Negatively Charged Red-Emitting Acridine Dyes for Facile Reductive Amination, Separation, and Fluorescent Detection of Glycans

Capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF) has become a key method in high-throughput glycan analysis. At present, CGE-LIF relies on the green fluorophore 8-aminopyrene-1,3,6-trisulfonic acid ( APTS ). However, APTS has moderate reactivity in labeling of glycans and a fixed selectivity profile. Here, we report synthesis of red-emitting and highly reactive fluorescent tags for glycan derivatization. The design is based on a 9-aminoacridine scaffold with various acceptor groups at C-2 (CN, SO 2 R) and a primary amino group at C-7 for conjugation via reductive amination. These reactive dyes exhibit absorption maxima close to 450 nm and emission above 600 nm. They readily undergo conjugation with reducing sugars at the desired 1:1 stoichiometry. The red emission of conjugates with a maximum at 610-630 nm can be observed under excitation with 488 nm light and detected separately from the APTS -labeled oligosaccharides. Phosphorylated 7,9-diaminoacridine-2-SO 2 R derivatives with variable amounts of negative charges provide high mobilities of glycoconjugates on polyacrylamide gel electrophoresis (PAGE), as compared with those of APTS . We further demonstrate their utility by labeling and separating a maltodextrin ladder and sialyllactose isomers. The new dyes are expected to cross-validate and increase the glycan identification precision in CGE-LIF and help to reveal "heavy" glycans, yet undetectable with the APTS label.