【作者】 司端运；

【导师】 钟大放；

【作者基本信息】 沈阳药科大学 ， 药物分析学， 2001， 博士

【摘要】 藤黄灰链霉菌（Streptomyces luteogriseus）是从土壤中分离出来的放线菌，其WC670号菌株的发酵液具有糖苷酶抑制活性。将发酵液经过提取和初步分离，得到次生代谢产物的有效部位称为WC670。WC670对甜菜等糖料作物显示出了良好的增糖效果；酶动力学实验发现对蔗糖转化酶和α-淀粉酶具有抑制作用；初步化学研究表明为一非常复杂的氨基寡糖类混合物，其中可能含有已知的α-淀粉酶抑制剂异戊他定类化合物。预计在农业和治疗糖尿病和肥胖症等方面具有开发潜力。 本论文的主要目标是利用现代高通量化学筛选技术，结合传统的化学结构研究方式，阐明WC670的化学组成；通过生物转化研究，丰富有关氨基寡糖类化学成分药物代谢方面的资料，也为简化氨基寡糖类复杂混合物的化学组成、促进WC670的深入开发提供思路和策略。 一、异戊他定系列化合物化学结构的深入研究 采用LC/MS技术研究WC670的化学组成，确认含有6个已知的异戊他定系列化合物。但在仔细分析它们的结构研究资料时，发现文献所建立起来的化学结构尚存在明显问题。以Spherisorb C₈柱（300×8.0mm，10μm）为固定相，不同比例的乙腈-1.5mmol/L氨水为流动相，206nm下检测，对收集到的色谱峰流分进行冷冻干燥，首次分离纯化出全部6个异戊他定已知化合物的单体。通过质谱分析和葡萄糖淀粉酶催化的水解反应特点，确定了结构的组成单元及其连接顺序；通过核磁共振光谱分析以及理化性质测试，分别修正了结构中的异戊酸应该与D单元葡萄糖的6位羟基连接成酯、存在还原性的葡萄糖末端、结构中的苷键全部呈α-（1→4）连接形式等，最终建立起了它们的准确结构，并重新命名为异戊他定M03、M13、M23、D03、D13、D23。 在结构修正工作的基础上，初步总结出了异戊他定类化合物的结构与色谱行为、电喷雾离子化质谱行为以及核磁共振光谱特征之间的规律，为进一步进行WC670的化学组成研究奠定了基础。 二、藤黄灰链霉菌发酵液中次生代谢产物的化学组成研究 采用LC/MS技术分析WC670样品时，发现除了已知的6个异戊他定化合物外，还存在葡萄糖个数、葡萄糖单元的连接顺序、伪三糖个数以及酯侧链形式等发生变化的成分。将几种变化因素进行组合，计算出可能存在组分的分子量和质谱检测时准分子离子及碎片离子的质荷比数值。以Spherisorb C₈柱（200×4.6mm，5μm）为固定相，乙腈-1.5mmol/L氨水（15∶85，v/v, 0.5ml/min）为流动相，n 藤黄灰链霉菌发酵液中次生代谢产物的研究设定质谱参数，对预测组分逐一检测。分析各色谱峰的保留时间和相应Cio质谱图的特征碎片离子，发现WC670中至少含有异戊酸酯、丁酸酯、异己酸酯和异己烯酸酯侧链的4大系列138种服绒类成分，其中132种是首次鉴定出的新化合物，推测出它们的可能结构，并分别命名为异戊他定类osovalertatins＊丁他定类比 人异己他定类＊sohex…s)和异己烯他定类Osohex聊－tatins)。通过Cio质谱图的离子强度数值，初步评价WC670中以 sed碳酯侧链、l《个伪三糖、伪三糖左侧为0—3个葡萄糖以躺三糖右侧3个葡萄糖时组合出的化合物为主要成分。 为了验证化学筛选结果的准确性，l从 WC670中制备出 ＊ 个新化合物的单体，通过核触振光谱分析等手段确认了它们的结构分别为异戊他定M33、异戊他定D33、异戊他定D43、异戊他定T03、异戊他定T13、丁他定M03、T他定M13、异己他定M33、异己他定M43、异己他定D23和异己他定D43，均与LCMS分析所提示的化学结构相一致。 分析比较 13 8种眼黝类成分在 CS反才柱上的色飘为，与化学结构之间存在如下的规律：O酯侧链对色渐为的影响最大。侧链中每增加 1个CHZ，化合物的保留能力大大增强，保留时间延长。②伪三糖和葡萄糖的数目增加，分子的极性加大，保留时间缩短。③位置异构体能得到较好的分离。当葡萄糖由连接于伪三糖右侧改变到左侧时，保留时间缩短。 氨基寡糖化合物以色谱流动相溶解后进行ESI－MS分析，主要得到准分子离子峰［M＋H 椭二个以上仲胺结构的化合物，还可以出现叭2切 峰。对仰卜田”和仰刊m”断CID分析，所得到的碎片离子都能够用于结构解析。分析比较138种撼髓类成分的Cio质谱图，总结出质谱断裂规律：①主要存在由于一个糖苦键或伪糖苦键断裂，产生包含有仲胺基团的碎片离子。②对阶二田h赫 cro分析时，可以产生带二个正电荷的碎片离子。＠鸡纳糖苦键易发生断裂，伪糖普键次之，葡萄糖旮键再次。鸡纳糖苦键断裂形成的碎片离子往往构咸Cio质谱图中的最强峰，容易识别，是解析各组成单元之间连接顺序的主要依据。④碎片离子o刁 厂说明结构中存在非还原性六碳糖末端，［M们I 厂说明存在还原性的六碳糖末端，阶H上 厂和 d 304说明伪三糖连接于分子的最左侧。⑤母离子及丰蹦大的碎片离子容易脱水，产生减少18 U的碎片离子。 寡糖类化合物分子大，碳氢数目多，且多数为连接一个氧的脂肪碳氢结构，N’－－?更多还原

