Identification of N-acylphosphatidylserine molecules in eukaryotic cells

While profiling the lipidome of the mouse brain by mass spectrometry, we discovered a novel family of N-acylphosphatidylserine (N-acyl-PS) molecules. These N-acyl-PS species were enriched by DEAE-cellulose column chromatography, and they were then characterized by accurate mass measurements, tandem mass spectrometry, liquid chromatography/mass spectrometry, and comparison to an authentic standard. Mouse brain N-acyl-PS molecules are heterogeneous and constitute about 0.1% of the total lipid. In addition to various ester-linked fatty acyl chains on their glycerol backbones, the complexity of the N-acyl-PS series is further increased by the presence of diverse amide-linked N-acyl chains, which include saturated, monounsaturated, and polyunsaturated species. N-Acyl-PS molecular species were also detected in the lipids of pig brain, mouse RAW264.7 macrophage tumor cells, and yeast, but not Escherichia coli. N-Acyl-PSs may be biosynthetic precursors of N-acylserine molecules, such as the recently reported signaling lipid N-arachidonoylserine from bovine brain. We suggest that a phospholipase D might cleave N-acyl-PS to generate N-acylserine, in analogy to the biosynthesis of the endocannabinoid N-arachidonoylethanolamine (anadamide) from N-arachidonoylphosphatidylethanolamine.     

鞭檐犁头尖中的苯丙素甙类化合物

从天南星科植物鞭檐犁头尖的根茎中分离得到3个化合物,运用波谱技术(UV,IR,^1HNMR,^13C NMR and 2D NMR)对它们的化学结构进行解析,分别鉴定为松柏甙(1),甲基松柏甙(2)和硝酸钾(3)。从鞭檐犁头尖中分离得到苯丙素甙类化合物尚属首次。     

Bacopaside I and II: two pseudojujubogenin glycosides from Bacopa monniera

Two saponins, designated as bacopaside I and II, have been isolated from Bacopa monniera Wettst. and their structures have been elucidated as 3-O-alpha-L-arabinofuranosyl-(1-->2)-[6-O-sulphonyl-beta-D-glucopyranosyl-(1-->3)]-alpha-L-arabinopyranosyl pseudojujubogenin (1) and 3-O-alpha-L-arabinofuranosyl-(1-->2)-[beta-D-glucopyranosyl (1-->3)]-beta-D-glucopyranosyl pseudojujubogenin (2) mainly on the basis of 2D NMR and other spectral analyses.     

Tannins from Bendo Eucalyptus

The gallic acid, (+)-catechin and myricetin glucoside have been isolated and indentified from Bendo Eucalyptus, and two kinds of dimeric flavan-3-ol B1 and B3 were isolated as their peracetates and identified by chromatography on TLC and 1H NMR spectroscopic cha- racteristics. It is showed that condensed tannin is a procyanidin with (+)-catechin as terminal units and (–)-epicatechin as extension units by hydrolyzation anthocyanidin reaction, phloroglucinol degradation and polarimetry, UV and IR spectrometry, HPLC, 3C NMR. Linkage is C4–C8. Degree of polymerization is about 4–5.     

Eleganoside-A, B and C from Pseudocalymma elegans, a native of Brazil

The toxic methanol-soluble part of Pseudocalymma elegans (leaves), a native of Brazil, yielded three new iridoidglucosides (1a-3a) as their acetate-derivatives (1-3) named eleganoside-A (1a), B (2a) and C (3a) which have been characterized with the aid of spectroscopic techniques, including 2D NMR.     

Dimeric anthocyanins from strawberry (Fragaria ananassa) consisting of pelargonidin 3-glucoside covalently linked to four flavan-3-ols

Flavanol-anthocyanin complexes were isolated by successive use of Amberlite XAD-7 chromatography, Sephadex LH-20 gel filtration and preparative HPLC from acidified, methanolic extract of strawberries (Fragaria ananassa Dutch.). These purple minor pigments were characterized by UV-Vis spectroscopy, 1D and 2D NMR techniques, and electrospray mass spectrometry to be catechin(4alpha --> 8)pelargonidin 3-O-beta-glucopyranoside (1), epicatechin(4alpha --> 8)pelargonidin 3-O-beta-glucopyranoside (2), afzelechin(4alpha --> 8)pelargonidin 3-O-beta-glucopyranoside (3) and epiafzelechin(4alpha --> 8)pelargonidin 3-O-beta-glucopyranoside (4). The stereochemistry at the 3- and 4-positions of the flavan-3-ol units was based on assumption of R-configuration at C-2. Each of the four pigments occurred in the NMR solvent as a pair of rotamers. Proved by cross-peaks in the 1H-1H NOESY NMR spectra of 1, 2 and 4, the two conformations within each rotameric pair were in equilibrium with each other. Even though 1 and 2 are based on a different aglycone, their structures may be similar to tentatively identified pigments, which have been assumed to contribute to the colour of red wines.     

