KAT ligation: potassium acyltrifluoroborate amide-forming ligation
Definition. KAT ligation is a chemoselective amide-forming reaction between a potassium acyltrifluoroborate (KAT) and an O-substituted hydroxylamine. It proceeds in water with unprotected peptides and requires no coupling reagent, which makes it useful for joining peptide segments, labelling biomolecules, and forming hydrogels.
What is a potassium acyltrifluoroborate (KAT)?
A potassium acyltrifluoroborate is an acylboron compound, R-C(=O)-BF3K, in which a trifluoroborate group is bonded directly to a carbonyl carbon. KATs are typically bench-stable salts. In KAT ligation the KAT serves as the acyl donor that becomes the carbonyl of the new amide bond.
How does KAT ligation form an amide bond?
The KAT reacts with an O-substituted hydroxylamine under aqueous, mildly acidic conditions to give an amide. Because neither partner reacts with the free amines, carboxylic acids, thiols, or alcohols of unprotected peptides, the reaction is chemoselective and needs no protecting groups or activating reagents.
How is KAT ligation used in chemical protein synthesis?
In chemical protein synthesis, peptide segments made by solid-phase peptide synthesis are joined chemoselectively to build proteins too long to make in one piece. KAT ligation provides an amide-forming disconnection that complements native chemical ligation. Its decisive advantage is concentration: whereas native chemical ligation and KAHA ligation need millimolar reactant concentrations, KAT ligation is fast in dilute solution. Schilling, Steiner, and Bode (Science 2026) used it to join segments at 100 to 200 micromolar, with about 50% conversion still reached at 20 micromolar, and assembled the 57-residue apolipoprotein C-I and the aggregation-prone immunoglobulin V domain of human PD-L2.
How can a KAT survive solid-phase peptide synthesis? (the ONNO ligand)
For years the obstacle to protein synthesis by KAT ligation was that a C-terminal KAT could not survive Fmoc solid-phase peptide synthesis: no protecting group masked both its reactive carbonyl and its trifluoroborate while tolerating repeated base and acid treatments. ONNO, a tetradentate ligand introduced by Schilling, Steiner, and Bode (Science 2026), chelates boron to mask the KAT as a bench-stable zwitterionic organoboron complex that survives Fmoc SPPS and trifluoroacetic acid cleavage, and is then removed under mild conditions with the commercial reagent DMPU-HF with full retention of configuration. ONNO can also act as a temporary hydrophilic handle that helps purify difficult hydrophobic segments.
How does KAT ligation compare with other chemoselective ligations?
Native chemical ligation joins a C-terminal thioester to an N-terminal cysteine. Serine/threonine ligation uses C-terminal salicylaldehyde esters and N-terminal serine or threonine. KAHA ligation joins alpha-ketoacids and hydroxylamines. KAT ligation is also amide-forming and hydroxylamine-based, but uses an acylboron acyl donor. Rate constants of several chemoselective ligations have been compared under stoichiometric aqueous conditions (ACS Chem. Biol., reference below).
How are KATs prepared?
One route converts carboxylic acids to KATs by activating them as isobutyl mixed anhydrides, borylating them with bis(pinacolato)diboron under copper catalysis, and treating the product with aqueous KHF2 (Tung, Schuhmacher, Schilling, Bode, Mankad, Angew. Chem. Int. Ed. 2021).
Research by Philipp E. Schilling
References
- Rapid Ligations with Equimolar Reactants in Water with the Potassium Acyltrifluoroborate (KAT) Amide Formation. J. Am. Chem. Soc. doi:10.1021/ja5018442
- Critical Evaluation and Rate Constants of Chemoselective Ligation Reactions for Stoichiometric Conjugations in Water. ACS Chem. Biol. doi:10.1021/cb5006728
- Synthesis of Acyltrifluoroborates. Org. Lett. doi:10.1021/ol300668m
- Synthesis of Biocompatible PEG Hydrogels by pH-Sensitive Potassium Acyltrifluoroborate (KAT) Amide Ligations. ACS Biomater. Sci. Eng. doi:10.1021/acsbiomaterials.5b00145
- Native chemical ligation. Science 1994. doi:10.1126/science.7973629
- Serine/threonine ligation. Proc. Natl. Acad. Sci. USA. doi:10.1073/pnas.1221012110
- KAHA ligation. Angew. Chem. 2006. doi:10.1002/ange.200503991