【Abstract】 The soil Actinomycete, Streptomyces luteogriseus strain 670, showed to produce secondary metabolites with the remarkably inhibitory activities against several saccharide hydrolases, such as sucrose invertase and ct-amylase. The effective fraction, WC670, was extracted and isolated from the culture filtrate of S. luteogriseus, which found to be a very complex mixture of aminooligosaccharides, possibly containing the known hypoglycemic agents, isovalertatins. An agricultural trial proved that WC670 could significantly increase the sucrose output of sugarbeet. Therefore, WC670 might be further developed in agriculture and in treating diabetes and obesity in medicine. The aims of this study were to interpret the chemical composition of WC670 by applying the advanced techniques of chemical screening and structural characterization, and to under- stand the biotransformation pattern of aminooligosaccharides by microorganisms and in rats. 1. Structural reinvestigation of the six known isovalertatins A preliminary LC/ESI-MS analysis confirmed that the WC670 contains all of the six known isovalertatins. However, when verifying their structures, we found several inconsistencies in the spectroscopic data. This encouraged us to reinvestigate their structures. B The WC670 sample was dissolved in water and used for separation by means of semi- preparative RP-HPLC on a Spheisorb C3 column where the mobile phase was ACN-1.5 mmol/L aqueous ammonia with UV detection at 206 nm, which yielded the six oligomers. Careful inspections of the CII) spectra and the hydrolysis results catalyzed by the glucoamylase led to the confirmation of the composed units and their linkage sequence. And, extensive analyses of the NMR data and the color reaction results made the revisions of their linkage positions and glycosidic bond configurations. Thus, their chemical structures were finally established. The names for the six oligomers were redesignated isovalertatins M03, M13, M23, D03, D13, and D23, respectively. 2. Compositional interpretation of the secondary metabolits produced by S. luteogrisew The ESI-MS analysis of WC670 sample illustrated the complexicity of the mixture. Besides the six known isovalertatins, there existed much more homologues with different numbers of glucose and pseudoU-isaccharide units, and variable linkage sequences and an esterized side chain. The combination of these changeable structural factors made the expectation of the possible compositions. And, by exploiting the summarized positive ESI-MS fragmentation patterns for isovalertatins, the miz values for the protonated molecular ions of the desired components and their potential CII) product ions derived from the single bond cleavages could also be predicted. Thus, by using a unique and active LCIESI-MSIMS procedure of analyzing - lv - ABSTRACT V these desired components through the SRM chromatograms and the corresponding CID spectra. we had totally identified the presence of 138 components. Of all the detected compositions, six isovalertatins are the known. And among the 132 newly characterized components, eleven oligomers, isovalertatins M33, D33, D43, 103, 113, butytatins M03, M13, and isohexanytatins M33, M43, D23, D43 had been prepared by RP-HIPLC methods. Their determined structures were a更多还原