Synthesis Of A Trisaccharide Repeating Unit Of The O-Antigen From Burkholderia Cenocepacia And Its Dimer

Carbohydrates are the most widely distributed and numerous biological macromolecules in nature.They are widely found in animals,plants and microorganisms and are essential substances that constitute organisms and store energies.The diversity of species and the complexity of structures make carbohydrates to be the biomolecule with the largest information capacity.For example,the amount of sialic acid residue on the cell surface can be used to determine the probability of cancer occurrence;the chemical structure of polysaccharides can determine human blood types.Furthermore,polysaccharides on the cell surface have important biological functions,which not only participate in various physiological and pathological processes,such as fertilization,growth,aging and disease occurrence,but also can be used as the ideal target antigens for development of vaccines due to their highly conservative expression.The Burkholderia cepacia complex(Bcc)studied in this thesis represents a group of Gram-negative bacteria with similar phenotype but different genotypes.It playes an important role in nature.On the one hand,the metabolism of Bcc can be used as bioremediation agent,sterilization and nitrogen fixation.On the other hand,Bcc has been characterized as a dangerous opportunistic human pathogen that easily infect people with low immunity and result in a clinically uncontrollable "cepacia" syndrome that cause further deterioration in patients with chronic fibrosis and induce pneumonia and septicemia.Bcc infections are difficult to cure because of high resistance to clinical antimicrobial drugs,thus new effetive treatments for Bcc infections are needed to be developed.It has been well established that polysaccharides(LPSs,CPSs)exposed on the cell surface are ideal source of antigens for vaccines development because of the relatively high conservation and immunogenicity.They can induce specific immune responses in the body.Unfortunately,the immune responses induced by such ploysaccharides are weak and T-cell independent,which are only provide immuno-protection for above 2 year old.Howerver,studies have shown that polysaccharide-protein conjugate can induce T-cell dependent and memorable immune responses.Thus far,many bacterial ploysaccahrides have been developed into antibacterial glycoconjugate vaccines.In recent years,oligosaccharide-based artificial semi-/full synthetic vaccines have been received more attention due to their well-defined structure and easy-to-control quality.In this thesis,we chemically synthesized a trisaccharide repeating unit of the O-antigen from B.cenocepacia and its dimer.Meanwhile,we conjugated the synthetic oligomers with carrier proteins(TT or BSA)using disuccinimidyl glutarate(DSG)as bifunctional linker to generate glycoconjugate for immunological study.Three chapters are included:The first chapter summarizes the research backgroud on pathogenic mechanism,characterization and function of toxicity factor of Burkholderia cepacia complex.Moreover,the synthetic progress on Bcc O-antigens were also reviewed.The second chapter is the synthesis of the repeating unit of O-antigen from Burkholderia cenocepacia and its dimer.Solvent and neighboring group participation effect were employed to improve the synthetic efficiency and to control ?,?-steroselectivity.Under inverse glycosylation condition,compounds 4 and 5 were condensed to construct the a-linked disaccharide 12 in good yield.At this stage,the 2'-azido group in 12 was converted into acetamido group using thioacetyl acid(AcSH)in pyridine,affording 13 in a high yield.Basic selective deacetylation of 13 in methanol afforded 14,which was served as a glycosyl acceptor to glycosylate with 6 under the promotion of TMSOTf,furnishing fully protected trisaccharide 3 in a high yield.The reaction between 3 and 3-azido-1-propanol proceeded smoothly in the presence of NIS and TMSOTf to produce 15 in a 76%yield.It was then subjected to globle deprotection in four steps to yield target 1.Also,15 was selectively dechloroacetylated with thiourea in methanol,generating trisaccharide acceptor 16.Next,a convergent[3+3]glycosylation strategy was attempted to construct hexasaccharide 2 using trisaccharide 3 as glycosyl acceptor and 16 as glycosyl donor,respectively.Unfortunately,this reaction was very sluggish and gave only a trace amount of the hexasaccharide detectable by MS.To increase the synthetic efficiency,we use different glycosyl donors,including phosphate,trichloroacetimidate but failed to accomplish the desired result.We assumed that this might be because of the low reactivity of the 4-OH group in 16 due to the presence of electron-withdrawing benzoyl groups on C-2 and C-3 positions.To verity the hypothesis,trisaccharide acceptor 27 containing a 2,3Rha-O-acetonide group was prepared and glycosylated with 3 via a highly convergent[3+3]strategy.Under the same conditions,the reactions proceeded smoothly to furnish 28 in 76%yield.Then,Global deprotection of 28 in four steps by the same sequence as described for 1 produced 2 in a high yield.In addition,both synthetic targets 1 and 2 carriered a 3-azidopropyl linker at their reducing end,allowing for their convenient attachment to functionalized biomolecules for the exploration of various biological problems,such as B.cenocepacia immunology and development of O-antigen-based vaccines against Bcc infections.All of the intermediates and target compounds involved in this paper have been confirmed by NMR and mass spectrometry.The third chapter described the preparation of Bcc oligosaccharide-protein conjugates.After hydrogenation of the azide group in compounds 1 and 2 to an amino group,the resulting oligosaccharides 29/30 were reacted with commercially available disuccinimidyl glutarate(DSG)to furnish the monoactibated esters 31/32,which were then individually mixed with TT or bovine serum albumin(BSA)in phosphate-buffered saline solution(PBS,pH 8.0,0.1 m),affording the desired conjugates 33-36 after dialysis against distilled water to remove any remaining free sugars and salts.The carbohydrate loading valuse of these conjugates were determined by MALDI-TOF MS analysis.The synthetic glycoconjugates will be used in future animal experiment for the immunological study.