Design And Synthesize Thioglycoside Heparin Sulfate With HPSE As The Target

Sugars provide energy for living organisms,polysaccharides also have unique biological activities in anti-cancer,neurodegenerative diseases,anti-hepatitis,cardiovascular diseases and anti-aging.Sulphur plays an important role in animal and plant proteins and enzymes,and thiosaccharides contribute to the rapid construction of innovative thiose derivatives with special biological activities and better targeting properties.Heparin sulfate（HS）was discovered for the first time as a by-product of commercial heparin production.Glucuronic acid（GlcA）is the first monosaccharide identified in HS,and Glucosamine（GlcN）is the second monosaccharide found in HS.HS exists in the body as the form of heparin sulfate polysaccharide.Heparan sulfate proteoglycans（HSPGS）in ECM on the cell surface shows complex multipotency and plays an important role in cell differentiation,migration,angiogenesis and cell signal regulation.HPSE is an endoglycosidase with unique ability to degrade HS,which helps to remodel extracellular matrix and regulate the bioavailability of HS-binding protein.HPSE catalyzes the cleavage of theβ-（1,4）-glycoside bond between GlcA and GlcN residues in HS,thus participating in many important biological functions.At present,cancer is the main cause of human morbidity and mortality.Studies have shown that the high expression of HPSE in cancer cells is closely related to the deterioration and metastasis of cancer,and it is a potential effective target for clinical treatment of cancer metastasis.In this study,HPSE is used as a target to study the anti-cancer drugs of HPSE inhibitors.HS is a sulfated disaccharide repetitive unit,in which GlcA is linked with GlcN at 1-4 sites.The linked O atom is the cleavage site of HPSE.On this basis,thioglycoside heparin sulfate was synthesized.Sulphur atom was used to replace oxygen atom in hydrolysis site,and the hydrolysis rate was reduced.In this paper,HS analogue heparin thioglycoside sulfate was synthesized by combining chemical and enzymatic chemistry to inhibit the activity of HPSE.With glucose as the starting material,the relationship between protective group or glycohydroxyl group and glucose was explored,which provided a theoretical basis for the structural modification of glucosamine.Three glycosyl donor intermediates were obtained by first-class protection and deprotection,benzoyl and benzyl protection of 1,2,3,6-OH and free 4-OH.Halogen I₂,p-toluene sulfonyl chloride and trifluoromethanesulfonic anhydride were used to activate free hydroxyl groups,respectively.The reactivity of two glycosyl donors,4-OH,was investigated.The results showed that 4-OH free glucose protected by benzoyl group could be activated successfully,and the active group trifluoromethane sulfonyl group was easy to leave,which could change the configuration.4-S-acetyl-1,2,3,6-tetra-O-benzoyl-β-D-pyran glucose was synthesized by KSAc substitution.The total yield of the compound was 2.7%.Based on the structure modification experiment of glucose,a sugar monomer of heparin thioglycoside sulfate skeleton can be obtained by using glucosamine as starting material,protecting 2-NH2 and 1,3,6-OH,dissociating 4-OH,reactivating 4-OH,and replacing 4-OH with SH.The selective protection of benzoyl group to glucosamine was also investigated in this part of the experiment.The results showed that the selectivity of benzoyl group to glucosamine hydroxyl was poor.In order to reduce the steric resistance,1,2,3 sites were chosen to be protected by acetyl group.6-OH is primary hydroxyl group with higher activity than 4-OH,but benzoyl chloride has lower selectivity to OH at the two sites.It was found that benzoyl cyanide can selectively protect 6-OH well,and then react with KSAc to obtain 4-S-acetyl-2-N-acetyl-1,3-di-O-acetyl-6-O-benzo yl-β-D-glucosamine by trifluoromethanesulfonic anhydride activation and configu ration modification.The compound was synthesized for the first time.After 8 steps,the total yield was 2.43%.The alkaline medium was deprotected.MS confirmed that the protective group could be successfully removed.With glucuronic acid as starting material,acetyl group protects 2,3,4-OH and 6-COOH,removes acetyl group from COOH,and replaces it with methoxy group protection to stabilize the structure.Bromine activates 1-OH and connects chromogenic groups.In the experimental part,three chromogenic groups p-nitrobenzyl alcohol,p-nitrophenol and p-methoxybenzyl alcohol were investigated.The results showed that the yield of p-nitrobenzyl alcohol was much higher than that of the other two chromogenic groups,up to 62.35%.In this part,com pound 1-O-p-nitrophenylmethyl-glucuronic acid was synthesized by six steps in total yield of 6.16%.The enzymatic synthesis of HPSE inhibitors was carried out through the a ction of a series of specific enzymes NahK,GlmU,PPA and pmsH2 to synthesize HPSE inhibitor skeleton by polymerizing thiosaccharides.The two modified sugar monomers were synthesized by enzymatic reaction,using glucosamine monomers as control substrates,providing energy substances ATP and UTP.Un der the catalysis of NahK,Glmu and PPA enzymes,UDP-GlcNHAc was constructed,and polymerization of two sugar monomers with glycoside polymerase pmsH2.The target compounds were initially confirmed by MS.The experimental results showed that glucosamine could be formed with the two sugar monomers.Glucuronic acid forms disaccharides,while 4-SH-GlcNHAc as a substrate,MS did not detect molecular weight conforming to thiodisaccharide structure,which needs further exploration and research.