【作者】 杨波；

【导师】 于广利；

【作者基本信息】 中国海洋大学 ， 药物化学， 2009， 博士

【摘要】 本论文以几种海洋硫酸半乳聚糖为研究对象，采用酸法和自由基氧化法和现代色谱分离技术获得了120种寡糖，通过还原胺化方法制备了30种拟糖脂生物探针，并运用生物质谱技术确定了它们的结构序列。首先，运用电喷雾碰撞诱导串联质谱(ES-CID-MS／MS)技术，分析了14种不同结构特征的具有代表性的卡拉胶寡糖的序列，总结了不同寡糖在质谱分析中产生的糖基碎片信息，发现并成功解决了硫酸寡糖质谱分析中硫酸根易脱落的问题，找到了不同结构的寡糖二级质谱碎片离子的断裂规律，从理论上解释了产生特殊离子碎片的原因，最终建立了硫酸半乳寡糖微量序列分析的质谱学方法。这些规律同样适用于其它硫酸寡糖序列分析。所建立的微量寡糖序列分析方法为卡拉胶类寡糖的构效关系研究提供了重要技术支持。以κ-、ι-和λ-卡拉胶、琼胶和脱硫λ-卡拉胶为原料，采用温和稀酸降解法制备了系列寡糖并用ES-CID-MS／MS技术确定了其序列。获得了90个寡糖单体，其中40种寡糖单体为首次报道。从位阻效应和环张力效应出发，以特异酸解条件制备了聚合度3～19的κ-卡拉胶奇数硫酸寡糖和聚合度3～19的琼胶奇数寡糖；根据3，6-内醚半乳糖的酸不稳定性，进而选用了还原性弱酸降解制备了聚合度2～24的κ-卡拉胶偶数硫酸寡糖醇与2～20的偶数琼胶寡糖醇系列寡糖；根据C2位硫酸基拉电子诱导效应及稀酸对α-1，3和β-1，4糖苷键水解选择性的差异，制备了聚合度2～20的ι-卡拉胶偶数硫酸寡糖及其糖醇，本技术具有拟α-琼胶酶和α-卡拉胶酶特异性降解特点，但却制备了结构不同于酶法制备的寡糖化合物，其成本又远低于生物酶法。含有3，6-内醚半乳糖的多糖易被稀无机酸和有机酸以及固体酸如各种酸性阳离子交换树脂降解而得到全部为奇数的罕见降解结果，在大量实验数据的基础上，首次阐明了这种能够特异性获得奇数寡糖的降解机理。在上述降解机理的指导下，以一种红藻多糖为原料，采用分步降解法制备了14种杂合寡糖并确定了它们的序列，为复杂红藻多糖的结构研究提供了方法学参考。此外，进一步评价了系列杂合硫酸半乳寡糖对血管紧张素转化酶(ACE)和β-分泌酶(BACE)抑制活性，结果显示：在648μg／mL下，组分M4具有一定抑制ACE活性，抑制率为10．4％，具有弱降压活性；在100μg／mL下，低分子量组分M1，M2和M3抑制BACE分别为42．5％，31．1％和47．6％，表明具有一定抗老年痴呆活性，但高分子量组分则无活性。该结果为从事其它硫酸半乳寡糖构效关系的研究、生物活性评价提供了基础。在对硫酸半乳多糖的自由基氧化降解实验中，采用正交实验设计法确定了自由基降解的最佳条件，获得了系列自由基降解产物，并通过质谱技术成功分析了复杂产物中的寡糖结构。实验结果表明，自由基降解是一种非特异性的降解方法，所得寡糖结构复杂，这为获得结构丰富多样且新颖的寡糖提供了有效方法。此外，通过对产物的分析发现，该方法没有硫酸根的脱落问题，能够较好的保留多糖分子中的硫酸酯基团，有利于某些生物活性的评价。通过此降解方法获得了3个系列30多种寡糖和杂合硫酸半乳寡糖组分，首次获得了结构新颖的κ-和ι-卡拉胶寡糖酸。为了深入研究寡糖与蛋白相互作用，快速筛选活性寡糖化合物，利用上述寡糖为原料，进一步采用还原氨胺化技术探讨了拟糖脂生物探针的合成，获得了30种拟糖脂，并运用生物质谱技术确定了它们的结构。这些寡糖脂为糖芯片的构建，以及进一步从事寡糖与蛋白之间的相互作用研究提供了基础。综上所述，本文以多种硫酸半乳寡糖为研究对象，系统建立了一种微量简便的生物质谱学方法，该方法能确定各种硫酸半乳寡糖的序列，并在对寡糖产物结构的序列分析的基础上，深入探讨了化学降解多糖的机理，创建了以化学手段进行可控、定向降解、规模化制备奇数与偶数卡拉胶寡糖方法。在此基础上，运用还原胺化法将系列寡糖合成了拟糖脂生物探针，为海洋硫酸寡糖芯片的建立和海洋糖药物的开发提供了理论与技术支持。更多还原

