Chemical synthesis of Burkholderia Lipid†A modified with glycosyl phosphodiester-linked 4-amino-4-deoxy-ß-L-arabinose and its immunomodulatory potential.

Modification of the Lipid A phosphates by positively charged appendages is a part of the survival strategy of numerous opportunistic Gram-negative bacteria. The phosphate groups of the cystic fibrosis adapted Burkholderia Lipid A are abundantly esterified by 4-amino-4-deoxy-ß-L-arabinose (ß-L-Ara4N), which imposes resistance to antibiotic treatment and contributes to bacterial virulence. To establish structural features accounting for the unique pro-inflammatory activity of Burkholderia LPS we have synthesised Lipid A substituted by ß-L-Ara4N at the anomeric phosphate and its Ara4N-free counterpart. The double glycosyl phosphodiester was assembled by triazolyl-tris-(pyrrolidinyl)phosphonium-assisted coupling of the ß-L-Ara4N H-phosphonate to α-lactol of ß(1→6) diglucosamine, pentaacylated with (R)-(3)-acyloxyacyl- and Alloc-protected (R)-(3)-hydroxyacyl residues. The intermediate 1,1'-glycosyl-H-phosphonate diester was oxidised in anhydrous conditions to provide, after total deprotection, ß-L-Ara4N-substituted Burkholderia Lipid A. The ß-L-Ara4N modification significantly enhanced the pro-inflammatory innate immune signaling of otherwise non-endotoxic Burkholderia Lipid A.

Synthesis of Neoglycoconjugates Containing 4-Amino-4-deoxy-l-arabinose Epitopes Corresponding to the Inner Core of Burkholderia and Proteus Lipopolysaccharides.

Disaccharides that contain 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) and d-glycero-d-talo-oct-2-ulosonic acid (Ko) substituted at the 8-position by 4-amino-4-deoxy-ß-l-arabinopyranosyl (Ara4N) residues have been prepared. Coupling an N-phenyltrifluoroacetimidate-4-azido-4-deoxy-l-arabinosylglycosyl donor to acetyl-protected allyl glycosides of Kdo and Ko afforded anomeric mixtures of disaccharide products in 74 and 90 % yield, respectively, which were separated by chromatography. Further extension of an intermediate Ara4N-(1→8)-Kdo disaccharide acceptor, which capitalized on a regioselective glycosylation with a Kdo bromide donor under Helferich conditions, afforded the branched trisaccharide α-Kdo-(2→4)[ß-l-Ara4N-(1→8)]-α-Kdo derivative. Deprotection of the protected di- and trisaccharide allyl glycosides was accomplished by TiCl(4)-promoted benzyl ether cleavage followed by the removal of ester groups and reduction of the azido group with thiol or Staudinger reagents, respectively. The reaction of the anomeric allyl group with 1,3-propanedithiol under radical conditions afforded the thioether-bridged spacer glycosides, which were efficiently coupled to maleimide-activated bovine serum albumin. The neoglycoconjugates serve as immunoreagents with specificity for inner core epitopes of Burkholderia and Proteus lipopolysaccharides.

Synthesis and antiviral activities of spacer-linked 1-thioglucuronide analogues of glycyrrhizin.

The influenza virus infection remains a significant threat to public health and the increase of antiviral resistance to available drugs generates an urgent need for new antiviral compounds. Starting from the natural, antivirally active compound glycyrrhizin, spacer-bridged derivatives were generated with improved antiviral activity against the influenza A virus infection. Simplified analogues of the triterpene saponin glycyrrhizin containing 1-thio-ß-D-glucuronic acid residues have been prepared in good yields by alkylation of 3-amino and 3-thio derivatives of glycyrrhetinic acid with a 2-iodoethyl 1-thio-ß-D-glucopyranosiduronate derivative. The spacer-connected 3-amino derivatives were further transformed into N-acetylated and N-succinylated derivatives. The deprotected compounds containing these carboxylic acid appendices mimic the glycon part of glycyrrhizin as well as the hemisuccinate derivative of glycyrrhetinic acid, carbenoxolone. Antiviral activities of the compounds were determined in a biological test based on influenza A virus-infected cells, wherein the 3-(2-thioethyl)-N-acetylamino- and 3-(2-thioethyl)-thio-linked glucuronide derivatives were effective inhibitors with IC(50) values as low as 54 µM.

Comparison of fluorescent labels for oligosaccharides and introduction of a new postlabeling purification method.

