Biosynthesis of thiophenes in Tagetes patula.

The biosynthesis of 5-(3-buten-1-ynyl)-2,2'-bithiophene was studied in root cultures of Tagetes patula. Organ cultures were grown with [U-13C(6)]glucose or [1-13C]glucose. The bithiopene and amino acids from cell protein were isolated and analysed by quantitative NMR spectroscopy. Retrobiosynthetic analysis establish acetyl-CoA or a closely related compound (e.g. malonyl-CoA) as building blocks and their orientations in the bithiophene. The data confirm a previously suggested biosynthetic route via long-chain fatty acids and polyacetylenes. However, a polyketide-like biosynthesis via a carbocyclic intermediate cannot be excluded.     

Acetylenes from the callus of Panax ginseng

Two new polyacetylenes were isolated from dried callus of Panax ginseng. The structures of the polyacetylenes were confirmed as heptadeca-3-oxo-4,6-diyne-9,10-diol and its dihydro derivative by their IR, ¹H NMR, ¹³C NMR and mass spectra, and some chemical reactions. The new acetylenes exhibited growth inhibition against Yoshida sarcoma cells in tissue culture.     

Chemosystematic investigations of irregular diterpenes in Anisotome and related New Zealand Apiaceae

A chemosystematic HPLC-UV and HPLC-MS investigation of New Zealand members of the Apiaceae was performed. Diterpenes were identified and quantified in methanolic extracts from subaerial parts of 28 taxa and 54 samples of Aciphylla, Anisotome, Apium, Gingidia, Lignocarpa, Oreomyrrhis, and Scandia. Six diterpenes (1-2, 4-7) and four polyacetylenes (8-11) were identified. The known compounds were the diterpenes anisotomenoic acid 1, anisotomene-1-ol 2, 16-acetoxyanisotomenoic acid 4 and anisotomene-1,12-diol 5; and the polyacetylenes falcarinol 8, falcarindiol 9, (+)-9(Z),17-octadecadiene-12,14-diyne-1,11,16-triol 10, and (+)-9(Z),17-octadecadiene-12,14-diyne-1,11,16-triol 1-acetate 11. New irregular diterpenes 13,14-dihydroanisotom-12E-ene-1,14-diol 6 and 14-methoxy-13,14-dihydroanisotom-12E-ene-1-ol 7 were isolated from A. haastii. Isomers of the new semi-synthetic diterpene 16-hydroxyanisotomenoic acid 3 were detected in extracts of Anisitone flexuosa. Structure elucidation was performed by HR mass spectrometry and 1D and 2D NMR spectroscopy. In crude extracts, compounds were identified by their HPLC retention times and their on-line HPLC-UV and MS spectra. Anisotomene diterpenes occurred in eight out of 16 species of the genus Anisotome, but were not detected in any of the other genera. In contrast, polyacetylenes were present in all the genera investigated.     

Lignans,phenylpropanoids and polyacetylenes from Chaerophyllum aureum L. (Apiaceae)

Sub-aerial parts of Chaerophyllum aureum L. yielded two polyacetylenes, falcarinol (1), falcarindiol (2), three lignans, namely nemerosin (3), deoxypodorhizone (4), deoxypodo-phyllotoxin (5), two phenylpropanoids, 1'-hydroxymyristicin (6) and its angeloyl ester (7). Compounds 6 and 7 were isolated for the first time from plant material and their structures were elucidated by means of extensive 1- and 2-dimensional NMR spectroscopy and high resolution mass spectrometry. In bioautographic tests on TLC plates the dichloromethane extract showed a significant antimicrobial activity. Falcarindiol was identified as the main active principle whereas the phenylpropanoids and lignans showed no activity.     

The influence of the fungal pathogen Mycocentrospora acerina on the proteome and polyacetylenes and 6-methoxymellein in organic and conventionally cultivated carrots (Daucus carota) during post harvest storage

Many carrots are discarded during post harvest cold storage due to development of fungal infections, caused by, e.g., Mycocentrospora acerina (liquorice rot). We compared the susceptibility of carrots grown under conventional and organic agricultural practices. In one year, organically cultivated carrots showed 3 × to 7 × more symptoms than conventionally cultivated, when studying naturally occurring disease at 4 and 6 months, respectively. On the other hand, we have developed a bioassay for infection studies of M. acerina on carrots and observed that organic roots were more susceptible after one month of storage than conventional ones, but no differences were apparent after four or six months storage. Levels of polyacetylenes (falcarinol, falcarindiol and falcarindiol-3-acetate) did not change, whereas the isocoumarin phytoalexin (6-methoxymellein) accumulated in infected tissue as well as in healthy tissue opposite the infection. The proteomes of carrot and M. acerina were characterized, the intensity of 33 plant protein spots was significantly changed in infected roots including up regulation of defence and stress response proteins but also a decrease of proteins involved in energy metabolism. This combined metabolic and proteomic study indicates that roots respond to fungal infection through altered metabolism: simultaneous induction of 6-methoxymellein and synthesis of defence related proteins.     

