First Chemical Synthesis Of The Tetrasaccharide Repeating Unit Of Lipopolysaccharide O-antigen Of Pseudomonas Aeruginosa IID 1001 (ATCC 27577)

Pseudomonas aeruginosa is a gram-negative bacterium and exists widely in nature.It is one of the most common opportunistic pathogens in the clinic,which usually causes various infections in the respiratory system,bloodstream,urinary tract,wounds,and burns.Respiratory system infection such as the pneumonia and cystic fibrosis,is a typical health issue with P.aeruginosa,.In addition,P.aeruginosa is a pathogen with serious antibiotic resistance,and its resistance rate has been increasing around the world,resulting in the rise of patient mortality and healthcare costs.The efficiency of conventional antibiotic therapy is gradually decreasing.In this context,there is an urgent need to develop an effective vaccine to prevent and treat P.aeruginosa infection.Lipopolysaccharide(LPS)is the major virulent factor of P.aeruginosa.Its O-antigen as an effective immunogen is structurally variant according to different serotypes.It was demonstrated that the polysaccharide-protein conjugate vaccine prepared by coupling natural O-antigens with immunogenic carrier proteins could elicit effective immune responses in animals and humans,indicating their potential applications in clinic against P.aeruginosa infection.Based on the above studies,we here designed and accomplished the first chemical synthesis of the tetrasaccharide hapten 2-1 related to the repeating unit of LPS O-antigen of P.aeruginosa IID 1001,hopefully to facilitate the subsequent vaccine development and investigation of novel diagnostic strategy.This paper includes:In chapter 1,we briefly summarized the research progresses of serotypes,epidemiology,antibiotic resistance,and vaccines of P.aeruginosa.In addition,the synthesis of related rare monosaccharides and their applications in oligosaccharide synthesis were also systematically reviewed.Accordingly,we proposed the research basis,objective,and specific experimental scheme.In chapter 2,we introduced the first chemical synthesis of the tetrasaccharide repeating unit of P.aeruginosa IID 1001 LPS O-antigen,the structure of which was identified as[→4)α-L-GalpNAc3AcA-(1→3)-β-D-Bacp2NR4NAc-(1→2)-α-L-Rhap-(1→6)-α-D-GlcpNAc-(l→].and an(S)-3-hydroxy-butyryl residue was linked to the 2-amino group of Bac.Assembly of the target tetrasaccharide 2-1 represents a notable synthetic challenge.Firstly,it contains rare monosaccharides D-Bac and L-GalNAcA.and their preparations in sufficient amounts from commercially available materials need multiple-step transformation.Secondly.two 1.2-cisglycosidic bonds are difficult to be constructed with high stereoselectivity.Especially.construction of the α-L-GalNAcA has not been achieved.Thirdly.the target 2-1 contains multiple functional groups,such as O-acetyl,N-acetyl,N-(S)-3-hydroxybutyryl,free amino.and carboxyl groups.Given the low reactivity of L-galacturonic acid as a glycosyl donor and the possible interference of the(S)-3-O-benzyl-butyryl side chain with the glycosylation reaction of adjacent hydroxyl group.a post-glycosylation oxidation and lipid chain modification strategy were envisioned to synthesize the target tetrasaccharide 2-1.At first.building blocks 2-4,2-5,2-6.and 2-7 were designed and efficiently synthesized.Next,these building blocks were consecutively assembled via stepwise glycosylation to furnish the tetrasaccharide 2-3 in high yields.The high stereoselectivity of the resultant α-D-GalN glycosidic bond was possibly owing to the synergistic α-directing effects of the steric β-facial shielding of donor 6-O-TBS and the TolSCl/AgOTf promotion system.The glycosyl linkage of L-GalN with exclusive α-specificity was constructed under the activation of NIS/TfOH at10℃ in a mixed dichloromethane-ether(v:v=1:1)solvent.This represented the first achievement of α-L-GalN linkage by chemical method and provided a solid basis for the synthesis of other oligosaccharides containing α-L-GalN unit.Besides,two 1,2-trans-glycosyl linkages were effectively constructed by using the neighboring group participation of the 2-OBz in 2-5 and the 2-N-Troc group in 2-6.respectively.After that,the TBS group of compound 2-3 was chemoselectively removed using TBAF and AcOH.and the resultant C6"’-OH was smoothly oxidized to uronic acid by using our modified TEMPO/BAIB oxidation protocol to give the desired carboxylate intermediate 2-34.Subsequently,the three azido groups were simutaneously converted to acetamido groups upon one-pot two-step transformation.Removal of the N-Troc protecting group with Zn-AcOH was followed by condensation with(S)-3-Obenzylbutyryl chloride to generate the tetrasaccharide 2-2.Finally,the target compounds 2-1 and 2-1a containing a migrated acetyl group were successfully obtained in 29%overall yield for eight steps by the Pd(OH)2-catalyzed hydrogenation to remove all of the Bn and Cbz groups.The structures of the synthetic intermediates and target molecules involved in this work were fully characterized by 1D-and 2D-NMR and HR-MS data.