First solid state alkaline-earth complexes of monensic acid A (MonH): crystal structure of [M(Mon)2(H (2)O)2] (M = Mg, Ca), spectral properties and cytotoxicity against aerobic Gram-positive bacteria.

Alkaline-earth metal complexes of the monoanionic form of the polyether ionophore monensin A were isolated for the first time in solid state and were structurally characterized using various spectroscopic methods (IR, NMR, FAB-MS). The stoichiometric reaction of monensic acid (MonH) with M(2+) (M = Mg, Ca) in the presence of an organic base leads to the formation of mononuclear complexes of composition [M(Mon)(2)(H(2)O)(2)]. The structures of magnesium (1) and calcium (2) monensin complexes in the solid state were established by single crystal X-ray crystallography. The complexes crystallize as [Mg(Mon)(2)(H(2)O)(2)]x5MeCN (1) and [Ca(Mon)(2)(H(2)O)(2)]xH(2)Ox5MeCN (2) in the monoclinic P21 space group. The alkaline-earth metal ion is placed in a distorted octahedral environment, defined by two monensin anions acting as bidentate ligands in the equatorial plane of the complex as well as by two water molecules occupying the axial positions of the inner coordination sphere. The bactericidal activity of 1 and 2 was evaluated against aerobic Gram-positive microorganisms applying the double layer agar hole diffusion method.

Phosphatidyl ethanolamine with increased polyunsaturated fatty acids in compensation for plasmalogen defect in the Zellweger syndrome brain.

To elucidate the neuropathological mechanism of Zellweger syndrome (ZS), we studied changes in the molecular species of glycerophospholipids in the cerebral tissue by thin-layer chromatography (TLC) and fast atom bombardment mass spectrometry (FABMS). First, we estimated the amount of plasmalogens by TLC. Plasmalogen-type phosphatidyl ethanolamine (PE) accounted for 30% of the total PE in the control brain, but was absent in the ZS brain. Plasmalogen-type phosphatidyl choline (PC) was undetectable in both control and ZS brains. Next, we analyzed plasmalogen-type PE by FABMS. Oleic (18:1), arachidonic (20:4) and docosapentanoic (22:5) acids were present in the control gray matter, but not in the ZS gray matter. In compensation for the defect of plasmalogen, the level of diacyl PE with polyunsaturated fatty acids, 20:4, 22:4, 22:5 and 22:6, was higher in the ZS brain than that in the control brain. These results indicate an alteration in the molecular species of PE, which may cause abnormal neural membrane fluidity and excessive vulnerability to oxygen stress.

Structural determination of sildenafil and its analogues in dietary supplements by fast-atom bombardment collision-induced dissociation tandem mass spectrometry.

Sildenafil and its analogues, which are used as illegal additives in several dietary supplements, were isolated by liquid-liquid extraction and column chromatography and analyzed by fast-atom bombardment mass spectrometry (FAB-MS). Structures of sildenafil and its derivatives were elucidated by FAB-tandem mass spectrometry (MS/MS) with exact mass measurement in the positive-ion mode. To find structurally diagnostic ions for the sildenafil analogues, authentic sildenafil was preferentially analyzed by high-energy collision-induced dissociation (CID)-MS/MS. The CID-MS/MS spectra of [M+H](+) precursor ions resulted in the formation of numerous characteristic ions via a series of dissociative processes. The product ions formed by CID provided important information on the modification of the piperazine ring, the phenylsulfonyl group and the pyrazolopyrimidine moiety of sildenafil. By interpreting their MS/MS spectra, the chemical structures of sildenafil analogues isolated from dietary supplements could be elucidated and fragmentation patterns were proposed. To clearly identify the sidenafil derivatives in dietary supplements, some of the derivatives such as acetildenafil, homosildenafil and hydroxyhomosildenafil which are not commercially available were synthesized and compared with their MS/MS spectra. In addition, high-resolution mass measurements were conducted to obtain the elemental compositions of sildenafil and its analogues.

Differentiation of the isomeric o-(m- and p-)nitro-(chloro- and bromo-)benzyl-2,4-(and 2,1-) disubstituted 2-thiocytosinium halides by electron impact ionisation and fast atom bombardment mass spectrometry.

