Studies On The Synthesis Of The 1, 3-substitutive Pyrazolones Reagents And Their Application In Saccharides' Derivatization And Analysis

With the development of molecular biology and cell biology, biological functions of carbohydrates as the third biomolecules in construction of life body have been increasingly recognized. Recent studies have already demonstrated that carbohydrates of glycoconjugates in vivo not only supply energy sources to life body, but also bear the weight of bioinformations several times more than nucleic acids and proteins as an important informational molecule. They play an important role in life movement. People's many vicious diseases such as tumor, which nosogenesis and development are closely concerned with the disorders and the expression amount of glycosylation in body. Therefore, study on the qualitative and quantitative analysis of glycans has considerable significance in illuminating glycan's biological functions and seeking useful information about some diseases. However, due to carbohydrates with complex structure, no template to synthetize and microamount for glycoprotein from biosystem, moderm analytical techniques face with a draconic challenge to carbohydrates'analysis. A main problem is that it is impossible for carbohydrates with strong hydrophilicity and no UV absorption and fluorescence group to meet the detection by high sensitivity techniques. Derivatization as a sample pre-processing technique can bring a UV or fluorescence group into glycan and make it detected by high sensitivity techniques. It plays an important role in research of the carbohydrates with some modern analytical techniques such as HPLC, CE and MS. Compared to reductive amination reagents using in base medium,1,3-substitutive pyrazolone derivatization reagents have some conspicuous merits such as free from acid-labile groups in carbohydrates and suitable for many kinds of glycans, due to the fact that their labeling reactions with saccharide are achieved in weakly alkaline medium. But the reagent variety is very few so far (only one named 1-phenyl-3-methyl-5-pyrazolone (PMP) used generally), and fail to meet the analysis of some complex saccharides. It is necessary to develop some new 1,3-substitutive pyrazolones reagents for saccharide derivatization. The research on PMP isotope labeling-mass spectrometry technique for glycan's quantitative analysis has not been reported so far, in spite of PMP being extensively applied in saccharides'study. Based on these facts, the synthesis and application of the 1,3-substitutive pyrazolones as derivatization reagents for saccharides' analysis were studied in this thesis. Results are summarized as followings:1.1-(d5) phenyl-3-methyl-5-pyrazolone (d5-PMP) and two PMP analogs named 1, 3-diphenyl-5-pyrazolone (DPP) and 1-(4-isopropyl) phenyl-3-methyl-5-pyrazolone (PPMP) were synthesized with satisfied yield under the conditions of glacial acetic acid as catalyst in solvent-free state at room temperature. All products were characterized by1HNMR, 13CNMR, MS and UV spectrum. The preliminary investigation in the reaction activity of the synthesized products labeling with lactose was also carried on. The results indicate that d5-PMP shows the highest reaction activity and obtained mainly dimolecular derivatives, DPP shows the lowest one and failed to as saccharide's derivatization reagent, and PPMP shows the second one following PMP and trended to generate unimolecule derivatives with better stability than dimolecular derivatives, no PPMP losing happened, and can be used to analyze the saccharides if the quantitative derivatization condition is optimized.2. Application of the d0/d5-PMP in relative quantitative analysis of saccharides with isotopic-tagging coupled with mass spectrometry was further studied.The method based on d0/d5-PMP stable isotopic labeling for quantitative analysis of N-linked glycans released from glycoprotein with mass spectrometry has been established for the first time. The basis of this method is the quantitative derivatization of free reducing glycans by d0/d5-PMP. The d0/d5-PMP derivatives for free N-linked glycans from two samples were mixed in equimolar ratio following by MS detection. The same glycan appears 10-Da difference doublet. This can be applied in qualitative analysis of glycans. Due to the isotopic glycans derivatives possessing the same ionization efficiency for their similar chemical characteristics, difference at relative abundances between d0-PMP derivatives and d5-PMP derivatives embody difference in quantity of glycans between two samples. It permits the relative quantitative analysis of glycans in glycomic studies. Application of this method on analysis of DP2-DP6 oligosaccharides from maltodextrin and N-linked glycans released from ribonuclease B and bovine fetuin by PNGase F demonstrated the 10-fold relative quantitative linear dynamic range, satisfied reproducibility (CV≤8.34%) and accuracy (relative error, RE≤5.1%) of the method. It has been successfully applied to compare the oligosaccharides in human milk versus in bovine milk qualitatively and quantitatively.The new method of O-glycans releasing and d0/d5-PMP stable isotopic labeling-mass spectrometry quantitative analysis has also been established for the first time. The new method is based on O-glycans (serine-or threonine-linked oligosaccharides) releasing and labeling with d0/d5-PMP stable isotope in the same base medium and can be come true simultaneously in one pot. Inβ-releasing reaction, we use ammoniae instead of 0.1 mol·L-1 NaOH aqueous solution and d0/d5-PMP instead of NaBH4 to release and label O-glycans in one pot. The derivatives were purified and mixed with equimolar ratio, then put into ESI-MS or MALDI-TOF-MS analysis. Application of this method on analysis of the O-glycans from bovine fetuin and porcine stomach mucin indicated the 10-fold relative quantitative linear dynamic range, satisfied reproducibility (CV≤7.48%) and accuracy (relative error, RE≤17.2%) of the method. In this method, O-glycans'releasing in alkaline medium and labeling with PMP were carried out in one pot reaction simultaneously. The sample's disposal procedures were greatly simplified. The method overcomes the shortage of generating variable mass difference among different glycans in the quantitative method based on permethylation isotope labeling, and can be applied to compare the O-glycans released from Fetuin or mucin type glycoprotein from two different sample source qualitatively and quantitatively. It provides a method reference for research of comparative glycomics.Such results indicate that the method based on d0/d5-PMP stable isotopic labeling can be applied in quantitative analysis of both N-glycans and O-glycans with mass spectrometry. Many glycans from unknown samples can be precisely analyzed qualitatively and quantitatively by it coupled with MS/MS2, HPLC, CE or their multiple techniques without further chemical analyses, because of PMP tag with strong hydrophobicity and UV absorption at 245 nm. This new method based on d0/d5-PMP stable isotopic labeling has considerable application values in research of comparative glycomic.3. The application of PPMP reagent in carbohydrates'derivatization and sugar composition analysis were studied first time.The methods with PPMP precolumn derivatization by HPLC coupled with on-line ESI-MS/MS2 techniques for the analysis of composition of monosacchrides have been established. Taking glucose as a study object, the best derivatization conditions such as molar ratio of sample to PPMP-1:6-7, reaction temperature-80℃, reaction time-70 min and derivatives'structure being mono-PPMP-derivatives have been determined. Compared with PMP, PPMP derivatization shows many merits such as mono-PPMP-derivatives with more chemical stability and better chromatographic separating property and excess reagent to be removed easier than PMP. Twelve mono-PPMP-derivatives of monosacchrides were successfully separated HPLC by and detected by on line ESI-MS/MS2. The characteristic MS/MS2 fragments for thirteen mono-PPMP-labeled monosaccharides were obtained and assigned. When the method was applied to analyze the monosaccharide composition of natural Spirulina polysaccharide SPPB-1, the reliable analysis result was obtained. In addition, presence of a reducing 6,7-deoxyheptanose residue was for the first time proved in Spirulina polysaccharide SPPB-1 by this method. The proposed method can be applied in routine analysis of monosacchrides from biological specimen due to PPMP derivatization with some good features such as rapid, accurate, reproducible, and sample treatment simple for analysis of saccharides.The methods with PPMP precolumn derivatization by HPLC coupled with on-line ESI-MS/MS2 techniques for the analysis of composition of oligosacchrides mixture have been also established. For matodextrin as study objects, the derivatization effectiveness of PPMP to oligosacchrides and derivatives'ESI-MS analysis were explored. The results indicated that mono-PPMP-derivatives are still principal product for oligosacchrides and the mono-PPMP-derivatives shows satisfactory response to ESI-MS both in positive-ion mode and in negative-ion mode. The feasibility of analysis to oligosacchrides mixture with PPMP pre-column derivatization by HPLC coupled with on-line ESI-MS/MS2 techniques. When the method was applied to analyze the chitooligosaccharides mixture sample with molecular weight less than 1500 Da by biomembrane filtering, the satisfied result was obtained. The method with high performance separation of HPLC and MS'high sensitivity and precise qualitation shows some merits as following:sample treatment simple, analysis fast and result accurate, and it can be taken as a reference method for research of oligosaccharides'quality control and structure-function relationship.