Biosynthetic studies of the glycopeptide teicoplanin by (1)H and (13)C NMR

The biosynthesis of the glycopeptide antibiotic teicoplanin was studied by growing a teicoplanin producing strain of Actinoplanes teichomyceticus (ATCC 31121) on glucose containing either 34.0% [1-(13)C]glucose or 9.7% [U-(13)C]glucose. The fractional enrichment pattern of teicoplanin produced in the medium containing [1-(13)C]glucose was obtained from a one-dimensional (13)C spectrum. The enrichment pattern showed characteristic peaks indicating that amino acids 3 and 7 are derived from acetate, whereas amino acids 1, 2, 4, 5, and 6 are derived from tyrosine. Multiplet structures in heteronuclear single quantum coherence spectra of teicoplanin produced in the medium containing [U-(13)C]glucose showed characteristic coupling patterns supporting these results. Fractional enrichment patterns and multiplet structures of the three sugars in teicoplanin showed that about 50% of the sugars have the same labeling pattern as the glucose substrate whereas the rest have a labeling pattern showing that they are reassembled, probably from precursors in the primary metabolism.     

Synthesis of the alpha-L-Araf-(1-->2)-beta-D-Galp-(1-->6)-beta-D-Galp-(1-->6)-[alpha-L-Araf-(1-->2)]-beta-D-Galp-(1-->6)-D-Gal hexasaccharide as a possible repeating unit of the cell-cultured exudates of Echinacea purpurea arabinogalactan.

For the characterization of the supposed epitope of an arabinogalactan, isolated from the extract of the cell-cultured Echinacea purpurea, the title hexasaccharide was synthesized. The whole synthetic route was based on the 6-O-(methoxydimethyl)methyl ether (MIP) protecting group strategy. 2-O-Benzyl-3,4-O-isopropylidene-6-O-(methoxydimethyl)methyl-beta-D-galactopyranosyl-(1-->6)-1,2:3,4-di-O-isopropylidene-alpha-D-galactopyranose was used to prepare the desired glycosyl donor and glycosyl acceptor both carrying a persistent O-benzyl group at position 2'. Reaction of the digalactose donor and the digalactose acceptor resulted in a beta-(1-->6)-linked galactose-containing tetrasaccharide in which OH-2' and OH-2"' were substituted with benzyl groups. Hydrogenolytic removal of the benzyl groups of the tetragalactose compound gave the diol aglycon which was diarabinosylated in one step to furnish the protected target compound, whose deprotection led to the title hexasaccharide. All of the synthesized compounds were characterized by 1H and 13C NMR spectra, as well as by MALDI-TOF mass-spectrometry measurements.     

Immunologically active (1-->3)-(1-->4)-alpha-D-glucan from Cetraria islandica.

A polysaccharide, Ci-3, resembling isolichenan except with a much higher degree of polymerization, has been isolated from the water extract, as well as from the alkali extract, of the lichen Cetraria islandica (L.) using ethanol fractionation, dialysis, ion-exchange chromatography and gel filtration. The mean M(r) of Ci-3 was determined to be 2000 kD, compared to 6-8 kD reported for isolichenan. The structure of Ci-3 was elucidated and found to be composed of (1-->3)- and (1-->4)-alpha-D-glucopyranosyl units in the ratio of 2:1, using methanolysis, methylation analysis, optical rotation and NMR spectroscopy. The immunomodulating activity of Ci-3 was tested in an in vitro phagocytosis assay and anti-complementary, and proved to be active in both tests.     

Comparative 1H NMR study of hydroxy protons in galabioside and its S-linked 4-thiodisaccharide analogue in aqueous solution

The NMR data obtained from hydroxy protons have been used to investigate the presence and absence of intramolecular hydrogen bonding in aqueous solutions of 2-(trimethylsilyl)ethyl galabioside (alpha-D-Galp-(1-->4)-beta-D-Galp-O(CH2)2SiMe3) and the S-linked 4-thiodisaccharide analogue. The data show that there is a weak hydrogen bond interaction between O-6H and O-2'H in galabioside, but not in the thio-analogue. The results are in good agreement with those reported for the substances in a Me2SO-d6 solution. It is also shown that the existence of a hydrogen bond can be quite easily monitored by comparing the NMR data of the hydroxy protons.     

Insight into multi-site mechanisms of glycosyl transfer in (1-->4)beta-D-glycans provided by the cereal mixed-linkage (1-->3),(1-->4)beta-D-glucan synthase.

