Multi-stage mass spectrometric studies of triterpenoid saponins in crude extracts from Acanthopanax senticosus harms

Three triterpenoid saponins in crude extracts from Acanthopanax senticosus Harms have been investigated by use of multi-stage mass spectrometry (MSn) combined with electrospray ionization (ESI), MSn spectra were applied to direct structure elucidation of these saponins in crude plant extracts, in positive and negative ion mode. The characteristic fragmentations of triterpenoid saponins are discussed. The method provides a means of rapid initial screening of crude plant extracts. Copyright (C) 1999 John Wiley & Sons, Ltd.

An acidic polysaccharide with xylose branches from Porphyra yezoensis

An acidic polysaccharide (PY3) was isolated from the hot water extract of the red algae Porphyra yezoensis by successive column chromatographies over DEAE-cellulose and Sephadex G-200. PY3 with an average molecular weight of 1.8x10(5) was demonstrated to be composed of galactose (Gal), 3,6-anhydrogalactose (3,6-AnGal), 6-OSO3-galactose (6-OSO3-Gal) and xylose (Xyl) in an approximate molar ratio of 25 : 15, 10, 1. In view of Smith degradation and methylation and on the basis of spectral evidence including those of IR, GC, GC-MS, and H-1 and C-13 NMR, the most probable repeating unit of PY3 could be proposed as [(1-->3)beta -D-Gal(1 --> 4)alpha -L-3,6-AnGal](3)-[(1 --> 3)beta -D-Gal(1 --> 4)alpha -L-6-OSO3-Gal](2) with a xylose moiety at the C-6 of one of every twenty-five beta -D-Gal residues. To our knowledge, PY3 was shown to be the first porphyran possessing occasional xylose branches.

5,7-Dihydroxy-5,6,7,8-tetrahydro-1H-azocin-2-one from a marine-derived Streptomyces sp.

In the course of a screening program, we have isolated the new natural product, 5,7-dihydroxy-5,6,7,8-tetrahydroazocin-2(IH)-one (1), from the staurosporine producing marine-derived Streptomyces sp. strain QD518. Here we report the isolation and structure elucidation of 1 and the artifacts 3 and 4 resulting from I by acid catalyzed intra- and inter-molecular reactions.

Identification of an unknown b-agonist in feed by liquid chromatography/bioassay/quadrupole time-of-flight tandem mass spectrometry with accurate mass measurement.

A new approach to the search for residues of unknown growth promoting agents such as anabolic steroids and -agonists in feed is presented. Following primary extraction and clean-up, samples are separated using gradient liquid chromatography (LC). The effluent is split towards two identical 96-well fraction collectors and an optional electrospray quadrupole time-of-flight mass spectrometry (QTOFMS) system for accurate mass measurement. One 96-well plate is used for a bioassay (enzyme-immuno assay, receptor assay) and will detect the bioactivity and position of the relevant peak in the chromatogram. The positive well in the second 96-well plate is used for identification by LC/QTOFMS/MS. The value of this LC/bioassay/QTOFMS/MS methodology is highlighted by the finding and structure elucidation of a new -agonist in a feed extract.

Investigation of the composition of linear alkylbenzenes with emphasis on the identification and quantitation of some trace compounds using GS/MS system in both electron impact and chemical ionization modes

