| Group A streptococcus(GAS)and group B streptococcus(GBS)are both important species of Gram-positive extracellular bacterial pathogens.GAS is often colonized in the throat or skin of human with various clinical manifestations.These clinical manifestations caused by GAS encompass invasive GAS infections and toxin-mediated diseases,including pharyngitis,pyoderma,bacteremia,scarlet fever,streptococcal toxic shock syndrome and so on.GAS is also responsible for a series of autoimmune sequelae,such as rheumatic fever,acute glomerulonephritis,rheumatic heart disease.Different from GAS,GBS were found to colonize the vagina,which may lead to intrauterine infection,pre-term delivery or stillbirth.Maternal colonization is likely to transmit GBS to their offspring as bacteria can spread through parturient canal,leading to severe bacterial infection in newborns,which is one of the main factors leading to neonatal death.Currently,antibiotics is the main treatment option for bacterial infection in the clinic,while the resistance to antibiotics is becoming more common and severe.Besides,asymptomatic carriers remain to be the main obstacle for infection control of GAS and GBS.These people do not display clinical symptoms,but are capable of transmitting the pathogens to susceptible groups.Vaccination is the most effective strategy to prevent the spread of pathogens.Therefore,it is critical and necessary to develop vaccines against GAS and GBS.Carbohydrates is one of the most important energy sources and core components of organisms,and also participates in a variety of biological processes.The cell wall-anchored carbohydrates in GAS is a kind of classical Lancefield antigen,which is highly conserved in various GAS serotypes.This prompted the consideration of GAS cell-wall polysaccharides as a potential GAS vaccine antigen.GBS can be classified into ten serotypes for its capsular polysaccharides,which is the major extracellular virulence factor of GBS.These capsular polysaccharides play an important role in helping bacterial evasion of the host's defense system.Both GAS cell wall polysaccharide and GBS capsule polysaccharide are ideal carbohydrate target antigens.Synthetic oligosaccharides with well-defined structure are important to vaccine development.Firstly,synthetic oligosaccharides provided the opportunity to clarify the structure-activity relationships between carbohydrate antigens and oligosaccharide-specific antibodies for vaccine design.Secondly,semi-synthetic glycoconjugate vaccines with chemically well-defined antigenic structures are able to confer highly reproducible biological properties and a better safety profile.Currently,there are several reports about studies of antigenic determinant and semi-antigen glycoprotein conjugate vaccines with GAS cell-wall oligosaccharide.However,the researches of GBS capsule polysaccharide are still in the stage of synthesis of oligosaccharide repeating unit of various serotypes.In this dissertation,we synthesized the structurally well-defined GAS oligosaccharides and conjugated them with different carrier proteins to provide olisaccharide-protein conjugates for immunological studies.In addition,we also synthesize the pentasaccharide repeating unit of type VI GBS capsular polysaccharide.This dissertation mainly includes three major parts:In chapter 1,the progress on synthetic and applied studies of oligosaccharide of GAS cell-wall polysaccharides and GBS capsular polysaccharides has been reviewed.In the aspect of synthesis of GAS cell-wall oligosaccharide,several groups have reported the synthesis of GAS cell-wall oligosaccharide with different glycosylation strategy.However,there are still some problems in the existing synthesis schemes.For example,the assembly of building block isn't efficient enough,and the preparation process of building block is complex.In the aspect of application of synthetic GAS oligosaccharide,the studies on structure-activity relationship have revealed that repeat unit of branch trisaccharide structure is the key antigen epitope in GAS cell-wall carbohydrates.The immunological studies proved that synthetic GAS oligosaccharides displayed similar immunogenicity to that of natural GAS cell-wall polysaccharides-based glycoconjugates.Both glycoconjugates could induce T cell-dependent carbohydrate-specific immune responses.In the aspect of synthetic oligosaccharide of GBS capsular polysaccharide,there several research groups have been reported oligosaccharide derivatives of the repeating unit of type ?a,?,?,?,?and VIII GBS capsular polysaccharide.But to date,there has been no reported about synthetic research on type Ib,?,?,and ? GBS capsular polysaccharide.In the aspect of application of these synthetic oligosaccharide,the immunological activity evaluation of glycoconjugates of type la GBS show that synthetic oligosaccharide of GBS capsular polysaccharide could be be utilized for development of anti-GBS vaccines.In chapter 2,the synthesis and immunological studies of GAS cell-wall oligosaccharide-streptococcal C5a peptidase conjugates was described.Firstly,we synthesized structurally well-defined mono-,di-,and trimers of the branched trisaccharide repeating unit of GAS cell-wall