Synthases of cellulose, chitin, hyaluronan, and all other polymers containing (1-->4)beta-linked glucosyl, mannosyl and xylosyl units have overcome a substrate orientation problem in catalysis because the (1-->4)beta-linkage requires that each of these sugar units be inverted nearly 180 degrees with respect to its neighbors. We and others have proposed that this problem is solved by two modes of glycosyl transfer within a single catalytic subunit to generate disaccharide units, which, when linked processively, maintain the proper orientation without rotation or re-orientation of the synthetic machinery in 3-dimensional space. A variant of the strict (1-->4)beta-D-linkage structure is the mixed-linkage (1-->3),(1-->4)beta-D-glucan, a growth-specific cell wall polysaccharide found in grasses and cereals. beta-Glucan is composed primarily of cellotriosyl and cellotetraosyl units linked by single (1-->3)beta-D-linkages. In reactions in vitro at high substrate concentration, a polymer composed of almost entirely cellotriosyl and cellopentosyl units is made. These results support a model in which three modes of glycosyl transfer occur within the synthase complex instead of just two. The generation of odd numbered units demands that they are connected by (1-->3)beta-linkages and not (1-->4)beta-. In this short review of beta-glucan synthesis in maize, we show how such a model not only provides simple mechanisms of synthesis for all (1-->4)beta-D-glycans but also explains how the synthesis of callose, or strictly (1-->3)beta-D-glucans, occurs upon loss of the multiple modes of glycosyl transfer to a single one.     

Comparative (1)H NMR and molecular modeling study of hydroxy protons of beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->O)(CH(2))(7)CH(3) analogues in aqueous solution

The (1)H chemical shifts, coupling constants, temperature coefficients, exchange rates, and inter-residual ROEs have been measured, in aqueous solution, for the hydroxy and amine/amide proton resonances of a set of beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->O)(CH(2))(7)CH(3) analogues. From the structural data, a few significant structural features could be ascertained, such as a preferential anti-conformation for the amide protons of the N-acetyl and N-propionyl groups. The introduction of systematic modifications at Gal 2-C and Gal 6-C resulted in alterations of the Gal 4-OH, Gal 3-OH, and GlcNAc 3-OH areas, since variations in chemical shifts and temperature coefficient were observed. In order to verify the possibility of hydrogen bonds, molecular dynamics simulations in the gas phase and explicit solvent were performed and correlated with the experimental data. A network of hydrogen bonds to solvent molecules was observed, but no strong intramolecular hydrogen bonding was observed.     

Total chemical synthesis and NMR characterization of the glycopeptide tx5a, a heavily post-translationally modified conotoxin, reveals that the glycan structure is alpha-D-Gal-(1-->3)-alpha-D-GalNAc.

The 13-amino acid glycopeptide tx5a (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* = 6-bromotryptophan and Thr* = Gal-GalNAc-threonine), isolated from Conus textile, causes hyperactivity and spasticity when injected intracerebral ventricularly into mice. It contains nine post-translationally modified residues: four cysteine residues, two gamma-carboxyglutamic acid residues, and one residue each of 6-bromotryptophan, 4-trans-hydroxyproline and glycosylated threonine. The chemical nature of each of these has been determined with the exception of the glycan linkage pattern on threonine and the stereochemistry of the 6-bromotryptophan residue. Previous investigations have demonstrated that tx5a contains a disaccharide composed of N-acetylgalactosamine (GalNAc) and galactose (Gal), but the interresidue linkage was not characterized. We hypothesized that tx5a contained the T-antigen, beta-D-Gal-(1-->3)-alpha-D-GalNAc, one of the most common O-linked glycan structures, identified previously in another Conus glycopeptide, contalukin-G. We therefore utilized the peracetylated form of this glycan attached to Fmoc-threonine in an attempted synthesis. While the result-ing synthetic peptide (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* =6-bromotryptophan and Thr* = beta-D-Gal-(1-->3)-alpha-D-GalNAc-threonine) and the native peptide had almost identical mass spectra, a comparison of their RP-HPLC chromatograms suggested that the two forms were not identical. Two-dimensional 1H homonuclear and 13C-1H heteronuclear NMR spectroscopy of native tx5a isolated from Conus textile was then used to determine that the glycan present on tx5a indeed is not the aforementioned T-antigen, but rather alpha-D-Gal-(1-->3)-alpha-D-GalNAc.     

Enzyme-linked immunosorbent assay specific for (1-->6) branched, (1-->3)-beta-D-glucan detection in environmental samples.

