| Bacterial infections have always been a threat to human health,and various effective measures have been developmed to prevent and treat bacterial infectious diseases.Currently,antibacterial drugs,such as antibiotics,are mainly used to kill bacterIal pathogens,however,many multidrug-resistant bacterial strains have emerged owing to the abuse of antibiotics.Therefore,development of more effective therapies to treat with such emergence is in demand.Carbohydrates are the most abundant and structurally diverse organic molecules in nature.The cell glycocalyx,which is a layer of carbohydrates that covers the surface of cells,is involved in various biological processes such as cell differentiation,recognition,adhesion,and even pathological development.Bacterial capsular polysaccharides are one of the cell glycocalyx that are not only principal virulent factors but also important characterization for bacterial serotypes.Many capsular polysaccharides are highly conserved and immunogenic.Thus,they were characterized as ideal target antigens for the development of clinically functional antibacterial vaccines.Several vaccines based on bacterial capular polysaccharides,such as Streptococcus pneumoniae vaccine,Haemophilus influenzae type B vaccine,and Neisseria meningitidis group C vaccine etc,have been licensed and commercialized to prevent bacterial infectious diseases.Compared with antibiotics,these bacterial vaccines can be widely vaccinated and long-term protection after inoculation.The first generation of S.pneumoniae vaccine is only a polysaccharide-based vaccine,which only produce a T cell-independent immune response owing to their weak immunogenicity of ploysaccharide.To improve its immunogenicity,the second generation of S.pneumoniae CPS-based glycoconjugate vaccines were devolped.However,the carbohydrates used in the preparation of the first and/or second generation of bacterial vaccines were usually extracted from bacterial cells and/or cell cultures,and they were heterogeneous that resulted in difficulties in quality control of synthetic glycoconjugate vaccines.Therefore,oligosaccharide fragments with well-defined structures by chemical synthesis have been used as haptens to conjugate with carrier protein to obtain glycoconjugates,leading to the third generation of semi-synthetic glycoconjugate vaccine.Pneumococcal serotype 3(SP3)is one of the most prevalent serotypes that can cause invasive pneumococcal disease(IPD),necrotizing pneumonia,and acute otitis media in adults.Although SP3 has been embodied in PCV13,its CPS conjugate exhibited a lower immunoglobulin G(IgG)response,which can affect the whole efficacy of PCV13.Thus,in this thesis,we designed and synthesized four homogenous and structurally defined SP3 CPS oligosaccharides antigens and conjugated these synthetic antigens with tetanus toxoid to form semi-synthetic vaccine candidates for preliminary immunological.Bacterial pore-forming toxins that secreted by infectious bacteria were associated with many diseases.They were not only the main virulence and pathogenic factors,but also participated in the growth and development of infectious bacteria.It has been further demonstrated that some of these toxins,including aerolysin,CAMP factor and many others,form porous oligomers on the surface of the host cell and then spontaneous inserted these porous oligomers into the cell membrane to breach the cell permeability barrier,trigger adversematerial exchanges,and finally cause cell death.The pore-forming toxins usually bind to glycosylphosphatidyliiositol(GPI)anchors present on the host cell surface to assist their formation of porous oligomers,because most of them are hydrophilic proteins and do not contain obvious pore-forming structures.The GPI anchors are a structurally diverse class of glycolipids that are ubiquitous in eukaryotic species and immobilize proteins or glycoproteins on the cell surface by inserting their lipid fractions into the cell membrane.Previous studies have shown that the CAMP factor can bind to both GPI and GPI fragments,and GPI derivatives without phosphoglyceride chain can inhibit the lysis of sheep red blood cells mediated by CAMP factor.Thus,a series of structurally well-defined GPI analogs were designed and synthesized in this thesis;moreover,and two BODIPY fluorescence-tagged GPI analogs were also prepared for found pore-forming toxin inhibitors.This thesis includes three major parts:In chapter 1,synthetic and other related studies of SP3 CPS oligosaccharides and fluorescent or biotin labled GPI derivatives have been detailly reviewed.SP3 CPS is a linear polysaccharide that is composed of a repeating disaccharide made of ?-linked D-glucuronmc acid(GlcA)and D-glucose(Glc).The synthesis of these oligosaccharids is divided into two ways:?The glucuronic acid building block was directly used to construct the oligosaccharide chains through a reasonable protection and glycosidation strategy,and finally deprotection to give the target compound.?Regioselective oxidation of multiple Glc units in complex glucosides to generate GlcA units in one step after using the glucose building block to construct the oligosaccharide chains to afford the desired products.For the synthesis of fluorescent or biotin labled GPI derivatives,fluorescent compounds or biotin derivatives were designed to contain a carboxyl group,which could linked with the free amino group in the GPI anchor via an amide bond formation.In their synthesis,the free 2-amino group in the glucosamine residue was protected by the Boc group before construction of the amide bond,which could be removed with mild acidic condition to give the target compound.Chapter 2 described the synthesis and immunological studies of SP3 CPS oligosaccharides and their glycoconjungates.Disaccharide thioglycoside SP-10 was synthesized starting from D-glucose through the preactivation protocol,and it was used as the common intermediate for generating other disaccharide and trisaccharide building blocks and for elongating the carbohydrate chain to achieve penta-,hexa-,hepta-,and octasaccharides SP-(1-4)through[3+2]?[4+2]?[3+4]and[4+4]glycosylations strategy,respectively.SP-(1-4)were linked with BSA and TT to afford glycoconjugates,respectively,using disuccinimide glutarate as the linker,and the carbohydrate loadings of glycoconjugates were determined by the differences of conjugate weights compared from MALDF-TOF mass spectra.Immunological evaluations of the glycoconjugates showed that they elicited robust T-cell-dependent immunoglobulin G antibody responses and the penta-and hexasaccharides glycoconjugates were proven to induce much stronger immune responses than the hepta-and octasaccharides glycoconjugates.The synthesis of a series of structurally well-defined GPI analogs and two BODIPY fluorescence-tagged GPI analogs were carried out in chapter 3.The glucosaminyl donor GI-24 and the optical inositol acceptor GI-38,which were synthesized starting from D-glucosamine and ?-D-methylglucoside,respectively,were coupled to afford the pseudodisaccharide GI-40.Subsequently,the fully protected GPI core pseudopentaose GI-57 was achieved from the trimannose glycoside donor GI-56,which was synthesized starting from D-mannose,and the pseudodisaccharide GI-40 through a[3+2]glycosylation strategy,and then phosphorylation of the 6-O position of Man-? and the glucosamine 2-amino protected with a Boc group.After catalytic hydrogenation,the resulting GPI derivative GI-62 coupled with the BODIPY derivative GI-66 containing a long chain of a carboxylic acid at the presence of HATU.Unfortunately,when we attempt to remove the Boc group in acid condition,the fluorescence compound was decomposed.We next replaced the Boc group with the base-removed Fmoc group to eventually the target compounds GI-14 and 15.On the other hand,the mono-,di-,and trimannoseglycoside donor(GI-46,86,89),which were synthesized starting from D-mannose,coupled with the pseudodisaccharide acceptor GI-40 through[1+2],[2+2],[3+2]glycosylation strategy to afford pseudo-tri,tetra,and pentasaccharide(GI-90,95,100),respectively.Phosphorylation after removed Man-I 2-O-acetyl or inositol 1-O-p-methoxybenzyl,and a series of structurally well-defined GPI derivatives GI-(1-13)were obtained.The synthetic targets GI-(1-13)and GI-14,15 will be used in find pore-forming toxin inhibitors. |
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