以HPSE为靶点设计合成硫代糖苷类硫酸肝素

【作者】 陈健；

【导师】 汤建；

【作者基本信息】 江苏大学 ， 中药学， 2019， 硕士

【摘要】 糖类为生命体提供能量,聚糖类还在抗肿瘤、神经退行性疾病上、抗肝炎、心血管疾病以及抗衰老方面有独特的生物活性。硫在动植物的蛋白质和酶中起着至关重要的作用,而硫代糖苷类有助于快速构建创新的硫糖衍生物,具有特殊的生物活性和较好的靶向特性。硫酸肝素（HS）是作为商业制造肝素时产生的副产物而被首次发现,由葡萄糖醛酸（GlcA）、艾杜醛酸（Idu A）与葡萄糖胺衍生物（Glc Nx）二糖重复单位组成。HS在体内是以硫酸肝素多糖的形式存在的,主要分布在细胞表面与细胞外基质（ECM）里,往往与蛋白共价结合为HS蛋白多糖（HSPGS）,表现了复杂的多效性,在细胞的分化、迁移、血管生成和细胞信号调节中发挥着重要的作用。乙酰肝素酶（HPSE）是一种具有独特降解HS能力的糖苷内切酶,有助于重塑细胞外基质和调节HS结合蛋白的生物利用率。HPSE催化裂解HS中GlcA与GlcN残基间β-（1,4）-糖苷键的裂解,参与了很多重要的生理与病理功能调控。研究表明,HPSE在肿瘤细胞中高表达,与肿瘤恶化转移密切有关,是临床上治疗癌症转移潜在有效靶点。本研究以HPSE为靶点,基于HPSE水解机制,设计与合成结构明确的HPSE硫酸肝素类肿瘤抑制剂。HS由是硫酸化的二糖重复单位,由GlcA和GlcN的1-4位相连,之间-O-则是HPSE的酶切位点,HPSE可以特异性水解HS。本研究拟以此为基础合成硫代糖苷硫酸肝素,用硫原子代替水解位点氧原子,降低水解速度,提高其对HPSE耐受性。硫代糖苷硫酸肝素各组成糖残基的合成主要采用主要是化学手段,然后采用酶催化制备HS类似物硫代糖苷硫酸肝素,期望发现新的结构新颖HPSE硫酸肝素抑制剂,主要取得研究结果如下:以葡萄糖为起始原料,探究糖不同羟基的反应强弱关系,为氨基葡萄糖的结构修饰提供理论基础。经过一系类保护和脱保护,用苯甲酰基和苄基保护1,2,3,6-OH,游离4-OH,得三种基于葡萄糖的中间体。采用卤素I₂,对甲苯磺酰氯和三氟甲磺酸酐分别活化游离羟基,考察两个糖基中间体4-OH的反应活性。结果显示苯甲酰基保护的4-OH游离葡萄糖可被成功活化,且活化基团三氟甲磺酰基易离去,能够达到改变构型的目的,经KSAc取代合成4-S-乙酰基-1,2,3,6-四-O-苯甲酰基-β-D-吡喃葡萄糖,该化合物经5步反应得到,总产率为2.70%,碱性介质脱保护,MS确认保护基可被成功脱除。以氨基葡萄糖为起始原料合成4-SH-D-氨基葡萄糖,保护2-NH₂和1,3,6-OH,游离4-OH,再活化4-OH,SH取代4-OH后可得硫代糖苷硫酸肝素骨架的一种糖单体。本部分实验同样考察了苯甲酰基对氨基葡萄糖的选择性保护,结果显示苯甲酰基对氨基葡萄糖羟基的选择性较差。实验选择1,2,3位用乙酰基保护,减小位阻。6-OH为伯羟基,活性较4-OH高,但苯甲酰氯对两个位置的OH选择性较差,实验发现苯甲酰氰可以较好的选择性保护6-OH,再经三氟甲磺酸酐活化和改变构型,与KSAc反应得4-S-乙酰基-2-N-乙酰基-1,3-二-O-乙酰基-6-O-苯甲酰基-β-D-氨基葡萄糖。该化合物为首次合成,经8步反应,总产率为2.43%,碱性介质脱保护,MS确认保护基可被成功脱除。以葡萄糖醛酸为起始原料,合成1-O-对硝基苯甲基-β-葡萄糖醛酸苷。乙酰基保护2,3,4-OH和6-COOH,脱除COOH上的乙酰基,换为甲基保护,使结构稳定。Br活化1-OH,接上芳基取代基团。实验部分考察了三种芳基取代基团对硝基苯甲醇,对硝基苯酚和对甲氧基苯甲醇,结果显示对硝基苯甲醇的产率远高于其他两种芳基基团,高达62.35%。本部分实验经过6步反应合成化合物1-O-对硝基苯甲基-葡萄糖醛酸,总产率为6.16%。HPSE抑制剂的酶化学合成。经一系列特异性酶NahK,GlmU,PPA,pmsH2的作用,聚合硫代糖合成HPSE抑制剂骨架。综合得到上述修饰后的两种糖单体,利用酶催化反应,以氨基葡萄糖单体为对照底物,提供能量物质ATP和UTP,在NahK,GlmU,PPA酶的催化作用下构建成UDP-GlcNHAc,再以糖苷聚合酶pmsH2连接聚合两种糖单体,经MS初步确认目标化合物,实验结果显示氨基葡萄能够与修饰的葡萄糖醛酸形成二糖,而4-SH-GlcNHAc作为底物,MS并未检测到符合硫代二糖结构的分子量,还需进一步探索和研究。更多还原

【Abstract】 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.更多还原