Optimized conditions for 2-aminobenzamide labeling and high-performance liquid chromatography analysis of N-acylated monosaccharides.

The monosaccharides GlcNAc (N-acetylglucosamine) and the home-made GlcNC16 (N-palmitoyl-D-glucosamine) were labeled with 2-AB (2-aminobenzamide) by reductive amination of the sugar. The aldehyde group of the monosaccharide reacts with the amino group of 2-AB, forming a Schiff base. In the second step, the Schiff base is reduced with sodium cyanoborohydride to yield a stable secondary amine. We describe here a simple and fast procedure. Previous studies reported the same labeling at high concentration (10−1 M) during 30 h with further purification steps. In the present paper all operations were carried out in an Eppendorf tube and the reaction medium was directly analyzed without purification. Using the described protocol, the whole procedure can be accomplished in less than 6 h at 65°C at very low concentration (10−4 M). For both GlcNC16 and GlcNAc, the 2-AB labeling conditions were optimized and, in addition, new conditions of high-performance liquid chromatography analysis were developed. These N-alkylated sugars were analyzed on reversed-phase HPLC with fluorimetric detection at excitation and emission wavelengths of 340 and 400 nm, respectively. The separation was achieved on a C18 column with a gradient mobile phase composed of water (0.1% formic acid)–methanol (volume varying) in less than 19 min with 12.5 and 18.3 min retention times for GlcNAc and GlcNC16, respectively. Positive-ion electrospray ionization mass spectrometry (ESI-MS) analysis enabled their structural determination. Copyright © 2009 John Wiley & Sons, Ltd.