Synthesis and characterization of polyacids from palm acid oil and sunflower oil via addition reaction

In this study aliphatic polyacids were synthesized using palm acid oil (PAO) and sunflower oil (SFO) via addition reaction technique. The synthesized materials were characterized using Fourier-transform infra-red (FTIR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF-MS) and thermo-gravimetric analysis (TGA). Mixing formic acid and hydrogen peroxide with PAO or SFO at the ratio 3:10:1 produced the lowest iodine value of 10.57 and 9.24 respectively, indicating the increase in epoxidization of both oils. Adding adipic acid to the epoxidized oils at a ratio of 1:10 increases the acid values of SFO and PAO to 11.22 and 6.73 respectively. The existence of multi-acid groups present in synthesized polyacid was confirmed by MALD-ToF-MS. This feature indicates a possible value to the biomaterials development.     

Structural studies utilizing 13C-enrichment of the O-antigen polysaccharide from the enterotoxigenic Escherichia coli O159 cross-reacting with Shigella dysenteriae type 4.

The structure of the O-antigen polysaccharide from Escherichia coli O159 has been determined using primarily NMR spectroscopy of the 13C-enriched polysaccharide. The sequence of the sugar residues could be determined by heteronuclear multiple bond connectivity NMR experiments. The polysaccharide is composed of a pentasaccharide repeating unit with the following structure: [sequence: see text] Matrix assisted laser desorption ionization mass spectrometry was performed on intact lipopolysaccharide and from the resulting molecular mass the O-antigen part was estimated to contain approximately 23 repeating units. Cross-reactivity of this O-antigen to that of Shigella dysenteriae type 4 was confirmed using enzyme-linked immunoabsorbant assay.     

Thallium-205 NMR determination of the thermodynamics of the interaction between the thallous ion and gramicidin dimers incorporated into micelles

A study has been made of the thermodynamics of the interaction between the thallous ion and gramicidin dimers incorporated into micelles using thallium-205 NMR spectroscopy. The chemical shift data obtained are interpreted interms of a model in which the dimer has only one tight binding site. The variation of the binding constant over the temperature range 303-323 K is used to determine the changes in enthalpy and entropy of binding giving values of -11.3 kcal/mole and -16 e.u. at 303 K, respectively.     

Hydrolysis of wheat bran and straw by an endoxylanase: production and structural characterization of cinnamoyl-oligosaccharides.

Hydrolysis of wheat bran and wheat straw by a 20.7 kDa thermostable endoxylanase released 35 and 18% of the cell-wall xylan content, respectively. Separation of the cinnamoyl-oligosaccharides (accounting for 6%) from the bulk of total oligosaccharides was achieved by specific anion-exchange chromatography. The cinnamoyl-oligosaccharides were further purified by preparative paper chromatography (PPC) and their molecular weight was determined by MALDI-TOF mass spectrometry. The partially purified hydrolysis end-products contained from 4 to 16 and from 4 to 12 pentose residues for wheat bran and straw, respectively, and only one cinnamic acid per molecule. The primary structure of the new feruloyl arabinoxylopentasaccharide from wheat bran hydrolysis, which has been determined using 2D NMR spectroscopy, is O-beta-D-xylopyranosyl-(1-->4)-O-[5-O- (feruloyl)-alpha-L-arabinofuranosyl-(1-->3)]-O-beta-D-xylopyranosy l-(1-->4) -O-beta-D-xylopyranosyl-(1-->4)-D-xylopyranose.     

Generalized ordering parameter S2 for N-H peptide bonding as measure of the conformational flexibility of proteins: comparison of algorithms of S2 calculation from molecular dynamics simulation data

NMR spectroscopy in combination with molecular dynamics (MD) simulation technique is a powerful tool for investigation of biomolecule conformations. In the present study we compare the two algorithms of calculation of the generalized order parameter S2 for the peptide N-H bond from MD simulation data. The program based on this algorithm has been written and the effect of an adjustible parameter on results of calculation was studied. HIV-1 protease MD simulation was performed during 7.36 ns using GROMACS 3.1.4. software package. The calculated order parameter values are in good agreement with those obtained by other authors using NMR.     