1,3,4-Oxadiazole-3(2H)-carboxamide derivatives as potential novel class of monoamine oxidase (MAO) inhibitors: synthesis, evaluation, and role of urea moiety

A new series of 1,3,4-oxadiazole-3(2H)-carboxamide derivatives have been synthesized by direct heterocyclization reaction of substituted benzoylisocyanate with various aroylhydrazones as novel monoamine oxidase inhibitors (MAOIs). The target molecules have been identified on the basis of satisfactory analytical and spectra (IR, (1)H NMR, (13)C NMR, and HR-MS) data. The newly synthesized compounds were evaluated for their MAO inhibitory activity by kynuramine fluorimetric assay method. The preliminary results showed that most of the compounds have moderate inhibitory activities toward MAO at the concentration of 10(-5)-10(-3)M. This work may provide a novel class of lead compounds with potential MAO inhibitions for further optimization.     

Backbone structure confirmation and side chain conformation refinement of a bradykinin mimic BKM-824 by comparing calculated (1)H, (13)C and (19)F chemical shifts with experiment

Calculated and experimental (1)H, (13)C and (19)F chemical shifts were compared in BKM-824, a cyclic bradykinin antagonist mimic, c[Ava(1)-Igl(2)-Ser(3)-DF5F(4)-Oic(5)-Arg(6)] (Ava=5-aminovaleric acid, Igl=alpha-(2-indanyl)glycine, DF5F=pentafluorophenylalanine, Oic=(2S,3aS,7aS)-octahydroindole-2-carboxylic acid). The conformation of BKM-824 has been studied earlier by NMR spectroscopy (M. Miskolzie et al., J. Biomolec. Struct. Dyn. 17, 947-955 (2000)). All NMR structures have qualitatively the same backbone structure but there is considerable variation in the side chain conformations. We have carried out quantum mechanical optimization for three representative NMR structures at the B3LYP/6-31G* level, constraining the backbone dihedral angles at their NMR structure values, followed by NMR chemical shift calculations at the optimized structures with the 6-311G** basis set. There is an intramolecular hydrogen bond at Ser(3) in the optimized structures. The experimental (13)C chemical shifts at five C(alpha) positions as well as at the Cbeta, Cgamma and Cdelta position of Ava(1), which forms part of the backbone, are well reproduced by the calculations, confirming the NMR backbone structure. A comparison between the calculated and experimental H(beta) chemical shifts in Igl(2) shows that the dominant conformation at this residue is gauche. Changes of proton chemical shifts with the scan of the chi(1) angle in DF5F(4) suggest that chi(1)180 degrees. The calculated (1)H and (13)C chemical shifts are in good agreement with experiment at the rigid residue Oic(5). None of the models gives accurate results for Arg(6), presumably because of its positive charge. Our study indicates that calculated NMR shifts can be used as additional constraints in conjunction with NMR data to determine protein conformations. However, to be computationally effective, a database of chemical shifts in small peptide fragments should be precalculated.     

Acute biochemical effects of La(NO3)3 on liver and kidney tissues by magic-angle spinning 1H nuclear magnetic resonance spectroscopy and pattern recognition

High-resolution magic-angle spinning (MAS) 1H nuclear magnetic resonance (NMR) spectroscopic and pattern recognition (PR) based methods have been applied to studies on the acute biochemical effects of La(NO3)3 on rats. Male Wistar rats were treated with various doses of La(NO3)3 (2, 10, and 50 mg/kg body weight), and MAS 1H NMR spectra of their intact liver and kidney tissues were analyzed using principal components analysis to extract metabolic information. The biochemical effects of La(NO3)3 were characterized by the increase of triglyceride and bile acid and the decrease of glycogen in liver tissue, together with a slight elevation of triglyceride level in kidney tissue. The target lesion of La(NO3)3 to liver was found by MAS NMR-PR methods. This study illustrated the power of the combination of MAS 1H NMR and pattern recognition for the analysis of biochemical effects of rare earths.     

