不同修饰模式的肝素寡糖的化学酶法合成及肝素酶的底物特异性研究

发布时间：2018-04-17 18:33

本文选题：肝素寡糖 + 化学酶法合成　；参考：《山东大学》2017年硕士论文

【摘要】：肝素和硫酸乙酰肝素(heparin sulfate,HS)是由氨基葡萄糖(GlcN)和葡萄糖醛酸(GlcA)或艾杜糖醛酸(IdoA)以1→4糖苷键连接而成的二糖单元组成的糖胺聚糖,二糖单元的多个位置可以被硫酸化修饰,因此结构极其复杂。肝素/HS中丰富的寡糖序列是其表现出多种多样的生物活性的结构基础。针对天然肝素的动物源性和结构不均一性造成的安全隐患,合成结构确定的肝素/HS寡糖受到越来越多重视。单纯化学法合成尽管进展迅速,但仍面临合成步骤多、产率低等挑战。最近发展的化学酶法策略因具有立体选择性强、产率高等优点,有望发展成为一种理想的肝素和HS寡糖的合成新技术。因此,本课题拟利用化学酶法合成具有不同硫酸化模式的肝素寡糖。此外,细菌来源的肝素酶(heparinases或heparin lyases)能够通过β-消除机制降解肝素及HS,是表征其结构和制备低分子量寡糖的重要工具酶。但是,之前的研究多以肝素及其衍生物为底物探究其催化机制和切割活性,结构不均一的多糖底物很可能对酶解活性产生干扰,极大增大了产物分析的难度,更难以清晰阐明肝素酶作用于不同切割位点时的差异。因此,本课题根据肝素酶可能的切割位点设计、合成一系列结构均一确定的HS寡糖作为底物,并深入探究三种肝素酶的底物适应性,为拓展肝素酶的应用奠定基础。本学位论文取得的成果及结论包括以下几个方面:1.糖基供体和硫酸基供体的规模化制备利用N-乙酰氨基葡萄糖(GlcNAc)或N-三氟乙酰氨基葡萄糖(GlcNTFA)、ATP、UTP为原料,在三种重组酶NahK、GlmU、PPA的共同催化作用下合成尿苷二磷酸(UDP)-GlcNTFA/UDP-GlcNAc;以尿苷二磷酸葡萄糖(UDP-Glc)为原料,由UDP-Glc脱氢酶、乳酸脱氢酶(LDH)催化合成UDP-葡萄糖醛酸(UDP-GlcA)。经强离子交换柱层析纯化后,糖基供体的纯度可达98%以上,制备规模可达克级。以Na2SO4、ATP为原料,利用ATP硫酸化酶、APS激酶催化合成硫酸基供体PAPS,纯化后的产物纯度达99%,制备规模达到克级。2.肝素六糖的合成、纯化及结构表征以对硝基苯-β-D-葡萄糖醛酸苷(GlcA-PNP)为起始原料,利用糖基转移酶KfiA或PmHS2的催化,在其非还原端交替添加GlcNTFA(GlcNAc)或GlcA得到HS六糖骨架,每步反应产率高达98%,经反相C18柱层析纯化,得到纯度高于90%的六糖骨架,合成规模达百毫克级。利用LiOH脱除三氟乙酰基后,以PAPS为硫酸基供体,由N-硫酸基转移酶(NST)催化合成N-硫酸化六糖。然后在C5异构化酶和2-O硫酸基转移酶(2-OST)的共同催化下,使糖链中介于两个N-硫酸化葡萄糖胺(GlcNS)的GlcA残基转变为2-硫酸化艾杜糖醛酸(IdoA2S)。最后由6-O硫酸基转移酶1/3(6-OST1/3)催化使寡糖的GlcNS或GlcNAc发生6-O-硫酸化修饰(GlcNS6S或GlcNAc6S)。三种硫酸化修饰模式的肝素寡糖产率分别为99%、72%、99%,经离子交换柱层析纯化后的寡糖纯度均高达99%,合成规模达到百毫克级。ESI-MS及NMR分析表明不同修饰模式的肝素六糖产物的结构均正确。3.肝素酶的底物特异性研究根据肝素酶可能的切割位点设计、并利用化学酶法合成了一系列结构确定的肝素及HS寡糖,HPLC分析其纯度90%,ESI-MS测定表明结构的正确性。以合成的寡糖为底物,利用HPLC分析测定肝素酶Ⅰ、Ⅱ、Ⅲ在不同条件下对它们的切割作用,以探究酶对不同底物的催化特异性。同时利用表面等离子共振(surface plasmon resonance,SPR)技术初步研究了肝素酶Ⅲ与不同HS寡糖底物的相互作用。研究结果表明:(1)肝素酶Ⅰ不能切割GlcN与非硫酸化糖醛酸之间的糖苷键位点(GlcN-GlcA或GlcN-IdoA),仅能切割-GlcNS-IdoA2S-,-GlcNS6S3S-IdoA2S-、-GlcNS-GlcA2S-之间的糖苷键,这一结果表明2-O-硫酸化糖醛酸(UA2S)是肝素酶Ⅰ切割必需的,而GlcN的6-O-或3-O-硫酸化修饰对切割作用影响不大。(2)肝素酶Ⅲ可以切割含主要切割位点(GlcNAc-GlcA或GlcNS-GlcA)的三糖底物(Tri-NAc或Tri-NS),但对Tri-NAc的催化效率显著高于Tri-NS。肝素酶Ⅲ能够耐受含不同修饰的GlcN(GlcNH2、GlcNAc6S及GlcNS6S)与GlcA之间的次要切割糖苷键,但催化效率显著低于对应的主要三糖底物,提示N-非取代或6-O-硫酸化修饰均会降低肝素酶Ⅲ对HS寡糖底物的反应活性。肝素酶Ⅲ对含IdoA的次要切割位点(GlcNS-IdoA)的反应效率在反应初期低于主要位点GlcNS-GlcA,但总体差别不大。HS寡糖中的IdoA2S大大降低肝素酶Ⅲ对其还原端相邻的GlcNS-GlcA位点的切割效率,但是对其非还原端的位点的影响不大。肝素酶Ⅲ对底物的特异性强弱与底物分子大小有关的,即其对分子量越大的底物切割效率越高。肝素酶Ⅲ对含有多个位点的HS寡糖中的切割次序为随机切割,这与内切酶的特性相一致;但是相比于还原端和非还原端,肝素酶Ⅲ对内部糖苷键具有更高的切割偏好性。SPR分析表明,单纯通过酶与底物间亲和力大小来判断酶对底物切割效率并不完全可取,因为寡糖底物结构、带电荷情况极其复杂,从而可能会导致寡糖底物与酶的错误结合而阻碍催化反应。(3)肝素酶Ⅱ能够切割含肝素酶Ⅰ和肝素酶Ⅲ作用位点的寡糖;除对含-GlcNS6S-GlcA-位点的寡糖切割效率高于肝素酶Ⅲ,肝素酶Ⅱ对其他含肝素酶Ⅲ作用位点寡糖的反应活性低于肝素酶Ⅲ;肝素酶Ⅱ对肝素酶Ⅰ的作用位点的催化效率与肝素酶Ⅰ相当,均高于对GlcNS-GlcA的作用。
[Abstract]:Heparin and heparan sulfate (heparin sulfate HS) is composed of glucosamine (GlcN) and glucuronic acid (GlcA) or iduronic acid (IdoA) in 1 to two sugar units from 4 glycosidic composition, multiple positions two sugar units can be sulfated modification, so the structure is extremely complex oligosaccharide sequence rich in /HS is the heparin exhibit structural basis for a variety of diverse biological activities. To analyze the safety of animal origin and structure of natural heparin heterogeneity caused by heparin, /HS oligosaccharide structure determination has been more and more attention. A simple chemical synthesis despite rapid progress, but still face synthesis multiple steps, low yield challenges. Chemoenzymatic strategy recently developed with stereo selectivity, high yield, is expected to become the new synthesis technology of a kind of ideal heparin