Quaternization of Vinyl/Alkynyl Pyridine Enables Ultrafast Cysteine‐Selective Protein Modification and Charge Modulation

Abstract

Quaternized vinyl‐ and alkynyl‐pyridine reagents were shown to react in an ultrafast and selective manner with several cysteine‐tagged proteins at near‐stoichiometric quantities. We have demonstrated that this method can effectively create a homogenous antibody–drug conjugate that features a precise drug‐to‐antibody ratio of 2, which was stable in human plasma and retained its specificity towards Her2+ cells. Finally, the developed warhead introduces a +1 charge to the overall net charge of the protein, which enabled us to show that the electrophoretic mobility of the protein may be tuned through the simple attachment of a quaternized vinyl pyridinium reagent at the cysteine residues. We anticipate the generalized use of quaternized vinyl‐ and alkynyl‐pyridine reagents not only for bioconjugation, but also as warheads for covalent inhibition and as tools to profile cysteine reactivity.

Modulation of charge transfer by N-alkylation to control photoluminescence energy and quantum yield

“…The compound produced by double N-alkylation displays enhanced photoluminescence quantum yields (PLQYs) and solubilities across a range of solvents compared to acidied quinine sulfate (H 2 Qn$SO 4 ), which is a common PLQY standard for characterizing blue emitters. Overall, this N-alkylation approach represents a simple, robust pathway for tuning the emission and functional properties of Qn and other tertiary amines [23][24][25][26] to impart functional properties such as improved PLQYs and, potentially, TADF emission.…”

Site-selective modification strategies in antibody–drug conjugates

“…7E), 98,99 and N-alkyl vinylpyridine salts (Fig. 7F) 100 and have been used to synthesise ADCs. Detailed reviews on cysteine-targeted protein modification are available elsewhere.…”

The mechanism behind enhanced reactivity of unsaturated phosphorus(v) electrophiles towards thiols

“…1 Selective labeling of cysteine's sulydryl group in the presence of other functional groups can be achieved with various types of reagents, 2 many of which are unsaturated electrophiles. In addition to classical maleimides 3 and exocyclic maleimide derivatives, 4 unsaturated carbon-carbon bonds can be activated towards thiol addition by adjacent carbonyl [5][6][7] or sulfonamide groups, 8 electron-withdrawing nitro 9 or tosyl groups, 10,11 via pyrimidine 12 and quaternary pyridinium 13 substituents or by means of ring-strain. 14,15 The phosphoryl (P]O) moiety is another electronwithdrawing moiety that can induce thiol addition to an adjacent vinyl or ethynyl group.…”