Chiral recognition in dipeptides containing 1-aminocyclopropane carboxylic acid or alpha-aminoisobutyric acid: NMR studies in solution.

Protected dipeptides containing 1-aminocyclopropane carboxylic acid (Ac3c) or alpha-aminoisobutyric acid (Aib) residues at the C-terminus and Phe, Val or Ala residues at the N-terminus displayed different proton NMR spectra for the pure enantiomers and the racemic mixtures in deuterochloroform (CDCl3) solution. An unequal mixture of enantiomers showed two sets of resonances (NMR nonequivalence), one corresponding to major and the other to minor enantiomer. The NMR nonequivalence was originated by the presence of the C-terminal Ac3c or Aib residues, which have been known for their unique spatial preferences in avoiding an extended (C5) conformation. When a C5 conformation favoring residue such as glycine was incorporated in place of Ac3c or Aib, negligible NMR nonequivalence was observed. The magnitude of the NMR nonequivalence depended on the side chain as well as on the protecting groups at N-terminus alpha-amino acid. For the same peptide, the magnitude of nonequivalence increased with increasing solution concentration and/or with decreasing the solution temperature. The NMR nonequivalence disappeared in polar solvent-like deuterated dimethylsulfoxide (DMSO-d6). A preference for hetero-chiral recognition leading to dimeric association under fast exchange conditions had been invoked to explain the observed phenomenon. The dipeptides thus prepared could well serve as 'model peptides' for the evaluation of any preparative methods.     

Green synthesis and biological evaluation of some novel azoles as antimicrobial agents

A series of novel fluorine-containing triazoles 3, thiadiazoles 4, and oxadiazoles 5 were synthesized from thiosemicarbazides 2. These reactions were carried out by green technique such as ultrasonication and microwave. All products have been characterized by IR, (1)H NMR, and Mass spectral study and screened for their antimicrobial activity.     

Dynamic structure of pharaonis phoborhodopsin (sensory rhodopsin II) and complex with a cognate truncated transducer as revealed by site-directed 13C solid-state NMR

We have recorded (13)C nuclear magnetic resonance (NMR) spectra of [3-(13)C]Ala, [1-(13)C]Val-labeled pharaonis phoborhodopsin (ppR or sensory rhodopsin II) incorporated into egg PC (phosphatidylcholine) bilayer, by means of site-directed high-resolution solid-state NMR techniques. Seven (13)C NMR signals from transmembrane alpha-helices were resolved for [3-(13)C]Ala-ppR at almost the same positions as those of bacteriorhodopsin (bR), except for the suppressed peaks in the loop regions in spite of the presence of at least three Ala residues. In contrast, (13)C NMR signals from the loops were visible from [1-(13)C]Val-ppR but their peak positions of the transmembrane alpha-helices are not always the same between ppR and bR. The motional frequency of the loop regions in ppR was estimated as 10(5) Hz in view of the suppressed peaks from [3-(13)C]Ala-ppR due to interference with proton decoupling frequency. We found that conformation and dynamics of ppR were appreciably altered by complex formation with a cognate truncated transducer pHtr II (1-159). In particular, the C-terminal alpha-helix protruding from the membrane surface is involved in the complex formation and subsequent fluctuation frequency is reduced by one order of magnitude.     

Synthetic, spectral, thermal and antimicrobial studies on some mixed 1,3-dithia-2-stannacyclopentane derivatives with dialkyldithiocarbamates

1,3-dithia-2-stannacyclopentane derivatives with dialkyldithiocarbamates of the types SCH(2)CH(2)SSn[S(2)CNR(2)]Cl (I) and SCH(2)CH(2)SSn[S(2)CNR(2)](2) (II) (where R = CH(3), C(2)H(5) and -CH(2)-CH(2)-) have been synthesized by the reaction of 2,2-dichloro-1,3-dithia-2-stannacyclopentane and sodium/ammonium salts of dialkyldithiocarbamates in 1:1 and 1:2 molar ratios, respectively, in anhydrous benzene. These newly synthesized derivatives have been characterized by elemental analyses (C, H, N, S and Sn), thermal [thermogravimetry (TG) and differential thermal analyses (DTA)] as well as spectral [UV, IR and multinuclear NMR ((1)H, (13)C and (119)Sn)] studies. The monodentate behaviour of the dialkyldithiocarbamate ligands was confirmed by IR and (119)Sn NMR spectral data and distorted tetrahedral structures have been suggested for both type (I) and (II) compounds. The free ligands and their tin complexes have also been screened for their antibacterial and antifungal activities. These results made it desirable to delineate a comparison between free ligands and their tin complexes. These exhibit higher antibacterial effect than some of the previously investigated antibiotics.     

