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

Abstract

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, SO2R) 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-SO2R 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.