Assessing Squarates as Amine-Reactive Probes

Abstract

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 bioconjugation handles. The data revealed that N-hydroxy succinimidyl esters react 4 orders of magnitude faster, consistent with their labeling promiscuity. Squarate reactivity can be tuned by a substitution pattern. Electron-withdrawing groups on the vinylogous ester or amide increase reaction rates. Dithionosquarates react more rapidly than squarates, while vinylogous thioester analogs, dithiosquarates, react more slowly. We assessed squarate selectively using the UDP-sugar processing enzyme GlfT2 from Mycobacterium tuberculosis, which possesses 21 surface-exposed lysines. The reaction predominately modified one lysine proximal to a binding site to afford covalent inhibition. These findings demonstrate the selectivity of squaric esters and squaramides, which is a critical feature for affinity-based chemoproteomic probes.

Emerging and Re-emerging Warheads for Targeted Covalent Inhibitors: An Update

“…A very interesting recent development that was published during the revision of this manuscript is the use of tunable squarate-based vinylogous esters or amides for lysine modification …”

Polysquaramides

“…Additionally, tripodal squaramide-based monomers self-assembled into fibrillar supramolecular polymers form self-healing hydrogels under physiological conditions and have been successfully utilized as 3D cell culture media . In addition to their self-assembly ability, squaramides function as effective gelators and covalent probes, as illustrated by their use in forming gelatin-based hydrogels and amine-reactive probes through efficient cross-linking and regioselective reaction of lysine residues with squarates.…”

Ex Situ Gaseous Reagent for Multicomponent Amine Bioconjugation

“…Amine−amine conjugation remains relatively rare as a result of the hetero-cross-linking issues. 16 Among reported examples, a squaric acid diester was used as a linchpin reagent for stepwise coupling of two amino groups (Figure 1a), 18 and orthophthalaldehyde allows for one-pot clamping of two amines (Figure 1b). 19 However, new methods of amine−amine crosslinking would expand the bioconjugation toolbox and provide new opportunities for the construction of complex bioconjugates.…”