Characterization of reactions between water soluble trialkylphosphines and thiol alkylating reagents: implications for protein-conjugation reactions

Water soluble trialkylphosphines such as tris(carboxyethyl)phosphine (TCEP) and trishydroxypropyl phosphine (THPP) are effective agents for reducing disulfide bonds in proteins and are increasingly becoming the reagents of choice for bioconjugation strategies which modify cysteine (thiol containing) amino acids. These reducing agents are often considered as being chemically compatible with thiol-alkylating groups such as maleimides and, as such, are often not removed prior to performing protein conjugation reactions. Here we demonstrate the rapid and irreversible reaction of both TCEP and THPP with derivatives of the commonly employed thiol alkylating groups, maleimide and vinyl sulphone. Mechanistic investigations revealed distinct differences between the reactions of TCEP and THPP with maleimide, leading to the production of either non-productive ylenes or succidimidyl derivatives, respectively. Importantly, we also demonstrate the incorporation of non-productive ylenes formed between maleimide and TCEP into the Pneumococcal capsular polysaccharide Pn6b following strategies employed towards the production of conjugate vaccines.