A short synthesis of D-glycero-D-manno-heptose 7-phosphate

D-glycero-D-manno-Heptopyranose 7-phosphate-an intermediate in the biosynthesis of nucleotide-activated heptoses-has been prepared in good overall yield from benzyl 5,6-dideoxy-2,3-O-isopropylidene-alpha-D-lyxo-(Z)-hept-5-enofuranoside by a short-step synthesis. Phosphitylation using the phosphoramidite procedure followed by in situ oxidation afforded the corresponding 7-O-phosphotriester derivative in high yield. Subsequent osmylation proceeded in good diastereoselectivity (4:1) to furnish the D-glycero-D-manno-configured derivative, which was separated from the L-glycero-L-gulo-isomer by chromatography. Hydrogenolysis led to simultaneous removal of the benzyl and isopropylidene groups and afforded the target compound in high yield, which serves as a substrate of bacterial heptose 7-phosphate kinases.     

Modifications of Bordetella bronchiseptica core lipopolysaccharide influence immune response without affecting protective activity

Bordetella bronchiseptica produces respiratory disease primarily in mammals including humans. Although a considerably amount of research has been generated regarding lipopolysaccharide (LPS) role during infection and stimulating innate and adaptive immune response, mechanisms involved in LPS synthesis are still unknown. In this context we searched in B. bronchiseptica genome for putative glycosyltransferases. We found possible genes codifying for enzymes involved in sugar substitution of the LPS structure. We decided to analyse BB3394 to BB3400 genes, closed to a previously described LPS biosynthetic locus in B. pertussis. Particularly, conservation of BB3394 in sequenced B. bronchiseptica genomes suggests the importance of this gene for bacteria normal physiology. Deletion of BB3394 abolished resistance to naive serum as described for other LPS mutants. When purified LPS was analyzed, differences in the LPS core structure were found. Particularly, a GalNA branched sugar substitution in the core was absent in the LPS obtained from BB3394 deletion mutant. Absence of GalNA in core LPS alters immune response in vivo but is able to induce protective response against B. bronchiseptica infection.     

A short synthesis of d-glycero-d-manno-heptose 7-phosphate

d-glycero-d-manno-Heptopyranose 7-phosphate—an intermediate in the biosynthesis of nucleotide-activated heptoses—has been prepared in good overall yield from benzyl 5,6-dideoxy-2,3-O-isopropylidene-α-d-lyxo-(Z)-hept-5-enofuranoside by a short-step synthesis. Phosphitylation using the phosphoramidite procedure followed by in situ oxidation afforded the corresponding 7-O-phosphotriester derivative in high yield. Subsequent osmylation proceeded in good diastereoselectivity (4:1) to furnish the d-glycero-d-manno-configured derivative, which was separated from the l-glycero-l-gulo-isomer by chromatography. Hydrogenolysis led to simultaneous removal of the benzyl and isopropylidene groups and afforded the target compound in high yield, which serves as a substrate of bacterial heptose 7-phosphate kinases.     

Efficient chemical synthesis of both anomers of ADP L-glycero- and D-glycero-D-manno-heptopyranose

A series of anomeric phosphates and ADP-activated L-glycero- and D-glycero-D-manno-heptopyranoses has been prepared in high overall yields, which provided model compounds and substrates in the elucidation of biosynthetic pathways and glycosyl transfer reactions of nucleotide-activated bacterial heptoses. The alpha-anomers of the heptosyl phosphates were obtained in high yield and selectivity using the phosphoramidite procedure, whereas the beta-phosphates were formed preferentially employing acylation of reducing heptoses with diphenyl phosphorochloridate. An efficient route to the formation of the nucleotide diphosphate sugars was elaborated by coupling of the O-acetylated phosphates with AMP-morpholidate followed by alkaline deprotection to furnish ADP-L- and D-glycero-alpha-D-manno-heptose in 84 and 89% yield, respectively. Deacetylation of the O-acetylated beta-configured ADP heptoses was conducted at strictly controlled conditions (-28 degrees C at pH 10.5) to suppress formation of cyclic heptose-1,2-phosphodiesters with concomitant release of AMP. Isolation of the unstable beta-configured ADP-heptoses by anion-exchange chromatography and gel-filtration afforded ADP L- and D-glycero-beta-D-manno-heptose in high yields.     