Linear alkylbenzenes, LAB, formed by the Alel3 or HF catalyzed alkylation of benzene are common raw materials for surfactant manufacture. Normally they are sulphonated using S03 or oleum to give the corresponding linear alkylbenzene sulphonates In >95 % yield. As concern has grown about the environmental impact of surfactants,' questions have been raised about the trace levels of unreacted raw materials, linear alkylbenzenes and minor impurities present in them. With the advent of modem analytical instruments and techniques, namely GCIMS, the opportunity has arisen to identify the exact nature of these impurities and to determine the actual levels of them present in the commercial linear ,alkylbenzenes. The object of the proposed study was to separate, identify and quantify major and minor components (1-10%) in commercial linear alkylbenzenes. The focus of this study was on the structure elucidation and determination of impurities and on the qualitative determination of them in all analyzed linear alkylbenzene samples. A gas chromatography/mass spectrometry, (GCIMS) study was performed o~ five samples from the same manufacturer (different production dates) and then it was followed by the analyses of ten commercial linear alkylbenzenes from four different suppliers. All the major components, namely linear alkylbenzene isomers, followed the same elution pattern with the 2-phenyl isomer eluting last. The individual isomers were identified by interpretation of their electron impact and chemical ionization mass spectra. The percent isomer distribution was found to be different from sample to sample. Average molecular weights were calculated using two methods, GC and GCIMS, and compared with the results reported on the Certificate of Analyses (C.O.A.) provided by the manufacturers of commercial linear alkylbenzenes. The GC results in most cases agreed with the reported values, whereas GC/MS results were significantly lower, between 0.41 and 3.29 amu. The minor components, impurities such as branched alkylbenzenes and dialkyltetralins eluted according to their molecular weights. Their fragmentation patterns were studied using electron impact ionization mode and their molecular weight ions confirmed by a 'soft ionization technique', chemical ionization. The level of impurities present i~ the analyzed commercial linear alkylbenzenes was expressed as the percent of the total sample weight, as well as, in mg/g. The percent of impurities was observed to vary between 4.5 % and 16.8 % with the highest being in sample "I". Quantitation (mg/g) of impurities such as branched alkylbenzenes and dialkyltetralins was done using cis/trans-l,4,6,7-tetramethyltetralin as an internal standard. Samples were analyzed using .GC/MS system operating under full scan and single ion monitoring data acquisition modes. The latter data acquisition mode, which offers higher sensitivity, was used to analyze all samples under investigation for presence of linear dialkyltetralins. Dialkyltetralins were reported quantitatively, whereas branched alkylbenzenes were reported semi-qualitatively. The GC/MS method that was developed during the course of this study allowed identification of some other trace impurities present in commercial LABs. Compounds such as non-linear dialkyltetralins, dialkylindanes, diphenylalkanes and alkylnaphthalenes were identified but their detailed structure elucidation and the quantitation was beyond the scope of this study. However, further investigation of these compounds will be the subject of a future study.

Isolation, purification and characterization of Protease Inhibitor from Moringa oleifera Lam.

Protease inhibitors are one of the most important tools of nature for regulating the proteolytic activity of their target proteases. They are synthesized in biological systems and they play a critical role in controlling a number of diverse physiological functions. The current investigation focused on the isolation, purification and characterization of a novel protease inhibitor from Moringa oleifera. The results obtained during the course of study opens new perspectives for the utilization of protease inhibitor from Moringa oleifera for various pharmaceutical, agricultural and food industries. The biological and physicochemical properties exhibited by the novel protease inhibitor from Moringa oleifera clearly testify its suitability for the development as a drug for application in pharmaceutical industries such as anticoagulant agent or biocontrol agent in agriculture and even as a food preservant. There is a scope for further research on the structure elucidation and protein engineering towards a wide range of further applications. Detailed structure/function analysis of these proteins is important to facilitate their use in genetic engineering for various applications.

Lipase production by marine fungus Aspergillus Awamori Nagazawa

The present study indicate the scope for the utilization of the marine fungus Aspergillus awamori Nagazawa BTMFW 032 for extracellular lipase production employing submerged fermentation. To the best of our knowledge this is the first report on lipase production by a marine fungus employing statistical modeling towards industrial production. The characterization of purified lipase produced by A. awamori showed stability in organic solvents, oxidizing agent and reducing agents, I,3-regiospecificity and hydrolytic activity. These properties make this lipase an ideal candidate for biocatalysis in organic media for the production of novel compounds such as biodiesel and sugar fatty esters. 91.4 % reduction in oil and grease content in ayurvedic oil by the treatment of A. awamori lipase indicates that there is a scope for this enzyme in the treatment of oil effluents and bioremediation. There is ample scope for further research on the biochemistry of the enzyme, structure elucidation and enzyme engineering towards a wide range of further applications, besides enriching scientific knowledge on marine enzymes.

