Rapid glycan labelling with fluorogenic molecular rotor dyes and nucleic acid stains responding to changes of the cell surface

Herein, we introduce a dual-labelling technique for covalent modification of cell surfaces with fluo-rescent molecular rotor (FMR) dyes and environmentally insensitive fluorescent dyes. For this pur-pose, we combined an improved method for chemical reactions on sialic acid residues with FMR-dye hydrazides including dyes commonly used to stain nucleic acids. Fluorescence of FMR dyes increases upon molecular crowding and with increasing viscosity providing options for fluorogenic labelling of cell surfaces and detecting changes of cell surfaces that, for example, affect molecular crowding or local viscosity. The method involves mild periodate oxidation of cell surface glycans followed by or-ganocatalyzed dye conjugation. Both steps are performed within a total of 15 min at 25 °C, avoiding cold stress of cells. We show labelling glycans with FMR dyes such as 9-(2-carboxy-2-cyanovinyl)julolidine (CCVJ) or Cyanine 3 (Cy3) and the nucleic acid stain thiazole orange (TO) pro-vides sufficiently strong signals to allow fluorescence microscopy imaging of surfaces of A549, HeLa and CEM cells. Measurements with mucin suggest that the close proximity to glycans induced upon formation of covalent bonds is very efficient in hindering FMR torsional motions. We demonstrate that the fluorescence of CCVJ and TO on cell surface glycans and isolated mucin is influenced by the action of the disulfide-cleaving agent TCEP. Co-labelling of cells with CCVJ and an environmentally insensitive AF568 dye revealed a TCEP-induced decrease in the intensity CCVJ fluorescence, which did not occur when the dye was studied in glycerol solution, consistent with a decrease of the fluores-cence lifetime determined by fluorescence lifetime imaging microscopy (FLIM). This indicates that glycan-bound FMR dyes respond to changes in friction that hinder dye intramolecular rotation, poten-tially enabling the detection of changes occurring to surfaces of epithelial cells during disease and therapeutic intervention.