Electron ionisation (EI) and fast atom bombardment (FAB) mass spectral fragmentations of nine 2,4-(and 2,1-) disubstituted o-(m- and p-)nitro-(chloro- and bromo-)-2-thiocytosinium halides are investigated. Fragmentation pathways, whose elucidation is assisted by accurate mass measurements and metastable transitions [EI-mass spectrometry (MS)], as well as FAB/collision-induced dissociation (CID) mass spectra measurements are discussed. The correlations between the abundances of the (C(11)H(10)N(4)SO(2))(+) 1-3; (C(11)H(10)N(3)SCl)(+) 4-6 and (C(11)H(10)N(3)SBr)(+) 7-9 ions and the selected fragment ions (EI- MS), as well as (C(18)H(16)N(5)SO(4))(+) 1-3; (C(18)H(16)N(3)SCl(2))(+) 4-6 and (C(18)H(16)N(3)SBr(2))(+) 7-9 ions and the selected ions (C(7)H(6)NO(2))(+) 1-3; (C(7)H(6)Cl)(+) 4-6; (C(7)H(6)Br)(+) 7-9 (FAB-MS) are discussed. The data obtained can be used for distinguishing isomers.

[Progress in the determination of glucosinolates].

The glucosinolates are a large group of hydrophilic, sulphur-containing plant secondary metabolites, occurring mainly in cruciferous family, which have recently attracted intense research interest because of their cancer chemoprotective attributes. Developing the analytical methods is the premises for studying the property of glucosinolates, so that much effort has been devoted to developing methods for efficient isolation and identification of them. In this article, the recent progress on analyzing of glucosinolates was reviewed.

Alterations in the molecular species of plasmalogen phospholipids and glycolipids due to peroxisomal dysfunction in Chinese hamster ovary-mutant Z65 cells by FABMS method.

Changes in the molecular species of lipids associated with Pex2 gene-mutation were investigated to elucidate the pathogeneses of peroxisome biogenesis disorders. Although no differences were observed in the concentrations of cholesterol and phosphatidyl choline between mutated Z65 and control CHO-K1 cells, the amounts of cholesterol esters and glycolipids in Z65 cells were twice those in CHO-K1 cells, but phosphatidyl ethanolamine (PE), particularly 1-O-octadec-1'-enyl-2-oleoyl PE, was absent in Z65 cells by FABMS. Enhanced synthesis of glycolipids in Z65 cells was associated with an abundance of lignoceric acid-containing ones, suggesting a role of glycolipids in the retention of longer saturated fatty acids.

Generation of a novel fluorescent product, monochlorofluorescein from dichlorofluorescin by photo-irradiationdagger.

Dichlorofluorescin (DCFH), a widely used fluorescent probe for reactive oxygen species (ROS) was decomposed completely and generated two distinct fluorescent products by photo-irradiation at 254 nm for 30 min. In the previous study, we had shown that one was dichlorofluorescein (DCF), a well known oxidized product of DCFH. In this study we investigated the other product and identified it as monochlorofluorescein (MCF) by 1H-NMR and fast atom bombardment/mass spectrum (FAB/MS) analyses. MCF was generated by photo-irradiation, but not by ROS. On the other hand, DCF was produced by both photo-irradiation and ROS. MCF showed similar fluorescent emission spectrum to DCF, however, its fluorescence intensity was more than that of DCF. The kinetic study suggested that MCF was not generated from DCF but from monochlorofluorescin, which might be generated from DCFH by photo-irradiation.

5-Hydroxy-seco-carotenoids from Pittosporum tobira.

Three 5-hydroxy-seco-carotenoids were isolated from seeds of Pittosporum tobira. These structures were determined to be (3S,3'S,5'?)-3,3'-di(tetradecanoyloxy)-5'-hydroxy-5,6,5',6'-diseco-beta,beta-carotene-5,6,6'-trione (1), (3S,5?,3'S,5'R,6'S,9'Z)-3-tetradecanoyloxy-5',6'-epoxy-5,3'-dihydroxy-5',6'-dihydro-5,6-seco-beta,beta-caroten-6-one (2), and (3S,5?,3'S,5'R,6'R)-3-tetradecanoyloxy-5,3',5'-trihydroxy-6',7'-didehydro-5',6'-dihydro-5,6-seco-beta,beta-caroten-6-one (3) based on analysis of UV-vis, IR, FAB MS, and NMR spectroscopic data.

Analysis of agaritine in mushrooms and in agaritine-administered mice using liquid chromatography-tandem mass spectrometry.

