Stepwise dual N-terminal modification of proteins via nickel(II)-mediated aldol and Pictet–Spengler reactions

The dual modification of proteins with two distinct artificial functionalities significantly broadens the scope and utility of protein conjugates. To achieve this, it is necessary to perform site-specific and stoichiometrically controlled modifications, for which the N-terminus is a promising target owing to its unique accessibility and chemical reactivity. In this study, a sequential N-terminal dual-modification strategy was developed that combines a nickel(II)-mediated aldol reaction (using imidazo[1,2-a]pyridine-2-carbaldehyde derivatives) with a subsequent Pictet–Spengler reaction (using 2-pyridinecarboxaldehyde derivatives) at the N-terminus. Both transformations employ structurally simple, readily accessible reagents and proceed under mild, non-denaturing aqueous conditions (pH 7 and 5.5, 37 °C). Using this approach, the dual modification of myoglobin and trastuzumab was achieved. The resulting trastuzumab conjugate, bearing Cy5 (a fluorescent dye) and monomethyl auristatin E (a potent cytotoxic agent), retained the ability to recognize human epidermal growth factor receptor 2 (HER2) and exhibited potent cytotoxicity against HER2-positive cancer cell lines. This sequential method established the N-terminus as a versatile alternative to cysteine for dual functionalization, enabling the creation of well-defined multifunctional protein conjugates using simple, modular chemistry.