Chatterjee D, Cho S N, Stewart C, Douglas J T, Fujiwara T, Brennan P J
Department of Microbiology, Colorado State University, Fort Collins 80523.
Carbohydr Res. 1988 Dec 1;183(2):241-60. doi: 10.1016/0008-6215(88)84078-3.
The trisaccharide segment, O-(3,6-di-O-methyl-beta-D-glucopyranosyl)-(1----4)-O-(2,3-di-O-methyl- alpha-L-rhamnopyranosyl)-(1----2)-3-O-methyl-L-rhamnopyranose, of the Mycobacterium leprae-specific phenolic glycolipid I has been synthesized as its 8-(methoxycarbonyl)octyl glycoside and coupled to a carrier protein, to produce a leprosy-specific neoglycoprotein, the so-called natural trisaccharide-octyl-bovine serum albumin (NT-O-BSA). Special features of the synthetic strategy were the use of silver trifluoromethanesulfonate (triflate) to promote glycosylation, resulting in the rhamnobiose in high yield and absolute stereospecificity. The terminal 3,6-di-O-methyl-D-glucopyranosyl group was introduced after O-deallylation of the rhamnobiose. Removal of protecting groups yielded the trisaccharide hapten suitable for coupling to carrier protein. Poly(acrylamide)-gel electrophoresis of the neoglycoprotein demonstrated its purity, and subsequent immunoblotting with a monoclonal antibody directed to the terminal 3,6-di-O-methyl-beta-D-glucopyranosyl epitope of the native glycolipid demonstrated its antigenicity. Comparative serological testing in enzyme-linked immunosorbent assays of NT-O-BSA, the corresponding disaccharide-containing products, and another trisaccharide-containing neoglycoprotein, O-(3,6-di-O-methyl-beta-D-glucopyranosyl)-(1----4)-O-(2,3-di-O- methyl-alpha-L-rhamnopyranosyl)-(1----2)-(3-O-methyl-alpha-L-rhamnopy ran osyl)- (1----4')-oxy-(3-phenylpropanoyl)-BSA (NT-P-BSA) [Fujiwara et al., Agric. Biol. Chem., 51 (1987) 2539-2547] against sera from leprosy patients and control populations showed concordance; the presence of the innermost sugar did not contribute significantly to sensitivity or specificity. The di- and tri-saccharide-containing neoantigens, on account of ready availability and solubility, provide greater flexibility than the native glycolipid for the serodiagnosis of leprosy.
麻风分枝杆菌特异性酚糖脂I的三糖片段，即O-(3,6-二-O-甲基-β-D-吡喃葡萄糖基)-(1→4)-O-(2,3-二-O-甲基-α-L-鼠李吡喃糖基)-(1→2)-3-O-甲基-L-鼠李吡喃糖，已被合成为其8-(甲氧羰基)辛基糖苷，并与载体蛋白偶联，以产生一种麻风特异性新糖蛋白，即所谓的天然三糖-辛基-牛血清白蛋白(NT-O-BSA)。合成策略的特点是使用三氟甲磺酸银(三氟甲磺酸盐)促进糖基化，从而以高产率和绝对立体特异性得到鼠李二糖。在对鼠李二糖进行O-去烯丙基化后引入末端3,6-二-O-甲基-D-吡喃葡萄糖基。去除保护基得到适合与载体蛋白偶联的三糖半抗原。新糖蛋白的聚丙烯酰胺凝胶电泳证明了其纯度，随后用针对天然糖脂末端3,6-二-O-甲基-β-D-吡喃葡萄糖基表位的单克隆抗体进行免疫印迹，证明了其抗原性。在酶联免疫吸附试验中对NT-O-BSA、相应的含二糖产物以及另一种含三糖的新糖蛋白O-(3,6-二-O-甲基-β-D-吡喃葡萄糖基)-(1→4)-O-(2,3-二-O-甲基-α-L-鼠李吡喃糖基)-(1→2)-(3-O-甲基-α-L-鼠李吡喃糖基)-(1→4')-氧基-(3-苯丙酰基)-BSA(NT-P-BSA)[藤原等人，《农业生物化学》，51(1987)2539-2547]针对麻风病人血清和对照人群进行的比较血清学检测显示结果一致；最内层糖的存在对敏感性或特异性没有显著贡献。由于易于获得和溶解，含二糖和三糖的新抗原在麻风病血清诊断方面比天然糖脂具有更大的灵活性。