A sensitive and specific method for quantifying a genotoxic hydrazine, agaritine, has been developed using liquid chromatography-electrospray ionization tandem mass spectrometry (MS). Synthetic agaritine was structurally assigned by (1)H, (13)C and two-dimensional nuclear magnetic resonance (NMR) analysis (heteronuclear multiple-bond correlation [HMBC] and heteronuclear multiple-quantum coherence [HMQC]), high-resolution fast-atom-bombardment (HR-FAB) MS. Agaritine was separated on an ODS column using 0.01% AcOH-MeOH (99:1) as an eluent with a simple solid-phase-extraction cleanup for mushroom samples and with acetonitrile and methanol deprotenization for plasma samples. There were no interference peaks in any of the mushrooms or mouse plasma samples. The recoveries of agaritine from the spiked mushroom samples and spiked mouse plasma were 60.3-114 and 74.4%, respectively. The intra-day precision values for the spiked mushrooms were 5.5 and 4.2%, and the inter-day precision values were 15.0 and 23.0%, respectively. The limit of quantification was 0.01 microg/g (in mushrooms) and 0.01 microg/ml (in plasma). A precursor ion scan confirmed that agaritine derivatives, which can exert a similar toxicity, were absent. These results indicate that this analytical method for quantifying agaritine could help to evaluate the risk of mushroom hydrazines to humans.

Three 1-thio-beta-D-glucopyranosides from the seeds of Afrostyrax lepidophyllus Mildbr.

Three new 1-thioglycosides namely methylthiomethyl 1-thio-beta-D-glucopyranoside (Afrostyraxthioside A), methylsulfonylmethyl 1-thio-beta-D-glucopyranoside (Afrostyraxthioside B) and methylsulfonylmethylthiomethyl 1-thio-beta-D-glucopyranoside (Afrostyraxthioside C) were isolated from the seeds of Afrostyrax lepidophyllus Mildbr. Their structures were mainly elucidated by using one- and two-dimensional NMR and mass spectroscopies and also by an efficient one-step synthesis. Moreover, Afrostyraxthiosides A, B and C constitute a new subclass of 1-thioglycosides isolated from natural sources.

Derivatization in mass spectrometry--8. Soft ionization mass spectrometry of small molecules.

This is the first of two reviews devoted to derivatization approaches for "soft" ionization mass spectrometry (FAB, MALDI, ESI, APCI) and deals, in particular, with small molecules. The principles of the main "soft" ionization mass spectrometric methods as well as the reasons for derivatizing small molecules are briefly described. Derivatization methods for modification of amines, carboxylic acids, amino acids, alcohols, carbonyl compounds, monosaccharides, thiols, unsaturated and aromatic compounds etc. to improve their ionizability and to enhance structure information content are discussed. The use of "fixed"-charge bearing derivatization reagents is especially emphasized. Chemical aspects of derivatization and "soft" ionization mass spectrometric properties of derivatives are considered.

Studies on high-energy collision-induced dissociation of endogenous cannabinoids: 2-arachidonoylglycerol and n-arachidonoylethanolamide in FAB-mass spectrometry.

Analysis of 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamide (anandamide) via alkali or alkaline earth metal-adduct high-energy collision-induced dissociation (CID) in fast-atom bombardment (FAB) ionization-mass spectrometry (MS) is described. The CID-MS/MS of the [2-AG+Li](+) or [2-AG+Na](+) ion undergoes charge-remote fragmentation (CRF), which is useful for the determination of the double-bond positions in the hydrocarbon chain, while the CID-MS/MS of the [2-AG-H+Cat](+) (Cat = Mg(2+), Ca(2+), Ba(2+)) ion provides an abundant fragment ion of the cationized arachidonic acid species, which is derived from cleaving the ester bond via a McLafferty-type rearrangement in addition to structurally informative CRF ions in small amounts. On the other hand, the CID-MS/MS spectra of anandamide cationized with both alkali metal (Li(+) or Na(+)) and alkaline earth metal (Mg(2+), Ca(2+), or Ba(2+)) show CRF patterns: the spectra obtained in lithium or sodium adduct are more clearly visible than those in magnesium, calcium, or barium adduct. The McLafferty rearrangement is not observed with metal-adduct anandamide. The characteristics in each mass spectrum are useful for the detection of these endogenous ligands. m-Nitrobenzyl alcohol (m-NBA) is the most suitable matrix. A lithium-adduct [2-AG+Li](+) or [anandamide+Li](+) ion is observed to be the most abundant in each mass spectrum, since the affinity of lithium for m-NBA is lower than that for other matrices examined.

