biostudies-literatureTaylor KINational Institute of Allergy and Infectious DiseasesNIAID NIH HHSNational Institute of General Medical SciencesNIGMS NIH HHSDivision of Graduate Education25056-25060https://www.ebi.ac.uk/biostudies/studies/S-EPMC10935565biostudies-literatureUnknown145(46)Probes that covalently label protein targets facilitate the identification of ligand-binding sites. Lysine residues are prevalent in the proteome, making them attractive substrates for covalent probes. However, identifying electrophiles that undergo amine-specific, regioselective reactions with binding site lysine residues is challenging. Squarates can engage in two sequential conjugate addition-elimination reactions with amines. Nitrogen donation reduces the second reaction rate, making the mono squaramide a mild electrophile. We postulated that this mild electrophilicity would demand a longer residence time near the amine, affording higher selectivity for binding site lysines. Therefore, we compared the kinetics of squarate and monosquaramide amine substitution to alternative amine biocoJournal of the American Chemical SocietyAssessing Squarates as Amine-Reactive Probes.PMC10935565R01 AI126592F31 GM148069AI-R01 1265922019281534Kiessling LLHo JSCarter AWTaylor KITrial HOfalseAssessing Squarates as Amine-Reactive Probes.Probes that covalently label protein targets facilitate the identification of ligand-binding sites. Lysine residues are prevalent in the proteome, making them attractive substrates for covalent probes. However, identifying electrophiles that undergo amine-specific, regioselective reactions with binding site lysine residues is challenging. Squarates can engage in two sequential conjugate addition-elimination reactions with amines. Nitrogen donation reduces the second reaction rate, making the mono squaramide a mild electrophile. We postulated that this mild electrophilicity would demand a longer residence time near the amine, affording higher selectivity for binding site lysines. Therefore, we compared the kinetics of squarate and monosquaramide amine substitution to alternative amine bioco2023-01-01T00:00:00Z2023 Nov2026-06-03T07:25:06.49Z2025-04-06T10:11:24.821ZS-EPMC109355653793880210.1021/jacs.2c05691