High-resolution NMR structure of an RNA model system: the 14-mer cUUCGg tetraloop hairpin RNA

We present a high-resolution nuclear magnetic resonance (NMR) solution structure of a 14-mer RNA hairpin capped by cUUCGg tetraloop. This short and very stable RNA presents an important model system for the study of RNA structure and dynamics using NMR spectroscopy, molecular dynamics (MD) simulations and RNA force-field development. The extraordinary high precision of the structure (root mean square deviation of 0.3 Å) could be achieved by measuring and incorporating all currently accessible NMR parameters, including distances derived from nuclear Overhauser effect (NOE) intensities, torsion-angle dependent homonuclear and heteronuclear scalar coupling constants, projection-angle-dependent cross-correlated relaxation rates and residual dipolar couplings. The structure calculations were performed with the program CNS using the ARIA setup and protocols. The structure quality was further improved by a final refinement in explicit water using OPLS force field parameters for non-bonded interactions and charges. In addition, the 2′-hydroxyl groups have been assigned and their conformation has been analyzed based on NOE contacts. The structure currently defines a benchmark for the precision and accuracy amenable to RNA structure determination by NMR spectroscopy. Here, we discuss the impact of various NMR restraints on structure quality and discuss in detail the dynamics of this system as previously determined.     

New finding of nalbuphine metabolites in men: NMR spectroscopy and UPLC–MS/MS spectrometry assays in a pilot human study

A safe and efficient semi-synthetic narcotic nalbuphine (NAL) which was broadly applied in analgesic therapy has long been considered to eliminate from human body via phase II conjugation. However, up to the present, neither the complete metabolic pathways nor the identified metabolites of NAL have been clarified in documented reports. In this study, four novel metabolites were discovered by incubating NAL with human liver microsomes. These metabolites were later quantified in blood samples from human volunteers treated with NAL. An accurate and precise new method for simultaneously determining NAL and its metabolites was also established. Their chemical structures were elucidated on the basis of one- and two-dimensional NMR spectroscopic analyses including ¹H–¹H correlation spectroscopy, nuclear overhauser enhancement spectroscopy, heteronuclear single-quantum correlation, and heteronuclear multiple bond correlation, and further confirmed by mass spectrometry. The analytical method was validated and applied successfully to a pilot human study with ultra-high performance liquid chromatography–tandem mass spectrometry employed with positive ion electrospray ionization via multiple reaction monitoring mode. This is the first report on the qualitative and quantitative analysis of NAL coupled with its two hydroxylated (3′-hydroxynalbuphine and 4′-hydroxynalbuphine) and two conjugated metabolites (nalbuphine-3-β-d-glucuronide and nalbuphine-6-β-d-glucuronide). The present method offers a rapid and simple way of performing pharmacokinetic studies of NAL, and assists in elucidating its metabolic pathway in humans.     

NMR methods for in situ biofilm metabolism studies

Novel procedures and instrumentation are described for nuclear magnetic resonance (NMR) spectroscopy and imaging studies of live, in situ microbial films. A perfused NMR/optical microscope sample chamber containing a planar biofilm support was integrated into a recirculation/dilution flow loop growth reactor system and used to grow in situ Shewanella oneidensis strain MR-1 biofilms. Localized NMR techniques were developed and used to non-invasively monitor time-resolved metabolite concentrations and to image the biomass volume and distribution. As a first illustration of the feasibility of the methodology an initial 13C-labeled lactate metabolic pathway study was performed, yielding results consistent with existing genomic data for MR-1. These results represent progress toward our ultimate goal of correlating time- and depth-resolved metabolism and mass transport with gene expression in live in situ biofilms using combined NMR/optical microscopy techniques.     

Improved segmental isotope labeling methods for the NMR study of multidomain or large proteins: application to the RRMs of Npl3p and hnRNP L

The study of multidomain or large proteins in solution by NMR spectroscopy has been made possible in recent years by the development of new spectroscopic methods. However, resonance overlap found in large proteins remains a limiting factor, making resonance assignments and structure determination of large proteins very difficult. In this study, we present an expressed protein ligation protocol that can be used for the segmental isotopic labeling of virtually any multidomain or high molecular mass protein, independent of both the folding state and the solubility of the protein fragments, as well as independent of whether the fragments are interacting. The protocol was applied successfully to two different multidomain proteins containing RNA recognition motifs (RRMs), heterogeneous nuclear ribonucleoprotein L and Npl3p. High yields of segmentally labeled proteins could be obtained, allowing characterization of the interdomain interactions with NMR spectroscopy. We found that the RRMs of heterogeneous nuclear ribonucleoprotein L interact, whereas those of Npl3p are independent. Subsequently, the structures of the two RRMs of Npl3p were determined on the basis of samples in which each RRM was expressed individually. The two Npl3p RRMs adopt the expected βαββαβ fold.     