Labeling of oligosaccharides with fluorescent dyes is the prerequisite for their sensitive analysis by high-performance liquid chromatography (HPLC). In this work, we present a fast new postlabeling cleanup procedure that requires no device other than the reaction vial itself. The procedure can be applied to essentially all labeling reagents. We also compare the performance of 15 different labels for N-glycan analysis in various analytical procedures. We took special care to prevent obscuring influences from incomplete derivatization and signal quenching by impurities. Procainamide emerged as more sensitive than anthranilic acid for normal-phase HPLC, but its chromatographic performance was not convincing. 2-aminopyridine was the label with the lowest retention on reversed-phase and graphitic carbon columns and, thus, appears to be most suitable for glycan fractionation by multidimensional HPLC. Most glycan derivatives performed better than native sugars in matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and electrospray ionization-MS (ESI-MS), but the gain was small and hardly sufficient to compensate for sample loss during preparation.

Synthesis and characterization of several carbamoyl- and methylcarbamoyl-substituted EMPO derivatives.

The spin trapping behavior of four novel carbamoyl-substituted EMPO derivatives, namely 5-carbamoyl-3,5-dimethyl-pyrroline N-oxide (CADMPO), 3,5-dimethyl-5-methylcarbamoyl-pyrroline N-oxide (DMMCAPO), 5-carbamoyl-3-ethyl-5-methyl-pyrroline N-oxide (CAEMPO), and 3-ethyl-5-methyl-5-methylcarbamoyl-pyrroline N-oxide (EMMCAPO), towards different oxygen- and carbon-centered radicals is described, the half lives of the respective superoxide adducts ranging from about 10 to 20 min. The most characteristic adducts were, however, formed from methyl, hydroxymethyl, hydroxyethyl, and carbon dioxide anion radicals.

Biosynthesis of dTDP-3-acetamido-3,6-dideoxy-alpha-D-glucose.

Derivatives of 3-amino-3,6-dideoxyhexoses are widespread in Nature. They are part of the repeating units of lipopolysaccharide O-antigens, of the glycan moiety of S-layer (bacterial cell surface layer) glycoproteins and also of many antibiotics. In the present study, we focused on the elucidation of the biosynthesis pathway of dTDP-alpha-D-Quip3NAc (dTDP-3-acetamido-3,6-dideoxy-alpha-D-glucose) from the Gram-positive, anaerobic, thermophilic organism Thermoanaerobacterium thermosaccharolyticum E207-71, which carries Quip3NAc in its S-layer glycan. The biosynthesis of dTDP-alpha-D-Quip3NAc involves five enzymes, namely a transferase, a dehydratase, an isomerase, a transaminase and a transacetylase, and follows a pathway similar to that of dTDP-alpha-D-Fucp3NAc (dTDP-3-acetamido-3,6-dideoxy-alpha-D-galactose) biosynthesis in Aneurinibacillus thermoaerophilus L420-91(T). The ORFs (open reading frames) of interest were cloned, overexpressed in Escherichia coli and purified. To elucidate the enzymatic cascade, the different products were purified by HPLC and characterized by NMR spectroscopy. The initiating reactions catalysed by the glucose-1-phosphate thymidylyltransferase RmlA and the dTDP-D-glucose-4,6-dehydratase RmlB are well established. The subsequent isomerase was shown to be capable of forming a dTDP-3-oxo-6-deoxy-D-glucose intermediate from the RmlB product dTDP-4-oxo-6-deoxy-D-glucose, whereas the isomerase involved in the dTDP-alpha-D-Fucp3NAc pathway synthesizes dTDP-3-oxo-6-deoxy-D-galactose. The subsequent reaction steps of either pathway involve a transaminase and a transacetylase, leading to the specific production of nucleotide-activated 3-acetamido-3,6-dideoxy-alpha-D-glucose and 3-acetamido-3,6-dideoxy-alpha-D-galactose respectively. Sequence comparison of the ORFs responsible for the biosynthesis of dTDP-alpha-D-Quip3NAc revealed homologues in Gram-negative as well as in antibiotic-producing Gram-positive bacteria. There is strong evidence that the elucidated biosynthesis pathway may also be valid for LPS (lipopolysaccharide) O-antigen structures and antibiotic precursors.

Spin trapping experiments with different carbamoyl-substituted EMPO derivatives.