GC/MS identification and quantification of constituents of bacterial lipids and glycoconjugates obtained after methanolysis as heptafluorobutyrate derivatives

In previous articles [Anal. Biochem. 284 (2000) 201; J. Lipid Res. 43 (2002) 794], we reported that the GC/MS identification and quantification of nearly all constituents of glycolipids could be obtained on the same sample in a single GC/MS analysis as heptafluorobutyrate derivatives of the products liberated using acid-catalyzed methanolysis. The same type of data could be obtained on glycoproteins and proteoglycans [Biochemistry 42 (2003) 8342]. These experiments were performed on material from higher organisms, and there was no evidence that bacteria-specific constituents could also be identified and quantified. The current article reports that the GC/MS analysis of compounds liberated by acid-catalyzed methanolysis as heptafluorobutyrate derivatives allows the simultaneous qualitative and quantitative determinations of pentoses, deoxyhexoses, hexoses, hexosamines, uronic acids, Kdo, Mur, heptose, Kdn, and neuraminic acid as well as of most fatty acids (including hydroxylated fatty acids). This approach provides a way of obtaining fingerprints of bacterial constituents and quantification of the overall effect of gene inactivation or of culture conditions.     

Comparison of Predicted Epimerases and Reductases of the Campylobacter jejuni d-altro- and l-gluco-Heptose Synthesis Pathways

Uniquely modified heptoses found in surface carbohydrates of bacterial pathogens are potential therapeutic targets against such pathogens. Our recent biochemical characterization of the GDP-6-deoxy-d-manno- and GDP-6-deoxy-d-altro-heptose biosynthesis pathways has provided the foundation for elucidation of the more complex l-gluco-heptose synthesis pathway of Campylobacter jejuni strain NCTC 11168. In this work we use GDP-4-keto,6-deoxy-d-lyxo-heptose as a surrogate substrate to characterize three enzymes predicted to be involved in this pathway: WcaG_NCTC (also known as Cj1427), MlghB (Cj1430), and MlghC (Cj1428). We compare them with homologues involved in d-altro-heptose production: WcaG₈₁₁₇₆ (formerly WcaG), DdahB (Cjj1430), and DdahC (Cjj1427). We show that despite high levels of similarity, the enzymes have pathway-specific catalytic activities and substrate specificities. MlghB forms three products via C3 and C5 epimerization activities, whereas its DdahB homologue only had C3 epimerase activity along its cognate pathway. MlghC is specific for the double C3/C5 epimer generated by MlghB and produces l-gluco-heptose via stereospecific C4 reductase activity. In contrast, its homologue DdahC only uses the C3 epimer to yield d-altro-heptose via C4 reduction. Finally, we show that WcaG_NCTC is not necessary for l-gluco-heptose synthesis and does not affect its production by MlghB and MlghC, in contrast to its homologue WcaG₈₁₁₇₆, that has regulatory activity on d-altro-heptose synthesis. These studies expand our fundamental understanding of heptose modification, provide new glycobiology tools to synthesize novel heptose derivatives with biomedical applications, and provide a foundation for the structure function analysis of these enzymes.     

Complete 6-Deoxy-D-altro-heptose Biosynthesis Pathway from Campylobacter jejuni: MORE COMPLEX THAN ANTICIPATED

The Campylobacter jejuni capsule is important for colonization and virulence in various infection models. In most strains, the capsule includes a modified heptose whose biological role and biosynthetic pathway are unknown. To decipher the biosynthesis pathway for the 6-deoxy-d-altro-heptose of strain 81-176, we previously showed that the 4,6-dehydratase WcbK and the reductase WcaG generated GDP-6-deoxy-d-manno-heptose, but the C3 epimerase necessary to form GDP-6-deoxy-d-altro-heptose was not identified. Herein, we characterized the putative C3/C5 epimerase Cjj1430 and C3/C5 epimerase/C4 reductase Cjj1427 from the capsular cluster. We demonstrate that GDP-6-deoxy-d-altro-heptose biosynthesis is more complex than anticipated and requires the sequential action of WcbK, Cjj1430, and Cjj1427. We show that Cjj1430 serves as C3 epimerase devoid of C5 epimerization activity and that Cjj1427 has no epimerization activity and only serves as a reductase to produce GDP-6-deoxy-d-altro-heptose. Cjj1430 and Cjj1427 are the only members of the C3/C5 epimerases and C3/C5 epimerase/C4 reductase families shown to have activity on a heptose substrate and to exhibit only one of their two to three potential activities, respectively. Furthermore, we show that although the reductase WcaG is not part of the main pathway, its presence and its product affect the outcome of the pathway in a complex regulatory loop involving Cjj1427. This work provides the grounds for the elucidation of similar pathways found in other C. jejuni strains and other pathogens. It provides new molecular tools for the synthesis of carbohydrate antigens useful for vaccination and for the screening of enzymatic inhibitors that may have antibacterial effects.     

