摘要
微量或超微量生物活性成分研究一直是人们关注的热点之一,建立与发展相关的分析方法具有良好的学术意义与应用价值,本论文围绕生物活性成分快速识别进行了如下的研究:
1.以微管蛋白作为靶分子并构建功能超滤装置,通过靶蛋白与具有抗肿瘤活性成分的相互作用而对这些生物活性成分加以识别;通过检测形成复合物前和形成复合物后的活性物质量的差异来确定活性物质与微管蛋白的相互作用能力,运用高效液相色谱/质谱联用技术(HPLC/MS~n)测定上述识别前后的指纹图谱并进行对照比较以发现差异,从中提取抗肿瘤活性成分的信息;并对未知活性分子进行在线结构鉴定。具体研究内容为:
(1)分别对临床用抑制微管聚合抗癌药物秋水仙碱、柔红霉素、促进微管聚合抗癌药物紫杉醇以及非抗癌药物酮洛酚进行了单一的识别研究,结果表明:微管蛋白对秋水仙碱、柔红霉素和紫杉醇有识别作用,而非抗癌药物酮洛酚则不被微管蛋白识别;同时,通过比较靶分子与生物活性小分子的结合摩尔比及结合率可判断紫杉醇与微管蛋白相互结合作用最强,秋水仙碱次之,柔红霉素较弱。该结果与这些药物的实际药用机制相符。
应用该方法进行了混合物的识别,结果表明活性成分能互不干扰的被一一识别,非活性成分则能被排除,这些研究为发展并建立微量水平上生物活性成分快速识别和分析一体化方法奠定了基础。
(2)对天然来源的一些潜在抗肿瘤活性成分白藜芦醇,植物多酚提取物进行了识别研究,结果表明:parthenocissin B,parthenocissin A,quadrangularin A和两个未知化合物的与微管蛋白相互作用较强,其中parthenocissin B的相互作用最强;而pallidol和Amurensin A的相互作用较弱。这些成份与微管蛋白作用时,体现了相互竞争的关系。识别实验的结果与体外活性实验结果一致,表明该识别途径可用于天然来源抗肿瘤生物活性的快速筛选;同时通过液相色谱质谱联用技术对两个未知活性成分进行了初步结构分析鉴定。
(3)对一些天然抗肿瘤衍生物如鬼臼毒素衍生物、毛兰素衍生物和cis-(±)-7-deoxynimbidiol进行了识别研究,结果表明:β构型鬼臼毒素自由基衍生物与微管蛋白的结合较α构型强。毛兰素自由基衍生物与微管蛋白的结合较母体强。不饱和自由基较饱和自由基强。cis-(±)-7-deoxynimbidiol与微管蛋白有较强的结合能力,而其氧化产物则与微管蛋白作用较弱。识别实验的结果与体外活性实验结果一致,该识别方法为药物合成中的活性评价提供了新途径。
2.发展了一种快速、灵敏、准确的鉴定荜茇酰胺类生物碱的液相色谱质谱联用分析方法。在对荜茇酰胺类生物碱单体进行二级质谱分析的基础上,总结出了基于该类化合物结构特征的质谱碎裂规律,根据这个规律,运用HPLC/ESI-MS/MS快速地鉴定了不皂化物中的42个酰胺类组分。14个为首次从该植物中分离得到的化合物,其中,6个是新化合物。二级质谱技术对于快速分析结构特征,锁定官能团和发现新结构类型尤其有效,离子萃取技术的运用有效的增加了灵敏度和选择性,为从天然来源有效成分中发现新型结构分子和指导靶向分离提供了简捷可靠的方法。
3.发展了一种快速、灵敏、有效的分析鉴定原料药中微量杂质结构的分析方法。在对西酞普兰进行多级质谱碎裂分析得出其碎裂机理的基础上,利用液相色谱/质谱联用对西酞普兰原料药中的一个微量未知杂质进行了在线的正离子模式下的多级质谱和离线高分辨质谱分析,直接推断出了该杂质的可能化学结构。杂质核磁共振数据完全证实了质谱推断的正确性,该杂质为新化合物。在美洛培南原料药中微量杂质结构分析时,通过高分辨质谱(FTICR-MS)和电喷雾离子阱多级质谱碎裂(ESI-MS~n)分析结果,对杂质的可能化学结构、杂质的产生过程和裂解机理进行了推测。本研究不仅对于原料药的杂质分析、质量控制具有重要作用,而且对其合成工艺的改进也具有重要的指导意义。
此外,论文还对现代质谱技术在活性筛选中应用进展进行了简单总结。
The study of biologically active constituents in the trace level is a challenging task. Therefore, setting up and developing its corresponding analytical methods is of great value in both academic research and application fields. The current dissertation focuses on the rapid recognition of biologically active molecules, and it is organized into three parts as follows.