【Abstract】 In the thesis, one hundred and twenty oligosacchandes were obtained fromseveral marine-derived sulfated galactans by mild acid hydrolysis and free-radicaloxidative degradation with modern chromatographic separation and purificationtechniques, and thirty neoglycolipids prepared by a reductive animation reaction.Their sequences and structures were determined by advanced mass spectrometry.Negative-ion electrospray tandem mass spectrometry with collision induceddissociation (ES-CID-MS/MS) was established for sequence determination ofmultiply sulfated carrageenan oligosaccharide fragments using twelve representativeoligosaccharides with different structural features. Carrageenan oligosaccharides withhomogeneous disaccharide-compositions were used to establish their fragmentationpattern which was then applied to sequence determination of unusual oligosaccharides.All sugar fragments of different oligosaccharide were collected and compared. Ashydrogen form of sulfate groups are labile and easily lost during collision-inducedassociation, we change all sulfated oligos to sodium form,and solved the big problemof sulfate-group existed in mother-ion unstable during MS/MS analysis. Theproduct-ion spectra of [M-Na]-feature an extensive series of B- and C-type glycosidiccleavages whereas the Y-type cleavage occurs mainly at the sulfated residues. Theprinciples established here was readily applicable to other types of sulfatedoligosaccharides. The analysis method for sequence determination established byES-CID-MS/MS provide a basis for the deep study on structure-function relationshipsof sulfated-oligosaccharides. Nine series of oligosaccharides were obtained fromκ-,ι-,λ-carrageenan andagarose by mild acid hydrolysis, whose sequences were determined by ES-CID-MS/MS. Eighty oligosaccharides were determined, and forty oligosaccharides werefirstly reported. Considering that the steric effect and ring tension, series ofodd-numberedκ-carrageenan oligosaccharides with degree of polymerization (dp) of3-25, and odd-number agar-oligosaccharides with dp3-17 were prepared from mildacid hydrolysis. Based on the reagional selection, and the liability of 3,6-anhydro-galatose,(AnG) even-numberκ-carra-oligosaccharide alcohol with dp2-24, andeven-number agar-oligosaccharides with dp 2-20, were prapared with reductive mildacid hydrolysis. Based on the effect of sulfate-group at C2 of AnG and selectedhydrolysis toβ-1,4-andα-1,3-glycosidic bond, series of even-numbered iota-carra-oligosaccharideswith dp2-20 and their alditols with dp2-20 were prepared by mildacid hydrolysis. The hydrolysis specificity is similar toα-agarase andα-carrageenase,but the cost is far lower than that of enzyme, especially for the structure ofoligosaccharides obtained are different from enzyme hydrolysate. The 3,6-anhydro-galactosecontaing polysaccharides can not only be hydrolyzed by inorganic acid, canalso hydrolyzed by other varies organic and strong-cation exchange resin. Thus, wepropose the point of view thatκ-carrageean and agarose can only get odd-numberoligosaccharide by mild acid hydrolysis, and iota-carrageenan can get even-numberoligosaccharide under the similar condition.Under the guidance of hydrolysis mechanism, fourteen hydride oligosaccharideswere obtained from FB1, a polysaccharide from red alga Furcellaria lumbricalis, bystep mild acid hydrolysis, and their structural sequence were also determined by massspectrometry technique. The results provide a good methodological reference for thestudy on structure of complex polysaccharides from red alga. Furthermore, theangiotensin-converting enzyme (ACE) inhibition, immunomudulation andβ-Secretase(BACE) inhibition activities of the low-molecular-weight hydride oligosaccharidesmixture fractions were evaluated in vitro. The result shows that fraction M4 has weakactivity in ACE inhibition assay, with the inhibition rate 10.4% at the testconcentration (648μg/mL). The lower molecular weight fractions M1, M2 and M3 appear to have certain activities in anti-Alzheimer’s disease assay, withβ-Secretaseinhibition rate 42.5%, 31.1% and 47.6% at the test concentration (100μg/mL),respectively. The higher molecular weight oligosaccharides fractions M4-M6 are noactive. These data provide a basis for the deep study on structure-function relation-shipsand bioassays of other sulfated galactans.The optimization conditions of free-radical degradation of carrageenan wereestablished on the basis of the systematical orthogonal experiment. Series ofoligosaccharides were obtained by free-radical degradation, whose structures wereanalyzed successfully by mass spectrometry technique. The result shows that thefree-radical degradation is a non-specificity hydrolytic method. Some novel structuresof oligosacchardes were acquired from the complex hydrolysate by low pressure gelpermeation chromatography. Under free-radical degradation, all sulfated groups werereserved and the reducing ends of part of oligosaccharides were being oxidized.Especially, the AnG at the reducing end was easily oxidized and part of hemiacetalwas changed to carboxyl group. Their degree of polymerization and structuralcharacterizations were determined by high-sensitivity electrospray ionization tandemmass spectrometry (ESI-MS) techniques. Those novel structures of oxidizedκ-carrageean were firstly reported. The tyrosine kinase inhibition activities of theoligosaccharides were evaluated in vitro. The results show that YB5 will be developedas potential anti-tumor drugs with nhibition rate 90% at the test concentration.In order to deeply study on oligosaccharides-protein relationship and screeningbioactivities of oligosaccharides, thirty neoglycolipids were obtained from by areductive animation reaction and their sequence and structure were identified withESI-CID-MS/MS technique. The results provide a good reference for synthesis ofsulfated-neoglycolipids, and importantly, a basis for the preparation of theoligosaccharide-chip and the deep study on oligosaccharide-protein interactions.In summary, a method for the determination of sequence of sulfated oligosaccharidesby MS in this thesis. Base on this technique, deeply study on themechanism of mild acid hydrolysis of galactan polysaccharides with highly ordereddisaccharide repeats leading to a complete series of exclusively odd-numbered oligosaccharides. Furthermore, thirty neoglycolipids were synthesized for glycomicroarryanalysis.更多还原