Ludger Ernst

Chemotypes of two pyrrolizidine alkaloid-containing Senecio species

Evaluation of the pyrrolizidine alkaloids (PAs) of more than 100 populations of Senecio jacobaea and of ca 25 populations of S. erucifolius from different geographic locations revealed the existence of different chemotypes. Two chemotypes were established within each species. They are for S. jacobaea, (a) ‘jacobine chemotype’ with jacobine and its derivatives as major PAs, representing the well known ‘classical’ PA pattern of this species. (b)‘erucifoline chemotype’, with erucifoline and O-acetylerucifoline as dominating PAs, and for S. erucifolius, (a) ‘erucifoline chemotype’, erucifoline and O-acetylerucifoline as major PAs. (b) ‘eruciflorine chemotype’, with eruciflorine (21-hydroxyintegerrimine) as dominating PA. Eruciflorine was identified as a novel PA. Its structure was elucidated by means of NMR and mass spectrometry. Individual populations representing the different chemotypes within the respective species are indistinguishable from their morphological characters.

Acquired and Partially De Novo Synthesized Pyrrolizidine Alkaloids in Two Polyphagous Arctiids and the Alkaloid Profiles of Their Larval Food-Plants

The profiles of pyrrolizidine alkaloids (PAs) in the two highly polyphagous arctiids Estigmene acrea and Grammia geneura and their potential PA sources in southeastern Arizona were compiled. One of four species of Boraginaceae, Plagiobothrys arizonicus, contained PAs; this is the first PA record for this plant species. The principle PA sources are Senecio longilobus (Asteraceae) and Crotalaria pumila (Fabaceae). The known PA pattern of S. longilobus was extended; the species was found to contain six closely related PAs of the senecionine type. Three novel PAs of the monocrotaline type, named pumilines A–C, were isolated and characterized from C. pumila, a species not studied before. The pumilines are the major PAs in the seeds, while in the vegetative organs they are accompanied by the simple necine derivatives supinidine and as the dominant compound subulacine (1β,2β-epoxytrachelanthamidine). In both plant species, the PAs are stored as N-oxides, except C. pumila seeds, which accumulate the free bases. Great variation in PA composition was observed between local populations of C. pumila. The PA profiles were established for larvae and adults of E. acrea that as larvae had fed on an artificial diet supplemented with crotalaria-powder and of G. geneura fed with S. longilobus. In both experiments, the larvae had a free choice between the respective PA source and diet or food plants free of PAs. The profiles compiled for the two species reflect the alkaloid profiles of their PA sources with one exception, subulacine could never be detected in E. acrea. Besides acquired PAs, insect PAs synthesized from acquired necine bases and necic acids of insect origin were detected in the two arctiid species. These insect PAs that do not occur in the larval food sources accounted for some 40–70% (E. acrea) and 17–37% (G. geneura) of total PAs extracted from the insects. A number of novel insect PAs were identified. Plant-acquired and insect PAs were found to accumulate as N-oxides. The results are discussed in relation to specific biochemical, electrophysiological, and behavioral mechanisms involved in PA sequestration by arctiids.

Identification and quantification of synthetic cannabinoids in 'spice-like' herbal mixtures: a snapshot of the German situation in the autumn of 2012.

Synthetic compounds mimicking cannabis-like effects are a recent trend. Currently, these so-called synthetic cannabinoids are the largest and fastest growing class of newly appearing designer drugs. Many national authorities are continuously adapting their regulations to keep pace with the permanently changing variety of compounds. We have analyzed eight herbal smoking blends containing synthetic cannabinoids. Altogether, nine compounds could be identified, namely AM-2201, AM-2201-pMe (MAM-2201), AM-1220, AM-1220-azepane, UR-144, XLR-11, JWH-122-pentenyl, AM-2232, and STS-135. Newly appearing compounds were isolated by column chromatography and their structures elucidated by 1D- and 2D-nuclear magnetic resonance (NMR) experiments. In addition, the compounds were investigated by electron ionization-mass spectrometry (EI-MS) and electrospray ionization-tandem mass spectrometry (ESI-MS/MS) to complete the physicochemical dataset. Based on the purified compounds a universal gas chromatography-mass spectrometry (GC-MS) method was developed for the identification and quantification of these compounds in commercial smoking blends. By applying this method, up to five different compounds could be found in such products showing total concentrations from 72 to 303 mg/g smoking blend while individual compounds ranged from 0.4 to 303 mg/g. (1)H NMR spectra of the chiral compounds AM-1220 and its azepane-isomer recorded in the presence of 1 equivalent of (R)-(+)-α-methoxy-α-trifluoromethylphenylacetic acid (MTPA, Moshers acid) showed them to be racemic mixtures.