and HS oligosaccharide. Therefore, this topic Oligs with different sulfate pattern synthesized by chemical enzymatic method. In addition, heparinase from bacteria (heparinases or heparin lyases) can eliminate the degradation mechanism of heparin and HS by beta, is an important tool for enzyme characterization of the structure and preparation of low molecular weight oligosaccharides. However, previous studies by heparin and its derivatives to explore the catalytic mechanism and substrate cleavage activity, structure is not a homogeneous polysaccharide substrate is likely to interfere with the enzymatic activity, greatly increase the difficulty of product analysis, the differences are more difficult to articulate heparanase in different cutting sites. Therefore, this topic according to the heparanase cleavage site may design, HS oligosaccharide the synthesis of a series of uniform structure identified as substrates, and explore the three types of heparin enzyme substrate adaptability, lay the foundation for the application of heparanase. And this thesis made a knot The theory includes the following aspects: the scale of 1. glycosyl donor and sulfate donor prepared using N- acetyl glucosamine (GlcNAc) or N- three fluoro acetyl glucosamine (GlcNTFA), ATP, UTP as raw materials, GlmU in the three kinds of recombinant enzyme NahK, common PPA catalyzed synthesis of uridine phosphate (two -GlcNTFA/UDP-GlcNAc; UDP) with uridine glucose two phosphate (UDP-Glc) as raw materials by UDP-Glc dehydrogenase, lactate dehydrogenase (LDH) catalyzes the synthesis of UDP- glucuronic acid (UDP-GlcA). The strong ion exchange column chromatography, the purity of sugar donor could reach above 98%, the preparation of the scale of up to Na2SO4. ATP grams, for the use of raw materials, ATP sulfurylase, APS kinase catalytic synthesis of sulfate radical donor PAPS, after purification the purity of the product reached 99%, the preparation reached.2. six grams of synthetic heparin sugar, purification and characterization of nitrobenzene to beta -D- glucuronide (GlcA-PNP) as starting The raw material, using catalytic glycosyltransferase KfiA or PmHS2, add GlcNTFA in the reducing end of alternate non (GlcNAc) or GlcA HS six sugar backbone, each reaction yield was 98%, by reversed-phase C18 column chromatography, six sugar backbone purity higher than 90%, the scale of hundreds of milligrams of synthesis. The use of LiOH removal of three fluoro acetyl, with PAPS as the sulfate donor by N- sulfotransferase (NST) catalytic synthesis of sulfated sugar. Then six N- transferase in C5 isomerization enzyme and 2-O sulfate (2-OST) Co catalyst, the sugar chain intermediary to the two N- sulfated glucose amine (GlcNS) GlcA residues into 2- sulfated iduronic acid (IdoA2S). By the end of 6-O sulfotransferase 1/3 (6-OST1/3) GlcNS or GlcNAc catalyst. The oligosaccharides of 6-O- sulfation (GlcNS6S or GlcNAc6S). Three kinds of sulfated modification mode of heparin oligosaccharides yield were 99%, 72%. 