Synthesis of Lipopolysaccharide O-antigens from Klebsiella pneumoniae Serotype O2a as Molecular Probes for Biosynthetic Studies

• Author / Creator

  Ryan P. Sweeney

• Many Gram-negative bacteria are pathogenic organisms that can cause disease and death. As antibiotic resistance and nosocomial disease occurrences both increase, these infections are becoming more problematic. These issues can be partially attributed to lipopolysaccharide (LPS), which is a major structural and immunomodulatory molecule found in the outer membrane of Gram-negative bacteria. The O-antigenic polysaccharide (O-PS), the terminal domain of LPS, is a major virulence factor. Understanding the biosynthesis of these molecules may allow for new treatments of these pathogenic diseases.
  One manner by which O-PS is biosynthesized is the ATP-binding cassette (ABC) transporter-dependent pathway. In this process, the full length O-PS is synthesized in the cytoplasm, and then the transporter flips it to the periplasmic side, prior to extension to full-length LPS. There are two prototypical systems in this pathway: 1) termination of chain extension by a modification (E. coli O9a) and 2) termination without a chain modification (K. pneumoniae O2a). While the glycosyltransferases (GTases) and chain length control mechanisms have been identified for E. coli O9a, many questions still remain in the K. pneumoniae O2a system. This includes: 1) What are the precise roles of the GTases involved in O-PS assembly?, 2) How is the chain length controlled?, and 3) What is the mechanism by which the O-PS chain is flipped across the membrane by the ABC transporter?. Investigating these questions requires access to structurally well-defined, synthetic glycans. This thesis focuses on the synthesis of glycan acceptors that will be used as molecular probes to further elucidate the K. pneumoniae O-PS biosynthetic pathway.
  In one investigation, three fluorescein-based probes, two disaccharides and one monosaccharide, were chemically synthesized. The challenge in generating the galactofuranose ring in these molecules was overcome using a kinetically controlled I2-catalyzed cyclization of D-galactose dithioacetal. Completely stereoselective glycosylations were achieved by the use of the appropriate donor: 2-O-benzoylated galactofuranoside donors gave the desired 1,2-trans- linkages, and DTBS-protected galactopyranoside donors gave the more challenging 1,2-cis- linkages. The monosaccharide probe was obtained via a concise, three-step synthetic route. Preliminary investigations with these probes have clarified the activities of the three GTases involved in its O-PS assembly in K. pneumoniae O2a.
  The second investigation centers on the synthesis of lipid-linked oligosaccharides from K. pneumonia O2a. Our targets are five molecules, differing in size from nine monosaccharide residues up to 41 monosaccharide residues. The highly convergent route developed gave the desired target backbones (up to the 33-mer) in substantial quantities. Conversion of these backbones to the final targets gave three of the target glycans (the 9-mer, 17-mer, and 25-mer). These synthetic glycans will be used to further elucidate the O-PS biosynthetic pathway found in K. pneumoniae O2a.

• Subjects / Keywords

  • Chemical Synthesis
  • Lipopolysaccharides
  • Klebsiella pneumoniae

• Graduation date

  Fall 2018

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R38K75C58

• License

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