Synthesis of donor substrates for 4-amino-4-deoxy-L-arabinose 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

Many Gram-negative bacteria have various efficient defense mechanisms against the immune system of their respective host at their disposal. Negatively charged groups in the outer membrane of the bacterial cell wall are targets of the innate immune response and a few antimicrobial drugs, which are therefore masked by positively charged aminosugars such as aminoarabinose leading to increasing antimicrobial resistance of these bacteria. The enzymes involved in the transfer of aminoarabinose have only been incompletely characterized since the activated sugar as long-chain lipid sugar-phosphate can only be isolated in tiny amounts from bacterial sources. Within the project, chemical synthesis of donor substrates has been carried out, aiming to explore simplified versions of the donor substrates which would still be accepted by the enzyme. A small library containing seven phosphodiester linked donor substrates was prepared as well as derivatives containing a carbon-bond at the reactive site of the carbohydrate. The latter compounds are inert to the enzymatic transfer reaction and could thus act as inhibitors thereby restoring activity of specific antibiotics. In collaboration with the group of Miguel Valvano (Queens University Belfast, UK), a transferase from pathogenic Burkholderia was expressed in Escherichia coli and tested as membrane preparation using the synthetic donor substrates and a lipopolysaccharide acceptor from E. coli. The enzymatic reaction was monitored by thin layer chromatography and products were characterized by high-resolution mass spectrometry. The transferase reaction was only productive with the -neryl derivative - confirming the high specificity of glycosyl transferases - leading to mono- and disubstituted products. The attachment site at the 1- and 4'-phosphates of the lipid A part of the acceptor was eventually proven after acidic cleavage of the Kdo-units. Thus, a readily accessible donor substrate is now available for further biochemical studies using additional aminotransferases and modified lipopolysaccharide acceptor derivatives as well as for detailed binding studies by NMR spectroscopy and x-ray crystallography. In summary, the project contributed substantially to the understanding of transfer mechanisms of phospholipid-activated carbohydrates onto bacterial acceptor substrates with potential implications for future therapies of infections caused by multiresistant Burkholderia and other Gram-negative pathogens.