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

1)不同末端官能团数和不同核心结构的碳硅烷树状大分子的制备。我们开发了第一代和第二代碳硅烷树状大分子，它们的结构中含有9、18和36个三糖，即球三烷基神经酰胺的糖部分，作为其功能末端单元（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，一个分子中至少含有6个三糖，就足以使这些SUPER TWIGs抑制Stx的这些生物活性。此外，我们发现SUPER TWIGs中存在的三糖的相对距离而不是三糖的数量在与stx的高亲和力结合中起重要作用。2)SUPER TWIGs在体内的抑制作用。在小鼠模型中，通过静脉注射Stx2致死剂量，观察SUPER TWIGs对Stx2致死性的抑制作用。在所有超级树枝发达在这项研究中,即SUPRER树枝(0)3(0)4(1)4(1)6,(1)9(2)18岁,和(2)36(括号里的数字表示一代数字,0,第一、第二代超级树枝有1、3、5线性硅原子,分别在核心结构),3,4,6,9日,18日和36三糖,分别在他们的结构中,只有超级树枝(1)6,(2)18能有效地抑制Stx2的杀伤力。这两种SUPER TWIGs的一个主要共同特征是它们的哑铃形状，在它们的结构两端分别以对称的方式含有3个和9个三糖。另一方面，使用Biacore系统测定的SUPER TWIGs(1)4、(1)6、(1)9、(2)18和(2)36与Stx1和Stx2的b亚基结合的解离常数（KD值）在相似的范围内都很低，表明SUPER TWIGs与b亚基的结合具有很高的亲和力。而零代SUPER TWIGs(0)3和(0)4的KD值非常高。这些结果表明，除了与Stx的高亲和力结合外，还需要另一个因素，如分子的哑铃形状，以使SUPER TWIGs在体内发挥有效的抑制作用。目前，SUPER TWIGs(1)6和(2)18在体内有效抑制Stx2致死率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可以作为治疗stx产生大肠杆菌o157:H7感染的药物。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