Engineered aldehyde dehydrogenases for amide bond formation.

Amide bond formation is widely used in pharmaceutical synthesis, typically involving stoichiometric coupling reagents to activate carboxylic acid substrates for a condensation reaction. As an alternative approach, we repurposed aldehyde dehydrogenases into oxidative amidases by creating a more hydrophobic and spacious catalytic pocket for amines to capture the thioester intermediate. This biocatalyst efficiently facilitates the formation of amide bonds between diverse aldehydes and amines. We also developed a two-step enzymatic cascade to synthesize amides from broadly available aliphatic alcohols. This biocatalytic strategy enabled the redesign of synthetic routes for five drug molecules. Our findings highlight the potential of oxidative amidases in advancing the synthesis of structurally diverse drug molecules through efficient amide bond formation. Editor's summary: Amides are a fundamental functional group in biochemistry and are found in many natural products and synthetic drugs. Coupling reactions to form amides typically rely on activation of carboxylic acids to serve as electrophiles with amine nucleophiles, but aldehydes could be used instead as electrophiles if coupled to an oxidation. Gao et al. identified aldehyde dehydrogenases with suitable initial activity and engineered them for greater efficiency. The authors achieved amide couplings directly for the more stable aromatic aldehydes or in a cascade reaction for aliphatic alcohols and demonstrate several alternative syntheses of drug molecules. —Michael A. Funk INTRODUCTION: Amide bonds are among the most common structural motifs in pharmaceuticals. One-hundred seventeen out of the top 200 small-molecule drugs by retail sales in 2023 contain at least one amide bond. Half of the chemical transformations used in medicinal and process chemistry focus on amide coupling reactions in drug development. However, conventional chemical synthesis typically relies on activated carboxylic acid derivatives produced using stoichiometric or large excess coupling reagents. These strategies generate considerable waste, limit substrate scope, and compromise atom economy. Developing more efficient and sustainable methods for amide synthesis is, therefore, highly desirable. In this work, we repurposed aldehyde dehydrogenases (ALDHs) into oxidative amidases (OxiAms), which directly form an amide by coupling aldehydes and amines through oxidative amidation. RATIONALE: ALDHs oxidize aldehydes to carboxylic acids through a thioester intermediate. We proposed that, by protein engineering, we might be able to intercept this reactive intermediate with amines before hydrolysis, thereby yielding amides directly. Enabling this chemistry requires redesigning the catalytic pocket. Guided by x-ray structural analysis, several conserved amino acid residues in the binding pocket were substituted to create a more spacious, hydrophobic environment, thereby enhancing amine binding and reactivity. RESULTS: The engineered OxiAms catalyze the formation of amide bonds between structurally diverse aldehydes and amines under mild aqueous conditions, using dioxygen as the sole oxidant. X-ray crystallography confirmed active site remodeling that accommodates amines and stabilizes the amidation pathway. OxiAms displayed broad substrate scope and high selectivity, producing amides as the predominant products. Numerous common functional groups are tolerated in this biocatalytic amide bond–forming reaction, including free alcohols, ketones, carboxylic acids, esters, amides, and olefins. Furthermore, coupling OxiAms with alcohol dehydrogenase enabled a two-step enzymatic cascade that directly converts alcohols into amides in one pot. The application of this approach to five amide-containing pharmaceutical molecules demonstrated alternative synthetic routes with improved atom and step economy compared with that of conventional methods. This strategy proved general across multiple ALDHs, highlighting the broad evolutionary accessibility of OxiAms in widely distributed ALDHs. CONCLUSION: Repurposing ALDHs yielded OxiAms as efficient biocatalysts for amide bond formation. This strategy provides a green, versatile platform for synthesizing structurally diverse amides, highlighting the potential of enzyme engineering to advance pharmaceutical manufacturing through biocatalytic synthesis. This work developed a protein engineering strategy to convert ALDHs into OxiAms that catalyze amide bond formation between diverse alcohols or aldehydes and amines.: Single-letter abbreviations for the amino acid residues referenced throughout the figure are as follows: E, Glu; T, Thr; R, Arg; L, Leu; G, Gly; M, Met. [ABSTRACT FROM AUTHOR]