Synthesis of heptose analogs as potential inhibitors of the bacterial heptose biosynthetic pathway

Lipopolysaccharides (LPS) are essential components of the outer leaflet of the bacterial cell membrane. Gene defects affecting the biosynthesis of the conserved inner-core region of LPS lead to decreased barrier performance of the membrane, an increase in permeability for hydrophobic antibiotics and a reduced bacterial virulence. The proposal is focused on the chemical synthesis of native phosphorylated compounds and stable derivatives of bacterial heptoses. The synthetic compounds should contribute towards a full understanding of the biosynthetic pathway involved in the assembly of the bacterial heptose region. In addition, compounds will be tested for potential inhibitory properties. In preliminary experiments novel details concerning the structure of the native substrates for bacterial heptosyl transferases could be obtained. Since the physiological substrate of gram-negative heptosyltransferases - beta- configured ADP-heptose - is rather unstable, stable analogs will be synthesized. The synthetic outline will comprise C-glycosides and CF2-glycosides of L-glycero- and D-glycero-D-manno-heptose as well as derivatives obtained by replacement of the 2-OH group with azide, fluorine or hydrogen substituents. The compounds will be transformed into phosphonic acid and phosphoric acid derivatives and subsequently converted into the nucleotide- activated sugars and tested as substrates / inhibitors for bacterial heptosyl transferases. The compounds will also be used for biochemical studies of the other 5 key enzymes involved in the biosynthesis of heptose, such as a novel - putative - kinase and phosphatase as well as the nucleotide-heptose-synthetase and epimerase. The latter enzyme has recently been crystallized and the x-ray structure has been determined. With the aid of the synthetic compounds, the determination of the crystal structure of the enzyme-substrate and enzyme-product complex will be attempted. The data to be obtained on the binding process of the substrate and the reaction mechanism in the active site of that enzyme should provide a reasonable model for the rational design of inhibitors, which may serve as novel lead compounds in antibacterial drug research.

Heptoses (seven carbon sugars) are common carbohydrates of Gram-negative bacteria with various physiological functions. Gene defects affecting the biosynthesis of the conserved inner-core region lead to decreased barrier performance of the outer cell wall membrane, an increase in permeability for hydrophobic antibiotics and a significantly reduced bacterial virulence. The project was focused on the chemical synthesis of native phosphate-containing compounds and stable derivatives of bacterial heptoses. In cooperation with the BOKU department of nanobiotechnology (FWF-projects by Dr. Paul Messner) the biosynthesis of bacterial heptoses was fully elucidated with the aid of synthetic compounds (comparison of synthetic products and purified bacterial extracts). Five key enzymes involved in the activation and transfer of the heptoses and their underlying genes were identified. For the first time, additional steps in the biosynthesis comprising a phosphate substitution at C-1 followed by hydrolysis of a second phosphate in the side chain were proven as well as the existence of two different biosynthetic pathways involved in the assembly of the bacterial heptoses. Furthermore, the genetic organization encoding these enzymes was shown for many pathogens such as Haemophilus influenzae, Neisseria meningitidis, Campylobacter jejuni, Helicobacter pylori, Mycobacterium tuberculosis and many other Enterobacteriaceae. Additionally to the synthesis of the biochemical intermediates, analogues were prepared since two of the activated heptoses are unstable and are hydrolyzed in a spontaneous reaction. The stable analogues contained a carbon- carbon bond replacing the oxygen-linkage at C-1 and allowed a general approach towards a new class of compounds mimicking activated sugar phosphates. In addition, deoxy-analogues of heptoses were prepared using an indium-catalyzed chain-elongation reaction. Furthermore, synthetic substrates have been subjected to crystallization studies in order to obtain three-dimensional structural data of enzyme-substrate and enzyme-product complexes and provide detailed knowledge on the binding process and reaction within the active site of the enzymes. The biochemical results pertaining to the inhibition of heptose transfer and substrate specifities of the epimerase have not been finished yet. These data should provide a reasonable model for the rational design of inhibitors as novel lead compounds in antibacterial drug research.