Josef Zapp

Biosynthesis of the isoprene units of chamomile sesquiterpenes

Abstract Chamomile sesquiterpenes were labelled with 13 C by injection of an [1- 13 C]glucose solution into the anthodia of the plant. The sesquiterpenes bisabololoxide A and chamazulene were isolated from the hydrodistillate of the labelled flowers. Analysis of the labelling patterns and absolute 13 C abundances using quantitative 13 C NMR spectroscopy showed that two of the isoprene building blocks were predominantly formed via the new triose\pyruvate pathway, whereas the third unit was of mixed origin, being derived from both the mevalonic acid pathway and the triose\pyruvate pathway.

Myxovirescin A biosynthesis is directed by hybrid polyketide synthases/nonribosomal peptide synthetase, 3-hydroxy-3-methylglutaryl-CoA synthases, and trans-acting acyltransferases.

Myxococcus xanthus DK1622 is shown to be a producer of myxovirescin (antibiotic TA) antibiotics. The myxovirescin biosynthetic gene cluster spans at least 21 open reading frames (ORFs) and covers a chromosomal region of approximately 83 kb. In silico analysis of myxovirescin ORFs in conjunction with genetic studies suggests the involvement of four type I polyketide synthases (PKSs; TaI, TaL, TaO, and TaP), one major hybrid PKS/NRPS (Ta‐1), and a number of monofunctional enzymes similar to the ones involved in type II fatty‐acid biosyntesis (FAB). Whereas deletion of either taI or taL causes a dramatic drop in myxovirescin production, deletion of both genes (ΔtaIL) leads to the complete loss of myxovirescin production. These results suggest that both TaI and TaL PKSs might act in conjunction with a methyltransferase, reductases, and a monooxygenase to produce the 2‐hydroxyvaleryl–S–ACP starter that is proposed to act as the biosynthetic primer in the initial condensation reaction with glycine. Polymerization of the remaining 11 acetates required for lactone formation is directed by 12 modules of Ta‐1, TaO, and TaP megasynthetases. All modules, except for the first module of TaL, lack cognate acyltransferase (AT) domains. Furthermore, deletion of a discrete tandem AT—encoded by taV—blocks myxovirescin production; this suggests an “in trans” mode of action. To embellish the macrocycle with methyl and ethyl moieties, assembly of the myxovirescin scaffold is proposed to switch twice from PKS to 3‐hydroxy‐3‐methylglutaryl–CoA (HMG–CoA)‐like biochemistry during biosynthesis. Disruption of the S‐adenosylmethionine (SAM)‐dependent methyltransferase, TaQ, shifts production toward two novel myxovirescin analogues, designated myxovirescin Qa and myxovirescin Qc. NMR analysis of purified myxovirescin Qa revealed the loss of the methoxy carbon atom. This novel analogue lacks bioactivity against E. coli.

A new Bacillus megaterium whole-cell catalyst for the hydroxylation of the pentacyclic triterpene 11-keto-β-boswellic acid (KBA) based on a recombinant cytochrome P450 system

The use of cytochromes P450 for the regio- and stereoselective hydroxylation of non-activated carbon atoms in biotechnological applications reflects an efficient and cost-effective alternative in comparison to classical organic chemistry. The prokaryotic cytochrome P450 CYP106A2 from Bacillus megaterium ATCC 13368 hydroxylates a variety of 3-oxo-Δ4 steroids and recently it was identified to carry out a one-step regioselective allylic hydroxylation of the diterpene abietic acid. The anti-inflammatory pentacyclic triterpene 11-Keto-β-boswellic acid (KBA) was found to be a further substrate of CYP106A2, being the first report of a pentacyclic triterpene conversion by a prokaryotic P450. The reaction products were analyzed by HPLC and the corresponding kinetic parameters were investigated. Structure determination of the main product by NMR revealed a 15α-hydroxylation of this substrate. In order to overcome the inability of a recombinant P450 whole-cell system in E. coli for the uptake of acids with terpene structure, we developed for the first time an expression system for cytochromes P450 in B. megaterium (strains MS941 and ATCC 13368). Interestingly, CYP106A2 was only successfully expressed in the plasmid-less B. megaterium strain MS941 but not in ATCC13368. This recombinant system, with the co-expressed heterologous redox chain of the P450, bovine adrenodoxin reductase (AdR), and bovine adrenodoxin (Adx), was applied for the whole-cell conversion of KBA. The formation of 15α-hydroxy-KBA was increased 15-fold in comparison with the naturally CYP106A2-expressing B. megaterium strain ATCC 13368.

