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HPLC-UV/PAD and HPLC-MS(n) analyses of leaf and root extracts of Vismia guineensis and isolation and identification of two new bianthrones.

In order to evaluate the possible use of the leaves instead of the roots of Vismia guineensis as a new source for the traditional use of this drug, the chemical composition of both organs were compared by HPLC-UV/PAD and HPLC-MS analyses. The leaves are analysed here for the first time. The results show the presence of five major classes of secondary metabolites having specific chromophores: anthraquinones, vismiones, flavonoids, xanthones and benzophenones. The molecular weights and characteristic fragments, compared with previous EI or HPLC-MS literature data, allowed the partial identification of the major peaks in the chromatograms. Six additional isomeric bianthrones and one anthraquinone were detected in the dichloromethane extract of the roots after long storage in solution; the targeted isolation of the bianthrones was performed and enabled the identification of two original C-geranyl derivatives. The chemical compositions of the extracts demonstrated that only a minority of the constituents is shared by both organs. Thus, in order to establish a definitive phytoequivalence, additional pharmacological investigations are required.

Anthracenes↗

Synthesis and anti-mildew activity of 5-(2-aroyl)aryloxymethyl-2-phenyl-1 ,3,4-oxadiazoles against downy mildew of pearl millet.

A manipulatively simple, rapid, high-yielding and environmentally benign method for the integration of a heterocyclic ring, 1,3,4-oxadiazole, at the benzophenone nucleus has been achieved through intramolecular cyclization of substituted aroylaryloxyacetohydrazides to substituted 5-(2-aroyl)aryloxymethyl-2-phenyl-1,3,4-oxadiazoles under solventless 'dry' conditions using montmorillonite K10 clay and microwave irradiation. A comparison is made of the microwave-accelerated reaction with conventional heating conditions. Certain of the derivatives tested showed significant anti-mildew activity against Sclerospora graminicola (Sacc) Schroeter, the downy mildew pathogen of pearl millet.

Biological Assay↗

Mass spectral fragmentation of (S,S)-2-substituted 4,4-diphenyl-3,1-oxazabicyclo[3.3.0]octanes: ring contraction of pyrrolidine and 1,3-oxazolidine in mass spectrometry.

The mass spectral behaviour of (S,S)-2-substituted 4,4-diphenyl-3,1-oxazabicyclo[3.3.0]octanes has been studied with the aid of mass-analyzed ion kinetic energy spectrometry and accurate mass measurements under fast atom bombardment (FAB) and electron impact (EI) ionization conditions. Under FAB ionization, all compounds show a tendency to form protonated aldehyde or benzophenone ions and to form protonated 1-azabicyclo[3.1.0]hexane ions, which can further lose an ethylene or cyclopropane from the pyrrolidine ring to produce protonated 1-azabicyclo[1.1.0]butane ions and 3H-azirine ions, respectively. Under EI ionization, a similar fragmentation to that under FAB ionization was observed. The title compounds also show a tendency to yield oxirane ions and oxirenium ions by loss of pyrrolidine and pyrrolidine plus H. Ring contractions of 1,3-oxazolidine by loss of an aldehyde or ketone and of pyrrolidine by loss of an ethylene or cyclopropane were observed under both FAB and EI ionization conditions.

Journal Article↗

Photoaffinity labeling and site-directed mutagenesis of rat squalene epoxidase.

Squalene epoxidase (SE) (EC 1.14.99.7) is a flavin-requiring, non-cytochrome P-450 oxidase that catalyzes the conversion of squalene to (3S)-2,3-oxidosqualene. Photolabeling and site-directed mutagenesis were performed on recombinant rat SE (rrSE) to elucidate the location and roles of active-site residues important for catalysis. Two new benzophenone-containing analogs of NB-598, a nanomolar inhibitor of vertebrate SE, were synthesized in tritium-labeled form. These photoaffinity analogs (PDA-I and PDA-II) became covalently attached to SE when irradiated at 360 nm. Lys-C digestion and HPLC purification of [3H]PDA-I-labeled rrSE resulted in isolation of a single major peptide. MALDI-TOF mass spectrometry of this peptide indicated a covalent adduct between PDA-I and a tripeptide, Asp-Ile-Lys, beginning at Asp-426 of rat SE. Based on the labeling results, three mutant constructs were made. First, the D426A and K428A constructs showed a 5- to 8-fold reduction in SE activity compared with wild-type enzyme, while little change was observed in the I427A mutant. Second, a set of five mutant constructs was prepared for the conserved region based on the structure of the flavoprotein p-hydroxybenzoate hydroxylase (PHBH). Compared with wild-type, D284A and D407A showed less than 25% SE activity. This reduction also appeared to correlate with reduced affinity of the mutant proteins for FAD. Finally, each of the seven Cys residues of rrSE were individually mutated to Ala. Three Cys substitutions had no effect on SE activity, and substitutions at Cys-500 and Cys-533 showed a 50% lower SE activity. Mutations at Cys-490 and Cys-557 produced proteins with negligible SE activity, implicating these residues as being either structurally or catalytically essential. Chemical modification of wildtype and Cys mutants with a thiol-modifying reagent support the existence of a disulfide bond between Cys-490 and Cys-557.

