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的微需氧细菌，它被认为是消化性溃疡的一个原因。在实验室中，利用含有血清和血液衍生物的复杂培养基进行热利杆菌的生长。相反，H.pyroli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，但有人认为，CD捕获了对热杆菌生长有害的物质。如果捕获的物质能够被鉴定出来，它将是开发热螺旋体生长抑制剂的一个很好的先导化合物。从这个角度来看，我们试图从用于培养H-pyroli的培养基的上清液中分离CD-guest复合物。然而，cd -客体配合物没有紫外线吸收，使其纯化非常困难。在本研究中，我们合成了三个β-CD衍生物，这将有助于cd -客体配合物的纯化。第一种化合物是生物素化的β-CD。合成该CD，通过亲和素柱纯化该配合物。更重要的是，该化合物高度不溶于水，因此，不能用于H.pyroli的培养。接下来，我们制备了携带4-溴- 2,3,5,6 -四氟苯甲酰基团的β-CD。溴由两种同位素组成，^<79>Br和^<81 >br，数量几乎相同。因此，该CD衍生物的洗脱分数将很容易通过从溴衍生的特征质谱来识别。虽然利用该CD成功培养了H.pyroli，但该CD衍生物的洗脱位置无法通过质谱确定。最后一个衍生物在β-CD上携带荧光素，也成功地用于H.pyroli的培养。培养后上清采用凝胶过滤层析分离。CD衍生物具有较强的荧光和紫外吸光度，可以清楚地确定其洗脱位置。用MALDI质量对含有CD导数的分数进行分析，发现信号比CD导数高约100个原子质量单位。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。我们现在正试图通过核磁共振来确定它的结构。综上所述，我们成功地建立了一种从pyroli芽孢杆菌培养上清中纯化CD-guest化合物的方法，该方法可用于鉴定pyroli芽孢杆菌生长抑制剂。这种方法也将适用于寻找其他细菌的抑制剂，如百日咳杆菌，也可以在含有β- cd的培养基中培养。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