polysaccharide.We designed and synthesized three building blocks,including reducing end disaccharide acceptor,trisaccharide thioglycoside intermediate,rhamnosyl donor.Both disaccharide acceptor and trisaccharide intermediate were prepared from common disaccharide synthon.The synthetic targets,tri-,hexa-and nonasaccharides were assembled by a convergent and efficient strategy,through[2+1]3 {2+[3+1]} and {[2+3]+[3+1]} glycosylation,respectively.Secondly,we synthesized a series of glycoconjugates with synthetic GAS oligosaccharides.Three groups of synthetic oligosaccharides with free amino group at the reducing end,were treated with disuccinimidyl glutarate to produce glycosyl activated esters.Then,these glycosyl activated esters were coupled with ScpA193,CRM 197,TT and BSA to afford conjugates for further immunological studies,respectively.The CRM 197 conjugates and TT conjugates were prepared as positive controls for ScpA193 conjugates,whereas BSA conjugates were prepared as coating antigens to detect carbohydrate antigen-specific antibodies.In addition,we chosed diethyl quadrate and HSA to prepare other trisaccharide conjugates for evaluating immunogenicity of glutaryl linker.Finally,we evaluated immunological activity of GAS oligosaccharide-ScpA193 conjugates.Compared with CRM 197 conjugates and TT conjugates,GAS oligosaccharide-ScpA193 conjugates not only elicited similar or stronger carbohydrate antigen-specific immune response,but also induced robust specific antibodies responses of wild-type ScpA.Moreover,the glutaryl linker did not induce significant immune responses These results suggested that GAS oligosaccharide-ScpA193 conjugates could function as a new anti-GAS bivalent vaccine candidate.In chapter 3,we introduced the synthetic studies of the pentasaccharide repeating unit of type VI GBS capsular polysaccharide.We have achieved the first chemical synthesis of the core pentasaccharides repeating unit of type VI GBS capsular polysaccharide.In the process of preparing building blocks,lactose was directly utilized to construct reducing end disaccharide acceptor,which simplified the process of glycosylation and preparation of building block.In the process of synthesizing the building block of lactose,we attempt to select the most optimum reaction conditions for introducing isopropylidene in lactose derivatives and get a good yield.The sialodisaccharide donor with difficult a-sialic acid linkage was synthesized by a reported method.A key thioglucoside building block with two temporary protecting groups was designed and synthesized for connection between the sialodisaccharide and reducing end disaccharide.Building blocks were assembled by a{2+[1+2]} glycosylation strategy.The desired 1,2-trans glycosidic bond was highly stereoselective to generate owing to the neighboring group participation effect by benzoyl groups present on the 2-positions of glycosyl donors.In the process of assembly of trisaccharide,the formation of the new ?-(1?3)-glucosyl linkage was synthesized by utilizing the different reactivity of 3,4-OH group in acceptor with reaction condition control.In the process of synthesis of pentasaccharides,various catalytic system was failed in glycosylation reaction of trisaccharide acceptor with sialodisaccharide thioglycoside donor.To address the issue,the thioglycoside in sialodisaccharide was converted into more reactive trichloroacetimidate as glycosyl donor.Finally,fully protected pentasaccharides was synthesized by glycosylation of trisaccharide acceptor with new synthetic imidate.In fully protected pentasaccharides,the TBDPS as a temporary protecting group could be selectively deprotected to enable carbohydrate chain elongation at this position.Finally,target pentasaccharide was obtained by four-step deprotections.In this dissertation,the GAS oligosaccharide-protein conjugates and pentasaccharides fragment of the repeating unit of type VI GBS capsular polysaccharide were chosed as main research target.The innovations of the research including following parts:(1)We designed structurally well-defined tri-,hexa-and nonasaccharides as haptens,which consisted of the branched trisaccharide repeating unit of GAS cell-wall polysaccharide.In order resolve the problem of low efficiency of assembly and long preparation process of building block in previous synthetic studies,we utilized the same disaccharide synthon for synthesis of reducing end disaccharide acceptor and trisaccharide building blocks,and synthesized target oligosaccharides by convergent and efficient strategy via[2+1],{2+[3+1]} and {[2+3]+[3+1]} glycosylation.(2)Based on the mechanism for glycoconjugate activation of adaptive immune,we synthesized GAS glycoconjugate with an enzymatically inactive ScpA mutant ScpA193 as carrier proteins.Preliminary immunological evaluations revealed that the new conjugates could concurrently induce carbohydrate antigen-specific and ScpA-specific antibodies.(3)We have achieved the first chemical synthesis of the pentasaccharides repeating unit of type VI GBS capsular polysaccharide by {2+[1+2]}glycosylation strategy,which provide a reference for other research groups. |
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