Redox ribonucleosides. Isolation and characterization of 5-hydroxyuridine, 8-hydroxyguanosine, and 8-hydroxyadenosine from Torula yeast RNA.

Three hydroxyribonucleosides catalyzing the oxido-reduction of NADH and K3F3(CN)6 were purified from Torula yeast RNA by a series of steps including sodium dodecyl sulfate/phenol extraction, nuclease P1 digestion, alkaline phosphatase digestion, anion-exchange chromatography, and high performance liquid chromatography on an ODS column. Analysis by fast atom bombardment-mass spectrometry and 1H and 13C NMR spectroscopy led to identification of the redox ribonucleosides as 5-hydroxyuridine, 8-hydroxyguanosine, and 8-hydroxyadenosine. Their mass spectra, chromatographic behavior, UV spectra, NMR spectra, and IR spectra were identical to those from natural and synthetic sources. Oxidoreduction activities were specific for K3Fe(CN)6 as the oxidant and NADH as the reductant; and their magnitudes decreased in the order 5-hydroxycytidine, 5-hydroxyuridine, 8-hydroxyguanosine, and 8-hydroxyadenosine. The fact that these nucleosides have redox activities suggests new functional roles for RNAs as catalysts.     

Biosynthesis of the trichothecene 3-acetyldeoxynivalenol. Identification of the oxygenation steps after isotrichodermin

Upon feeding an excess of the substrate isotrichodermin, five tricyclic metabolites accumulated in Fusarium culmorum cultures. These compounds were also identified as transient intermediates of trichothecene biosynthesis by kinetic pulse labeling. Their structures were characterized by spectroscopic techniques (1H NMR, 13C NMR, 2H NMR, and nuclear Overhauser effect difference experiments) as: 1, 15-deacylcalonectrin; 2, calonectrin; 3, 7-hydroxyisotrichodermin; 4, 8-hydroxyisotrichodermin; and 5, 7, hydroxycalonectrin. Four of these metabolites (1-4) were rigorously proven to be biosynthetic precursors to 3-acetyldeoxynivalenol. Indeed, their deuteriated derivatives were shown to be incorporated very efficiently into 3-acetyldeoxynivalenol by 2H-NMR. In addition, our experimental data suggests that the first oxygenation step after isotrichodermin is at C-15, producing 15-deacylcalonectrin.     

Sequencing microbial copolymers of 3-hydroxybutyric and 3-mercaptoalkanoic acids by NMR, electrospray ionization mass spectrometry, and size exclusion chromatography NMR

Copolymers of 3-hydroxybutyrate (3HB) and 3-mercaptopropionate (3MP) or 3-mercaptobutyrate (3MB) units and minor amounts of 3-hydroxypropionate (3HP), 3-hydroxyvalerate (3HV), or 3-mercaptovalerate (3MV) were investigated regarding their microstructure by NMR, electrospray ionization mass spectrometry, and size exclusion chromatography NMR. These copolymers were produced by Ralstonia eutropha strain H16 when cells were cultivated in a mineral salts medium with gluconate as a carbon source for growth and 3MP or 3MB as precursor substrates for incorporation of 3-mercaptoalkanoates. Mass spectrometry analysis of partially methanolyzed or pyrolyzed samples proved the presence of true copolymers or terpolymers. (13)C NMR spectroscopy of intact polymer samples, with values of average block length and degree of randomness deviating from a random sequence model, suggested microblock structures; however, composition analysis by (1)H NMR of fractions obtained by size exclusion chromatography showed significant variations with molecular weight, revealing the presence of blends of poly(3HB-co-3MP-co-3HP) or poly(3HB-co-3MB) with poly(3HB). The experimental NMR carbonyl dyad signal intensities were satisfactorily matched by a random sequence model when the presence of poly(3HB) was taken into account.     

