Investigation Of Specificities Of Chemical Hydrolysis Of Galactans And Sequence Analysis Of Their Oligosaccharides And Synthesis Of Their Neoglycopid Probes Towards Construction Of Marine-Derived Oligosaccharide-Chips

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.