Protease Production by haloarchaea Natrinema sp. BTSH10 isolated from salt pan of South India

The present study led to the recognition of Natrinema sp. BTSH 10 isolated from saltern ponds, as an ideal candidate species for production of gelatinase, which was noted as a halozyme capable of showing enzyme activity in the presence of 15% NaCl. Results obtained during the course of the present study indicated potential for application of this enzyme in industrial catalysis that are performed in the presence of high concentrations of salt. The enzyme characteristics noted with this gelatinase also indicate the scope for probable applications in leather industry, meat tenderization, production of fish sauce and soy sauce. Since halophilic proteases are tolerant to organic solvents, they could be used in antifouling coating preparations used to prevent biofouling of submarine equipments. The gelatinase from haloarchaea could be considered as a probable candidate for peptide synthesis. However, further studies are warranted on this haloarcheal gelatinase particularly on structure elucidation and enzyme engineering to suit a wide range of applications. There is immense scope for developing this halozyme as an industrial enzyme once thorough biochemistry of this gelatinase is studied and a pilot scale study is conducted towards industrial production of this enzyme under fermentation is facilitated. Based on the present study it is concluded that haloarchaea Natrinema sp. that inhabit solar saltern ponds are ideal source for deriving industrially important halozymes and molecular studies on enzymes are prerequisite for their probable industrial applications. This is the first time this species of archaea is recognized as a source of gelatinase enzyme that has potential for industrial applications.

N-H...S=C hydrogen bond in O-alkyl N-methoxycarbonyl thiocarbamates, ROC(S)N(H)C(O)O)CH(3) (R = CH(3)(-), CH(3)CH(2)-)

Pure O-methyl N-methoxycarbonyl thiocarbamate CH(3)OC(S)N(H)C(O)OCH(3) (I) and O-ethyl N-methoxycarbonyl thiocarbamate, CH(3)CH(2)OC(S)N(H)C(O)OCH(3) (II), are quantitatively prepared by the addition reaction between the CH(3)OC(O)NCS and the corresponding alcohols. The compounds are characterized by multinuclear ((1)H and (13)C) and bi-dimensional ((13)C HSQC) NMR, GC-MS and FTIR spectroscopy techniques. Structural and conformational properties are analyzed using a combined approach involving crystallographic data, vibration spectra and theoretical calculations. The low-temperature (150 K) crystal structure of II was determined by X-ray diffraction methods. The substance crystallizes in the monoclinic space group P2(1)/n with a = 4.088(1)angstrom. b = 22.346(1)angstrom, c = 8.284(1)angstrom, beta = 100.687(3)degrees and Z = 4 molecules per unit cell. The conformation adopted by the thiocarbamate group -OC(S)N(H)- is syn (C=S double bond in synperiplanar orientation with respect to the N-H single bond), while the methoxycarbonyl C=O double bond is in antiperiplanar orientation with respect to the N-H bond. The non-H atoms in II are essentially coplanar and the molecules are arranged in the crystal lattice as centro-symmetric dimeric units held by N-H center dot center dot center dot S=C hydrogen bonds Id(N center dot center dot center dot S) = 3.387(1)angstrom, <(N-H center dot center dot center dot S) = 166.4(2)degrees]. Furthermore, the effect of the it electronic resonance in the structural and vibrational properties is also discussed. (C) 2009 Elsevier Ltd. All rights reserved.

