Efficient Protein–Protein Couplings Mediated by Small Molecules under Mild Conditions

Abstract Protein–protein coupling reactions under physiological conditions that do not impact the three‐dimensional structures of the proteins are in high demand. Owing to the combination of phenylsulfonyl and aldehyde groups in 5‐fluoro‐4‐(phenylsulfonyl)picolinaldehyde (FPPA), the fluorine substituent shows high reactivity toward free thiols. In FPPA, the fluorine is more reactive than phenylsulfonyl for free thiols. Thus the first quantitative nucleophilic substitution can be followed by selective substitution of phenylsulfonyl by an additional thiol or cyclization of aldehyde with a 1,2‐aminothiol molecule. The FPPA mediated protein–protein coupling proceeds efficiently under mild conditions, resulting in stable protein conjugates. This coupling method has negligible 3D structural perturbations on the target proteins, and it produces overall intact, nearly traceless, and native structural folds of proteins. It is highly suitable for reconstruction of proteins that are difficult to make and segmental isotopic labeling of multidomain proteins.