Synthesis of novel cyclodextrin derivatives and its application to search for the inhibitor of H.pyroli growth

幽门螺杆菌是一种革兰氏阴性微需氧细菌，已被认为是消化性溃疡的原因之一。通过使用含有血清和血液衍生物的复合培养基来进行霍乱杆菌的实验室培养。取而代之的是，羊肚菌可以在含有β-环糊精的培养基中生长。这一事实的原因尚不清楚，但可能是Cd捕获了对鹿蹄疫病菌生长有害的物质。如果捕获的物质能够被识别出来，它将是一种很好的先导化合物，用于开发抑制羊肚菌生长的药物。从这一角度出发，我们试图从用于培养鹿蹄草的培养上清液中分离出Cd-客体复合体。本研究合成了三种β-Cd的衍生物，为Cd-客体络合物的纯化提供了方便。第一个化合物是生物素化的β-CD。用亲和素柱层析的方法合成了该CD，并对其进行了纯化。Ho…更严重的是，该化合物在水中高度不溶，不能用于鹿蹄疫杆菌的培养。接下来，我们制备了带有4-溴-2，3，5，6-四氟苯甲酰基的β-CD。溴由两种同位素^&lt；79&gt；bR和^&lt；81&gt；br组成，含量几乎相同。因此，这种CD衍生物的洗脱部分将很容易通过来自溴的特征质谱图来识别。虽然用该CD成功地培养了猪瘟杆菌，但该CD衍生物的洗脱位置不能被质谱学鉴定。最后一种在β-CD上携带荧光素的衍生物也成功地用于霍乱弧菌的培养。用凝胶过滤层析分离培养上清液。通过其较强的荧光和紫外光吸收，清晰地确定了CD衍生物的洗脱位置。用MALDI质谱仪对含Cd衍生物的组份进行分析，观察到比Cd衍生物高出约100个原子质量单位的信号。这一结果可能表明，CD衍生物捕获了一种分子量约为100的物质。综上所述，我们成功地建立了一种从鹿蹄疫杆菌培养上清液中分离纯化CD客体化合物的方法，为鉴定鹿蹄疫杆菌的生长抑制物奠定了基础。这种方法也适用于寻找其他细菌的抑制剂，如百日咳杆菌，它也可以在含β-CD的培养基中培养。较少

Helicobacter pyroli is a gram-negative microaerobic bacterium, which has been proposed to be a cause of peptic ulcers. Laboratory growth of H.pyroli is carried out by using complex media containing serum, and blood derivatives. In stead, H.pyroli can be grown in media containing β-cyclodextrin. The reason of this fact is unknown yet, but it is suggested that CD traps the substances that is harmful for H.pyroli growth. If the trapped material can be identified, it will be a good lead compound for the development of the inhibitor of H.pyroli growth. From this point of view, we tried to isolate the CD-guest complex from the supernatant of the media used for the culture of H-pyroli. However, CD-guest complex had no UV absorption, causing its purification extremely difficult.In this research, we synthesized three β-CD derivatives, which will facilitate the purification of CD-guest complex. First compound is biotinylated β-CD. This CD is synthesized to purify the complex by avidin column. Ho … More wever, this compound was highly insoluble in water and thus, could not be used for the culture of H.pyroli. Next, we prepared β-CD carrying 4-bromo-2, 3, 5, 6-tetrafluorobenzoyl group. Bromine is composed of two isotopes, ^<79>Br and ^<81>Br with almost the same amount. Thus, elution fraction of this CD derivative will be easily identified by the characteristic mass spectrum derived from bromine. Although the culture of the H.pyroli using this CD was successful, the elution position of this CD derivative could not be identified by the mass spectrum. The last derivative, which carries fluorescein on β-CD, was also successfully used for the culture of H.pyroli. The post-cultural supernatant was separated by gel-filtration chromatography. The elution position of the CD derivative was clearly identified by its strong fluorescence and UV absorbance. The analysis of fraction containing the CD derivative by MALDI mass showed that the signal, which is about 100 atomic mass unit higher than the CD derivative was observed. This result might show that the CD derivative traps a substance, which has a molecular weight of about 100. We are now trying to identify its structure by ^1H-NMR.In conclusion, we succeeded in establishing a method to facilitate the purification of CD-guest compound from the cultural supernatant of H.pyroli, which will be useful to identify the growth inhibitor of H.pyroli. This method will be also applicable to find the inhibitor of other bacteria, such as B.pertussis, which can also be cultured in β-CD-containing media. Less