Synthesis and Characterization of Homoleptic and Heteroleptic Cobalt, Nickel, Copper, Zinc and Cadmium Compounds with the 2-(Tosylamino)-N-[2-(tosylamino)benzylidene]aniline Ligand

The electrochemical oxidation of anodic metal (cobalt, nickel, copper, zinc and cadmium) in an acetonitrile solution of the Schiff-base ligand 2-(tosylamino)-N-[2-(tosylamino)-benzylidene] aniline (H(2)L) afforded the homoleptic compounds [ML]. The addition of 1,1-diphenylphosphanylmethane (dppm), 2,2`-bipyridine (bipy) or 1,10-phenanthroline (phen) to the electrolytic phase gave the heteroleptic complexes [NiL(dppm)], [ML(bipy)] and [ML(phen)]. The crystal structures of H(2)L (1), [NiL] (2), [CuL] (3), [NiL(dppm)] (4), [CoL(phen)] (5), [CuL(bipy)] (6) and [Zn(Lphen)] (7) were determined by X-ray diffraction. The homoleptic compounds [NiL] and [CuL] are mononuclear with a distorted square planar [MN(3)O] geometry with the Schiff base acting as a dianionic (N(amide)N(amide)N(imine)O(tosyl)) tetradentate ligand. Both compounds exhibit an unusual pi-pi stacking interaction be-tween a six-membered chelate ring containing the metal and a phenylic ring of the ligand. In the heteroleptic complex [NiL(dppm)], the nickel atom is in a distorted tetrahedral [NiN(3)P] environment defined by the imine, two amide nitrogen atoms of the L(2-) dianionic tridentate ligand and one of the phosphorus atoms of the dppm molecule. In the other heteroleptic complexes, [CoL(phen)], [CuL(bipy)] and [ZnL(phen)], the metal atom is in a five-coordinate environment defined by the imine, two amide nitrogen atoms of the dianionic tridentate ligand and the two bipyridine or phenanthroline nitrogen atoms. The compounds were characterized by microanalysis, IR and UV/Vis (Co, Ni and Cu complexes) spectroscopy, FAB mass spectrometry and (1)H NMR ([NiL] and Zn and Cd complexes) and EPR spectroscopy (Cu complexes).

Cyclocreatine, an anticancer and neuroprotective agent. Spectroscopic, structural and theoretical study

The structural, spectroscopic and theoretical study of cyclocreatine (1-carboxymethyl-2-iminoimidazolidine, CyCre) has been performed prompted by the biological relevance of the molecule and its potential role as a ligand in biometalic compounds. The crystal structure of CyCre has been determined by X-ray diffraction methods. The compound crystallizes as a zwitterion in the monoclinic system, space group P2(1)/c. The crystal is further stabilized by a network of N-H center dot center dot center dot O bonds. Infrared and Raman spectra of the solid, electronic spectra of aqueous solutions at different pH values and (1)H and (13)C NMR spectra have been recorded and analyzed. Band assignments were accomplished with the help of theoretical calculations. Optimized molecular geometries, harmonic vibrational frequencies and molecular electrostatic potentials were calculated using methods based on the density functional theory. (C) 2010 Elsevier B.V. All rights reserved.

Solvothermal Preparation of Drug Crystals: Didanosine

For the first time, crystals of suitable size for X-ray diffractometry structure determination (Dian important anti-HI V drug were prepared under solvothermal conditions. In this study, the crystal structure of didanosine (2`,3`-dideoxyinosine, ddI) in the form of a hydrate was determined using single-crystal X-ray diffractometry. Powder X-ray diffraction analysis revealed that the solid-state phase of the drug incorporated into pharmaceutical solid dosage forms is isostructural to the solvothermally prepared ddI material, even though they do not exhibit an identical chemical composition due to different water fractions occupying hydrophobic channels formed within the crystal lattice. Two ddI conformers are present in the structure, in agreement with a previous structure elucidation attempt. Concerning the keto enol equilibrium of ddI, our crystal data and vibrational characterizations by Fourier transform infrared (FTIR) and FT-Raman spectroscopy techniques were conclusive to state that both conformers exist in the keto form, contrary to solid-state NMR spectroscopic assignments that suggested ddI molecules occur as enol tautomers. In addition, characterizations by thermal (differential scanning calorimetry) and spectroscopic techniques allowed us to understand the structural similarities and the differences related to the hydration pattern of the nonstoichiometric hydrates.

