Denitration of carbohydrate α-nitroepoxides by nucleophiles

Several nucleophiles were separately treated with methyl and phenyl 2,3-anhydro-4,6-O-benzylidene-3-deoxy-3-nitro-β-d-allopyranoside, to give 2-substituted aldos-3-ulose derivatives. In the latter case, the subsequent β-elimination of the aglyconic phenyl group always occurred to afford the corresponding glycal. Reaction mechanisms thereof are also discussed.     

Reactions of Re(III) with α-diketones: Oxidative addition

The reactions of [ReCl₃(CH₃CN)(PPh₃)₂] with benzil PhC(O)C(O)Ph, and with a natural 1,2-naphthoquinone derivative, β-lapachone (Lap), result in oxidative addition with the formation of Re(V) complexes with stilbenediolate, [ReCl₃(PhC(O)C(O)Ph)(PPh₃)] (1) and with a reduced semiquinonic form of lapachone, [Re^IVCl₃(Lap⁻)(PPh₃)] (2). The structures of both compounds were established by X-ray crystallography.     

Comparison of the carbohydrate and amino acid composition of normal and S-variant α-1-antitrypsin

α-1-Antitrypsin has been isolated and purified from the serum of an individual with the Pi S phenotype whose serum contains only 50–60% as much α-1-antitrypsin as normal M-type serum. The preparation was homogeneous by the criteria of sodium dodecyl sulfate polyacrylamide gel electrophoresis and sedimentation equilibrium ultracentrifugation. When analyzed in the ultracentrifuge, the S-type α-1-antitrypsin exhibited a molecular weight of 47500 which was essentially the same as that of the M-type (47300) and the Z-type (47500) α-1-antitrypsin. The S-type α-1-antitrypsin contains 15.2% carbohydrate consisting of 16.4 residues/mol of N-acetylglucosamine, 7.8 residues/mol of mannose. 6.7 residues/mol of galactose and 7.1 residues/mol of sialic acid which is essentially the same as the carbohydrate composition of the M-type α-1-antitrypsin. In addition, M- and S-type α-1-antitrypsin have very similar amino acid compositions.     

Synthesis and carbohydrate binding studies of fluorescent α-amidoboronic acids and the corresponding bisboronic acids

Fluorescent boronic acids are very useful for the design and synthesis of carbohydrate sensors. In an earlier communication, we first described the effort of developing water soluble fluorescent α-amidoboronic acids, which change fluorescence upon sugar binding. In this report, we describe a general method of functionalizing such boronic acids and their applications in the preparation of bis-α-amidoboronic acids with significantly enhanced binding for oligosaccharides as compared to their monoboronic acid counterparts. The advantages of good water solubility, easy modification to generate diversity, and modularity in synthesis will make α-amidoboronic acids very useful building blocks for future synthesis of boronic acid-based fluorescent sensors.     

The carbohydrate moiety of α-galactosidase from Trichoderma reesei

a-Galactosidase from Trichoderma reesei is a glycoprotein that contains O- and N-linked carbohydrate chains. There are 6 O-linked glycans per protein molecule that are linked to serine and threonine and can be released by b-elimination. Among these are monomers: D-glucose, D-mannose, and D-galactose; dimers: a1-6 D-mannopyranosyl- a-D-glycopyranoside and a1-6 D-glucopyranosyl- a-D-galactopyranoside and one trimer: a-D-glucopyranosyl- a1-2 D-mannopyranosyl- a1-6 D-galac-topyranoside. N-linked glycans are of the mannose-rich type and may be released by treating the protein with Endo- b-N-acetyl glycosaminidase F or by hydrozinolysis. The enzyme was deglycosylated with Endo- b- N-acetyl glycosaminidase F as well as with a number of exoglycosidases that partially remove the terminal residues of O-linked glycans. The effect of enzymatic deglycosylation on the properties of a-galactosidase has been considered. The effects of tunicamycin and 2-deoxyglucose on the secretion and glycosylation of the enzyme during culture growth have been analysed. The presence of two glycoforms of a-glactosidase differing in the number of N-linked carbohydrate chains and the microheterogeneity of the carbohydrate moiety of the enzyme are described. This revised version was published online in August 2006 with corrections to the Cover Date.     

