Aldol reactions using a dual catalytic system of copper and aldehyde catalysts enabled labeling of protein N-termini (Scheme 14A). Site-specific protein labeling methods can be advantageous for producing well-defined protein conjugate compared to chemoselective approaches and are useful for various applications such as single-molecule localization microscopy, 206 preparation of polyethylene glycol-tagged (PEGylated) therapeutic proteins, 207 and production of antibody-drug conjugates. 208 N-terminal amino groups can be attractive sitespecific modification handles, as its decreased basicity compared to lysine amines can be utilized for N-terminal labeling through pH control. While there is a repertoire of N-terminal selective methods to date, Hanaya and co-workers reported a catalytic variant by utilization of copper and aldehyde catalysts activating N-terminal α-proton. The copper-catalyzed aldol reaction was proposed to occur through activation of an N-terminal amino acid by the copper and aldehyde catalysts, forming a nucleophilic Cu(II)-enolate intermediate (Scheme 14B). This nucleophilic activation of the protein would be followed by electrophilic activation of another aldehyde molecule, eventually leading to aldol-type reactions between the activated species to produce the product with a stable C-C bond. Interestingly, in contrast to other acid catalysis earlier, other metal catalysts such as Sc(OTf)3 did not function as effective catalysts for the aldol reaction, perhaps indicating the importance of the subtle control of Lewis acidity and affinity toward certain ligands in this catalytic system. The aldehyde catalyst/reagent (2-pyridinecarboxaldehyde) was previously reported to form a hydrolytically unstable imidazolidinone product on N-termini of proteins, and the catalytic aldol process can be useful to produce more stable reaction products by alteration of the outcome of the aldehyde reaction with N-terminal amines by introduction of the copper catalyst (Scheme 14C). Whereas the hydrolytically unstable N-terminal product can be of use for reversible elimination of the chemical modification in a certain context, 212 this catalytic example represents the importance to develop an alternative approach for production of protein conjugate with a different property. The same research group recently reported another copper(II)-mediated N-terminal modification by leveraging the hydrolytically stable intermediate (Scheme 14A). Kanemoto and co-workers reported copper-catalyzed [3+2] cycloaddition between metalated azomethine ylide (bidentate) on the glycine Nterminus of peptides and maleimides in organic solvents. Hanaya and co-workers expanded the scope of this approach to proteins by the use of pyridyl-aldehydes, allowing reactions in the aqueous buffer through formation of a more stable intermediate, tridentate azomethine ylide (Scheme 14B, 14C). The reaction was applicable to peptides and proteins with various N-terminal amino acid residues. Site-specific modification of an antibody, trastuzumab with this modification method prepared antibodydrug conjugates with uniform drug-antibody ratio (DAR), which was applied for mice cancer models. Scheme 14. Dual catalytic aldol reaction (left) and copper-catalyzed cycloaddition (right). (A) Schematic illustrations of copper-/aldehyde-catalyzed aldol-reaction (left) and copper-catalyzed cycloaddition (right). The pyridyl-aldehyde acts as both the reagent and catalyst for the aldol reaction. (B) Catalysis mechanism of the copper-catalyzed aldol reaction (left) and copper-catalyzed cycloaddition (right). The pyridyl-aldehyde acting as a catalyst is depicted in red and the one as a regent in black. (C) Comparison of reaction products for N-terminal modification by choice of a labeling reagent and catalyst. Top: Alteration of the reaction product by the absence (reversible condensation reaction, 211,212 left) and presence (Irreversible aldol reaction, right) of the copper catalyst. Bottom: Irreversible copper-catalyzed cycloaddition.