| The existence of carboxyl and sulfate groups in acidic polysaccharides affectstheir dissolution in methylation solvent, so the methylation levels tend to be lower.The presence of carboxyl groups in uronic acids lead to acidic polysaccharides noteasy to hydrolysis and gasification, which eventually leading to the judgment of GC-MS is not very accuracy and it restricts the application of methylation method in theacidic polysaccharides. The related problems in methylation analysis of acidicpolysaccharides were studied in this paper, chondroitin sulfate (CS) which containinguronic acid and sulfate groups was used as raw material, reducing conditions wereoptimized to determine the best uronic acid reducing conditions, desulfurizationmethod of acidic polysaccharides containing sulfate groups and its methylationdissolution issues were discussed. And through comparative study, the optimalmethylation conditions of acidic polysaccharides were determined. It lays thefoundation for structural analysis of acidic polysaccharides.Firstly, CS was chosen to modify and determine the optimal reduction conditionsof uronic acid in acidic polysaccharides. Through comparing the level of reductionrate of the sodium and hydrogen form CS samples at different buffer systems, atcontrolled pH environment, respectively. The results show in the modified system of75%THF-0.1mol/L MES (THF-MES) buffer, EDC-NaBH4was used to reducecarboxyl-group of uronic acid under the condition of pH4.75and50℃, hydrogenacidic polysaccharides reduction rate is more98%, obviously higher than Sodium acidpolysaccharide；The advantages and disadvantages of different reduction detectivemethod were also compared in this paper, the results showed that fast performanceliquid chromatography (HPLC) method is an ideal uronic acid reduction rate detectionmethod. The method was applied to different structures of acidic uronic reduction, itis not only suitable for CS, but also adapted to hyaluronic acid, heparin, marinepolymannuronic acid and fucoidan uronic acid, suggesting that it is universal for analysis different of structure of acidic polysaccharides, in particular to provide apremise in determining the polysaccharide connection in methylation.Methylation dissolution issues of acidic polysaccharides containing sulfategroups and its desulfurization method were discussed in this paper. By comparing theCS containing sulfate salt formation with a different base, found the best conditionsfor CS dissolving is to be made into the form of triethylamine salt, the methylation ofCS can reach40%of the full extent of methylation. By comparing differentdesulfurization methods, found that PMA method has the best desulfurizationefficiency, and lowest degradation rate. After reduction and PMA desulfurization, GC-MS showed the ion response was significantly improved. It is suitable for acidicpolysaccharides methylation containing carboxyl groups and sulfate groups, whichwill help further structural study. And in this paper we found that the full-methylationpolysaccharides containing the sulfate groups can dissolved in the water layer also,through compareing the GC-MS analysis of the polysaccharides dissolved inchloroform layer and the aqueous layer, we can get the structure information moreaccurately.In this paper, by validated different structure of acidic polysaccharide containinguronic acids, it shows improved uronic acids reduction method is an ideal method, forreduce acidic polysaccharide containing uronic acids as well as provide a usefulreference for analysis acidic polysaccharide structure. Resolved the problem that theacidic polysaccharides containing uronic acidic occurring β-elimination impact thedetermining of the structure, and the presence of sulfate groups constrains thedissolution in the methylation lays a good foundation for methylated acidicpolysaccharides in the future research, provides a basis for study structure-activityrelationship and research of marine carbohydrate-based drugs. |
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