Protein bioconjugation is an important technique for various applications such as protein-based therapeutics, imaging, diagnostics, and biochemical investigations. Bioconjugation is a chemical strategy to form a covalent linkage between a substrate and desired functionality. In particular, the creation of a cleavable linkage has drawn significant attention for numerous biological applications, but the formation of a unique chemical linkage on a complex biomolecule is of challenge, especially in aqueous reaction media. The Ohata Lab has been focusing on the development of novel bioconjugation processes in nonaqueous media that would enable chemical reactions of biomolecules unfavored in an aqueous medium. We discovered that a formyl-methyl phosphonium reagent induces a condensation reaction with alkylamine groups of peptides and proteins in ionic liquids, which forges the imine/enamine linkage stable even in aqueous media. The use of ionic liquids is crucial in this process because of the inert nature of the formyl-phosphonium compound in an aqueous environment and the unsuitability of organic solvents for the study of proteins. In congruence with the known inertness of the formyl-phosphonium reagent in aqueous media, the phosphonium-peptide condensation reaction proved sensitive to the concentration of water in the reaction solution. A variety of phosphonium derivatives have been screened such as ketone- or ester-based reagents, and it was identified that the aldehyde-based phosphonium is the only reagent capable of causing this specific chemical transformation. This reaction was also tested on several peptide substrates containing an alkylamine (N-terminus and side chain of lysine residues), demonstrating its broad substrate scope. The current efforts are focused on the sensitivity of the enamine/imine linkage toward various chemical stimuli including biologically relevant small molecules.

Formation of enamine/imine phosphonium linkage on peptides and proteins through ionic liquid-based bioconjugation

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