyzing the cyclopropanation of (Z)-enol acetates with excellent activities and selectivities. We conducted docking studies and molecular dynamics simulations to understand how active-site residues distinguish between the substrate isomers and enable the enzyme to perform these distinct transformations with such high selectivities. Computational studies suggest the observed enantio- and diastereoselectivities are achieved through a stepwise pathway. These biotransformations streamline the synthesis of chiral 1,2,3-polysubstituted cyclopropanes from readily available mixtures of (Z/E)-olefins, adding a new dimension to classical cyclopropanation methods.

Enantio- and Diastereoenriched Enzymatic Synthesis of 1,2,3-Polysubstituted Cyclopropanes from (Z/E)-Trisubstituted Enol Acetates.

In nature and synthetic chemistry, stereoselective [2+1] cyclopropanation is the most prevalent strategy for the synthesis of chiral cyclopropanes, a class of key pharmacophores in pharmaceuticals and bioactive natural products. One of the most extensively studied reactions in the organic chemist's arsenal, stereoselective [2+1] cyclopropanation, largely relies on the use of stereodefined olefins, which require elaborate laboratory synthesis or tedious separation to ensure high stereoselectivity. Here we report engineered hemoproteins derived from a bacterial cytochrome P450 that catalyze the synthesis of chiral 1,2,3-polysubstituted cyclopropanes, regardless of the stereopurity of the olefin substrates used. Cytochrome P450 BM3 variant IC-G3 exclusively converts ( Z )-enol acetates to enantio- and diastereoenriched cyclopropanes and in our model reaction delivers a leftover ( E )-enol acetate with 98% stereopurity, using whole Escherichia coli cells. IC-G3 was further engineered with a single mutation to enable the biotransformation of ( E )-enol acetates to α -branched ketones with high levels of enantioselectivity while simultaneously catalyzing the cyclopropanation of ( Z )-enol acetates with excellent activities and selectivities. We conducted docking studies and molecular dynamics simulations to understand how active-site residues distinguish between the substrate isomers and enable the enzyme to perform these distinct transformations with such high selectivities. Computational studies suggest the observed enantio- and diastereoselectivities are achieved through a stepwise pathway. These biotransformations streamline the synthesis of chiral 1,2,3-polysubstituted cyclopropanes from readily available mixtures of ( Z/E )-olefins, adding a new dimension to classical cyclopropanation methods.

High Throughput Screening with SAMDI Mass Spectrometry for Directed Evolution.

Advances in directed evolution have led to an exploration of new and important chemical transformations; however, many of these efforts still rely on the use of low-throughput chromatography-based screening methods. We present a high-throughput strategy for screening libraries of enzyme variants for improved activity. Unpurified reaction products are immobilized to a self-assembled monolayer and analyzed by mass spectrometry, allowing for direct evaluation of thousands of variants in under an hour. The method was demonstrated with libraries of randomly mutated cytochrome P411 variants to identify improved catalysts for C-H alkylation. The technique may be tailored to evolve enzymatic activity for a variety of transformations where higher throughput is needed.