本文選題：肝素寡糖 + 化學酶法合成��； 參考：《山東大學》2017年碩士論文

【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糖苷鍵連接而成的二糖單元組成的糖胺聚糖,二糖單元的多個位置可以被硫酸化修飾,因此結(jié)構極其復雜。肝素/HS中豐富的寡糖序列是其表現(xiàn)出多種多樣的生物活性的結(jié)構基礎。針對天然肝素的動物源性和結(jié)構不均一性造成的安全隱患,合成結(jié)構確定的肝素/HS寡糖受到越來越多重視。單純化學法合成盡管進展迅速,但仍面臨合成步驟多、產(chǎn)率低等挑戰(zhàn)。最近發(fā)展的化學酶法策略因具有立體選擇性強、產(chǎn)率高等優(yōu)點,有望發(fā)展成為一種理想的肝素和HS寡糖的合成新技術。因此,本課題擬利用化學酶法合成具有不同硫酸化模式的肝素寡糖。此外,細菌來源的肝素酶(heparinases或heparin lyases)能夠通過β-消除機制降解肝素及HS,是表征其結(jié)構和制備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為底物探究其催化機制和切割活性,結(jié)構不均一的多糖底物很可能對酶解活性產(chǎn)生干擾,極大增大了產(chǎn)物分析的難度,更難以清晰闡明肝素酶作用于不同切割位點時的差異。因此,本課題根據(jù)肝素酶可能的切割位點設計、合成一系列結(jié)構均一確定的HS寡糖作為底物,并深入探究三種肝素酶的底物適應性,為拓展肝素酶的應用奠定基礎。本學位論文取得的成果及結(jié)論包括以下幾個方面:1.糖基供體和硫酸基供體的規(guī)模化制備利用N-乙酰氨基葡萄糖(GlcNAc)或N-三氟乙酰氨基葡萄糖(GlcNTFA)、ATP、UTP為原料,在三種重組酶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)為原料,由UDP-Glc脫氫酶、乳酸脫氫酶(LDH)催化合成UDP-葡萄糖醛酸(UDP-GlcA)。經(jīng)強離子交換柱層析純化后,糖基供體的純度可達98%以上,制備規(guī)�？蛇_克級。以Na2SO4、ATP為原料,利用ATP硫酸化酶、APS激酶催化合成硫酸基供體PAPS,純化后的產(chǎn)物純度達99%,制備規(guī)模達到克級。2.肝素六糖的合成、純化及結(jié)構表征以對硝基苯-β-D-葡萄糖醛酸苷(GlcA-PNP)為起始原料,利用糖基轉(zhuǎn)移酶KfiA或PmHS2的催化,在其非還原端交替添加GlcNTFA(GlcNAc)或GlcA得到HS六糖骨架,每步反應產(chǎn)率高達98%,經(jīng)反相C18柱層析純化,得到純度高于90%的六糖骨架,合成規(guī)模達百毫克級。利用LiOH脫除三氟乙酰基后,以PAPS為硫酸基供體,由N-硫酸基轉(zhuǎn)移酶(NST)催化合成N-硫酸化六糖。然后在C5異構化酶和2-O硫酸基轉(zhuǎn)移酶(2-OST)的共同催化下,使糖鏈中介于兩個N-硫酸化葡萄糖胺(GlcNS)的GlcA殘基轉(zhuǎn)變?yōu)?-硫酸化艾杜糖醛酸(IdoA2S)。最后由6-O硫酸基轉(zhuǎn)移酶1/3(6-OST1/3)催化使寡糖的GlcNS或GlcNAc發(fā)生6-O-硫酸化修飾(GlcNS6S或GlcNAc6S)。三種硫酸化修飾模式的肝素寡糖產(chǎn)率分別為99%、72%、99%,經(jīng)離子交換柱層析純化后的寡糖純度均高達99%,合成規(guī)模達到百毫克級。ESI-MS及NMR分析表明不同修飾模式的肝素六糖產(chǎn)物的結(jié)構均正確。3.肝素酶的底物特異性研究根據(jù)肝素酶可能的切割位點設計、并利用化學酶法合成了一系列結(jié)構確定的肝素及HS寡糖,HPLC分析其純度90%,ESI-MS測定表明結(jié)構的正確性。以合成的寡糖為底物,利用HPLC分析測定肝素酶Ⅰ、Ⅱ、Ⅲ在不同條件下對它們的切割作用,以探究酶對不同底物的催化特異性。同時利用表面等離子共振(surface plasmon resonance,SPR)技術初步研究了肝素酶Ⅲ與不同HS寡糖底物的相互作用。研究結(jié)果表明:(1)肝素酶Ⅰ不能切割GlcN與非硫酸化糖醛酸之間的糖苷鍵位點(GlcN-GlcA或GlcN-IdoA),僅能切割-GlcNS-IdoA2S-,-GlcNS6S3S-IdoA2S-、-GlcNS-GlcA2S-之間的糖苷鍵,這一結(jié)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-硫酸化修飾對切割作用影響不大。(2)肝素酶Ⅲ可以切割含主要切割位點(GlcNAc-GlcA或GlcNS-GlcA)的三糖底物(Tri-NAc或Tri-NS),但對Tri-NAc的催化效率顯著高于Tri-NS。肝素酶Ⅲ能夠耐受含不同修飾的GlcN(GlcNH2、GlcNAc6S及GlcNS6S)與GlcA之間的次要切割糖苷鍵,但催化效率顯著低于對應的主要三糖底物,提示N-非取代或6-O-硫酸化修飾均會降低肝素酶Ⅲ對HS寡糖底物的反應活性。肝素酶Ⅲ對含IdoA的次要切割位點(GlcNS-IdoA)的反應效率在反應初期低于主要位點GlcNS-GlcA,但總體差別不大。HS寡糖中的IdoA2S大大降低肝素酶Ⅲ對其還原端相鄰的GlcNS-GlcA位點的切割效率,但是對其非還原端的位點的影響不大。肝素酶Ⅲ對底物的特異性強弱與底物分子大小有關的,即其對分子量越大的底物切割效率越高。肝素酶Ⅲ對含有多個位點的HS寡糖中的切割次序為隨機切割,這與內(nèi)切酶的特性相一致;但是相比于還原端和非還原端,肝素酶Ⅲ對內(nèi)部糖苷鍵具有更高的切割偏好性。SPR分析表明,單純通過酶與底物間親和力大小來判斷酶對底物切割效率并不完全可取,因為寡糖底物結(jié)構、帶電荷情況極其復雜,從而可能會導致寡糖底物與酶的錯誤結(jié)合而阻礙催化反應。(3)肝素酶Ⅱ能夠切割含肝素酶Ⅰ和肝素酶Ⅲ作用位點的寡糖;除對含-GlcNS6S-GlcA-位點的寡糖切割效率高于肝素酶Ⅲ,肝素酶Ⅱ?qū)ζ渌嗡孛涪笞饔梦稽c寡糖的反應活性低于肝素酶Ⅲ;肝素酶Ⅱ?qū)Ω嗡孛涪竦淖饔梦稽c的催化效率與肝素酶Ⅰ相當,均高于對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.

【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R914

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