Preparation Of Series Of Heparan Sulfate And Its Structural Characterization By IPRP-LC-MS IT TOF

Heparin is a glycosaminoglycan drugs that with strong anticoagulant effect,it is the top of choice for the treatment of thromboembolic diseases at present.In addition,heparin has also been applied to the anti-inflammatory,anti-viral,anti-cancer and other fields.However,the bleeding side effects become the biggest obstacle of heparin when it is used for any other purposes than anticoagulant.Previous studies had found that the sulfate content and location of heparin structure plays an important role in its anticoagulant activity.In order to reduce the anticoagulant activity of heparin and explore new polysaccharide drug,purposeful and appropriate chemical modification to simplify the structure of heparin chain is very important.This article focused on the prqparation of series of desulfated heparin,and the establishment of a standard qualitative and quantitative analysis method of heparin/heparan sulfate cleavage disaccharide,and then applied the standard method to the structural characterization of desulfated heparin derivative.This thesis includes four chapters as follows:In chapter 1,the basic structure,function and application of heparin/heparan sulfate;the structure and function of heparin derivatives,as well as the preparation method and application of desulfated heparin derivative were introduced in detail.In this part,the preparation and structural characterization methods of heparin/heparan sulfate disaccharide were mainly introduced.Besides,the aim and significance of this thesis were also briefly presented.In chapter 2,preparation of three series(NHSO3 series,NAc series and NH3+ series)of desulfated heparin,which were derivatived by the method of progressively desulfurization and DeNS re-acetylation of commercial heparin sodium.Then depolymerization of the prepared desulfated heparin derivative using the enzymatic method and HNO2 chemical cleavage method,respectively.Finally,the structural of the desulfated heparin derivative preliminary,was analysised by strong anion exchange high performance liquid chromatography(SAX-HPLC)In chapter 3,a rapid qualitative and quantitative analytical method was established to analyze the twelve kinds of heparin/heparan sulfate disaccharides which include four N-unsubstituted glucose amine heparin disaccharides,the method is based on ion-pair reversed-phase liquid chromatography-ion trap/time-of-flight mass spectrometry(IPRP-LC-IT TOF MS)technology,Different chromatographic and mass spectrometric detection conditions were tested to optimize the analytical method.By comparing with the reported methods,our method can detect biological samples quickly and accurately in qualitative and quantitative detection with high accuracy,good repeatability and wide range of application.In chapter 4,.the three series of desulfated heparin derivative prepared in chapter 2 were characterized using the method established in chapter 3.The results shown that the progressively desulfurization reaction introduced in this thesis can effectively prepared desulfated heparin derivative that with specific disaccharides composition.To further illustrate the advantages of the disaccharides detection method established herein,we compared the results of disaccharides detection before and after the desalted and deprotein by gel column.The results illustrated that the standard method established in this thesis is specificity and wide range of application.