A convenient synthesis of GDP D-glycero-alpha-D-manno-heptopyranose

GDP D-glycero-alpha-D-manno-Heptopyranose has been prepared in good overall yield from 2,3,4,6,7-penta-O-acetyl-D-glycero-D-manno-heptopyranose by a short-step synthesis. Phosphitylation using the phosphoramidite procedure afforded the alpha-anomer in high selectivity. Subsequent oxidation and partial deprotection gave the acetylated phosphate derivative, which was subjected to the coupling reaction with GMP-morpholidate to furnish the acetylated heptose nucleoside diphosphate in good yield. De-O-acetylation and final purification afforded the target GDP D-glycero-alpha-D-manno-heptopyranose, which serves as the substrate of the heptosyl transferase in Aneurinibacillus thermoaerophilus DSM 10155 and occurs as an intermediate in the biosynthesis of GDP 6-deoxy-heptose in Yersinia pseudotuberculosis.     

Synthesis,kinetics and inhibition of Escherichia coli Heptosyltransferase I by monosaccharide analogues of Lipid A

Gram-negative bacteria comprise the majority of microbes that cause infections that are resistant to pre-existing antibiotics. The complex cell wall architecture contributes to their ability to form biofilms, which are often implicated in hospital-acquired infections. Biofilms promote antibiotic resistance by enabling the bacteria to survive hostile environments such as UV radiation, pH shifts, and antibiotics. The outer membrane of Gram-negative bacteria contains lipopolysaccharide (LPS), which plays a role in adhesion to surfaces and formation of biofilms. The main focus of this work was the synthesis of a library of glycolipids designed to be simplified analogues of the Lipid A, the membrane embedded portion component of LPS, to be tested as substrates or inhibitors of Heptosyltransferase I (HepI or WaaC, a glycosyltransferase enzyme involved in the biosynthesis of LPS). Fourteen analogues were synthesized successfully and characterized. While these compounds were designed to function as nucleophilic substrates of HepI, they all demonstrated mild inhibition of HepI. Kinetic characterization of inhibition mechanism identified that the compounds exhibited uncompetitive and mixed inhibition of HepI. Since both uncompetitive and mixed inhibition result in the formation of an Enzyme-Substrate-inhibitor complex, molecular docking studies (using AutoDock Vina) were performed, to identify potential allosteric binding site for these compounds. The inhibitors were shown to bind to a pocket formed after undergoing a conformational change from an open to a closed active site state. Inhibition of HepI via an allosteric site suggest that disruption of protein dynamics might be a viable mechanism for the inhibition of HepI and potentially other enzymes of the GT-B structural class.     

Synthesis of a deoxy analogue of ADP L-glycero-D-manno-heptose

Starting from l-lyxose, indium-mediated chain elongation with allyl bromide followed by acetylation and oxidative cleavage of the double bond and deprotection afforded 2-deoxy-l-galacto-heptose as a 2-deoxy analogue of the bacterial carbohydrate l-glycero-d-manno-heptose in good overall yield. For the synthesis of the ADP-activated derivative, the 2-deoxy-heptose was O-acetylated and transformed into the anomeric bromide derivative, which was then converted into the acetylated heptopyranosyl phosphate by reaction with tetrabutylammonium phosphate. Deprotection and separation of the anomeric phosphates furnished 2-deoxy-beta-l-galacto-heptopyranosyl phosphate. Coupling of the acetylated heptosyl phosphate with AMP morpholidate afforded the acetylated ADP derivative in good yield. Removal of the acetyl groups gave the target compound ADP 2-deoxy-l-galacto-heptopyranose, which may serve as substrate analogue of bacterial ADP heptosyl transferases for biochemical and crystallographic studies.