Engineering radical enzymes for asymmetric olefin hydroalkylation

The National Institute of General Medical Sciences awarded Michigan State University $335,924 on April 1, 2026, under the Biomedical Research and Research Training program (CFDA 93.859) to engineer glycyl radical enzyme hydroalkylases for asymmetric C-C bond formation in pharmaceutical synthesis.

The project develops biocatalytic methods to form C(sp3)–C(sp3) bonds stereoselectively from simple, achiral starting materials, addressing the synthesis of sp3-rich compounds needed for therapeutic discovery. The work investigates native substrate scopes of two glycyl radical enzyme hydroalkylases—one functionalizing toluene and one functionalizing n-hexadecane—and engineers variants through genetic manipulation to expand their synthetic utility. Enzymatic hydroalkylation directly activates C-H bonds without pre-functionalization and can form up to three contiguous stereocenters in a single step, potentially accelerating medicine discovery by enabling rapid generation of complex intermediates.

Performance occurs at Michigan State University in East Lansing, Michigan, with a period of performance from April 1, 2026, through March 31, 2031. The award is a project grant, the standard assistance type for NIGMS investigator-initiated research funding.

Abstract

The synthesis of biologically active molecules through selective C-C bond-forming reactions is central to the discovery of new therapeutics. Though forging C-C bonds is often more challenging at C(SP3)-centers than C(SP2)-centers, especially when new stereocenters emerge, clinical success is correlated with SP3-richness. Consequently, developing new methods to rapidly generate complex, SP3-rich compounds from simple starting materials presents an opportunity for synthetic innovation to accelerate the discovery of new medicines. Nature constructs bioactive molecules using enzymes to form C(SP3)-C(SP3) bonds stereoselectively, and some of these enzymes have been harnessed for pharmaceutical synthesis. However, nature offers many synthetically useful enzymes that have not been explored for use in biocatalysis. Here, hydroalkylase enzymes will be developed for synthetic applications to form new intermolecular C(SP3)-C(SP3) bonds from simple, achiral starting materials. Enzymatic hydroalkylation can directly activate C-H bonds without pre-functionalization and has the potential to form up to three contiguous stereocenters in one step. Glycyl radical enzyme (GRE) hydroalkylases can convert C(SP3)-H bonds directly into new C-C bonds using simple amino acid-based radicals. The potential to use GRE hydroalkylases for biocatalysis has been noted for decades, but an inability to install the amino acid-based radicals in vitro severely limited their application. Recently, a platform to overcome this limitation was developed, enabling the generation of amino acid-based radicals within purified enzyme. Theme 1 of this proposal builds on this advance to investigate the native substrate scopes of two GRE hydroalkylases: one that functionalizes toluene and one that functionalizes N-hexadecane. These two wild-type enzymes will be used as starting points to engineer variants through genetic manipulation to broaden substrate scope, enhance selectivity, and discover non-native radical transformations. Theme 2 aims to uncover the molecular mechanisms underlying radical installation, substrate specificity, selectivity, and hydroalkylation. To probe these mechanisms, a range of structural, biochemical, and biophysical methods will be used. These fundamental studies will inform enzyme engineering efforts and deepen current mechanistic understanding of gres, which play key roles in human health due to their abundance in the GUT microbiome. Additionally, gres have significant environmental implications, as they enable microbes to metabolize crude oil. Overall, the themes within this proposal will develop enzymatic methods to convert simple achiral starting materials into complex products in a single step and mechanistically interrogate gres. These goals aim to advance efficient, high- throughput methods for designing SP³-rich, stereochemically defined compounds, expand understanding of gres, and pave the way for breakthroughs in drug discovery and synthetic routes to therapeutically relevant molecules.