The spin trapping behavior of five carbamoyl-substituted EMPO derivatives, 5-aminocarbonyl-5-methyl-pyrroline N-oxide (CAMPO (AMPO)), 5-aminocarbonyl-5-ethyl-pyrroline N-oxide (CAEPO), 5-aminocarbonyl-5-propyl-pyrroline N-oxide (CAPPO), 5-aminocarbonyl-5-n-butyl-pyrroline N-oxide (CABPO), and 5-aminocarbonyl-5-n-pentyl-pyrroline N-oxide (CAPtPO), toward different oxygen- and carbon-centered radicals is described, the stabilities of the superoxide adducts ranging from about 8 to 17min.

Structural characterization of the acid-degraded secondary cell wall polymer of Geobacillus stearothermophilus PV72/p2.

The secondary cell wall polymer (SCWP) from Geobacillus stearothermophilus PV72/p2, which is involved in the anchoring of the surface-layer protein to the bacterial cell wall layer, is composed of 2-amino-2-deoxy- and 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2-deoxy-D-mannose, and 2-acetamido-2-deoxy-D-mannuronic acid. The primary structure of the acid-degraded polysaccharide--liberated by HF-treatment from the cell wall--was determined by high-field NMR spectroscopy and mass spectrometry using N-acetylated and hydrolyzed polysaccharide derivatives as well as Smith-degradation. The polysaccharide was shown to consist of a tetrasaccharide repeating unit containing a pyruvic acid acetal at a side-chain 2-acetamido-2-deoxy-alpha-D-mannopyranosyl residue. Substoichiometric substitutions of the repeating unit were observed concerning the degree of N-acetylation of glucosamine residues and the presence of side-chain linked 2-acetamido-2-deoxy-beta-D-glucopyranosyl units: [Formula: see text].

Synthesis of spacer-containing chlamydial disaccharides as analogues of the alpha-Kdop-(2-->8)-alpha-Kdop-(2-->4)-alpha-Kdop trisaccharide epitope.

On the basis of high-resolution crystal structures of the antigen binding fragment of the chlamydia-specific monoclonal antibody S25-2 in complex with the trisaccharide alpha-Kdop-(2-->8)-alpha-Kdop-(2-->4)-alpha-Kdop and part structures thereof, seven modified alpha-Kdop-(2-->8)-alpha-Kdop disaccharide derivatives were synthesized starting from the protected disaccharide allyl ketoside 1. Hydroboration and subsequent oxidation as well as ozonolysis, respectively, followed by Wittig-reaction for chain elongation were used to install a terminal carboxylic group on spacer entities of various chain lengths. Furthermore, addition of methyl 2-thioacetate to the allyl group furnished the corresponding thioether derivative. Standard deprotection gave the target disaccharides as simplified trisaccharide analogues, which will be used to probe the contribution of the proximal carboxylic group in the binding of chlamydia-specific di- and trisaccharide-reactive monoclonal antibodies.

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.

Genome Analysis and Characterisation of the Exopolysaccharide Produced by Bifidobacterium longum subsp. longum 35624™.

The Bifibobacterium longum subsp. longum 35624™ strain (formerly named Bifidobacterium longum subsp. infantis) is a well described probiotic with clinical efficacy in Irritable Bowel Syndrome clinical trials and induces immunoregulatory effects in mice and in humans. This paper presents (a) the genome sequence of the organism allowing the assignment to its correct subspeciation longum; (b) a comparative genome assessment with other B. longum strains and (c) the molecular structure of the 35624 exopolysaccharide (EPS624). Comparative genome analysis of the 35624 strain with other B. longum strains determined that the sub-speciation of the strain is longum and revealed the presence of a 35624-specific gene cluster, predicted to encode the biosynthetic machinery for EPS624. Following isolation and acid treatment of the EPS, its chemical structure was determined using gas and liquid chromatography for sugar constituent and linkage analysis, electrospray and matrix assisted laser desorption ionization mass spectrometry for sequencing and NMR. The EPS consists of a branched hexasaccharide repeating unit containing two galactose and two glucose moieties, galacturonic acid and the unusual sugar 6-deoxy-L-talose. These data demonstrate that the B. longum 35624 strain has specific genetic features, one of which leads to the generation of a characteristic exopolysaccharide.

Spin adduct formation from lipophilic EMPO-derived spin traps with various oxygen- and carbon-centered radicals.