An unambiguous structural elucidation of a 1,3-β-d-glucan obtained from liquid-cultured Grifola frondosa by solution NMR experiments

Grifolan LE (GRN-LE), a purified β-d-glucan, which is obtained from liquid-cultured Grifola frondosa, exhibits various biological activities, including antitumor effects. Significant progress has been made in the study of these effects. However, an unambiguous structural characterization of GRN-LE using NMR spectroscopy has not been carried out as yet. It is well accepted that the biological effects of a β-glucan depend on its primary structure, conformation, and molecular weight. In the present study, we unambiguously elucidate the primary structure of GRN-LE using NMR spectroscopy. The data presented here reveal that GRN-LE comprises a 1,3-β-d-glucan backbone with a single 1,6-β-d-glucosyl side branching unit on every third residue.     

Characterization of O-acetyl-(4-O-methylglucurono)xylan isolated from birch and beech

The structures of water-soluble birch and beech xylans, extracted from holocellulose using dimethyl sulfoxide, were determined employing 1H and 13C NMR spectroscopy together with chemical analysis. These polysaccharides were found to be O-acetyl-(4-O-methylglucurono)xylans containing one 4-O-methylglucuronic acid substituent for approximately every 15 D-xylose residues. The average degree of acetylation of the xylose residues in these polymers was 0.4. The presence of the structural element -->4)[4-O-Me-alpha-D-GlcpA-(1-->2)][3-O-Ac]-beta-D-Xylp-(1--> was demonstrated. Additional acetyl groups were present as substituents at C-2 and/or C-3 of the xylopyranosyl residues. Utilizing size-exclusion chromatography in combination with mass spectroscopy, the weight-average molar masses (and polydispersities) were shown to be 8000 (1.09) and 11,100 (1.08) for birch and beech xylan, respectively.     

Reaction of iron(III) with theaflavin: complexation and oxidative products

Theaflavins are a family of compounds, whose chemistry has been sparsely investigated. They can comprise up to 40% the dry weight of black tea. They are known to chelate metals, however very little knowledge exists on the mechanisms involved. There is some correlation between both of these areas in that following degradation of the iron theaflavin complex, subsequent redox reactions may lead to the formation of similar products on both occasions. The interaction of iron(III) with theaflavin at pH < 3.0 is investigated by means of liquid chromatography mass spectroscopy (LC-MS), stopped flow spectroscopy and multivariate data analysis. Iron theaflavin complexes are formed which subsequently decay to form a number of oxidative species. The difficulties involved in the elucidation of the structure of polymeric phenolic compounds from black tea has been highlighted by numerous authors. The intermediates and major low molecular weight oxidised theaflavin products from the reaction of excess iron with theaflavin have been detected and identified using multivariate data analysis of diode array spectroscopic data. It is not possible to characterise the extremely polar high molecular weight oxidation products obtained from polyphenol oxidation. High performance liquid chromatography (HPLC) and electrospray mass spectroscopy (ES-MS) detected the low molecular weight oxidised theaflavin species present in the system. Enzymatic oxidation of theaflavin using peroxidase (POD) resulted in the formation of one major low molecular weight species oxidative product, which was fully characterised using nuclear magnetic resonance spectroscopy (NMR), high performance liquid chromatography (HPLC), electrospray mass spectroscopy (ES-MS), UV-visible (UV-Vis) and Fourier transform infra-red spectroscopy (FT-IR). The major objective of this work is to investigate the reaction of iron(III) with theaflavin and to add some insight into the mechanistic interaction of iron(III) with this family of compounds.     

Neuropeptide Y. Optimized solid-phase synthesis and conformational analysis in trifluoroethanol.

The 36-amino-acid neuropeptide Y (human), which is one of the most potent vasoconstrictors and which exhibits a number of other biological functions, has been synthesized using automated peptide synthesis. The optimized method, using 9-fluorenylmethoxycarbonyl protecting and single-step coupling, yielded the crude product in 90% purity allowing for single-step reversed-phase HPLC purification to greater than 98% purity and a high overall yield (50%). The hormone was characterized by several chromatographic methods, ion-spray mass spectroscopy and Edman degradation. The conformation of human neuropeptide Y was examined by CD, NMR and computer simulations. The CD measurements in trifluoroethanol/water (9:1) show a large percentage of alpha-helix. Variation of concentration, from 0.5 microM increasing up to the 1 mM used for NMR measurements, indicates no evidence for aggregation. In the same solvent system, the NMR line widths were very broad and therefore the resonance assignment was achieved with the exclusive use of two-dimensional NOE spectra. The 248 clearly distinguishable NOEs from the NMR study were used in distance geometry calculations and the resulting structures were refined with restrained molecular dynamics. The results indicate an alpha-helix extending from Arg19 to Gln34. For the N-terminal half of the molecule no regular structure was observed.     