Structural analysis of the carbohydrate moieties of α-l-fucosidase from human liver

Acid -l-fucosidase (EC 3.2.1.51) was obtained from human liver and purified to homogeneity. The enzyme consists of four subunits; each of these has a molecular mass of 50 kD_a and bears oneN-linked carbohydrate chain. The structures of these chains were studied at the glycopeptide level by methylation analysis and 500-MHz¹H-NMR spectroscopy. Oligomannoside-type chains andN-acetyllactosamine-type chains are present in an approximate ratio of 31. While the oligomannoside-type chains show some heterogeneity in size (Man_5–8GlcNAc₂), theN-acetyllactosaminetype chains are exclusively bi-(2–6)-sialyl, bi-antennary in their structure.These observations on the carbohydrate moieties of -l-fucosidase substantiate our hypothesis [Overdijket al. (1986) Glycoconjugate J 3:339–50] with respect to the relationship between the oligosaccharide structure of lysosomal enzymes and their residual intracellular activity in I-cell disease. For the series of enzymes examined so far, namely, -N-acetylhexosaminidase, -l-fucosidase and -galactosidase, the relative amount ofN-acetyllactosamine-type carbohydrate increases, while the residual intracellular activity in I-cell disease tissue decreases in this order. The system which is responsible for preferentially retaining hydrolases with (non-phosphorylated) oligomannoside-type chains both in I-cells and in normal cells has yet to be identified.     

Structure of the carbohydrate moiety of the α1-acid glycoprotein of human plasma

Periodate oxidation and Smith degradation of the carbohydrate moiety obtained by hydrazinolysis of the α₁-acid glycoprotein of human plasma have been studied, and an average structure for the polysaccharide has thereby been obtained. The inner core of the polysaccharide appears to be O-(2-acetamido-2-deoxy-d-glucopyranosyl)-(1→3)-O-d-mannopyranosyl)-(1→4)-O-(2-acetamido-2-deoxy-d-glucopyranosyl)- (1→3)-O-d-mannopyranosyl-(1→4)-2-acetamido-2-deoxy-d-glucose, to which several oligosaccharides are attached. To the residue of d-mannose that is linked to the reducting 2-acetamido-2-deoxy-d-glucose residue, equimolar amounts of O-sialyl-(2→3)- O-d-galactopyranosyl-(1→3)-O-(2-acetamido-2-deoxy-d-glucopyranosyl)- and O-l-fucopyranosyl-(1→3)-O-d-galactopyranosyl-(1→4)-O-(2-acetamido-2-deoxy-d-glucopyranosyl)-chains are attached at positions 2, and/or 4, and/or 6. An O-sialyl-(2→6)-d-galactopyranose residue is attached at position 6 of the second residue of 2-acetamido-2-deoxy-d-glucose. An O-sialyl-(2→4)-d-galactose residue is attached at positions 3 or 4 of the third residue of 2-acetamido-2-deoxy-d-glucose, and an O-d-galactopyranosyl-(1→6)-O-(2-acetamido-2-deoxy-d-glucopyranosyl)- (1→6)-O-d-mannopyranosyl chain is attached to the same residue of 2-acetamitado-2-deoxy-d-glucose at position 4 or 3.     

Maternal effects and carbohydrate changes of Pinus pinaster after inoculation with Fusarium circinatum

Key message

Carbohydrate differences in offspring as a consequence of maternal effects explain transgenerational tree-pathogen interactions.

