Total Synthesis Of The Capsular Oligosaccharides Of Vibrio Vulnificus BO62316 And Types ?A And ? Group B Streptococcus

Vibrio vulnificus is a halophilic Gram-negative bacterium and ubiquitously present in marine environments worldwide.It is a very dangerous human pathogen that can cause severe to life-threatening septicemia and necrotizing soft-tissue infection.Typically,the infection progresses rapidly,and the mortality rate for primary sepsis is>50%.V.vulnificus infection is generally treated with antibiotics,whilst surgical debridement is usually required for patients with soft-tissue infection.Sometimes,amputation of affected limbs is necessary.Therefore,the development of prophylactic vaccines has become an urgent strategy to control V.vulnificus infection.Capsular polysaccharides(CPSs),as the major virulent factor,can protect V.vulnificus by conferring resistance to phagocytosis and other bactericidal activities of the host immune system.V.vulnificus CPSs contain a number of rare sugars that are not present in human cell surface glycans,making them excellent haptens for developing the related glycoconjugate vaccines.It was revealed that V.vulnificus biotype 1 strains B062316 and M06-24 are the predominant pathogenic strains that account for approximately one-third of the clinical infections.Their CPSs can be used as potential useful carbohydrate antigens for the preparation of anti-V.vulnificus M06-24 and B062316 vaccines.Based on the above-mentioned research background,we designed and synthesized a tetrasacchaide fragement corresponding to the repeating unit of V.vulnificus BO62316 CPS and explored the preliminary immunological evaluation of semi-synthetic glycoconjugates associated with V.vulnificus BO62316 and M06-24.Group B Streptococcus(GBS)is one of the leading bacterial causes of sepsis,meningitis,pneumonia and abortion among neonates and pregnant women.GBS expresses CPSs on cell surfaces as the major virulence factor,which can assist bacterium to evade the host's innate defense mechanisms.According to the structurally unique CPSs,GBS can be classified into at least 10 serotypes(?a,?b,and?-?),and type ?a,the second predominant serotype,is responsible for 23%of the total GBS infection.Recently,it was revealed by several groups that the serotype specific GBS CPSs are effective target antigens for the development of bacteria diagnosis and glycoconjugate vaccines.It is difficult to derive homogeneous CPS fragments from natural sources owing to their structural microheterogeneity.Consequently,chemical synthesis as an effective method to obtain homogeneous oligosaccharides has attracted significant attentions.The synthesis of GBS-related oligosaccharides is very useful for the development of carbohydrate-based conjugate vaccines and sensitive GBS diagnosis strategies.Based on the above-mentioned research background,the pentasacchaide repeating unit of type ?a GBS CPS and the dimer that contain highly branched glycan motifs were designed and chemically synthesized based on on-site assembly of the sialotrisaccharide branches.The synthetic oligosaccharides could facilitate the development of the related glycoconjugate vaccines and explore the structure-activity relationship of antigenic determinant.The morbidity caused by type ? GBS is relatively low,accounting for about 1%.Since not all detections employ type ? antiserum in GBS serotyping,its real incidence may be underestimated.For the same reason,distribution of GBS serotypes in many regions has been significantly revised.In addition,serological study showed that type VII GBS CPS has a unique antigentic determinant and cannot cross-react with antisera induced by other serotypes.Therefore,type VII GBS CPS-related carbohydrate antigens should be contained in an effective GBS multivalent glycoconjugate vaccine.Based on the above-mentioned research background,the hexasacchaide repeating unit of type VII GBS CPS and the dimer were designed and chemically synthesized through efficient glycosylation strategies,in order to facilitate the preparation of the related glycoconjugate vaccine and explore the structure-activity relationship of antigenic determinant.The research of this dissertation includes the following