Reaction conditions for conjugation of two fluorescent ortho-substituted aniline derivatives, 2-amino benzamide (2-AB) and 2-anthranilic acid (2-AA), to N- and O-glycans have been investigated. Conjugation conditions for attaching 2-AB and 2-AA to core-fucosylated and non-fucosylated glycans were developed using complex N-glycans radiolabeled at the nonreducing terminus with [3H]C6-galactose. Optimal conditions for each of the following reaction parameters were experimentally defined: [glycans], [2-AB] or [2-AA], solvent and acid composition, temperature and time of Schiff's base formation, nature of reductant, and temperature and time of reduction. Using the optimized reaction conditions it has been shown with several standard glycans and glycoprotein-derived glycan libraries that (i) molar labeling efficiencies are high and essentially independent of the amount of glycans; (ii) negligible (< 2 mol%) desialylation occurs during conjugation; (iii) glycan labeling is nonselective, i.e., independent of glycan structure; and (iv) insignificant fluorescent or chemical "blank" is recovered during the glycan-labeling and purification protocol. Labeling glycan pools with 2-AB or 2-AA therefore allows representative glycan profiles to be obtained and also allows relative molar quantitation of individual glycans in a pool. The 2-AB label is compatible with several chromatographic means for separation of carbohydrates including Bio Gel P4 gel permeation, high-performance anion-exchange chromatography with fluorescence detection, and a variety of HPLC procedures, as well as with mass spectrometric methods including matrix-assisted laser desorption-mass spectrometry and electrospray-mass spectrometry. The 2-AA label is particularly well-suited for electrophoretic separations by polyacrylamide gel electrophoresis. These fluorophores show high intrinsic sensitivity and thus facilitate very sensitive analysis of protein glycosylation.

中文翻译：

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使用2-氨基苯甲酰胺和邻氨基苯甲酸的聚糖的非选择性和高效荧光标记

研究了将两种荧光邻位取代苯胺衍生物2-氨基苯甲酰胺（2-AB）和2-邻氨基苯甲酸（2-AA）与N-和O-聚糖缀合的反应条件。使用在非还原末端用[3H] C6-半乳糖放射性标记的复合N-聚糖，开发了将2-AB和2-AA与核心岩藻糖基化和非岩藻糖基化聚糖连接的结合条件。实验确定了以下每个反应参数的最佳条件：[聚糖]，[2-AB]或[2-AA]，溶剂和酸组成，席夫碱形成的温度和时间，还原剂的性质以及温度和时间减少。使用优化的反应条件，已经证明了几种标准的聚糖和糖蛋白衍生的聚糖文库：（i）摩尔标记效率很高，并且基本上与聚糖的量无关；（ii）在缀合过程中发生的微不足道的（<2 mol％）脱唾液酸化作用；（iii）聚糖标记是非选择性的，即不依赖于聚糖结构；（iv）在聚糖标记和纯化方案中回收了微不足道的荧光或化学“空白”。因此，用2-AB或2-AA标记聚糖库可以得到代表性的聚糖谱，并且还可以对库中的各个聚糖进行相对摩尔定量。2-AB标签与多种用于分离碳水化合物的色谱方法兼容，包括Bio Gel P4凝胶渗透，高效的阴离子交换色谱，具有荧光检测，多种HPLC程序以及质谱分析方法，包括基质辅助激光解吸质谱和电喷雾质谱。2-AA标记特别适合通过聚丙烯酰胺凝胶电泳进行电泳分离。这些荧光团显示出很高的内在敏感性，因此有助于对蛋白质糖基化进行非常灵敏的分析。