Programmable amide bond formation using de novo ligases

Amide bond formation is of central importance in the chemical industry, underpinning the synthesis of small bioactive molecules, macrocycles, peptides and their conjugates. Conventional methods of amide synthesis rely on stoichiometric coupling agents and costly protecting group manipulations. While biocatalysis can alleviate some of these challenges, enzymes typically operate across a narrow range of substrates and their adaptation to new coupling partners requires extensive engineering. Here we show that de novo amide ligases featuring a cysteine catalytic triad can be programmed computationally to operate across diverse substrate classes with high selectivity. We first designed complementary peptide ligases to selectively functionalise either the N-terminus or a single lysine residue of a polypeptide sequence containing 36 competing lysines. Crystal structures of both ligases show excellent agreement with the designs, with structural modelling suggesting that amidation outcompetes hydrolysis because the catalytically competent geometry forms only upon acceptor binding. We then extended our approach to the synthesis of small bioactive molecules by designing stereoselective ligases that mediate a key amide bonding forming step in the synthesis of the preventative HIV therapeutic lenacapavir. These results establish de novo enzyme design as a programmable platform for achieving selective amide bond formations across a wide range of molecular targets.

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Publication Details

Journal
bioRxiv (Cold Spring Harbor Laboratory)
Published
2026-11-11
DOI
https://doi.org/10.64898/2026.11.08.765178
Primary Topic
Enzyme Catalysis and Immobilization
Type
preprint
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preprint

Programmable amide bond formation using de novo ligases

Timo Flügel, Asim K. Bera, Kieran Didi, Andrew C. Hunt et al.
bioRxiv (Cold Spring Harbor Laboratory)
Enzyme Catalysis and Immobilization
preprint

Programmable amide bond formation using de novo ligases

Timo Flügel, Asim K. Bera, Kieran Didi, Andrew C. Hunt, Luke D. Lavis, Donald Hilvert, Maximilian Gantz, Alex Kang, David A. Baker, Sambashiva Banala, Anthony P. Green, Belen Sotomayor, Rafael I. Brent, Johnny Mendoza, H. Jones, Stanislav S. Moroz, Nathaniel Corley, Saman Salike, Maxwell W. Kazman, Jasper Butcher, Seth Michael Woodbury, Xinting Li, Hannah Nguyen, Zefei Xu, Abhishek Singh, Christina Savvides
preprint en

Abstract

Amide bond formation is of central importance in the chemical industry, underpinning the synthesis of small bioactive molecules, macrocycles, peptides and their conjugates. Conventional methods of amide synthesis rely on stoichiometric coupling agents and costly protecting group manipulations. While biocatalysis can alleviate some of these challenges, enzymes typically operate across a narrow range of substrates and their adaptation to new coupling partners requires extensive engineering. Here we show that de novo amide ligases featuring a cysteine catalytic triad can be programmed computationally to operate across diverse substrate classes with high selectivity. We first designed complementary peptide ligases to selectively functionalise either the N-terminus or a single lysine residue of a polypeptide sequence containing 36 competing lysines. Crystal structures of both ligases show excellent agreement with the designs, with structural modelling suggesting that amidation outcompetes hydrolysis because the catalytically competent geometry forms only upon acceptor binding. We then extended our approach to the synthesis of small bioactive molecules by designing stereoselective ligases that mediate a key amide bonding forming step in the synthesis of the preventative HIV therapeutic lenacapavir. These results establish de novo enzyme design as a programmable platform for achieving selective amide bond formations across a wide range of molecular targets.

bioRxiv (Cold Spring Harbor Laboratory)
Howard Hughes Medical Institute (US), Janelia Research Campus (US), University of Washington (US), University of Manchester (GB), ETH Zurich (CH), Laboratory of Organic Chemistry (CH)
Enzyme Catalysis and Immobilization
DOIOpen Access PDFOpenAlex
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