NMR Analyses of the Interaction between the FYVE Domain of Early Endosome Antigen 1 (EEA1) and Phosphoinositide Embedded in a Lipid Bilayer

Phosphoinositides (PIs) are crucial lipid components of membranes and are involved in a number of cellular processes through interactions with their effector proteins. Recently, we have established a lipid-protein nanoscale bilayer (nanodisc) containing PIs, hereafter referred to as PI-nanodisc and demonstrated that it could be used for both qualitative and quantitative evaluations of protein-membrane interactions. Here, we report further NMR analyses for obtaining structural insights at the residue-specific level between PI-binding effector protein and PI-nanodisc, using the FYVE domain of early endosome antigen 1 (EEA1), denoted as EEA1 FYVE, and PI(3)P-nanodisc as a model system. We performed a combination of the NMR analyses including chemical shift perturbation, transferred cross-saturation, and paramagnetic relaxation enhancement experiments. These enabled an identification of the interaction surface, structural change, and relative orientation of EEA1 FYVE to the PI(3)P-incorporated lipid bilayer, substantiating that NMR analyses of protein-membrane interactions using nanodisc makes it possible to show the residue-specific interactions in the lipid bilayer environment.     

Membrane channel forming polypeptides. 270-MHz proton magnetic resonance studies of the aggregation of the 11-21 fragment of suzukacillin in organic solvents

270-MHz 1H NMR studies of the 11-21 suzukacillin fragment Boc-Gln-Aib-Leu-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-OMe (11-G) and its analogue Boc-Ala-Aib-Leu-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-OMe (11-A) have been carried out in CDCl3 and (CD3)2SO. The NH chemical shifts and their temperature coefficients have been measured as a function of peptide concentration in both solvents. It is established that replacement of Gln by Ala is without effect on backbone conformation. Both peptides adopt highly folded 310 helical conformations stabilized by seven intramolecular 4 leads to hydrogen bonds. Nonlinear temperature dependences are demonstrated for free NH groups in the Gln(1) peptide. Aggregation is mediated by intermolecular hydrogen bonds formed by solvent-exposed NH groups. A major role for the Gln side chain in peptide association is suggested by differences in the NMR behavior of the Gln(1) and Ala(1) peptides. For the Gln(1) peptide in CDCl3, the carboxamide side chain carbonyl group forms an intramolecular hydrogen bond to the peptide backbone, while the trans side chain NH shows evidence for intermolecular interactions. In (CD3)2SO, the cis carboxamide NH is involved in intermolecular hydrogen bonding. The possible role of the central Gln residue in stabilizing aggregates of peptide channel formers is discussed, and a model for hexameric association is postulated.     

Synthesis, stereochemistry and in vitro antimicrobial evaluation of novel 2-[(2,4-diaryl-3-azabicyclo[3.3.1]nonan-9-ylidene)hydrazono]-4-phenyl-2,3-dihydrothiazoles

2-[(2,4-Diaryl-3-azabicyclo[3.3.1]nonan-9-ylidene)hydrazono]-4-phenyl-2,3-dihydrothiazoles (3a-3k) have been synthesized by the cyclization of 2-[(2,4-diaryl-3-azabicyclo[3.3.1]nonan-9-one thiosemicarbazones with phenacyl bromide and characterized by analytical (melting point and elemental analysis) and spectral (IR, (1)H NMR, (13)C NMR, D(2)O exchange, NOESY and mass) techniques. The novel Hantzsch products (3a-3k) were screened for their in vitro antibacterial and antifungal activities against some selected microorganisms. Structure activity relationship (SAR) for the reported compounds was studied by comparing their MIC values with standard drugs (Streptomycin and Amphotericin B). The results show that 3e against Escherichia coli and Cryptococcus neoformans3i against Bacillus Subtilis, 3b against Aspergillus flavus, and 3k against Rhizopus sp. were found to show significant growth inhibition.     

Synthesis and evaluation of new carbonic anhydrase inhibitors

A series of new sulfamide derivatives have been synthesized, their structures were confirmed by (1)H NMR and ESI-MS. Some target compounds were assessed by the tool of Dock6, and inhibition effects of all the new compounds on carbonic anhydrase II have been investigated. In addition, some compounds have been investigated for their antihypoxic effects in mice. Results indicated that nine target compounds exhibit as effectively as acetazolamide and 10 compounds have more potent inhibition effects on carbonic anhydrase II than acetazolamide. Three of them (I-8, I-18 and I'-3) can prolong markedly the survival time of mice in hypoxia, which are worth carrying out further studies.