Structure of a streptococcal adhesin carbohydrate receptor

Interactions between complementary protein and carbohydrate structures on different genera of human oral bacteria have been implicated in the formation of dental plaque. The carbohydrate receptor on Streptococcus sanguis H1 (one of the primary colonizing species) that is specific for the adhesin on Capnocytophaga ochracea ATCC 33596 (a secondary colonizer) has been isolated from the streptococcal cell wall, purified, and structurally characterized. The hexasaccharide repeating unit of the polysaccharide was purified by reverse-phase, amino-bonded silica, and gel permeation high performance liquid chromatography. Earlier studies established that the repeating unit was a hexasaccharide composed of rhamnose, galactose, and glucose in the ration of 2:3:1, respectively. In the present study, determination of absolute configuration by gas chromatography of the trimethylsilyl (+)-2-butyl glycosides revealed that the rhamnose residues were of the L configuration while the hexoses were all D. 252Californium plasma desorption mass spectrometry of the native, the acetylated and the reduced and acetylated hexasaccharide determined that the molecular mass of the native hexasaccharide was 959, and that the 2 rhamnose residues were linked to each other at the nonreducing terminus of the linear molecule. Methylation analysis revealed the positions of the glycosidic linkages in the hexasaccharide and showed that a galactose residue was present at the reducing end. The structural characterization of the hexasaccharide was completed by one and two dimensional 1H and 13C NMR spectroscopy. Complete 1H and 13C assignments for each glycosyl residue were established by two-dimensional (1H,1H) correlation spectroscopy, homonuclear Hartmann-Hahn, and (13C,1H) correlation experiments. The configurations of the glycosidic linkages were inferred from the chemical shifts and coupling constants of the anomeric 1H and 13C resonances. The sequence of the glycosyl residues was determined by a heteronuclear multiple bond correlation experiment. These data show that the structure of the hexasaccharide repeating unit derived from the cell wall polysaccharide of S. sanguis H1 is: alpha-L-Rhap-(1----2)-alpha-L-Rhap-(1----3)-alpha-D-Galp- (1----3)-beta-D-Galp-(1----4)-beta-D-Glcp-(1----3)-alpha/beta-D-Gal.     

Accumulation of N-acyl-ethanolamine phospholipids in rat brains during post-decapitative ischemia: a 31p NMR study

Phosphorus-31 nuclear magnetic resonance (31P NMR) spectroscopy has been used to study accumulation of N-acyl-ethanolamine phospholipids in rat brains during post-decapitative ischemia. Lipids were extracted from rat brain homogenates and the extracts were thoroughly washed with aq. potassium ethylenediaminetetraacetic acid (EDTA). The lower organic phases were isolated and evaporated to dryness under a stream of nitrogen and the lipids were redissolved in CDCl3-CH3OH-H2O 100.0:29.9:5.2 (v/v/v) for NMR analysis. Increasing the period of post-decapitative ischemia resulted in an accumulation of two signals in the NMR spectra at 0.18 and 0.22 ppm (relative to the chemical shift of 1,2-diacyl-sn-glycero-3-phosphocholine (PCDIACYL) at -0.84 ppm). These signals were identified as originating from 1,2-diacyl-sn-glycero-3-phospho-(N-acyl)-ethanolamine (NAPEDIACYL) and 1-(1'-alkenyl)-2-acyl-sn -glycero-3-phospho-(N-acyl)-ethanolamine (NAPEPLAS), respectively, by spiking with authentic materials. Additionally, the identification was verified by thin-layer chromatography, which also showed the accumulation of N-acyl-ethanolamine phospholipids. The use of K-EDTA instead of the commonly used Cs-EDTA in the preparation of the NMR samples allowed the separation of the chemical shifts of N-acyl-ethanolamine phospholipids from those of the ethanolamine phospholipids. Moreover, the chemical shift of cardiolipin was moved from 0.15 ppm observed with Cs-EDTA to about 0.31 ppm with K-EDTA.The present study demonstrates that it is possible to detect and quantify post-decapitative accumulation of NAPE subclasses (NAPEDIACYL and NAPEPLAS) in rat brains by the use of 31P NMR spectroscopy.