Affinity Labels↗

Interaction between caltropin and the C-terminal region of smooth muscle caldesmon.

Caltropin (CaT) binds caldesmon (CaD) in a Ca(2+)-dependent manner with an affinity higher than that of calmodulin (CaM). Photo-crosslinking between CaT and a benzophenone-labeled C-terminal CaD fragment (27K) results in a 35-kDa protein that corresponds to the 1:1 adduct between CaT and 27K. In the absence of Ca2+, no crosslinking is obtained. This result is similar to that obtained with CaM and 27K. The apparent affinity of CaM for GS17C, a CaM-binding peptide of CaD, is weakened by CaT, suggesting CaT competes with CaM for the peptide. In contrast to CaM, CaT does not induce changes in the tryptophan fluorescence of GS17C. Thus although the two Ca(2+)-binding proteins behave similarly, there are differences in their interactions with CaD.

Animals↗

Oligosaccharyltransferase: a complex multisubunit enzyme of the endoplasmic reticulum.

The attachment of N-linked oligosaccharide chains to proteins is an important cotranslational process. These chains can, in some cases, serve to stabilize the protein, while in other cases they function as recognition elements. A key enzyme in the N-glycosylation process is oligosaccharyltransferase (OT). In yeast this enzyme, which is found in the endoplasmic reticulum, consists of nine different transmembrane protein subunits. Our general aim is to learn more about the functions of the multiple subunits of yeast OT and their mode of interaction with each other. Using a combination of biochemical and genetic techniques the subunit Ost1p has been shown to recognize Asn-X-Ser/Thr glycosylation sites. The principle tool used in the identification process was a benzophenone-based glycosylation site peptide that was shown to be crosslinked to Ost1p. Our current objective is to identify the domain in the primary structure that is involved in recognition of the glycosylation site sequence. By use of bifunctional crosslinkers, the possible interaction of Ost1p with other subunits of OT will be studied. This work and other studies on the OT subunits are concisely summarized.

Endoplasmic Reticulum↗

Ketone EC50 values in the Microtox test.

The Microtox EC50 values for the following ketones are reported in the following homologous series: straight chain methyl ketones (acetone, 2-butanone, 2-pentanone, 2-hepatonone, 2-octanone, 2-decanone, and 2-tridecanone); methyl ketones substituted at one alpha carbon (3-methyl-2-butanone; 3,3-dimethyl-2-butanone); methyl substituted at two alpha carbons (2,4-dimethyl-3-pentanone; 2,2,4,4-tetramethyl-3-pentanone); phenyl groups replacing methyl in acetone (acetophenone; benzophenone); methyl groups substituted at the alpha carbons of cyclohexanone; and 2,3- 2,4-, and 2,5-hexanediones, most for the first time. While there were linear relationships between log EC50 and MW for the straight chain methyl ketones, and for methyl substitution at the alpha carbon for methyl ketones, there were no other linear relationships. As molecular weight increased, the EC50 values of soluble ketones decreased; as distance between two carbonyl groups decreased so too did EC50 values. Thus, for the ketones the geometry around the carbonyl group is an important determinant of toxicity as well as MW, water solubility, and octanol/water coefficient.

Ketones↗

A modification to the COSMIC parameterisation using ab initio constrained potential functions.

The H..H non-bonded potential employed in the current COSMIC force field has been contrasted with H..H potentials used in a number of other force fields. Initial conversion of the variety of functions to a Morse format, achieved using a simple graphical fitting procedure, allowed a direct comparison to be made, showing the COSMIC potential to differ considerably from the other potentials. This difference was reflected in the failure of COSMIC to reproduce ab initio and experimental energies for molecules with significant H..H interactions, with particular reference to the energy curves of benzophenone and diphenyl ether. Considerable improvement in these energies is produced by the use of a Morse function originally based on the H..H potential used in MM3.

