Highly Reactive and Tracelessly Cleavable Cysteine‐Specific Modification of Proteins via 4‐Substituted Cyclopentenone

Abstract

Ar apid and cysteine-specific modification of proteins using 4-substituted cyclopentenone via aM ichael addition tandem elimination reaction was developed. Compared to the classical method, this reaction featured fast kinetics with as table product. More importantly,t his conjugation could be tracelessly removed by exchange with aM ichael addition donor.T he conjugation and regeneration process not only exhibited little change to the structures or conformations of the proteins but also exhibited little disturbance to their biological functions,s uch as their enzymatic activities.

Thia-Michael addition: the route to promising opportunities for fast and cysteine-specific modification

“…The original protein can be released with a Michael addition donor demonstrating 4-substituted cyclopentenones as traceless, removable and valuable bioconjugation tools for the controlled release of a conjugate. 80 5-Methylene pyrrolones 80 are highly cysteine-specific thia-Michael acceptors that afford stable conjugates at neutral pH although they are cleavable in alkaline buffer ( pH 9.5) or via thiol exchange at pH 7.5. Zhou et al introduced cysteine specific and tracelessly removable bioconjugation via Michael addition to 5-methylene pyrrolones.…”

“…The original protein can be released with a Michael addition donor demonstrating 4-substituted cyclopentenones as traceless, removable and valuable bioconjugation tools for the controlled release of a conjugate. 80…”

Protein Modification at Tyrosine with Iminoxyl Radicals

“…Reversible SPMs, mimicking biochemical PTMs, however, remain less explored. Examples of reversible SPMs have been mostly limited to Michael addition/retro-Michael reaction [26][27][28][29][30][31][32][33][34] or alkylation/dealkylation at Cys [35][36][37] ; nevertheless, this chemistry has recently expanded to His 38 (Figure 1a). The use of a pHsensitive linker 39,40 and dynamic imine formation [41][42][43][44][45][46][47] at Lys have been demonstrated more recently, the latter of which was extended to N-terminal Cys modifications 48,49 (Figure 1b).…”

Chemoselective Copper-Mediated Radical Modification of Selenocysteines in Peptides and Proteins

“…[32][33][34][35][36][37][38][39] Similar to Cys, Sec's inherent nucleophilicity makes it a good target for chemical modification in peptides and proteins. 9,11,15,[40][41] Meanwhile, Sec is readily oxidized to diselenide (Se-Se) or selenylsulfide (Se-S), hence a reducing reagent, such as DTT or tris(2carboxyethyl)phosphine (TCEP), is typically needed to generate free selenol in aerobic conditions (Fig. 1a).…”