# Development and characterization of biocatalysts mediating Wolff rearrangements

> **NIH NIH F32** · CALIFORNIA INSTITUTE OF TECHNOLOGY · 2020 · $58,964

## Abstract

PROJECT SUMMARY/ABSTRACT
 Enzymes are widely used as biocatalysts during the production of pharmaceuticals and other industrially
important small molecules. In addition to operating under mild reaction conditions, enzymes are highly
selective, generating enantioenriched products and minimizing side-reactions. Directed evolution is a powerful
strategy for biocatalyst development, and recently it has been applied towards the engineering of hemoproteins
to mediate non-natural reactions involving carbenes. However, most of these enzymes form carbenes only
from α-diazoester substrates, limiting their synthetic potential. The focus of this proposal is to expand on the
synthetic and mechanistic scope of hemoproteins by evolving them to catalyze additional non-natural reactions
and to characterize the determinants of chemoselectivity of these enzymes. The specific aims are (1) to evolve
hemoprotein variants capable of catalyzing stereoselective Wolff rearrangements of α-diazocarbonyl
substrates, (2) to synthesize a panel of α-diazocarbonyl compounds and use them to screen for hemoproteins
useful for the production of therapeutically relevant small molecules, and (3) to biochemically characterize the
factors underlying the chemo-, regio-, and enantioselectivity of several different carbene transferases. The
Wolff rearrangement is a synthetically powerful tool, and biocatalysts capable of mediating asymmetric Wolff
rearrangements would be valuable for constructing small molecules important to human health. Screening of
known hemoprotein carbene transferases and directed evolution of new variants will be used to develop
biocatalysts mediating this rearrangement. Syntheses of additional substrates will be performed to accelerate
screening and to tune biocatalysts to act on industrially important small molecules using adaptive evolution.
Furthermore, hemoprotein carbene transferases catalyze a wide range of reactions, and biochemical
characterization and molecular dynamics simulations of these highly chemoselective enzymes will reveal the
underlying features that distinguish these proteins. Identifying the molecular mechanisms influencing selectivity
for these enzymes will empower future engineering efforts for developing biocatalysts catalyzing additional
reactions of biological and biomedical importance.

## Key facts

- **NIH application ID:** 9993008
- **Project number:** 1F32GM134566-01A1
- **Recipient organization:** CALIFORNIA INSTITUTE OF TECHNOLOGY
- **Principal Investigator:** Benjamin Joseph Levin
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $58,964
- **Award type:** 1
- **Project period:** 2020-04-01 → 2021-03-19

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9993008

## Citation

> US National Institutes of Health, RePORTER application 9993008, Development and characterization of biocatalysts mediating Wolff rearrangements (1F32GM134566-01A1). Retrieved via AI Analytics 2026-06-12 from https://api.ai-analytics.org/grant/nih/9993008. Licensed CC0.

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