Development and evaluation of new cyclooctynes for cell surface glycan imaging in cancer cells

Two reagents have been synthesized for selective labeling of cell surface azidoglycans, an unusually stable version of a dibenzocyclooctyne (TMDIBO) and a third-generation difluorinated cyclooctyne (DIFO3). Both syntheses are efficient with minimal purification, and the dibenzocyclooctyne is stable under basic and acidic conditions. Flow cytometric measurements with azidosugar labeled cancer cells, in which these reagents were linked to the fluorophore Alexa Fluor 647, gave a signal-to-background ratio of up to 35 with TMDIBO as compared to ≈10 for DIFO3 and ≈5 for a phosphine reagent. TMDIBO-based probes should have applications in molecular imaging of cell surface glycans in vivo.