Synthetic cannabinoids in “spice-like” herbal blends: First appearance of JWH-307 and recurrence of JWH-018 on the German market

Herbal smoking blends, available on the German market were analyzed and several known synthetic cannabinoids were identified (JWH-122 and JWH-018). In addition, we isolated a new active ingredient by silica gel column chromatography and elucidated the structure by nuclear magnetic resonance (NMR) methods. The compound was identified as JWH-307, a synthetic cannabinoid of the phenyl-pyrrole subclass with known in vitro binding affinities for cannabinoid receptors. To date, this is the first appearance of this subclass of cannabimimetics in such products. JWH-307 has been further characterized by gas chromatography accurate mass spectrometry (GC-HRMS), electrospray tandem mass spectrometry (ESI-MS/MS), ultraviolet (UV) and infrared (IR) spectroscopy. JWH-018 was among the first compounds banned by many countries world-wide including Germany. The identification of JWH-018 was striking, since this is the first report where JWH-018 recurred on the German market thus violating existing laws. A generic method was established to quantify synthetic cannabinoids in herbal smoking blends. Quantification was achieved using an isotopically labeled standard (JWH-018-D(3)). JWH-018 was found at a level of 150 mg/g while JWH-122 and JWH-307 occurred as a mixture at a total level of 232 mg/g.

Synthesis of galanthamine and related alkaloids - new approaches. I.

Abstract Modifications of protecting groups and of the oxidative coupling conditions lead to pure crystalline intermediates in the synthesis of galanthamine derivatives and gave dienone A in better yields than reported before. The E -configuration of amide 3′ in crystalline state has been determined by X-ray diffraction. Streptomyces affinis 6737 reduces A to the optically active (−)-epigalanthamine derivative C, whose absolute configuration was determined by Bijvoets method. Nematospora corylii CBS 2608 reduces to racemic B. With Ashbya gossypii IFO 1355 a mixture of racemic B and optically active C is obtained. some other microbial transformations are described.

Enantioselective reduction of acetyldimethylphenylsilane by Trigonopsis variabilis (DSM 70714)

SummaryGrowing and resting cells of the yeast Trigonopsis variabilis (DSM 70714) can be used for the enantioselective reduction of the organosilicon compound acetyldimethylphenylsilane (1) to give optically active (R)-(1-hydroxyethyl)dimethylphenylsilane [(R)-2] in good yields. The enantiomeric purity of the isolated product was determined to be 62–86% ee depending on the substrate concentration used. Both substrate and product caused an inhibition of the reaction at concentrations higher than 0.35 and 0.5 g/l, respectively. Besides, higher substrate and product concentrations led to increased formation of the by-product 1,1,3,3-tetramethyl-1,3-diphenyldisiloxane. Considering the limiting substrate and product concentrations, it was possible to use the same biomass at least 5 times without significant loss of enzyme activity. 3-Methyl-3-phenyl-2-butanone (5) and acetyldimethylphenylgermane (7), which represent carbon and germanium analogues of 1, were also found to be accepted as substrates by Trigonopsis variabilis (DSM 70714). The reduction rates of the silicon (1) and germanium compound (7) were much higher than the transformation rate of the corresponding carbon analogue 5.