(1-->3)-beta-D-Glucans have been recognized as a potential causative agent responsible for bioaerosol-induced respiratory symptoms observed in both indoor and occupational environments. A specific enzyme immunoassay was developed to quantify (1-->6) branched, (1-->3)-beta-D-glucans in environmental samples. The assay was based on the use of a high-affinity receptor (galactosyl ceramide) specific for (1-->3)-beta-D-glucans as a capture reagent and a monoclonal antibody specific for fungal cell wall beta-D-glucans as a detector reagent. The assay was highly specific for (1-->6) branched, (1-->3)-beta-D-glucans (such as that from Saccharomyces cerevisiae) and did not show any response at 200 ng/ml to curdlan, laminarin, pustulan, dextran, mannan, carboxymethyl cellulose, and endotoxins. The detection level was 0.8 ng/ml for baker's yeast glucan and Betafectin. A coefficient of variation of 7.8% was obtained for (1-->3)-beta-D-glucans in house dust samples. Metal working fluids spiked with (1-->3)-beta-D-glucans inhibited the glucan assay. Because the assay is specific for (1-->6) branched, (1-->3)-beta-D-glucans and is sensitive and reproducible, it will be useful for the investigation of health effects from exposure to this class of biologically active molecules.     

An efficient and stereoselective synthesis of beta-D-Arap-(1-->2)-beta-D-Galp-(1-->3)-beta-D-Galp-(1-->4)-alpha-D-Manp, a tetrasaccharide fragment of Leishmania major lipophosphoglycan.

A tetrasaccharide fragment of Leishmania major lipophosphoglycan (which seems to be involved in a biological mechanism for the parasite transmission) has been synthesised using the thioglycoside, trichloroacetimidate and halide-exchange glycosylation procedures and step-wise chain elongation strategy.     

1H and 51V NMR studies of the interaction of vanadate and 2-vanadio-3-phosphoglycerate with phosphoglycerate mutase.

The formation of complexes of vanadate with 2-phosphoglycerate and 3-phosphoglycerate have been studied using 51V nuclear magnetic resonance spectroscopy. Signals attributed to two 2,3-diphosphoglycerate analogues, 2-vanadio-3-phosphoglycerate and 2-phospho-3-vanadioglycerate, were detected but were not fully resolved from signals of inorganic vanadate and the anhydride formed between vanadate and the phosphate ester moieties of the individual phosphoglycerates. Equilibrium constants for formation of the two 2,3-bisphosphate analogues were estimated as 2.5 M-1 for 2-vanadio-3-phosphoglycerate and 0.2 M-1 for 2-phospho-3-vanadioglycerate. The results of the binding study are fully consistent with non-cooperativity in the binding of vanadiophosphoglycerate to the two active sites of phosphoglycerate mutase (PGM). 2-Vanadio-3-phosphoglycerate was found to bind to the dephospho form of phosphoglycerate mutase with a dissociation constant of about 1 x 10(-11) M at pH 7 and 7 x 10(-11) M at pH 8. Three signals attributed to histidine residues were observed in the 1H NMR spectrum of phosphoglycerate mutase. Two of these signals and also an additional signal, tentatively attributed to a tryptophan, underwent a chemical shift change when the vanadiophosphoglycerate complex was bound to the enzyme. The results obtained here are in accord with these vanadate-phosphoglycerate complexes being much more potent inhibitors of phosphoglycerate mutase than either monomeric or dimeric vanadate. The dissociation constant of 10(-11) M for 2-vanadio-3-phosphoglycerate is about 4 orders of magnitude smaller than the Km for PGM, a result in accordance with the vanadiophosphoglycerates being transition state analogues for the phosphorylation of PGM by 2,3-diphosphoglycerate.(ABSTRACT TRUNCATED AT 250 WORDS)     

1H NMR study of the exchangeable protons of the duplex d(GCGTTGCG).d(CGCAACGC) containing a thymine photodimer.

A comparison is presented of the imino proton NMR spectra of the double stranded octamer d(GCGTTGCG).d(CGCAACGC) and the same octamer in which the two central thymine residues occur as a cis-syn thymine dimer. Except for the terminal base pairs all imino protons were detected and assigned in the NMR spectrum. The spectra show that in the thymine dimer duplex, contrary to common belief, all base pairs occur in a hydrogen bonded form, although the hydrogen bonds of the two central AT base pairs are substantially weakened. The melting temperature decreases about 13 degrees C on thymine dimer formation.     

