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, 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.

Fluorescence Assisted Capillary Electrophoresis of Glycans Enabled by the Negatively Charged Auxochromes in 1‐Aminopyrenes

“…For the PSU dye, that would enable to collect more light, which is now distributed between green and yellow detectors, and probably fully avoid the cross‐talk with the APTS detection window. If so, PSU or acridine dyes [15] could be used not only as brighter or “rapid” (swifter moving) alternatives to APTS (which is now possible), but also for creating new internal standards for glycan analysis based on natural carbohydrates and compatible with APTS in one run.…”

“…The bright and negatively charged fluorescent dyes suitable for reductive amination of carbohydrates are rare. Rhodamines and cyanines decolorize in reduction with borohydrides; [14] 2,9‐diaminoacridines [15] and 2‐aminoacridones [16] have been applied successfully but proved to be rather “dark”; with large Stokes shifts, low absorption and low fluorescence quantum yields. The positively charged aminopyrene sulfonamides APTMP and APTDP (Figure 1) have been reported very recently [17] .…”

Liquid chromatography and capillary electrophoresis in glycomic and glycoproteomic analysis

“…Fomin et al [ 161 ] introduced new fluorescent dyes for capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF) analysis of glycans. The reactive dyes were based on phosphorylated 7,9-diaminoacridine derivatives with variable number of negative charges and the derivatized analytes showed higher mobilities then the APTS-labeled ones.…”

Synthesis of new 1H‐spiro[acridine‐9,3′‐indoline]‐1,2′(2H,10H)‐dione derivatives using aqueous ethanol as a reaction medium

“…This has also been used in reductive amination, separation, fluorescent detection of glycan, etc. [22] Reviews on the discovery of newer spiroacridine-based compounds, their properties, and uses as therapeutic agents are available in the literature [23,24]. The cyclocondensation reactions for the synthesis of spirocompounds involving Knoevenagel and Michael reactions with isatin related compounds as starting material are reported [25][26][27][28].…”