Algorithms↗

[Skin tanning and light protection-theory, agents and analytic aspects. 1. Skin tanning and skin tanning agents].

The different approaches to achieving skin tanning with emphasis on natural tanning, due to UV radiation of the sun or solaria, and artificial tanning through tannings agents are described. In contrast to the artificial tan the natural tan is a protection against UV radiation and is caused by pigmentation (melanin formation). The artificial tanning is based on a chemical reaction usually by means of dihydroxy acetone (melanoidin formation). Reaction mechanisms by both agents are reported. The determination of dihydroxy acetone in cosmetic tanning preparations by TLC, GC, HPLC, enzymatic tests and by a new method for capillary GC including sample preparation is described. When exceeding the natural protection mechanisms the skin should be protected by physico-chemical measures. The active principles in sunscreens are the UV filtering substances. UV filters permitted in the EG are listed. 2-Hydroxy benzophenone is used as an example to explain the mechanism of UV filtering substances. Analysis of UV filtering substances in cosmetic sunscreens by TLC, GC, HPLC is described as well as a fast and simple method for separation of lipophilic UV filters followed by capillary GC determination.

Chromatography, Gas↗

Photochemical transformation of the DDT and methoxychlor degradation products, DDE and DMDE, by sunlight.

DDE and DMDE, degradation products of the pesticides DDT and methoxychlor, rapidly undergo an unusual photoisomerization in solution when exposed to sunlight. The isomerization involves the exchange of a vinyl chlorine and an ortho aromatic hydrogen. Other photoproducts identified were corresponding benzophenones and 1,1-diaryl-2-chloroethylenes. Quantum yields for the reactions were measured and then used to compute sunlight photolysis half-lives for DMDE and DDE. Although both compounds absorb only the short-wavelength ultraviolet component of sunlight, their photolysis was found to be surprisingly rapid. During summer at latitude 40 degrees N, the photolysis half-lives near the surface of a water body are one hour and one day for dissolved DMDE and DDE, respectively. Photolysis of the DDE photoisomers is about an order of magnitude slower than that of DDE, suggesting that they may accumulate under environmental conditions. The DDE photoisomers photocyclize to form chlorinated dibenzofulvene and dichlorofluorenone. Neither DDE nor its photoisomers photoreact in solution to form PCB's. The environmental significance of these results is discussed, and its is suggested that the persistence of DDE in inland surface waters may be related to its tendency to sorb onto sediments and biota where not light is present.

Dichlorodiphenyl Dichloroethylene↗

New prostaglandin synthetase inhibitors--di-and triphenylacrylonitriles.

16 triphenylacrylonitriles (TPE) or diphenylacrylonitriles (DPE) were synthesized by condensation of various benzophenones or benzaldehydes with various phenylacetonitriles. The pharmacological potency of these compounds was studied by incubation of bovine seminal vesicle microsomes and PG-RIA. The results showed that the potency of inhibition of PG biosynthetase of DPE was stronger than that of TPE. Compounds with electron-releasing functional groups were proved to be more effective than those with electron-attracting groups. Compound DPE-9 was the most active one, the potency of which was 40 times stronger than that of naproxen. The structure of some compounds has been analysed by X-ray diffraction. The relationship between structure and activity was investigated by means of X-ray diffraction, UV and NMR.

Acrylonitrile↗

Design of histamine H3-receptor agonists and antagonists.

The development of highly potent and selective ligands for the characterization of histamine H3-receptors is reviewed. In the field of agonists stereoselectively methylated derivatives of the natural ligand are found to have the desired pharmacodynamic properties. Pharmacokinetic properties could be improved by forming bioreversible azomethine prodrugs of the primary amine with benzophenone derivatives. In the antagonist field a number of new leads belonging to different chemical classes are discovered. Potential compounds for drug development are identified. The radiolabelled probe [125I]iodoproxyfan shows high potency and selectivity in functional and binding studies. It is a useful compound for binding assays as well as for the detection and localization of histamine H3-receptors.

Animals↗

Metabolism and pharmacokinetics of metaclazepam (Talis), Part III: Determination of the chemical structure of metabolites in dogs, rabbits and men.