Abstract

The expression of disease is increasingly recognised as being influenced by maternal effects, given that environmental conditions experienced by mother trees affect tolerance in offspring. It is hypothesised that plant carbohydrates could mediate transgenerational tree-pathogen interactions. The carbohydrate content of Pinus pinaster seedlings obtained from two contrasting maternal environments was studied and seedlings from the two environments were challenged with Fusarium circinatum. The representative mid-infrared spectra of samples in the range of the carbohydrates diagnosed higher proportion of methylesterified pectic polysaccharides and lower proportion of nonesterified pectic polysaccharides for inoculated than for control seedlings. Total carbohydrate content of seedlings from the unfavourable environment did not differ much from total carbohydrate content of seedlings from the favourable maternal environment. However, glucose was 13 % higher and uronic acids 11 % lower in seedlings from the favourable environment after inoculation in comparison to seedlings from the unfavourable maternal environment which had their carbohydrate contents unaltered after inoculation. It is concluded that plant carbohydrates mediate transgenerational tree-pathogen interactions.     

Reaction of Toxic Bicyclic Phosphates with Acetylcholinesterases and α-Chymotrypsin

Some toxic bicyclic phosphates (BPs) inhibited acetylcholinesterases (AChEs), but the activity was very weak. Even the most potent inhibitor, 4-nitro BP, inhibited bovine erythrocyte and housefly head AChEs by only 37 and 38 per cent, respectively, at 1.5 mm. Kinetic analysis indicated that the poor inhibitory activity of 4-nitro BP is ascribed not only to the low affinity for AChEs but also to its poor phosphorylating ability. Similar findings were obtained in the case of the reaction of BPs with the serine enzyme α-chymotrypsin. Despite the relatively high reactivity in an alkaline solution, BPs are much less active than other bioactive organophosphorus esters in phosphorylating a general-base-catalyzed hydroxyl group. This fact suggests that the toxic action of BPs does not result from the phosphorylation of a critical site in biological systems.     

Quantification of lignin–carbohydrate linkages with high-resolution NMR spectroscopy

A quantitative approach to characterize lignin–carbohydrate complex (LCC) linkages using a combination of quantitative ¹³C NMR and HSQC 2D NMR techniques has been developed. Crude milled wood lignin (MWLc), LCC extracted from MWLc with acetic acid (LCC-AcOH) and cellulolytic enzyme lignin (CEL) preparations were isolated from loblolly pine (Pinus taeda) and white birch (Betula pendula) woods and characterized using this methodology on a routine 300 MHz NMR spectrometer and on a 950 MHz spectrometer equipped with a cryogenic probe. Structural variations in the pine and birch LCC preparations of different types (MWL, CEL and LCC-AcOH) were elucidated. The use of the high field NMR spectrometer equipped with the cryogenic probe resulted in a remarkable improvement in the resolution of the LCC signals and, therefore, is of primary importance for an accurate quantification of LCC linkages. The preparations investigated showed the presence of different amounts of benzyl ether, γ-ester and phenyl glycoside LCC bonds. Benzyl ester moieties were not detected. Pine LCC-AcOH and birch MWLc preparations were preferable for the analysis of phenyl glycoside and ester LCC linkages in pine and birch, correspondingly, whereas CEL preparations were the best to study benzyl ether LCC structures. The data obtained indicate that pinewood contains higher amounts of benzyl ether LCC linkages, but lower amounts of phenyl glycoside and γ-ester LCC moieties as compared to birch wood.     

Reactions of Tp–Os nitrido complexes with the nucleophiles hydroxide and thiosulfate

The reaction between TpOs(N)Cl₂ (1) [Tp = hydrotris(1-pyrazolyl)borate] and aqueous (ⁿBu₄N)(OH) in THF-d₈ forms the nitrosyl complex TpOs(NO)Cl₂ (5) among other products, suggesting an initial hydroxide attack at the nitrido ligand. In contrast, the reaction of the acetate complex TpOs(N)(OAc)₂ (2) with NaOH in Me₂CO/H₂O yields the osmium bis-hydroxide complex TpOs(N)(OH)₂ (3), which has been structurally characterized by single-crystal X-ray diffraction. Acetate for hydroxide exchange could occur by ligand substitution or by nucleophilic attack at the carbonyl carbon of the acetate ligands (saponification). Reacting 2 with Na¹⁸OH in H₂¹⁸O/CD₃CN yields predominantly doubly ¹⁸O-labeled TpOs(N)(¹⁸OH)₂ (3-¹⁸O₂) and unlabeled acetate, by ESI/MS and ¹³C{¹H} NMR. This indicates that hydroxide reacts by substitution rather than by attack at the ligand. The reaction of 2 with the softer nucleophile thiosulfate occurs at the nitrido ligand, giving the thionitrosyl complex TpOs(NS)(OAc)₂ (4). Reacting 4 with NaOH in (CD₃)₂CO/D₂O also generates the bis-hydroxide complex 3.     

