Maury Delphine, Couderc François, Czaplicki Jerzy, Garrigues Jean Christophe, Poinsot Véréna
Laboratoire des Interactions Moléculaires et Réactivités Chimique et Photochimique, 118 route de Narbonne, Toulouse cedex 09, France.
Biomed Chromatogr. 2010 Apr;24(4):343-6. doi: 10.1002/bmc.1299.
The monosaccharides GlcNAc (N-acetylglucosamine) and the home-made GlcNC(16) (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 degrees C at very low concentration (10(-4) M). For both GlcNC(16) 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 C(18) 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.
单糖N-乙酰葡糖胺（GlcNAc）和自制的N-棕榈酰-D-葡糖胺（GlcNC(16)）通过糖的还原胺化反应与2-氨基苯甲酰胺（2-AB）进行标记。单糖的醛基与2-AB的氨基反应，形成席夫碱。在第二步中，席夫碱用氰基硼氢化钠还原，生成稳定的仲胺。我们在此描述一种简单快速的方法。先前的研究报道在高浓度（10⁻¹ M）下进行30小时的相同标记，并伴有进一步的纯化步骤。在本文中，所有操作均在微量离心管中进行，反应介质无需纯化即可直接分析。使用所描述的方案，整个过程在65℃下以非常低的浓度（10⁻⁴ M）可在不到6小时内完成。对于GlcNC(16)和GlcNAc，优化了2-AB标记条件，此外，还开发了高效液相色谱分析的新条件。这些N-烷基化糖在反相高效液相色谱上进行分析，荧光检测的激发波长和发射波长分别为340和400 nm。在C₁₈柱上进行分离，流动相为水（0.1%甲酸）-甲醇（体积可变）的梯度洗脱，分离时间不到19分钟，GlcNAc和GlcNC16的保留时间分别为12.5和18.3分钟。正离子电喷雾电离质谱（ESI-MS）分析能够确定它们的结构。
