Design And Chemoenzymatic Synthesis Of Novel Reversible Anticoagulant Heparin Dodecasaccharide

Cardiovascular disease is a major disease threatening human health.With the advent of aging society in China,the incidence of cardiovascular disease,especially vascular embolism,has been increasing year by year,and antithrombotic drugs have become the key drugs for the prevention and treatment of cardiovascular diseases.Heparin,the most important anticoagulant in clinical practice,is widely used to treat and prevent arteriovenous thrombosis.Heparin is a member of glycosaminoglycan and is composed of alternately disaccharide units formed by glucuronic acid(GlcA)or iduronic acid(IdoA)residues glucosamine(GlcN)residues connected by 1-4 glycosidic bonds,with variable sulfation at each residue.It exerts its anticoagulant effect relying on the AT affinity-binding pentasaccharide sequence.The traditional unfractionated heparin is extracted from porcine intestinal mucosa as raw material and low molecular weight heparin is obtained by chemical or enzymatic depolymerization,and both are heterogeneous mixtures with risks of viral infection,adulteration and introduction of impurities due to their animal origin.In addition,the chemically synthesized fondaparinux sodium has the disadvantages of a large number of synthetic steps,a very low total yield and no protamine reversible activity.Therefore,it is of great significance to establish a novel and more efficient strategy to prepare non-animal-origin heparin compounds with well-defined structure and reversible anticoagulant activity.The most promising strategy is chemoenzymatic synthesis in vitro by simulating the in vivo biosynthetic pathway of heparin,that is,the de novo synthesis is started from commercial GlcA-PNP as the initial acceptor and using the glycosyl donors and sulfate donors as raw materials,and carried out by using glycosyltransferase,sulfotransferase and isomerase as catalyst and combining with mild chemical methods to finally yield homogeneous compounds,Compared with chemical synthesis,chemoenzymatic synthesis has the advantages of fewer steps,higher regioselectivity and higher yield.Heparin compounds with anticoagulant activity include at least one unique AT-binding pentasaccharide sequence(-GlcNS6S-GlcA-GlcNS3S6S-IdoA2S-GlcNS6S-)to specifically inhibit the activity of F X a.The sequence is introduced into appropriate highly sulfated saccharide to obtain a long-chain heparin compound that will be expected to be protamine-reversible anticoagulant activity.Accordingly,this study was based on the chemoenzymatic synthesis technology early established by our research group,and intended to use the unique substrate specificity of 2-O-sulfotransferase(2-OST)to synthesize novel heparin oligosaccharides containing rare 2-O sulfated GlcA residues(GlcA2S).Then,a novel heparin dodecasaccharide containing four continuous GlcA2S and AT-binding pentasaccharide sequence was designed and synthesized using reasonable chemoenzymatic routes.Finally,its in vitro anticoagulant activity and reversible activity by protamine were determined.The achievements and conclusions of my thesis include the following aspects:1.Large scale preparation of glycosyl donors and sulfate donorThe synthesis of heparin saccharide chains requires the rare saccharide donors UDP-GlcNTFA and UDP-GlcA,and the sulfate modification requires the sulfate donor PAPS.Firstly,the three donors were synthesized using the robust enzymatic or chemoenzymatic synthesis methods.The enzymes included UDP-GlcDH(that was crude enzyme prepared by step-by-step salting out from bovine liver tissue homogenate extracts),and NahK,GlmU,PPA,KAST/APSK(that were expressed in recombinant E.coli and purified by affinity chromatography).In this thesis,the reaction system and ion exchange purification system were scaled up for preparation of glycosyl donors and sulfate donor in gram scale,and the purity was more than 95%.2.Synthesis of novel heparin oligosaccharides containing continuous GlcA2SGlcA-PNP monosaccharide as the starting material