Part I: A functional ultra-filtration device was constructed using tubulin as the target protein. The potential anti-tumor compounds were recognized according to the bio-affinity interactions between the target protein and the bioactive constituents. The interactions were evaluated using the quantity difference of bioactive compounds before and after the formation of the complexes. In addition, the liquid chromatography tandem multi-stage mass spectrometry (LC/MS~n) approach was applied to provide the fingerprint spectra of the screening process, to obtain specific information on potential anti-tumor compounds, as well as to analyze and identify unknown compounds. The detailed investigations are concluded in the following:
1) The polymerization and depolymerization of microtubule drugs such as colchicine, taxol, and daunorubicin with other anti-tumor mechanisms and ketoprofen with no anti-tumor activity were screened, respectively. The results indicated that there were interactions among colchicine, taxol, and daunorubicin, while the interaction between tubulin and ketoprofen was not observed. Moreover, comparing the binding molar ratio and binding efficiency of the target protein and the bioactive compounds, it was presumed that taxol had the strongest interaction with tubulin. Meanwhile, the interaction between colchicine and daunorubicin with tubulin was relatively weak. The results matched very well the actual cases. Then mixtures were subjected to this rapid recognition process, and the results indicated that all of the anti-tumor constituents could be exactly recognized without any disturbance, and the inactive compounds were excluded. Overall, all of the above works provide a good foundation for the subsequent development of a rapid recognition method at trace level.
2) The proposed screening method was then applied to some potential anti-tumor natural polyphenol extracts. The results indicated that parthenocissin B, parthenocissin A, quadrangularin A, and two unknown compounds had a relatively strong interaction with tubulin. Among them, parthenocissin B exhibited the strongest interaction. Meanwhile, interactions among tubulin, pallidol A, and murensin A were evaluated as relatively weak. All the results are consistent with those obtained from the vitro experiments, indicating that the proposed approach could potentially be applied for the rapid recognition of naturally occurring anti-tumor constituents. In addition, the two unknown compounds were identified by the LC/MS~n technique.
3) Some natural anti-tumor derivatives such as the spin-labeled derivatives of podophyllotoxin, erianin derivatives, and cis- (±)-7-deoxynimbidiol were subjected to recognition investigation. The results indicated that theβ-configuration spin-labeled derivatives of podophyllotoxin presented stronger interactions with tubulin thanα-configuration. Referring to the erianin derivertives, it is observed that the spin-labeled derivatives exhibited a stronger interaction than erianin, and unsaturation spin-labeled derivatives are stronger than the saturated ones. In addition, cis-(±)-7-deoxynimbidiol was observed to have a relatively strong interaction with tubulin, while the interaction of its oxidizing product was relatively weaker. All the results are consistent with those from the vitro experiments, indicating that the proposed method is a novel and effective approach for evaluating the bioactivities in drug synthesis.
Part II: A rapid, sensitive, and exact HPLC/ MS/MS method for the structural analysis of amides from Piper Longum. on-line has been developed. A total of 42 amides were rapidly identified, of which 14 were found in this plant for the first time, and 6 were new compounds on the basis of the MS/MS results. In the determination of the structure of unknown amides, MS/MS data provided more information on the molecular structure by clearly defining the category of observed product ions, especially for minor constituents, helping locate characteristic functionalities, discovering a new skeleton, and providing guidance for herbal isolation and preparation. Furthermore, the extracted ion chromatography (EIC) and constant neutral losses extracted technique effectively increased sensitivity and selectivity especially for minor unknown components, which will also provide a highly effective approach for phytochemical investigations.
Part III: A rapid, sensitive, and practical method for the structural elucidation of the trace impurities in bulk drugs by HPLC-MS~n and FTICR-MS analysis has been developed. Using this method, the fragmentation behavior of citalopram was investigated, and an unknown impurity was rapidly elucidated and further confirmed by NMR experiments after preparative isolation. Also, the proposed structure, formed mechanism, and fragmentation pathway of an unknown impurity in bulk drug meropenem was rapidly characterized. This method is of importance for the control of the quality of bulk medicine and the synthesis process.
In addition, the application and developments of modern mass spectrometric technique in screening have also been summarized briefly in this dissertation.
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