Interaction of α-tocopherol quinone, α-tocopherol and other prenyllipids with photosystem II

We have found that plastoquinone-A (PQ-A) and α-tocopherol (α-Toc) increased the reduction level of the high-potential form of cytochrome b-559 (cyt. b-559 HP) and α-tocopherol quinone (α-TQ) decreased the level of this cytochrome form in Scenedesmus obliquus wild-type, while the investigated prenyllipids were not active in the restoration of the cyt. b-559 HP form in Scenedesmus PS28 mutant and Synechococcus 6301 (Anacystis nidulans) where the cyt. b-559 HP form is naturally not present. Among the tested prenyllipids, α-TQ quenched fluorescence in thylakoids of S. obliquus wild-type, the PS28 mutant and tobacco to the highest extent, while PQ-A was less effective in this respect. α-Tocopherol showed the opposite effect to α-TQ and it was rather small. The fluorescence quenching measurements of thylakoids in the presence of DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) showed that the α-Toc and FCCP (carbonylcyanide-p-trifluoromethoxy-phenyl-hydrazone) did not quench non-photochemically chlorophyll fluorescence while PQ-9 and α-TQ were effective fluorescence quenchers at higher concentrations (> 15 μM). However, at the lower α-TQ concentrations where its effective fluorescence quenching was found in DCMU-free samples, there was nearly no quenching effect by α-TQ observed in DCMU-treated thylakoids. This suggested a specific, not non-photochemical, DCMU sensitive, fluorescence quenching of photosystem II (PSII) at low α-TQ concentrations which is probably connected with the cyclic electron transport around PSII and might have a function of excess light energy dissipation. The effects of α-TQ on PSII resembled those of FCCP under many respects which might suggest similar mechanism of action of these compounds on PSII, i.e. the catalytic deprotonation and/or redox changes of some components of PSII such as the water splitting system, tyrosine D, Chl_z or cytochrome b-559.     

The carbohydrate–polypeptide linkages, the amino acid sequences of the peptides adjacent to some of these bonds, and the composition and size of the carbohydrate units of α1-acid glycoprotein

1. The glycopeptides derived from a proteolytic digest of sialic acid-free α₁-acid glycoprotein were separated on a DEAE-cellulose column into five main fractions. 2. The average molecular weight of these glycopeptides was 2400, except for one fraction whose molecular weight was 3100. The average molecular weight of the sialic acid-free carbohydrate units was found to be 2200. From these data and the carbohydrate content of the native protein and the assumed molecular weight of 44000, it was concluded that α₁-acid glycoprotein probably possesses five carbohydrate units. The sialic acid-containing carbohydrate units of this glycoprotein have an average molecular weight of 3000, except for one unit the molecular weight of which is significantly higher. 3. The N-, non-N- and C-terminal amino acids of the main glycopeptides were determined. Aspartic acid and threonine occur in most peptides. Alanine, glycine, proline, serine and lysine were present in varying amounts. Traces of other amino acids were also found. 4. The amino acid sequence of three main glycopeptides was established and indicated that these glycopeptides are located at different positions of the polypeptide chain of the glycoprotein. These sequences are: Asp(NH₂)-Pro-Lys; Thr-Asp(NH₂)-Ala; Asp(NH₂)-Gly-Thr. 5. From the results of a series of chemical reactions (periodate oxidation, hydrazinolysis, dinitrophenylation, mild acid hydrolysis) it was shown that the hydroxyl group of the N-terminal threonine and the -amino group of lysine are free and that the β-carboxyl group of aspartic acid is present as amide. It was concluded that this amide group is involved in the carbohydrate–polypeptide linkages of at least four carbohydrate units of α₁-acid glycoprotein. 6. The carbohydrate composition of the sialic acid-free glycopeptides was determined in terms of moles of neutral hexoses, glucosamine and fucose/mole. 7. Fucose, at least to the larger part, is not linked to sialic acid, and its (glycosidic) linkage is significantly more stable toward acid hydrolysis than the bond of the sialyl residues. 8. Heterogeneity of the carbohydrate units of α₁-acid glycoprotein was found with regard to size and to content of fucose and sialic acid.     

