Unexpected Reactivityof 1,3,4-Thiadiazole- N -oxides withStrained Alkynes Enables Enhanced AntibodyLoading

Abstract Protein bioconjugates are at the forefront of precision medicine, enabling the design of highly selective drugs for diagnostic and therapeutic applications. While increasing the number of payloads attached to a protein is a proven strategy to enhance the potency of a bioconjugate, the introduction of additional conjugation sites often risks compromising the protein’s targeting properties. In this study, we investigate the potential of 1,3,4-thiadiazole-N-oxides (TNO), a heterocyclic scaffold exhibiting excellent stability in biological media. We demonstrate that this reactive partner undergoes an unprecedented double addition reaction with bicyclo[6.1.0]non-4-yne derivatives. This unique reactivity is harnessed to achieve the site-specific attachment of two probes per conjugation site on a monoclonal antibody. Importantly, this strategy preserves antibody targeting properties, as confirmed by in vivo imaging studies. These findings establish TNO and the Strain-Promoted Alkyne-1,3,4-Thiadiazole-N-Oxide coupling (SPATOC) reaction as valuable new tools in the bioconjugation arsenal, expanding the possibilities for precise and efficient protein modification.