Structural determination of saponins extracted from starfish by fast atom bombardment collision-induced dissociation mass spectrometry.

Three saponins were extracted and isolated from starfish by reversed-phase high performance liquid chromatography (HPLC), and analyzed by fast atom bombardment mass spectrometry (FAB-MS). Their molecular weight information could be obtained by the presence of abundant [M+Na]+ ions and weak [M+H]+ ions in FAB-MS spectra. Moreover, high resolution mass measurements of their [M+Na]+ ions were performed at the resolution of 10000 to elucidate the element composition of extracted saponins. The collision-induced dissociation (CID) of sodium-adducted molecules [M+Na]+ yielded diverse product ions via dissociated processes. In the collision-induced dissociation (CID)-MS/MS analysis of [M+Na]+ ion, the sulfate-containing saponins produced characteristic ions such as SO4Na+, [NaHSO4+Na]+, [M+Na-sugar]+ and [M+Na-2sugar]+ ions, whereas the sulfate-free compound showed characteristic ions produced by cleavage of sugar moiety and side chain of aglycone. The fragmentation patterns could provide information on the linkage position of sugar groups in aglycone and sulfate groups.

Cardiolipin, alpha-D-glucopyranosyl, and L-lysylcardiolipin from gram-positive bacteria: FAB MS, monofilm and X-ray powder diffraction studies.

Cardiolipin preparations from Streptococcus B, Listeria welshimeri, Staphylococcus aureus, and a glucosyl and lysyl derivative of cardiolipin were analysed for fatty acid composition and fatty acid combinations. Three different fatty acid patterns are described and up to 17 molecular species were identified in Streptococcus B lipids by high resolution FAB MS. The physicochemical properties of these lipids were characterised in the sodium salt form by monofilm experiments and X-ray powder diffraction. All lipids formed stable monofilms. The minimal space requirement of unsubstituted cardiolipin was dictated by the fatty acid pattern. Substitution with L-lysine led to a decrease of the molecular area, substitution with D-glucopyranosyl to an increase. On self assembly at 100% relative humidity, all preparations adopted lamellar structures. They showed a high degree of order, in spite of the heterogeneous fatty acid compositions and numerous fatty acid combinations. The repeat distances in lamellar fluid phase varied between 4.99 and 5. 52 nm, the bilayer thickness between 3.70 and 4.46 nm. Surprising were the low values of sorbed water per molecule of the glucosyl and lysyl derivatives which were 58 and 60%, compared with those of the respective cardiolipin. When Na(+) was replaced as counterion by Ba(2+), the bilayer structure was retained, but the lipids were in the lamellar gel phase and the fatty acids were tilted between 32 and 53 degrees away from the bilayer normal. Wide angle X-ray diffraction studies and electron density profiles are also reported. Particular properties of glucosyl cardiolipin are discussed.

Mass spectral behavior of some homoleptic and mixed aryldichalcogenide bis(diphenylphosphino)ferrocenenickel(II), palladium(II), and platinum(II), and bis(diisopropylphosphino)ferrocenepalladium(II) complexes.

The mass spectral behavior of a number of organometallic complexes containing the Group 10 metals Ni, Pd, and Pt, together with various thiolate ligands were studied. For Pd, two main types of complexes, differing by the substituents on the phosphorus atom were studied. Types I and II were substituted with bis(diphenylphosphino)ferrocene and bis(diisopropylphosphino)ferrocene ligands, respectively. The Ni complexes, except for one, and the Pd Type I complexes had no molecular radical cations (M(+.)) in their EI spectra. On the other hand, all the Pt complexes showed intense M(+.) ions in their EI spectra indicating that these complexes were more stable as radical cations than those of Ni and Pd. The FAB and MALDI spectra of all the complexes displayed intense quasi-molecular ions (MH(+)) and the fragmentations in both modes were similar. The MALDI spectra of several complexes displayed only M(+.) ions while one gave evidence of both MH(+) and M(+.) ions. Several Pd Type II complexes yielded intense M(+.) in their EI spectra.

A new matrix metalloproteinase-9 inhibitor 3,4-dihydroxycinnamic acid (caffeic acid) from methanol extract of Euonymus alatus: isolation and structure determination.

