Modular CuAAC-Based Synthesis of <i>C</i> -Functional Cyclam-Picolinate Chelators for Antibody Conjugation

A modular strategy was developed for the bifunctionalization of TE1PA, a cyclam-based copper chelator used in nuclear medicine, via Cu(I)-catalyzed Azide-Alkyne Cycloaddition (CuAAC) and thiol-maleimide chemistry, to enable site-selective bioconjugation. An alkyne-functionalized TE1PA derivative was synthesized in high yield using a Boc/Teoc-protected intermediate and was subsequently coupled to azido-PEG₃-biotin and azido-PEG<i>_n</i>-maleimide linkers (<i>n</i> = 3 or 6), affording triazole-linked conjugates in 50 to 82% overall yields. The maleimide-TE1PA derivatives were conjugated to trastuzumab via cysteine residues, yielding well-defined immunoconjugates with chelator-to-antibody ratios (CARs) ranging from 2.2 to 3.8 and satisfactory yields (59-78%). In contrast, analogous conjugates prepared from the conventional <i>p-</i>NH₂-Bn-TE1PA using peptide coupling showed lower CARs (0.9-1.8) and lower isolated yields (7-14%). The influence of linker length and structure was evaluated, revealing that PEG₆ chains and triazole-containing linkers provided higher conjugation efficiency. The results demonstrate the advantage of CuAAC-based bioconjugation for generating TE1PA-trastuzumab conjugates and support the use of these new chelators for copper-64/copper-67 theranostic applications.