1H NMR studies of lambda cro repressor. 2. Sequential resonance assignments of the 1H NMR spectrum

The cro repressor protein from bacteriophage lambda has been studied in solution by two-dimensional nuclear magnetic resonance spectroscopy (2D NMR). Following the approach of Wüthrich and co-workers [Wüthrich, K., Wider, G., Wagner, G., & Braun, W. (1982) J. Mol. Biol. 155, 311-319], individual spin systems were identified by J-correlated spectroscopy (COSY) supplemented, where necessary, by relayed coherence transfer spectroscopy (RELAY). Nuclear Overhauser effect spectroscopy (NOESY) was used to obtain sequence-specific assignments. From the two-dimensional spectra, the peptide backbone resonances (NH and C alpha H) for 65 of the 66 amino acids were assigned, as well as most of the side chain resonances. The chemical shifts for the assigned protons are reported at 35 degrees C in 10 mM potassium phosphate, pH 6.8, and in 10 mM potassium phosphate, pH 4.6, 0.2 M KCl, and 0.1 mM EDTA. Small shifts were observed for some resonances upon addition of salt, but no major changes in the spectrum were seen, indicating that no global structural change occurs between these ionic strengths. NOE patterns characteristic of alpha-helices, beta-strands, and turns are seen in various regions of the primary sequence. From the location of these regions the secondary structure of cro in solution appears to be virtually identical with the crystal structure [Anderson, W. F., Ohlendorf, D. H., Takeda, Y., & Matthews, B. W. (1981) Nature (London) 290, 754-758]. Missing assignments include the Pro-59 resonances and the peripheral protons of the eight lysine, the three arginine, and three of the five isoleucine residues.     

Conformational studies of the glycopeptide Ac-Tyr-[Man5GlcNAc-beta-(1-->4)GlcNAc-beta-(1-->Ndelta)]-Asn-Leu-Thr-Se r-OBz and the constituent peptide and oligosaccharide

Glycopeptides of desired structure can be conveniently prepared by the coupling of reducing oligosaccharides to aspartic acid of peptides via their glycosylamines formed in the presence of saturated aqueous ammonium hydrogen carbonate. The resulting oligosaccharide chains are N-linked to asparagine as in natural glycoproteins, allowing different peptide oligosaccharide combinations to be analysed for conformational effects. In the present paper, a pentapeptide of ovalbumin was coupled to Man5GlcNAc2 oligosaccharide and the glycopeptide and the two parent compounds compared by NMR ROESY experiments and molecular dynamics simulations. Despite the small size of the peptide, conformational effects were observed suggestive of the oligosaccharide stabilising the peptide in solution and of the peptide influencing oligosaccharide conformation. These effects are relevant to the function of glycosylation and the enzymic processing of oligosaccharide chains.     

1H NMR studies of lac-operator DNA fragments.

The hydrogen-bonded imino protons of a 14 base pair double-stranded DNA fragment comprising one half of the lac operator of E. coli were investigated by 360 MHz H NMR. From combined melting studies of this synthetic 14 b.p. fragment and its two constituent 7 b.p. fragments a nearly complete assignment for the low-field proton resonances was obtained. The experimental spectra are compared with calculated spectra and with the spectrum of a 51 b.p. DNA restriction fragment from E. coli containing the complete lac operator. Structural information on these oligonucleotides is presented. This study is a prerequisite for future 1H NMR investigations of the interaction of the lac operator with the lac repressor.     

Solid-state NMR studies of the prion protein H1 fragment.

Conformational changes in the prion protein (PrP) seem to be responsible for prion diseases. We have used conformation-dependent chemical-shift measurements and rotational-resonance distance measurements to analyze the conformation of solid-state peptides lacking long-range order, corresponding to a region of PrP designated H1. This region is predicted to undergo a transformation of secondary structure in generating the infectious form of the protein. Solid-state NMR spectra of specifically 13C-enriched samples of H1, residues 109-122 (MKHMAGAAAAGAVV) of Syrian hamster PrP, have been acquired under cross-polarization and magic-angle spinning conditions. Samples lyophilized from 50% acetonitrile/50% water show chemical shifts characteristic of a beta-sheet conformation in the region corresponding to residues 112-121, whereas samples lyophilized from hexafluoroisopropanol display shifts indicative of alpha-helical secondary structure in the region corresponding to residues 113-117. Complete conversion to the helical conformation was not observed and conversion from alpha-helix back to beta-sheet, as inferred from the solid-state NMR spectra, occurred when samples were exposed to water. Rotational-resonance experiments were performed on seven doubly 13C-labeled H1 samples dried from water. Measured distances suggest that the peptide is in an extended, possibly beta-strand, conformation. These results are consistent with the experimental observation that PrP can exist in different conformational states and with structural predictions based on biological data and theoretical modeling that suggest that H1 may play a key role in the conformational transition involved in the development of prion diseases.     

