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.

Figure 1.

Amine modification reactions and second-order rate constants: (A) first and second amidation of squarate, 1, with benzylamine, (B) acylation of benzylamine with succinimidyl ester 4, and (C) addition of benzylamine to dichlorotriazine 6. Rates are also shown in Table S1.

Figure 2.

Hammett plot from squarates 8a-p (Table S2) reacting with benzylamine in d₆-DMSO to afford derivatives 9a-p. The substitution constant, σ, is plotted with the log of the relative reactivity of a given compound to phenyl-squaramide 8a. ρ = 0.71.

Figure 3.. Compounds used to examine the binding of UDP-derivatives to the glycosyltransferase GlfT2

(A) Native substrate donor, UDP-Galf and analogs: electrophilic squaric ester 12 or dichlorotriazine 13. Model of GlfT2 (PDB: 4FIY) with lysines covalently modified by 12 (B) highlighted in blue and 13 (C) highlighted in gray.

Figure 4.

(A) Relative activity of GlfT2 samples treated with compound 12 at various concentrations and durations. (B) GlFT2 treated with compounds 12 and UMP to obtain 80% inhibition with enzyme activity assessed before and after dialysis.