In a previous paper [Cha, B.Y., Park, C.J., Lee, D.G., Lee, Y.C., Kim, D.W., Kim, J.D., Seo, W.G., Moon, S.K., Kim, C.H., 2003. Inhibitory effect of methanol extract from Euonymus alatus on matrix metalloproteinase-9, J. Ethnopharm. 85, 163-167], methanol extracts prepared from stems of Euonymus alatus showed a strong inhibitory effect of matrix metalloproteinase (MMP)-9 activity, which is known to be involved in tumor cell invasion and metastasis, in a concentration-dependent manner on zymography. Assay guided fractionation led to the isolation of a caffeic acid (CA) as the compound responsible for the anti-MMP-9 activity. CA was finally obtained by reversed-phase HPLC, and its structure was elucidated by fast atom bombardment mass spectrometry and tandem mass spectrometry. The purified CA inhibited MMP-9 activity with the IC50 of 10-20 nM.

Determination of amide hydrogen exchange by mass spectrometry: a new tool for protein structure elucidation.

A new method based on protein fragmentation and directly coupled microbore high-performance liquid chromatography-fast atom bombardment mass spectrometry (HPLC-FABMS) is described for determining the rates at which peptide amide hydrogens in proteins undergo isotopic exchange. Horse heart cytochrome c was incubated in D2O as a function of time and temperature to effect isotopic exchange, transferred into slow exchange conditions (pH 2-3, 0 degrees C), and fragmented with pepsin. The number of peptide amide deuterons present in the proteolytic peptides was deduced from their molecular weights, which were determined following analysis of the digest by HPLC-FABMS. The present results demonstrate that the exchange rates of amide hydrogens in cytochrome c range from very rapid (k > 140 h-1) to very slow (k < 0.002 h-1). The deuterium content of specific segments of the protein was determined as a function of incubation temperature and used to indicate participation of these segments in conformational changes associated with heating of cytochrome c. For the present HPLC-FABMS system, approximately 5 nmol of protein were used for each determination. Results of this investigation indicate that the combination of protein fragmentation and HPLC-FABMS is relatively free of constraints associated with other analytical methods used for this purpose and may be a general method for determining hydrogen exchange rates in specific segments of proteins.

On the presence of phosphorylated sphingoid bases in rat tissues. A mass-spectrometric approach.

A simple and straightforward procedure to analyze phosphorylated sphingoid bases has been developed. After phase separation of lipid extracts under alkaline conditions, the compounds were quantitatively recovered in the aqueous upper phase. Following a clean-up of the aqueous phase on C18-solid phase extraction columns, the amino-group of the bases was derivatized by means of phenylisothiocyanate addition. FAB-MS of the phenylthiocarbamate derivatives of sphingenine- and sphinganine-phosphate in the negative mode revealed the expected pseudo-molecular ions (M-1) at 513 m/z and 515 m/z, respectively. Moreover, a typical fragmentation pattern, characterized by the loss of the phenylthiocarbamate moiety (m/z = 135), was observed. When applied to rat tissues, the presence of sphingenine-phosphate in brain, kidney and liver could easily be demonstrated. Highest levels, amounting to 5 nmol/g of wet weight, were present in brain.

Mass spectrometric evidence for a disulfide bond in aequorin regeneration.

Tryptic digests of purified recombinant apoaequorin were analyzed, before and after reduction with DTT, by fast atom bombardment mass spectrometry. The results showed that apoaequorin contains a disulfide bond between Cys145 and Cys152 and that the reduction of this bond is involved in the regeneration of aequorin.

Novel modification of ceramide: rat glioma ganglioside GM3 having 3-O-acetylated sphingenine.

A novel O-acetylated GM3 containing 3-O-acetyl 4-sphingenine was isolated with one having a non-acetylated base from transplanted rat glioma tissue. The presence and position of the acetyl group were estimated by one- and two-dimensional proton nuclear magnetic resonance, and fast atom bombardment-mass spectrometries. In addition, the O-acetyl GM3 showed higher immunological activity toward anti-melanoma antibody in the presence of non-acetylated GM3 in complement-dependent liposome lysis than did non-acetylated or acetylated GM3 alone in the liposome, suggesting enhancement of immunological reactivity of the intact tumor cells by a small amount of O-acetyl GM3.