Cinnamate:CoA Ligase Initiates the Biosynthesis of a Benzoate-Derived Xanthone Phytoalexin in Hypericum calycinum Cell Cultures

Although a number of plant natural products are derived from benzoic acid, the biosynthesis of this structurally simple precursor is poorly understood. Hypericum calycinum cell cultures accumulate a benzoic acid-derived xanthone phytoalexin, hyperxanthone E, in response to elicitor treatment. Using a subtracted complementary DNA (cDNA) library and sequence information about conserved coenzyme A (CoA) ligase motifs, a cDNA encoding cinnamate:CoA ligase (CNL) was isolated. This enzyme channels metabolic flux from the general phenylpropanoid pathway into benzenoid metabolism. HcCNL preferred cinnamic acid as a substrate but failed to activate benzoic acid. Enzyme activity was strictly dependent on the presence of Mg2+ and K+ at optimum concentrations of 2.5 and 100 mm, respectively. Coordinated increases in the Phe ammonia-lyase and HcCNL transcript levels preceded the accumulation of hyperxanthone E in cell cultures of H. calycinum after the addition of the elicitor. HcCNL contained a carboxyl-terminal type 1 peroxisomal targeting signal made up by the tripeptide Ser-Arg-Leu, which directed an amino-terminal reporter fusion to the peroxisomes. Masking the targeting signal by carboxyl-terminal reporter fusion led to cytoplasmic localization. A phylogenetic tree consisted of two evolutionarily distinct clusters. One cluster was formed by CoA ligases related to benzenoid metabolism, including HcCNL. The other cluster comprised 4-coumarate:CoA ligases from spermatophytes, ferns, and mosses, indicating divergence of the two clades prior to the divergence of the higher plant lineages.

Pyrrolizidine Alkaloids in the Food Chain: Development, Validation, and Application of a New HPLC-ESI-MS/MS Sum Parameter Method

Contamination of food and feed with pyrrolizidine alkaloids is currently discussed as a potential health risk. Here, we report the development of a new HPLC-ESI-MS/MS sum parameter method to quantitate the pyrrolizidine alkaloid content in complex food matrices. The procedure was validated for honey and culinary herbs. Isotopically labeled 7-O-9-O-dibutyroyl-[9,9-(2)H2]-retronecine was synthesized and utilized as an internal standard for validation and quantitation. The total pyrrolizidine alkaloid content of a sample is expressed as a single sum parameter: retronecine equivalents (RE). Ld/Lq for honey was 0.1 μg RE/kg/0.3 μg RE/kg. For culinary herbs, 1.0 μg RE/kg/3.0 μg RE/kg (dry weight, dw) and 0.1 μg RE/kg/0.3 μg RE/kg (fresh weight, fw) were determined, respectively. The new method was applied to analyze 21 herbal convenience products. Fifteen products (71%) were pyrrolizidine alkaloid positive showing pyrrolizidine alkaloid concentrations ranging from 0.9 to 74 μg RE/kg fw.

Microbial oxidation of tricyclic sesquiterpenoids containing a dimethylcyclopropane ring

Abstract Calarene was oxidized by Bacillus megaterium and Diplodia gossypina to give allylic calarenols and calarendiols in which either of the geminal methyl groups of the cyclopropane ring was hydroxylated. Globulol was hydroxylated faster and in higher yields than calarene by both strains. In the case of this compound, vicinal diols were formed and, again, either of the geminal methyl groups was oxidized. Only bacterial strains caused 9-hydroxylation. Mycobacterium smegmatis produced globulol-14-exo-14-oic acid in good yields. Of the geminal methyl groups, the one in the exo-orientation was attacked preferentially by all of the strains tested. Some of the metabolites formed are stereoisomers of known natural products.

A novel entry into a new class of cyclophane derivatives: synthesis of (±)-[2.2]paracyclophane-4-thiol ☆

Abstract Two syntheses of (±)-[2.2]paracyclophane-4-thiol have been accomplished in two and five steps, with overall yields of 46 and 31%, respectively, from [2.2]paracyclophane. The strategy involves two key reactions: (a) a lithium aluminium hydride reduction of the disulphide of [2.2]paracyclophane and (b) the use of a Newman–Kwart reaction for the conversion of [2.2]paracyclophane-4-ol to [2.2]paracyclophane-4-thiol.