99%, by ion exchange In column chromatography the purity of oligosaccharides were as high as 99%, reached 100 mg.ESI-MS synthesis and NMR analysis show that the research of substrate specificity of heparin six sugar products of different structure modification patterns are correct.3. heparanase cleavage sites of heparanase may according to the design, and the use of chemical enzyme heparin and HS oligosaccharide determines a series of the structure of the synthetic method, HPLC analyzed the purity of 90%, ESI-MS showed the validity of the structure. In the synthesis of oligosaccharides as substrate, determination of heparinase I, II III by HPLC, cutting effect on them in different conditions, to explore the catalytic enzyme on different substrates specificity. At the same time using surface plasmon resonance (surface plasmon resonance, SPR) a preliminary study on the technology of the interaction of heparin with different enzyme III HS oligosaccharide substrate. The results show that: (1) heparinumheparinase I cannot cut between GlcN and non sulfated uronic acid The glycosidic bond sites (GlcN-GlcA or GlcN-IdoA), only can cut -GlcNS-IdoA2S-, -GlcNS6S3S-IdoA2S-, the glycosidic bond between -GlcNS-GlcA2S-, the results show that 2-O- sulfated uronic acid (UA2S) is heparinumheparinase I cut required, and 6-O- or 3-O- GlcN sulfuric acid modification on cutting little effect. (2) heparinase III can cut major cleavage sites (GlcNAc-GlcA or GlcNS-GlcA) of the three sugar substrates (Tri-NAc or Tri-NS), but the catalytic efficiency of Tri-NAc was significantly higher than that of Tri-NS. heparanase III can be tolerated with different modified GlcN (GlcNH2, GlcNAc6S and GlcNS6S) between GlcA and the secondary cutting glycosidic bond, but the catalytic efficiency was significantly lower than that of the main three sugar the substrate corresponding to the N- or 6-O- to replace the non sulfated modification will decrease the reaction activity of heparinase III on HS oligosaccharide substrates. Heparinase III on secondary cutting sites containing IdoA (GlcNS-IdoA) reaction efficiency In the initial stage of the reaction is lower than the main site of GlcNS-GlcA, but little difference in overall.HS oligosaccharide IdoA2S can reduce the cutting efficiency of heparinase III GlcNS-GlcA loci on the reducing end of adjacent, but has little effect on the non reducing end of the site. The heparinase III of the substrate specificity and strong weak substrate molecular size, i.e. the greater the molecular weight of the substrate cutting efficiency is higher. The heparinase III on the cutting order of HS oligosaccharides containing multiple sites in random cutting, consistent with the characteristics of this enzyme; but compared to the reducing end and non reducing end, heparinase III has the.SPR preference analysis indicates that the higher cutting the internal glycosidic bond, only by the affinity between enzyme and substrate size to determine the enzyme cutting efficiency is not entirely desirable, because the oligosaccharide substrate structure, charge is extremely complex, which may lead to oligosaccharides and substrate The combination of enzyme catalytic reaction. The error block (3) heparinase II could cut oligosaccharides containing heparin and heparinase I enzyme III interaction sites; in addition to containing -GlcNS6S-GlcA- oligosaccharide on the cutting efficiency is higher than that of heparinase III, the reaction activity of heparinase II on other sites of action of heparinase III containing oligosaccharides than heparinase III; the catalytic efficiency and heparinase I heparanase II binding sites of heparinase I quite was higher than that of the GlcNS-GlcA.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R914

【参考文献】