four parts.In chapter 1,we briefly reviewed the epidemiology,pathogenic mechanism and typing of two pathogenic bacteria,V.vulnificus and GBS,and systematically summaried the progress on synthetic and applied studies of related oligosaccharides.Accordingly,the basis,objective,and specific experimental scheme for each research project are proposed.In chapter 2,we have described the first synthesis of the tetrasacchaide repeating unit of V.vulnificus BO62316 CPS and the immunological evaluation of semi-synthetic glycoconjugates related to V.vulnificus B062316 and M06-24.The repeating tetrasacchaide contains a trisacchaide main chain,3-?-D-FucpNAc-(1?3)-?-D-GalApN Ac-(1?3)-?-L-QuipN Ac-(1?,and a monosaccharide branch,?-L-RhapNAc,attached to the GalANAc 4-O-position.Assembly of the target tetrasaccharide 2-1 has several synthetic challenges.Firstly,all of the aminosaccharide building blocks are rare and commercially unavailable.Secondly,all the glycosyl linkages are ?-linked that are difficult to construct in a sterically controlled manner.Thirdly,assembly of the 3,4-branched ?-D-GalN motif and its oxidation to uronic acid are usually problematic.Fourthly,the orthogonal protection of acetylamino and amino groups are required.Through orthogonal installation of different functional groups and evaluation and comparison of various glycosylation methods/conditions and glycan assembly strategies,we employed[3+1]convergent glycosylation strategy to construct tetrasaccharide intermediate 2-2 in high yiled by using a 3,4-branched trisaccharide as the glycosyl donor.Each glycosylation reaction afforded excellent to exclusive ?-selectivity.Furthermore,the TEMPO/BAIB oxidation protocol was optimized for efficient conversion of ?-D-GalN to ?-D-GalAN in a series of disaccharides,trisaccharides and tetrasaccharides.Then,the modified TEMPO/BAIB oxidation protocol was applied to the oxidation of tetrasaccharide 2-35,and the desired carboxylate 2-36 was obtained in an 81%yield.Finally,the fully protected tetrasaccharide intermediate 2-36 was converted into the target compound 2-1 with an overall yiled of 58%through four-step deprotection reactions including removal of isopropylidene group,reduction of azido groups,regioselective N-acetylation and hydrogenolysis.With the synthetic tetrasaccharide haptens 2-37 and 2-1 of V.vulnificus in hand,they were covalently coupled with carrier protein diphtheria toxin mutant(CRM197)and bovine serum albumin(BSA)through DSG,generating the target glycoconjugates 2-40-43.The oligosaccharide/protein molar ratio of these conjugates were determined by MALDI-TOF MS analysis,and an average of 3-4 molecules of synthetic tetrasaccharide were loaded onto one molecule of carrier protein.Using BSA glycoconjugates 2-42 and 2-43 as capture antigens,enzyme-linked immunosorbent assay(ELISA)was empolyed to detect the carbohydrate antigen-specific antibodies in the antiserum of mice immunized with CRM197 glycoconjugates 2-40 and 2-41,respectivly.Immunological studies revealed that the CRM197 glycoconjugates 2-40 and 2-41 were immunogenic and could provoke robust antigen-specific T-cell-dependent responses.In addition,it was found that there was a weak cross-reactivity between the serotypes of V.vulnificus.It was concluded that both synthetic tetrasaccharides 2-37 and 2-1 may be employed as useful haptens for the development of V.vulnificus glycoconjugate vaccine.In chapter 3,the total synthesis of the pentasaccharide repeating unit of type ?a GBS CPS and the dimer were introduced.This repeating unit comprises a disaccharide backbone,4-?-D-Galp-(1?4)-?-D-Glcp-(1?,and a sialotrisaccharide branch,?-NeupNAc-(2?3)-?-D-Galp-(1?4)-?