Rapid Identification Of Biologically Active Molecules

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.