was elongated by adding alternately GlcNTFA or GlcA residue to the non-reducing end under the specific catalysis of glycosyltransferases KfiA or PmHS2,so as to obtain gram-grade heparin pentasaccharide backbone GlcA-GlcNTFA-GlcA-GlcNTFA-GlcA-PNP,and the reaction rate in each step was higher than 95%.The backbone oligomer was treated with LiOH to remove N-TFA followed by modification using the N-sulfotransferase(NST)to obtain N-sulfated pentasaccharide GlcA-GlcNS-GlcA-GlcNS-GlcA-PNP,with a reaction rate of higher than 99%.Next,it was found for the first time that to the N-sulfated pentasaccharide was efficiently modified by 2-OST into a novel heparin pentasaccharide GlcA-GlcNS-GlcA2S-GlcNS-GlcA2S-PNP containing two continuous GlcA2S residues,with a reaction rate of higher than 95%.Then,the saccharide chain extension,N-sulfation modification,and 2-OST enzymatic modification were rationally combined to successfully obtain a novel heparin heptasaccharide containing continuous three GlcA2S(GlcA-GlcNS-GlcA2S-GlcNS-GlcA2S-GlcNS-GlcA2S-PNP)and a novel heparin nonasaccharide containing four continuous GlcA2S(GlcA-GlcNS-GlcA2S-GlcNS-GlcA2S-GlcNS-GlcA2S-GlcNS-GlcA2S-PNP),with reaction rate of higher than 95%for each step.Finally,we successfully synthesized two novel heparin oligosaccharides containing both IdoA2S and GlcA2S abbreviated as GlcA-GlcNS-IdoA2S-GlcNS-GlcA2S-GlcNS-GlcA2S-PNP)and GlcA-GlcNS-GlcA2S-GlcNS-IdoA2S-GlcNS-GlcA-PNP.All of the intermediate compounds and target compounds were purified by ion exchange column chromatography,and the purity was higher than 95%by HPLC detection.ESI-MS and NMR were used analysis of the structures of target compounds.3.Synthesis of novel heparin dodecasaccharide 12-merThe resulting heparin nonasaccharide containing four continuous GlcA2S was enzymatically extended to undecasaccharide,followd by chemical de-N-TFA and enzymatic N-sulfated modification to obtain GlcA-GlcNS-GlcA-(GlcNS-GlcA2S)4-PNP.The N-sulfated product was modified catalyzed by C5-isomerase(C5-epi)and 2-OST in one step to GlcA-GlcNS-IdoA2S-(GlcNS-GlcA2S)4-PNP with a reaction rate of 91.16%.After enzymatic addition GlcNTFA to non-reducing end,de-N-TFA and N-sulfated modification with a reaction rate higher than 95%,the synthetic GlcNS-GlcA-GlcNS-IdoA2S-(GlcNS-GlcA2S)4-PNP was modified by 6-OST1/3 or 3-OST1 in turn,with 65%and 99%reaction rates respectively,to finally obtain the novel rare heparin dodecasaccharide containing AT-binding pentasaccharide sequence and continuous GlcA2S residues abbreviated as GlcNS6S-GlcA-GlcNS6S3S-IdoA2S-GlcNS6S-GlcA2S-(GlcNS6S-GlcA2S)3-PNP.The molecular weight was determined by ESI-MS,and its structure was preliminarily characterized by heparinase cleavage and analysis.4.Determination of in vitro anticoagulant activity and protamine reversibility of the novel heparin dodecasaccharide 12-merAccording to chromogenic substrate assay,The IC50 values of anti-FXa activity of 12-mer,fondaparinux sodium,unfractionated heparin and enoxaparin were 32.36ng/mL,14.41ng/mL,379.9ng/mL,92.7 ng/mL,respectively.The molar IC50 value of anti-FXa activity of 12-mer(7.9nM)was equivalent to that of fondaparinux sodium(8.4nM),and it had no obvious anti-FIIa activity.These results indicated that the anticoagulant activity of 12-mer,similar to that of fondaparinux sodium,only depended on the unique AT-binding sequence instead of the rare GlcA2S-containing long-chain sequence.Next,the effect of of protamine at different concentrations on anti-FXa activity of 12-mer was determined.The results showed that the anti-Xa activity of the synthetic 12-mer could be completely reversed by protamine which was similar to that of unfractionated heparin.In contrast,enoxaparin can only be reversed by 50%,and fondaparinux sodium was not reversed at all.Therefore,a novel heparin dodecasaccharide with specific,potent and completely reversible anti-Xa activity was successfully synthesized for the first time in this project.It is a new anticoagulant and antithrombotic drug candidate with research and development potential.