Free radicals are involved in the onset of many diseases, therefore the availability of adequate spin traps is crucial to the identification and localization of free radical formation in biological systems. In recent studies several hydrophilic compounds of 2-ethoxycarbonyl-2-methyl-pyrroline-N-oxide (EMPO) have been found to form rather stable superoxide spin adducts with half-lives up to twenty minutes at physiological pH. This is a major improvement over DMPO (t1/2=ca. 45 s), and even over DEPMPO (t1/2=ca. 14 min), the best commercially available spin trap for the unambiguous detection of superoxide radicals. In order to allow the detection of superoxide and also other radicals in lipid environment a series of more lipophilic derivatives of EMPO was synthesized and their structure unambiguously characterized by 1H and 13C NMR spectroscopy. In this way, six different compounds with a n-butyl group in position 5 and either an ethoxy- (EBPO), propoxy- (PBPO), iso-propoxy- (iPBPO), butoxy- (BBPO), sec-butoxy- (sBBPO) or tert-butoxycarbonyl group (tBBPO) in position 5 of the pyrroline ring were obtained and fully analytically characterized (NMR, IR). The stability of the superoxide adducts of all investigated spin traps were comparable with EMPO (t1/2=ca. 8 min), except for the two compounds bearing an additional methyl group in position 3 or 4 of the pyrroline ring, 5-butyl-5-ethoxycarbonyl-3-methyl-pyrroline-N-oxide (BEMPO-3) and 5-butyl-5-ethoxycarbonyl-4-methyl-pyrroline-N-oxide (BEMPO-4), of which the superoxide adducts were stable for more than 30 min. Spin adducts of other carbon- and oxygen-centered radicals were also investigated.

The Drosophila melanogaster homologue of the human histo-blood group Pk gene encodes a glycolipid-modifying alpha1,4-N-acetylgalactosaminyltransferase.

Insects express arthro-series glycosphingolipids, which contain an alpha1,4-linked GalNAc residue. To determine the genetic basis for this linkage, we cloned a cDNA (CG17223) from Drosophila melanogaster encoding a protein with homology to mammalian alpha1,4-glycosyltransferases and expressed it in the yeast Pichia pastoris. Culture supernatants from the transformed yeast were found to display a novel UDP-GalNAc:GalNAcbeta1,4GlcNAcbeta1-R alpha-N-acetylgalactosaminyltransferase activity when using either a glycolipid, p-nitrophenylglycoside or an N-glycan carrying one or two terminal beta-N-acetylgalactosamine residues. NMR and MS in combination with glycosidase digestion and methylation analysis indicate that the cloned cDNA encodes an alpha1,4-N-acetylgalactosaminyltransferase. We hypothesize that this enzyme and its orthologues in other insects are required for the biosynthesis of the N5a and subsequent members of the arthro-series of glycolipids as well as of N-glycan receptors for Bacillus thuringiensis crystal toxin Cry1Ac.

Spin trapping of superoxide, alkyl- and lipid-derived radicals with derivatives of the spin trap EPPN.

The N-t-butyl-alpha-phenylnitrone derivative N-2-(2-ethoxycarbonyl-propyl)-alpha-phenylnitrone (EPPN) has recently been reported to form a superoxide spin adduct (t(1/2)=5.25 min at pH 7.0), which is considerably more stable than the respective N-t-butyl-alpha-phenylnitrone or 5,5-dimethylpyrroline N-oxide adducts (t(1/2) approximately 10 and 45s, respectively). In continuation of our previous studies on structure optimization of 5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide derivatives, a series of six different EPPN derivatives was synthesized and characterized by 1H NMR, 13C NMR and IR spectroscopy. The ethoxy group of EPPN was replaced by a propoxy, iso-propoxy, n-butoxy, sec-butoxy, and tert-butoxy moiety, as well as the phenyl by a pyridyl ring. Electron spin resonance spectra and stabilities of the superoxide adducts of the propoxy derivatives were found to be similar to those of the respective EPPN adduct, whereas the electron spin resonance spectra of the superoxide adducts of N-2-(2-ethoxycarbonyl-propyl)-alpha-(4-pyridyl) nitrone and the butoxy derivatives were accompanied by decomposition products. In contrast to the 5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide series, no significant improvement of the superoxide adduct stability could be obtained when the ethoxy group was replaced by other substituents. Carbon centered radical adducts derived from methanol, ethanol, formic acid and linoleic acid hydroperoxide were more stable than those of 5,5-dimethylpyrroline N-oxide, whereas among the alkoxyl radicals only the methoxyl radical adduct could be detected.