The metabolism of 7-bromo-1-methyl-2-methoxymethyl-5-(2'-chlorophenyl)-2, 3-dihydro-1H-1,4-benzodiazepine (metaclazepam, Talis) in animals and men is described. Based upon mass spectrometry fifteen metabolites could be identified. Qualitative and quantitative differences in the biotransformation products of metaclazepam in comparison with the well known metabolites of other drugs in the 1,4-benzodiazepine class could be demonstrated. Metabolites with a benzodiazepine-2-one structure representing the most characteristic feature of other 1,4-benzodiazepines and their metabolites, were found in trace amounts only. The major metabolic pathways of metaclazepam led via stepwise demethylation of the O-methyl and/or the N-methyl group to O-demethyl-metaclazepam (M 2), N-demethyl-metaclazepam (M 7) and bis-demethyl-metaclazepam (M 6). Further aromatic hydroxylation yielded the metabolite M 1. Two metabolites with amino-benzophenone structure (M 5, M 8) which are in general known to result from other 1,4-benzodiazepines could be detected. Additionally a 3-oxo-benzodiazepine (M 4) was found. Minor biotransformation pathways led to a chlorophenyl-bromo-benzodiazepine (M 9) by loss of the side chain from bis-demethyl-metaclazepam and N-demethyl-metaclazepam. By further oxidation and degradation the 2-oxo-benzodiazepine M 10 and the dihydro-quinazoline M 12 were formed. The respective N-methylated metabolites M 13 and M 16 were possibly generated by the same pathway. Still open is the formation of M 15, a 1-methyl-3-hydroxy-4-(2'-chlorophenyl)-6-bromo-1,2-dihydroquinoline and M 11, a 2-methyl-4-(2'-chlorophenyl)-6-bromo-quinazoline. The substitution of bromine by a hydroxyl group during the formation of M 14 can be explained by a NIH-shift mechanism. Quantitative investigations show that the methoxymethyl side chain in the benzodiazepine ring system of metaclazepam acts as an effective barrier with respect to the metabolic attack at position two. We assume that this barrier only can be overcome by complete side chain degradation. This multi-step reaction can hardly compete with more favourable and faster conjugation and elimination processes.

Adult↗

Study on the mechanism of photosensitive dermatitis caused by ketoprofen in the guinea pig.

To investigate the mechanism on photosensitive dermatitis caused by ketoprofen (KP) in humans, the following experiments were performed by topical application on guinea pigs. The phototoxicity study involving treatment with 10% solution of KP, its enantiomers (R-KP and S-KP), loxoprofen, and flurbiprofen revealed no phototoxic reactions. In the photoallergenicity study, KP and its enantiomers (0.5-2% solution) induced skin reaction at all dosages; however, loxoprofen and flurbiprofen (1-5% solution) did not induce such a photoallergenic reaction. These results suggest that the chemical structure of the benzophenone chromophore in KP would be one of the important factors for induction of the photoallergy since both loxoprofen and flurbiprofen do not possess this structure and hence lack photoallergenic potential. Furthermore, to assess time profiles of KP concentration in the skin and plasma, guinea pigs received a repeated topical application of R-KP and S-KP at a dosage of 40 mg/kg over a period of 3 days. Plasma KP concentrations were extremely low as compared to skin KP concentrations and were not detected at 72 h after the final dosing. At 24 h after the final dosing, KP concentrations in the skin with R-KP and S-KP treatment were 187.4 and 254.7 microg/g, respectively, and their half-lives were 80.5 and 84.4 h, respectively. KP concentrations at 336 h after final dosing were 11.3 microg/g for R-KP and 15.7 microg/g for S-KP treatment. The acylglycerol-combined KP concentrations at 336 h were 2% or less as compared to KP concentrations with R-KP and S-KP treatment. There were no differences in KP concentrations in the skin between R-KP and S-KP and in combined KP concentrations between the enantiomers. The present study indicates that photosensitive dermatitis after topical application of KP in humans, caused by photoallergenicity and not phototoxicity, can be reproduced in the animal testing, and suggests that the skin reaction may be caused by the long period of retention of KP in the skin.

Administration, Topical↗

A study on photolinkers used for biomolecule attachment to polymer surfaces.