Determination of the structure of the carbohydrate chains of acid α-glucosidase from human placenta

Acid α-glucosidase (α-d-glucoside glucohydrolase, EC 3.2.1.20) from human placenta (70 and 76 kDa) was found to contain 4 N-glycosidic carbohydrate chains per molecule. Sugar analysis of purified enzyme revealed the presence of mannose, N-acetylglucosamine and fucose at a molar ratio of 5.0:2.0:0.6. In addition, trace amounts of galactose and N-acetylneuraminic acid were detected. The sugar chains were liberated from the polypeptides by the hydrazinolysis procedure and subsequently fractionated by gel filtration and HPLC. Purified compounds were investigated by 500-MHz ¹H-NMR spectroscopy. Oligomannoside-type chains of intermediate size, e.g., Man₅GlcNAcGlcNAc-ol and Man₇GlcNAcGlcNAc-ol, and N-type chains of smaller size e.g., Man_2–3GlcNAc[Fuc]_0–1GlcNAc-ol, were demonstrated to be present at a ratio of 2:3. In addition, a small amount of sialylated N-acetyllactosamine-type chains has been found. The possible biosynthetic route of the fucose-containing small-size chains is discussed.     

Analysis of the substrate specificity of α-L-arabinofuranosidases by DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis

Applied Microbiology and Biotechnology - Carbohydrate-active enzyme discovery is often not accompanied by experimental validation, demonstrating the need for techniques to analyze substrate...     

Interaction of α-thrombin and prethrombin 2, with phosphatidylserine-containing monolayers

Prothrombin activation complex is located at a phospholipid surface on activated platelets. To see whether the thrombin domain of the molecule plays a role in the interaction with lipids, we investigated the direct interaction of human α-thrombin and its precursor prethrombin 2 with phospholipid monolayers of varous compositions (PS/PC). Adsorption of the labeled proteins was determined by surface radioactive measurements. Penetrations of the proteins in the lipid layer was inferred from capacitance variation of the monolayer measured by a palarography. Disulfide bridges reduced at the electrode were determined by cycle voltametry.In all the cases studied, although in different manners thrombin was found both to adsorb and penetrate the lipid layer, whereas prethrombin 2 did not penetrate pure phosphatidylcholine (PC). In the case of thrombin but not of prethrombin 2, penetration is accompanied by S-S reduction which is maximum at 10 per cent of phosphatidylserine (PS). This indicate a different orientation for prethrombin 2 and thrombin in the lipid layer. This observation might be of importance for the comprehension of the architecture of the prothrombin might be of for the regulation of thrombin formation within the complex.     

Protein and carbohydrate moieties of a preparation of β-lactamase II

1. A crystalline preparation of beta-lactamase II has been separated into two moieties by gel filtration on a column of Sephadex G-100. 2. The first moiety consisted mainly of carbohydrate and showed virtually no beta-lactamase activity. 3. The second moiety was a protein of molecular weight 22500, which was enzymically active. 4. The protein moiety, like the original protein-carbohydrate complex, required Zn(2+) for beta-lactamase activity. It did not differ significantly from the complex in its behaviour to a number of cephalosporin substrates, but was less stable to heat than the complex. 5. About 30% of the total beta-lactamase activity was lost when the protein-carbohydrate complex was separated into the two moieties. This activity was regained when the protein and carbohydrate moieties were mixed, but the mixture did not show the heat stability of the original complex.