DEVELOPMENT OF A VEROTOXIN INHIBITOR WlTH ORIENTED CARBOHYDRATES BY USING CARBOSILANE DENDRIMERS

1)具有不同末端官能基数目及不同核心结构之碳矽烷树状大分子之制备。我们开发了第一代和第二代碳硅烷树枝状聚合物，其具有9，18和36个三糖基，糖部分的globotriaosyl神经酰胺，作为功能性末端单元在其结构中（称为SUPER TWIG）。研究了这些化合物对放射性标记的Stx与靶细胞的结合活性和Stx的细胞毒活性的抑制作用。我们发现，在一个分子中存在的至少6个三联体足以使这些SUPER TWIGs对Stx的这些生物活性产生抑制作用。此外，我们发现SUPER TWIGs中存在的三肽的相对距离而不是三肽的数量在与Stx的高亲和力结合中起重要作用。2）SUPER TWIGs的体内抑制作用。在小鼠模型中研究SUPER TWIGs对Stx 2致死率的抑制作用 ...更多信息 其中静脉内施用致死剂量的Stx 2，有或没有每个SUPER TWIG。在本研究开发的所有SUPER TWIGs中，i. e. SUPRER TWIG（0）3、（0）4、（1）4、（1）6、（1）9、（2）18和（2）36（括号中的数字表示代数;第0代、第1代和第2代SUPER TWIG在芯结构中分别具有1个、3个和5个线性硅原子），其具有3个、4个、6个、9个、18个，和36个三聚体中，只有SUPER TWIGs（1）6和（2）18能有效抑制Stx 2的致死性。这两个SUPER TWIG的主要共同特征之一是它们的哑铃形状，其中它们分别在其结构的两端以对称的方式具有3个和9个三角形。另一方面，通过Biacore系统测定的SUPER TWIG（1）4、（1）6、（1）9、（2）18和（2）36与Stx 1和Stx 2的B亚基结合的解离常数（KD值）在类似范围内非常低，表明这些SUPER TWIG以高亲和力与B亚基结合。相反，SUPER TWIG（0）3和（0）4（零代SUPER TWIG）的KD值非常高。这些结果表明，SUPER TWIGs在体内的有效抑制作用不仅需要与Stx的高亲和力结合，而且还需要另一个因素，例如分子的哑铃形状。目前，SUPER TWIGs（1）6和（2）18在体内有效抑制Stx 2致死性的确切机制尚不清楚，我们的研究结果表明，SUPER TWIGs（1）6和（2）18可作为治疗产Stx大肠杆菌感染的药物。coli O 157：H7。少

1) Preparation of carbosilane dendrimers with different numbers of terminal functional unit and different core structures. We developed the first and the second generation of carbosilane dendrimers which have 9, 18 and 36 trisaccharides, sugar moiety of globotriaosyl ceramide, as functional terminal units in their structures (referred to as SUPER TWIG). Inhibitory effects of these compounds on the binding activity of radio-labeled Stx to target cells and on the cytotoxic activity of Stx were investigated. We found that at least 6 trisaccharides present in one molecule are enough for the inhibitory effects of these SUPER TWIGs on these biological activities of Stx. Furthermore, we found that relative distances of the trisaccharides present in SUPER TWIGs rather than the number of trisaccharides play an important role in the high affinity binding to Stx.2) Inhibitory effects of SUPER TWIGs in vivo. Inhibitory effects of SUPER TWIGs on the lethality of Stx2 were investigated in mice model … More , in which lethal dose of Stx2 was intravenously administered with or without each SUPER TWIG. Among all the SUPER TWIGs developed in this study, i. e. SUPRER TWIG (0)3, (0)4, (1)4, (1)6, (1)9, (2)18, and (2)36 (the numbers in parentheses indicate the generation numbers; the zero, the first, and the second generation of the SUPER TWIGs have one, three, and five linear silicon atoms, respectively, in the core structures), which have 3, 4, 6, 9, 18, and 36 trisaccharides, respectively, in their structures, only SUPER TWIGs (1)6 and (2)18 could effectively inhibited the lethality of Stx2. One of the major common characteristics of these two SUPER TWIGs is their dumbbell shape, in which they have 3 and 9 trisaccharides, respectively, at the both end of their structures in a symmetrical manner. On the other hand, dissociation constants (KD value) of SUPER TWIGs (1)4, (1)6, (1)9, (2)18, and (2)36 for the binding to the B-subunits of Stx1 and Stx2 ,which were determined by using Biacore system, were very low in a similar range, suggesting that these SUPER TWIGs bind to the B-subunits with high affinities. In contrast, KD values of SUPER TWIGs (0)3 and (0)4, the zero generation SUPER TWIG, was very high. These results indicate that not only the high affinity binding to Stx but also another factor, such as the dumbbell shape of the molecule, are required for the efficient inhibitory effects of the SUPER TWIGs in vivo. At present, the precise mechanisms by which SUPER TWIGs (1)6 and (2)18 but not others effectively inhibit the lethality of Stx2 in vivo remains to be elucidated, our findings indicate that SUPER TWIGs (1)6 and (2)18 can be therapeutic agents against infections of Stx-producing E. coli O 157:H7. Less