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

1)制备不同末端官能团数目和不同核心结构的碳硅烷树枝状大分子。我们开发了第一代和第二代碳硅烷树枝状大分子，它们含有9、18和36个三糖，糖基部分为球状三糖神经酰胺，作为其结构中的功能末端单元(称为超级枝条)。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对STX的细胞毒活性的抑制作用。我们发现，一个分子中至少有6个三糖足以抑制这些超级小枝对STX的这些生物活性的抑制作用。此外，我们还发现超级小枝中三糖的相对距离而不是三糖的数量对超级小枝体内与Stx2)高亲和力结合的抑制作用起着重要作用。用小鼠…模型研究了超级枝对Stx2致死率的抑制作用更多，其中致命剂量的Stx2被静脉注射，每根超级树枝都有或没有。在本研究开发的所有超级枝条中，超级枝条(0)3、(0)4、(1)4、(1)6、(1)9、(2)18和(2)36(括号中的数字表示世代数；第0代、第1代和第2代超级枝条的核心结构中分别含有1个、3个和5个线性硅原子)，它们的结构中分别含有3、4、6、9、18和36个三糖，只有超级枝条(1)6和(2)18能够有效地抑制Stx2的致死。这两个超级树枝的一个主要共同特征是它们的哑铃形状，在它们的结构的两端分别有3和9个三糖，对称的方式。另一方面，用Biacore系统测定的超级枝(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)在相似的范围内很低，这表明这些超级枝与B亚基具有很高的亲和力。而零世代超级枝(0)3和(0)4的Kd值很高。这些结果表明，不仅需要与STX的高亲和力结合，还需要另一个因素，如分子的哑铃形状，以有效地抑制体内的超级树枝。目前，Super twigs(1)6和(2)18在体内有效抑制Stx2致死作用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可以作为治疗产生STX的大肠杆菌O157：H7感染的药物。较少

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