Andreas Hofinger

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.

Cellulose solutions in N-methylmorpholine-N-oxide (NMMO) – degradation processes and stabilizers

Efficient stabilization of cellulose solutions in NMMO(1) against side reactions and their harmful effects meansprevention of both homolytic and heterolytic side reactions, which is mainlyaccomplished by trapping radicals, formaldehyde, andN-(methylene)iminium ions (5). Whileradical trapping is commonly reflected by the antioxidativeefficiency, the effectivity against heterolyticdegradationin the Lyocell dope can be expressed by the newly introduced term‘formaldehyde trapping capacity’ (FTC). Propyl gallate (PG,4), the most widely applied Lyocell stabilizer nowadays, actsas a phenolic antioxidant, and is finally oxidized to a deeply colored, highlyconjugated chromophore (11) via ellagicacid (10). It was demonstrated that 4 is alsoa quencher of formaldehyde and N-(methylene)iminium ions,both in organic solutions of NMMO and in Lyocell dope. The processes of radicaltrapping and scavenging of HCHO/5 are competitive in the caseof propyl gallate. A novel oxa-chromanol derivative, PBD (14),was designed as stabilizer for Lyocell solutions. In analogy to propyl gallate,PBD acts as a scavenger of all three dangerous species, namely HCHO,5 and radicals. Upon oxidation by radical species, PBDreleasesacetaldehyde which acts as a very efficient HCHO trap. Thus, in contrast topropyl gallate, radical trapping and HCHO trapping are not competitive. Boththeantioxidative efficiency and the capacity to trap HCHO and 5are higher for PBD as compared to propyl gallate. In preliminary stabilizertesting, mixtures of PBD and PG proved to be especially effective.

On the conformation of the cellulose solvent N-methylmorpholine-N-oxide (NMMO) in solution

Abstract The N-oxide group of the cellulose solvent N-methylmorpholine-N-oxide showed a strong preference for the axial position compared with N-methyl as determined by NMR experiments and computational studies. In solvents with negligible solvent–solute interaction, about 95% of the NMMO molecules obtained a typical chair conformation with an axial N–O (1) while 5% had an equatorial N–O (2) at room temperature (25 °C). Other conformations (boat and twist) are energetically largely disfavored. N-Benzylmorpholine-N-oxide was prepared as reference compound possessing 100% axial N–O. Aprotic solvents of increasing polarity slightly shifted the conformation equilibrium towards the more polar conformer 2. The effect of protic solvents, capable of forming H-bonds, was more pronounced, with water increasing the percentage of 2 to 25% of the total population. Addition of sugar model compounds reversed this effect, so that formation of 2 was suppressed and exclusively 1 was found, indicating a strong interaction between the latter conformer and the carbohydrate.

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.

Synthesis and characterization of EMPO-derived 5,5-disubstituted 1-pyrroline N-oxides as spin traps forming exceptionally stable superoxide spin adducts

Abstract EMPO [5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide] is a highly hydrophilic cyclic nitrone spin trap, whose superoxide adduct is considerably more stable (t1/2=8.6 min) than DMPO (5,5-dimethyl-1-pyrroline Noxide, t1/2=45 s). EPR spectra of spin adducts of EMPO and its derivatives are very similar to those of the respective DMPO spin adducts, in contrast to the rather complex spectra obtained using DEPMPO [5- (diethoxyphosphoryl)-5-methyl-1-pyrroline Noxide]. Several EMPO derivatives, with both the ethoxycarbonyl group and the methyl group at position 5 of the pyrroline ring being replaced by other substituents, were synthesized and characterized by 1H and 13C NMR spectroscopy. Thus, a series of derivatives was obtained that exhibit large differences in the stability of their superoxide adducts, ranging from less than one to more than 25 min. The stability of the superoxide adducts was mainly determined by the steric environment of the nitroxyl group: in compounds with less bulky 5-alkoxycarbonyl substituents the nitroxyl group is sterically less shielded, which resulted in a lower stability of the superoxide adducts. The spin density distribution, as obtained from DFT computations, was found to be nearly identical for all compounds, so that in contrast to the steric influences the spin density did not seem to be a crucial factor for the stability of the superoxide adducts.

Molecular Basis of S-layer Glycoprotein Glycan Biosynthesis in Geobacillus stearothermophilus

The Gram-positive bacterium Geobacillus stearothermophilus NRS 2004/3a possesses a cell wall containing an oblique surface layer (S-layer) composed of glycoprotein subunits. O-Glycans with the structure [→2)-α-l-Rhap-(1→3)-β-l-Rhap-(1→2)-α-l-Rhap-(1→]n = 13-18, a2-O-methyl group capping the terminal repeating unit at the nonreducing end and a →2)-α-l-Rhap-[(1→3)-α-l-Rhap]n = 1-2(1→3)- adaptor are linked via a β-d-Galp residue to distinct sites of the S-layer protein SgsE. S-layer glycan biosynthesis is encoded by a polycistronic slg (surface layer glycosylation) gene cluster. Four assigned glycosyltransferases named WsaC-WsaF, were investigated by a combined biochemical and NMR approach, starting from synthetic octyl-linked saccharide precursors. We demonstrate that three of the enzymes are rhamnosyltransferases that are responsible for the transfer of l-rhamnose from a dTDP-β-l-Rha precursor to the nascent S-layer glycan, catalyzing the formation of the α1,3- (WsaC and WsaD) and β1,2-linkages (WsaF) present in the adaptor saccharide and in the repeating units of the mature S-layer glycan, respectively. These enzymes work in concert with a multifunctional methylrhamnosyltransferase (WsaE). The N-terminal portion of WsaE is responsible for the S-adenosylmethionine-dependent methylation reaction of the terminal α1,3-linked l-rhamnose residue, and the central and C-terminal portions are involved in the transfer of l-rhamnose from dTDP-β-l-rhamnose to the adaptor saccharide to form the α1,2- and α1,3-linkages during S-layer glycan chain elongation, with the methylation and the glycosylation reactions occurring independently. Characterization of these enzymes thus reveals the complete molecular basis for S-layer glycan biosynthesis.

The structure of the fructan sinistrin from Urginea maritima

The structure of sinistrin from red squill (Urginea maritima) was determined by methylation analysis and 13C NMR spectroscopy, using the fructans from Pucinella peisonis and quack-grass (Agropyron repens) as reference substances. Application of the reductive cleavage method showed that, of the beta-D-fructofuranosyl residues in sinistrin, 33% were 1-linked, 19% were 6-linked, 25% were terminal, and 19% were 1,6-linked. The average dp was 31 and, of the 3.24% of alpha-D-glucopyranosyl residues, 0.54% were terminal and 2.70% were 6-substituted. The fructan of quack grass was also highly branched with a (2-->6)-linked backbone, terminal alpha-D-glucopyranosyl residues, and a dp of approximately 45. The fructan from Pucinella peisonis was slightly branched, with a dp of approximately 10 and a (2-->6)-linked backbone.

Biosynthesis of dTDP-3-acetamido-3,6-dideoxy-α-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.

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.

Biosynthesis of dTDP-3-acetamido-3,6-dideoxy-{alpha}-D-glucose (dTDP-{alpha}-D-Quip3NAc)

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.