Thermal analysis of prednicarbate and characterization of thermal decomposition product

In the present work, the thermal behavior of prednicarbate was studied using DSC and TG/DTG. The solid product remaining at the first decomposition step of the drug was isolated by TG, in air and N(2) atmospheres and was characterized using LC-MS/MS, NMR, and IR spectroscopy. It was found that the product at the first thermal decomposition step of prednicarbate corresponds to the elimination of the carbonate group bonding to C(17), and a consequent formation of double bond between C(17) and C(16). Structure elucidation of this degradation product by spectral data has been discussed in detail.

Mixed-Ligand Complexes of Technetium and Rhenium with Tridentate Benzamidines and Bidentate Benzoylthioureas

Mixed-ligand complexes of technetium(V) or rhenium(V) containing tridentate N-[(dialkylamino)(thiocarbonyl)]benzamidine (H(2)L(1)) and bidentate N,N-dialkyl-N`-benzoylthiourea (HL(2)) ligands were formed in high yields when (NBu(4))[MOCl(4)] (M = Tc or Re) or [ReOCl(3)(PPh(3))(2)] was treated with mixtures of the proligands. Other approaches for the synthesis of the products are reactions of [MOCl(L(1))] complexes with HL(2) or compounds of the-composition [ReOCl(2)(PPh(3))(L(2))] with H(2)L(1). The resulting air-stable [MO(L(1))(L(2))] complexes possess potential for the development of metal-based radiopharmaceuticals. [TcO(L(1))(L(2))] complexes are readily reduced by PPh3 with formation of [Tc(L(1))(L(2))(PPh(3))]. The resulting Tc(III) complexes undergo two almost-reversible oxidation steps corresponding to one-electron transfer processes. ((C) Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009)

Analysis of Bothrops jararacussu venomous gland transcriptome focusing on structural and functional aspects: I - gene expression profile of highly expressed phospholipases A(2)

Snake venom glands are a rich source of bioactive molecules such as peptides, proteins and enzymes that show important pharmacological activity leading to in local and systemic effects as pain, edema, bleeding and muscle necrosis. Most studies on pharmacologically active peptides and proteins from snake venoms have been concerned with isolation and structure elucidation through methods of classical biochemistry. As an attempt to examine the transcripts expressed in the venom gland of Bothrops jararacussu and to unveil the toxicological and pharmacological potential of its products at the molecular level, we generated 549 expressed sequence tags (ESTs) from a directional cDNA library. Sequences obtained from single-pass sequencing of randomly selected cDNA clones could be identified by similarities searches on existing databases, resulting in 197 sequences with significant similarity to phospholipase A(2) (PLA(2)), of which 83.2% were Lys49-PLA(2) homologs (BOJU-1), 0.1% were basic Asp49-PLA(2)s (BOJU-II) and 0.6% were acidic Asp49-PLA(2)s (BOJU-III). Adjoining this very abundant class of proteins we found 88 transcripts codifying for putative sequences of metalloproteases, which after clustering and assembling resulted in three full-length sequences: BOJUMET-I, BOJUMET-II and BOJUMET-III; as well as 25 transcripts related to C-type lectin like protein including a full-length cDNA of a putative galactose binding C-type lectin and a cluster of eight serine-proteases transcripts including a full-length cDNA of a putative serine protease. Among the full-length sequenced clones we identified a nerve growth factor (Bj-NGF) with 92% identity with a human NGF (NGHUBM) and an acidic phospholipase A2 (BthA-I-PLA(2)) displaying 85-93% identity with other snake venom toxins. Genetic distance among PLA(2)s from Bothrops species were evaluated by phylogenetic analysis. Furthermore, analysis of full-length putative Lys49-PLA(2) through molecular modeling showed conserved structural domains, allowing the characterization of those proteins as group II PLA(2)s. The constructed cDNA library provides molecular clones harboring sequences that can be used to probe directly the genetic material from gland venom of other snake species. Expression of complete cDNAs or their modified derivatives will be useful for elucidation of the structure-function relationships of these toxins and peptides of biotechnological interest. (C) 2004 Elsevier SAS. All rights reserved.