Copper(II)-mediated N-terminal modification of proteins with maleimide derivatives

Maleimide derivatives are privileged reagents for the chemical modification of proteins via the Michael addition of cysteine due to their selectivity, reaction rate, and commercial availability. Since accessible free cysteine is rarely found in natural proteins, an alternative target for maleimide derivatives is highly desirable for direct bioconjugation. In this study, we developed a copper(II)-mediated [3+2] cycloaddition reaction with maleimide and 2-pyridinecarboxaldehyde (2-PC) derivatives as an operationally simple and powerful method for the N-terminal modification of peptides and proteins. This method utilizes commercially available maleimides to attach diverse functionalities to various N-terminal amino acids. The combined use of copper(II) ions and heteroaromatic aldehydes is key to the selective formation of azomethine intermediates at the N-terminus in aqueous media under mild conditions, achieving high N-terminal selectivity. We demonstrate the preparation of a ternary protein complex cross-linked at the N-termini and dually modified trastuzumab equipped with monomethyl auristatin E (MMAE), a cytotoxic agent, and a Cy5 fluorophore (MMAE–Cy5–trastuzumab). The MMAE–Cy5–trastuzumab retained human epidermal growth factor receptor 2 (HER2) recognition activity and exerted cytotoxicity against HER2-positive cells. Furthermore, MMAE–Cy5–trastuzumab helped successfully visualize HER2-positive cancer cells in mouse tumors. This straightforward method will enhance the accessibility and utility of protein conjugates with defined structures across a diverse spectrum of researchers.