Warner T G
Department of Pediatrics, University of Tennessee, Memphis 38163.
Biochem Biophys Res Commun. 1987 Nov 13;148(3):1323-9. doi: 10.1016/s0006-291x(87)80277-2.
A photoreactive, potent, competitive inhibitor of the human lysosomal neuraminidase in cultured skin fibroblasts has been prepared. The starting material, 2,3 dehydro-N-acetyl neuraminic acid methyl ester, was selectively tosylated at the C-9 position with tosyl chloride and subsequently peracetylated with acetic anhydride. The tosyl group was displaced with potassium thio acetate in dimethylformamide at 60 degrees C for 80 min. 4-fluoro-3-nitrophenylazide was incorporated by reaction with the thio acetate product and equimolar sodium methoxide in methanol followed by reacetylation. Base hydrolysis gave the final product, 9-S-(4-azido-2-nitrophenyl)-5-acetamido-2,6 anhydro-2,3,5,9-tetradeoxy-9-thio-D-glycero-D-galacto-non-2-enonic acid (W5). The yields at each step were 50-70%. Competitive inhibition kinetics were observed when W5 was tested with the fibroblast neuraminidase using 4-methylumbelliferyl-N-acetyl-neuraminic acid as substrate giving an apparent Ki of about 10 microM. These results suggest that the terminal hydroxyl group at C-9 may not be important in the recognition and binding of the substrate by the enzyme. Also, the compounds prepared here may be useful as photoaffinity probes or ligands for affinity chromatography for purification.
已制备出一种对培养的皮肤成纤维细胞中的人溶酶体神经氨酸酶具有光反应性、强效且具有竞争性的抑制剂。起始原料2,3-脱氢-N-乙酰神经氨酸甲酯在C-9位用对甲苯磺酰氯进行选择性甲苯磺酰化，随后用乙酸酐进行全乙酰化。在60℃下，对甲苯磺酰基在二甲基甲酰胺中被硫代乙酸钾取代80分钟。通过与硫代乙酸产物和等摩尔甲醇钠在甲醇中反应，然后再乙酰化，引入4-氟-3-硝基苯叠氮化物。碱水解得到最终产物9-S-(4-叠氮基-2-硝基苯基)-5-乙酰氨基-2,6-脱水-2,3,5,9-四脱氧-9-硫代-D-甘油-D-半乳糖-壬-2-烯酸（W5）。每一步的产率为50-70%。当以4-甲基伞形酮基-N-乙酰神经氨酸为底物，用W5对成纤维细胞神经氨酸酶进行测试时，观察到竞争性抑制动力学，表观Ki约为10 microM。这些结果表明，C-9位的末端羟基在酶对底物的识别和结合中可能不重要。此外，这里制备的化合物可用作光亲和探针或用于亲和色谱纯化的配体。