Synthesis of [26-2H(3)]brassinosteroids

A number of [26-2H(3)]brassinosteroids were prepared for biochemical studies. The parent, nondeuterated compounds were considered to be biosynthetic intermediates in brassinosteroid biosynthesis. Claisen rearrangement was used to construct the steroidal side chain. Deuterium was introduced by reducing the corresponding intermediates with lithium aluminium deuteride.     

Synthesis and 1H NMR spectroscopic characterization of trans-[Pt(NH3)2[d(ApGpGpCpCpT)-N7-A(1),N7-G(3)]]

The reaction of trans-[Pt(NH3)2Cl2] with the sodium salt of [d(ApGpGpCpCpT)]2 in aqueous solution at 37 degrees C was monitored by reversed-phase high-performance liquid chromatography and UV spectroscopy. Two intermediates, most likely monofunctional adducts, were observed, which subsequently formed one predominant single-stranded product, as well as several polymeric species proposed to be interstrand cross-linked products. The single-stranded adduct was structurally characterized by 1H NMR spectroscopy. From the pH dependence of the chemical shifts, two-dimensional homonuclear chemical shift correlation (COSY) spectroscopy, and one- and two-dimensional nuclear Overhauser effect (NOESY) experiments, the platinum(II) moiety was found to be coordinated to the N7 positions of adenine(1) and guanine(3), with the intervening guanine(2) base destacked from its neighboring residues. This intrastrand 1,3 adduct induces changes in the backbone torsion angles and causes the deoxyribose ring of adenine(1) to switch from a C2'-endo to a predominantly C3'-endo conformation. The other deoxyribose rings retain B DNA type conformations. The structure of trans-[Pt(NH3)2[d(ApGpGpCpCpT)-N7-A(1),N7-G(3)]] differs from those previously reported for cis-DDP 1,2- and 1,3-intrastrand oligonucleotide adducts but is consistent with the structures of trans-DDP 1,3-intrastrand adducts of two previously reported trinucleotides.     

Model studies for molybdenum enzymes. Reduction of cytochrome c complexes by mu-oxo-bis[oxodihydroxo(L-cysteinato)molybdate(V)]

The reduction by mu-oxo-bis[oxodihydroxo(L-cysteinato)molybdate(V)] (I) of cytochrome c complexes in which the methionine-80 ligand has been replaced by N3-, CN-, or imidazole has been investigated. The N3- and CN-substituted species are reduced by I in a first-order reaction that appears to proceed via a slow dissociation of N3- or CN-, followed by rapid reduction of native cytochrome c. At low concentrations of I, reduction of the imidazole-cytochrome complex occurs by the same mechanism, while, at higher concentrations of I, direct reduction by I and by a monomeric Mo(V) complex in equilibrium with I appears to occur, although at much lower rates than with native cytochrome c. Potentiometric measurements of E degrees for the cytochrome c complexes with N3- and imidazole indicate the lack of reducibility, or reduction in rate relative to native cytochrome c, is not due to thermodynamic reasons. In the case of the CN- complex, E degrees may be too low for direct reduction by I. The effects on the reduction rates are attributed to a conformational change accompanying the replacement of the methionine-80 ligand, which makes the exposed heme edge less available to attack by outer sphere reductants.     

1H NMR studies of the reactions of copper(I) and copper(II) with D-penicillamine and glutathione.

Reactions of copper ions with D-penicillamine (PSH) have been monitored by 1H NMR spectroscopy in the presence and absence of glutathione (GSH) under aerobic as well as anaerobic conditions. In D2O solution at pD = 7.4, PSH coordinates to Cu+ to form PS(-)-Cu+ under argon atmosphere as revealed from the broadening of each signal. In the presence of dioxygen, the complex was converted to the well-characterized purple cluster species consisting of Cu+, Cu2+, and PS2-. Addition of GSH into this solution quickly decomposed the cluster by the reduction of Cu2+ to Cu+. The cluster species was, however, reproduced after several hours because of the oxidation of Cu+ back to Cu2+. The solution containing both PSH and GSH formed three possible disulfides, PSSP, GSSG, and PSSG, under aerobic conditions. Addition of Cu+ to this solution again produced the purple cluster through several redox reactions. On the basis of these results, it was concluded that the co-existence of PSH and/or PSSP with Cu+ and/or Cu2+ leads to the formation of the stable cluster species regardless of the presence or absence of the other thiols such as GSH. This must be one of the reasons why PSH works in living cells as an effective drug for the Wilson disease.