Chemical synthesis of lipopolysaccharide derived from klebsiella pneumoniae for conjugate vaccine development

Abstract

Klebsiella pneumoniae (K. pneumoniae), a member of Gram-negative bacterium (GNB) belonging to Enterobacteriaceae family, is a commonly isolated nosocomial pathogens that can cause pneumonia, bloodstream infections and sepsis. Infections caused by carbapenem-resistant K. pneumoniae (CR-Kp) are particularly problematic due to a lack of therapeutic options leading to a 50% mortality. Capsular polysaccharide (CPS) and lipopolysaccharide (LPS) are two virulence factors of K. pneumoniae that provide attractive starting points for vaccine development. The large number (79) of serotypes, however, has complicated CPS vaccine development. In contrast, only nine serotypes of LPS have been identified to date. In addition, only two core types have been identified making the LPS core an attractive target for the development of a broad acting vaccine for K. pneumoniae. The chemical synthesis of an outer core tetrasaccharide is described in Chapter 2. The compound contains three amino groups in different forms, a Kdo residue which is a common component of inner core oligosaccharides, and a thiol-containing linker at the reducing end for protein conjugation. A two-carbon extension strategy was employed to synthesize a Kdo building block. The installation of challenging α-galactoside/α-Kdo linkages was accomplished by remote neighboring group participation. The subsequent Chapter 3 describes the synthesis of a pentasaccharide derived from the outer core of K. pneumoniae containing a nonstoichiometric L,D-heptose substituent. A crowded 4,5-branched Kdo residue was installed by a block synthetic strategy, and a heptosyl donor was prepared from D-mannose with excellent L-diastereoselectivity by using a one-carbon elongation approach. The immunological evaluation of the two conjugates is ongoing and will reveal the role of the heptoside in eliciting relevant immune responses. In Chapter 4, the synthesis of an inner core pentasaccharide derived from K. pneumoniae is described. Compared to the diversity of CPS, the inner core has only one type among K. pneumoniae making it attractive for the development of a broad acting vaccine. Galacturonic acid is expressed by the bacteria to provide negative charge since the phosphoryl modifications are absent in the inner core. To reveal the effects of the acidic substituent for eliciting immune response, a substructure lacking galacturonic acid was also synthesized containing a crowded 3,4-branched L,D-heptoside. The tetra- and pentasaccharide have been conjugated to carrier proteins, and future studies will focus on examining antigenic properties by mouse immunizations.