Synthesis of donor substrates for 4-amino-4-deoxy-L-arabiose transferases

Many Gram-negative bacteria have various efficient defense mechanisms against the immune system of their respective host at their disposal. The outer membrane of the bacterial cell wall exerts a protective function and is characterized by the presence of numerous negatively charged substituents such as sugar acids and sugar phosphates. The barrier function of the cell membrane, however, is counteracted by positively charged proteins (cationic antimicrobial peptides) provided by the innate immune system. Conversely, by decorating their membrane with positively charged aminosugars such as aminoarabinose, bacteria become resistant. These modifications are relevant in plant-pathogenic Burkholderia, being used in agricultural applications and which have meanwhile become major human pathogens of increasing clinical importance. Colonization of the lung by B. cepacia strains leads to dysfunction of the respiratory tract and to the lethal “cepacia syndrome”. In addition, Burkholderia strains are notoriously multiresistant against many common antibiotics. The enzymes involved in the transfer of aminoarabinose onto the bacterial lipopolysaccharide have only been incompletely characterized. This is also the case for other enzymes of the biosynthetic pathway, which generate the activated form of aminoarabinose as sugar-phosphate lipids. Since isolation of the substrates from native sources only generates tiny amounts, the project aims to prepare the native substrates, inhibitors as well as fluorescence-labeled derivatives by chemical synthesis. The central synthetic steps will first be studied using simplified lipids and then transferred to the synthesis of the complex, long-chain lipids (hepta- and undecaprenol containing 35 and 55 carbon atoms). In addition, carbon-connected derivatives (C-glycosyl phosphonates, monofluoro-C-phosphonates) are planned, which could inhibit the transfer reaction, thereby restoring efficacy of antimicrobial peptides. The synthetic substrates should also help to clarify, if one or two different aminoarabinosyl transferases are present. The synthetic compounds will be tested in collaboration with Miguel Valvano (Queens University Belfast, UK), who is a leading expert in the microbiology and genetics of Burkholderia. Appropriate inhibitors and substrates will also be used in binding studies (NMR spectroscopy, crystallography). In summary, the project should contribute substantially to the understanding of transfer mechanisms of phospholipid-activated carbohydrates onto bacterial acceptor substrates with far-reaching implications for future therapies of infections caused by multiresistant Burkholderia and other Gram-negative pathogens