Spin adducts of several N-2-(2-alkoxycarbonyl-propyl)-alpha-pyridylnitrone derivatives with superoxide, alkyl and lipid-derived radicals.

Several derivatives of N-t-butyl-alpha-phenylnitrone (PBN) such as N-2-(2-ethoxycarbonyl-propyl)-alpha-phenylnitrone (EPPN) have recently been reported to form superoxide spin adducts (t(1/2) ca. 2-7 min at pH 7.0), which are considerably more stable than their respective PBN or DMPO adducts (t(1/2) ca. 10 and 45 s, respectively). In continuation of our studies on structure optimization of EPPN derivatives, a series of 12 novel spin traps with 2-, 3- and 4-pyridinyl substituents was synthesized and fully characterized by 1H NMR, 13C NMR and IR spectroscopy. In addition to the replacement of the phenyl ring by a 2-, 3- or 4-pyridinyl substituent, the ethoxy group of the parent compound EPPN was replaced by either a propoxy, iso-propoxy, or cyclopropylmethoxy moiety. Superoxide adducts of all PPyN derivatives were considerably more stable than those of the respective EPPN derivatives with half-lives ranging from about 6 to 11 min. In addition, alkoxyl radical adducts were also considerably more stable than those of the EPPN series. Hydroxyl radical adducts were not detected, on the other hand, very stable spin adducts were formed from a series of carbon centered radicals, e.g. from the methyl or hydroxymethyl radical. The novel spin traps are offering an alternative to PBN or POBN, especially where the higher stability of oxygen-centered radical adducts is of major importance. All of them can easily be synthesized from commercially available compounds in two or three steps.

A general, selective, high-yield N-demethylation procedure for tertiary amines by solid reagents in a convenient column chromatography-like setup.

[reaction: see text] A traditional preparative chromatographic column can be used to achieve quantitative N-demethylation of tertiary N-methylamines and alkaloids. The filling is the crucial part and is loaded with different solid reagents in three reaction zones. The parent compound is charged on the column, and the neat N-demethylated secondary amine leaves the column some minutes later.

On a novel chromanone-naphthalenetrione rearrangement related to vitamin E.

4-Oxo-alpha-tocopherol (7) was synthesized in an efficient three-step procedure starting from alpha-tocopheryl acetate (1b) and rearranged under physiological conditions into naphthalenetrione 4, a minor vitamin E metabolite. The rearrangement involves a [4 + 2]-cycloaddition as the key step. [reaction: see text]

Synthesis of neoglycoproteins containing O-methylated trisaccharides related to excretory/secretory antigens of Toxocara larvae.

The disaccharides allyl beta-D-galactopyranosyl-(1-->3)-2-acetamido-2-deoxy-beta- and alpha-D-galactopyranoside 10a and 10b and the trisaccharides allyl 2-O-methyl-alpha-L-fucopyranosyl-(1-->2)-beta-D-galactopyranosyl-(1-->3)-2-acetamido-2-deoxy-beta- and alpha-D-galactopyranoside 18a and 18b have been prepared using stepwise assembly of the sugar units. The glycosidic linkages were formed employing the trichloroacetimidate procedure for the attachment of the galactopyranosyl residue and N-iodosuccinimide/triflic acid activation of an ethyl 1-thiofucopyranoside donor for fucosylation. Deprotection furnished the allyl glycosides which were converted into cysteamine-spacered ligands, activated with thiophosgene and subsequently linked to bovine serum albumin. The neoglycoproteins serve as immunoreagents to determine epitope specificities of monoclonal antibodies directed against highly immunogenic O-glycans located at the surface of Toxocara larvae.

Kinetic and chemical studies on the isomerization of monosaccharides in N-methylmorpholine-N-oxide (NMMO) under Lyocell conditions.

The Lyocell process is a modern and environmentally fully compatible industrial fiber-making technology. Cellulosic pulp is dissolved without chemical derivatization in a melt of N-methylmorpholine-N-oxide monohydrate (NMMO). In the present work, the reactions of monosaccharides under Lyocell conditions were investigated in detail, using capillary zone electrophoresis as the analytical technique to clarify the composition of reaction mixtures and to follow the kinetics. Under Lyocell conditions, xylose and glucose undergo two competitive reactions: rapid conversion to nonreducing products, and complete isomerization involving the whole carbohydrate backbone, via ketose intermediates. Sugar acids are present in minor amounts only, as demonstrated by employing isotopically labeled material for NMR techniques.

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.