-D-GlcpNAc-(1?,linked to the main chain 3'-O-position.For the synthesis of the repeating unit dimer,two branched trisaccharide building blocks,donor 3-7 and acceptor 3-6,were separately assembled according to the same glycosylation sequence that was to glycosylate the C3-OH of Gal unit prior to its C4-OH.Subsequently,after screening various reaction conditions,p-TolSCl/AgOTf catalyst was found efficient for the[3+3]convergent glycosylation to construct the pivotal hexasaccharide 3-19 with two branched trisaccharides.Moreover,encouraged by this result,we employed[1+2+3]one-pot two-step glycosylation strategy to achieve the efficient construction of hexasaccharide 3-19 via a preactivation-based glycosylation manner.Compared with the step by step synthesis strategy,the one-pot synthesis procedure could avoid the separation and purification of intermediates and improve the whole synthetic efficiency.With the hexasaccharide acceptor and the sialodisaccharide donor in hand,we carried out[2×2+6]dual glycosylation and obtained the fully protected decasaccharide intermediate 3-3.This accomplishment further reflected the great potential and general applicability of the on-site elongation strategy for synthesizing oligosaccharides containing sialotrisaccharide when it was located at a relatively unreactive position.Subsequently,the target decasaccharide 3-2 was obtained in a 40%overall yield through a five-step deprotection procedures to remove carboxylate,carbamate,amido,benzylidene,benzyl,azido,and acyl groups,and N-acetylation of the engaged amino intermediate.To facilitate the in-depth analysis of structure-activity relationship,we completed the synthesis of the pentasaccharide repeating unit 3-1 through a[2+3]glycosylation strategy.Thereafter,the pentasaccharide intermediate 3-23 was converted into 3-1 with an overall yield of 41%in six steps according to the similar deprotection protocol as described for 3-4.The reducing end of 3-1 and 3-2 were equipped with a free amino group,which could be coupled with other molecules for the development of anti-?a GBS glycoconjugate vaccines.In chapter 4,we have described the first synthesis of the hexasaccharide repeating unit of type ? group B Streptococcus CPS and the dimer.The repeating unit contains a trisacchaide backbone,4-?-D-Glcp-(1?4)-?-D-Galp-(1?4)-?-DGlcp-(1?,and a sialotrisaccharide branch,?-NeupNAc-(2?3)-?-D-Galp-(1?4)-?D-GlcpNAc-(1?,linked to the main chain 6"-O-position.Proviously,our group developed an efficient ?-glucosylation method that was achieved based on the synergetic ?-directing effects of the TolSCl/AgOTf catalysis system and the steric?-shielding assistance or remote acyl participation of protecting groups at the 6-O-position of glucosyl donor.Inspired by this method,we employed[2+1+1+2]one-pot three-step glycosylation strategy to efficiently construct the fully protected hexasaccharide intermediate 4-3 in an excellent overall yield(52%).The stereoselectivity and regioselectivity of each glycosylation were exclusive,and no other isomers were formed.The target hexasaccharide 4-1 was eventually obtained in 49%yield for five steps.After global deprotection to remove protecting groups in 4-3 and N-acetylation of the engaged amino intermediate.By using the preactivation-based glycosylation method similar to that used for preparing 4-3,the backbone hexasaccharide 4-14 was obtained in a 54%overall yield via the[1+2+1+2]one-pot three-step glycosylation.These reactions were highly stereoselective,and no other stereoisomers were generated.The construction of two?-glucosyl linkages and one ?-glucosyl linkage was achieved in one-pot manner.After finished the syntheses of hexasaccharide acceptor and the sialotrisaccharide donor,we applied[2×3+6]dual glycosylation strategy to synthesis the fully protected dodecasaccharide intermediat 4-4.The efficient synthesis of 4-4 demonstrated that when the sialotrisaccharide was linked with high reactive primary hydroxyl group,it could be pre-assembled and then emplolyed as a building block to achieve efficient synthesis of complex oligosaccharides.On the contrary,this sialotrisaccharide should be avoided to directly couple with unreactive acceptor.The global deprotection of 4-4 containing a series of functional groups was accomplished by a five-step deprotection protocal as described for 4-3 with slight modification of conditions,such as exended reation time and so on,to deal with the increased steric hindrance.The target dodecasaccharide 4-2 was obtained in 41%overall yield.The aminoethyl linker installed at the downstream end of 4-1 and 4-2 would facilitate further regioselective conjugation or immobilization with other moleculars,useful for the development of anti-GBS glycoconjugate vaccine and novel GBS detection strategy.