The use of photolinkers (photoactivatable heterobifunctional crosslinkers) is a popular method to attach biomolecules to polymer surfaces. This study addresses the selection of photolinker and the adjustment of reaction conditions, such as the concentration of biomolecule applied, and irradiation time. The influence of these variables are investigated for four prominent photolinkers: ketyl-reactive benzophenone (BP) and anthraquinone (AQ), nitrene-reactive nitrophenyl azide (NPA), and carbene-reactive phenyl-(trifluoromethyl)diazirine (PTD). The influence of substrate material is discussed, and three different polymers served as representative substrates: poly(methyl methacrylate) (PMMA), polystyrene (PS), and a cycloolefin copolymer (COC). We compared the overall photolinking efficiency of all photolinkers with respect to the polymer substrate they are applied to, and we found considerable differences for certain photolinker/substrate combinations. Of all photolinkers and substrates tested, PTD as photolinker and COC as substrate showed the highest photolinking efficiencies and fastest reaction times. For this study DNA oligonucleotides were chosen as a model system of biomolecular probes, and fluorescence detection of DNA microarrays served as method of detection.

Cross-Linking Reagents↗

Isolation and characterization of a novel polychlorinated biphenyl-degrading bacterium, Paenibacillus sp. KBC101.

The biphenyl-utilizing bacterial strain KBC101 has been newly isolated from soil. Biphenyl-grown cells of KBC101 efficiently degraded di- to nonachlorobiphenyls. The isolate was identified as Paenibacillus sp. with respect to its 16S rDNA sequence and fatty acid profiles, as well as various biological and physiological characteristics. In the case of highly chlorinated biphenyl (polychlorinated biphenyl; PCB) congeners, the degradation activities of this strain were superior to those of the previously reported strong PCB degrader, Rhodococcus sp. RHA1. Recalcitrant coplanar PCBs, such as 3,4,3',4'-CB, were also efficiently degraded by strain KBC101 cells. This is the first report of a representative of the genus Paenibacillus capable of degrading PCBs. In addition to growth on biphenyl, strain KBC101 could grow on dibenzofuran, xanthene, benzophenone, anthrone, phenanthrene, naphthalene, fluorene, fluoranthene, and chrysene as sole sources of carbon and energy. Paenibacillus sp. strain KBC101 presented heterogeneous degradation profiles toward various aromatic compounds.

Bacillaceae↗

Biphenyl synthase, a novel type III polyketide synthase.

Biphenyls and dibenzofurans are the phytoalexins of the Maloideae, a subfamily of the economically important Rosaceae. The carbon skeleton of the two classes of antimicrobial secondary metabolites is formed by biphenyl synthase (BIS). A cDNA encoding this key enzyme was cloned from yeast-extract-treated cell cultures of Sorbus aucuparia. BIS is a novel type III polyketide synthase (PKS) that shares about 60% amino acid sequence identity with other members of the enzyme superfamily. Its preferred starter substrate is benzoyl-CoA that undergoes iterative condensation with three molecules of malonyl-CoA to give 3,5-dihydroxybiphenyl via intramolecular aldol condensation. BIS did not accept CoA-linked cinnamic acids such as 4-coumaroyl-CoA. This substrate, however, was the preferential starter molecule for chalcone synthase (CHS) that was also cloned from S. aucuparia cell cultures. While BIS expression was rapidly, strongly and transiently induced by yeast extract treatment, CHS expression was not. In a phylogenetic tree, BIS grouped together closely with benzophenone synthase (BPS) that also uses benzoyl-CoA as starter molecule but cyclizes the common intermediate via intramolecular Claisen condensation. The molecular characterization of BIS thus contributes to the understanding of the functional diversity and evolution of type III PKSs.

Acyltransferases↗

Benzoic acid biosynthesis in cell cultures of Hypericum androsaemum.

Biosynthesis of benzoic acid from cinnamic acid has been studied in cell cultures of Hypericum androsaemum L. The mechanism underlying side-chain shortening is CoA-dependent and non-beta-oxidative. The enzymes involved are cinnamate:CoA ligase, cinnamoyl-CoA hydratase/lyase and benzaldehyde dehydrogenase. Cinnamate:CoA ligase was separated from benzoate:CoA ligase and 4-coumarate:CoA ligase, which belong to xanthone biosynthesis and general phenylpropanoid metabolism, respectively. Cinnamoyl-CoA hydratase/lyase catalyzes hydration and cleavage of cinnamoyl-CoA to benzaldehyde and acetyl-CoA. Benzaldehyde dehydrogenase finally supplies benzoic acid. In cell cultures of H. androsaemum, benzoic acid is a precursor of xanthones, which accumulate during cell culture growth and after methyl jasmonate treatment. Both the constitutive and the induced accumulations of xanthones were preceded by increases in the activities of all benzoic acid biosynthetic enzymes. Similar changes in activity were observed for phenylalanine ammonia-lyase and the xanthone biosynthetic enzymes benzoate:CoA ligase and benzophenone synthase.

Acetates↗