Identification of CYP106A2 as a Regioselective Allylic Bacterial Diterpene Hydroxylase

The cytochrome P450 monooxygenase CYP106A2 from Bacillus megaterium ATCC 13368 catalyzes hydroxylations of a variety of 3‐oxo‐Δ4‐steroids such as progesterone and deoxycorticosterone (DOC), mainly in the 15β‐position. We combined a high‐throughput screening and a rational approach for identifying new substrates of CYP106A2. The diterpene resin acid abietic acid was found to be a substrate and was docked into the active site of a CYP106A2 homology model to provide further inside into the structural basis of the regioselectivity of hydroxylation. The products of the hydroxylation reaction were analyzed by HPLC and the Vmax and Km values were calculated. The corresponding reaction products were analyzed by NMR spectroscopy and identified as 12α‐ and 12β‐hydroxyabietic acid. CYP106A2 was therefore identified as the first reported bacterial cytochrome P450 diterpene hydroxylase. Furthermore, an effective whole‐cell catalyst for the selective allylic 12α‐ and 12β‐hydroxylation was applied to produce the hydroxylated products.

Phase I and II metabolites of speciogynine, a diastereomer of the main Kratom alkaloid mitragynine, identified in rat and human urine by liquid chromatography coupled to low- and high-resolution linear ion trap mass spectrometry

Mitragyna speciosa (Kratom in Thai), a Thai medical plant, is misused as herbal drug of abuse. Besides the most abundant alkaloids mitragynine (MG) and paynantheine (PAY), several other alkaloids were isolated from Kratom leaves, among them the third abundant alkaloid is speciogynine (SG), a diastereomer of MG. The aim of this present study was to identify the phase I and II metabolites of SG in rat urine after the administration of a rather high dose of the pure alkaloid and then to confirm these findings using human urine samples after Kratom use. The applied liquid chromatography coupled to low- and high-resolution mass spectrometry (LC-HRMS-MS) provided detailed information on the structure in the MS(n) mode particularly with high resolution. For the analysis of the human samples, the LC separation had to be improved markedly allowing the separation of SG and its metabolites from its diastereomer MG and its metabolites. In analogy to MG, besides SG, nine phase I and eight phase II metabolites could be identified in rat urine, but only three phase I and five phase II metabolites in human urine. These differences may be caused by the lower SG dose applied by the user of Kratom preparations. SG and its metabolites could be differentiated in the human samples from the diastereomeric MG and its metabolites comparing the different retention times determined after application of the single alkaloids to rats. In addition, some differences in MS(2) and/or MS(3) spectra of the corresponding diastereomers were observed.

ent-Clerodane diterpenes and other constituents from the liverwort Adelanthus lindenbergianus (Lehm.) Mitt.

Eleven ent-clerodanes, 13-hydroxy-cis-ent-cleroda-3,14-diene, 15-hydroxy-cis-ent-cleroda-3,13(E)-diene, 1beta,12:15,16-diepoxy-cis-ent-cleroda-13(16),14-dien-18alpha,6alpha-olide, 8beta,12:15, 16-diepoxy-cis-ent-cleroda-13(16),14-dien-18alpha,6alpha-olide, 1beta,16:15,16-diepoxy-cis-ent-cleroda-12,14-dien-18alpha,6alpha-olide, 7beta,12:8beta,12-diepoxy-15-hydroxy-cis-ent-cleroda-13-en-16,15:18alpha,6alpha-diolide, 7beta,12:8beta,12-diepoxy-16-hydroxy-cis-ent-cleroda-13-en-15,16:18alpha,6alpha-diolide, 1alpha-acetoxy-8beta,12-epoxy-15-hydroxy-cis-ent-cleroda-13-en-16,15:18alpha,6alpha-diolide, 1beta,12-epoxy-16-hydroxy-cis-ent-cleroda-13-en-15,16:18alpha,6alpha-diolide, 8beta,12-epoxy-15-hydroxy-trans-cleroda-13-en-16,15:18alpha,6alpha-diolide, 8beta,12-epoxy-16-hydroxy-trans-cleroda-13-en-15,16:18alpha,6alpha-diolide along with the known clerodane diterpenes anastreptin and orcadensin have been isolated from the liverwort Adelanthus lindenbergianus (Lehm.) Mitt. Furthermore, three eudesmane sesquiterpenes together with the known (-)-1beta,10-epoxyaristolan, 3,4-seco-4(23),20(29)-lupadien-3,28-dicarboxylic acid dimethyl ester and two acetophenone derivatives were identified by spectroscopic methods, essentially MS and NMR experiments.

Use of liquid chromatography coupled to low- and high-resolution linear ion trap mass spectrometry for studying the metabolism of paynantheine, an alkaloid of the herbal drug Kratom in rat and human urine

AbstractThe Thai medicinal plant Mitragyna speciosa (Kratom in Thai) is misused as a herbal drug of abuse. During studies on the main Kratom alkaloid mitragynine (MG) in rats and humans, several dehydro analogs could be detected in urine of Kratom users, which were not found in rat urine after administration of pure MG. Questions arose as to whether these compounds are formed from MG only by humans or whether they are metabolites formed from the second abundant Kratom alkaloid paynantheine (PAY), the dehydro analog of MG. Therefore, the aim of the presented study was to identify the phase I and II metabolites of PAY in rat urine after administration of the pure alkaloid. This was first isolated from Kratom leaves. Liquid chromatography–linear ion trap mass spectrometry provided detailed structure information of the metabolites in the MSn mode particularly with high resolution. Besides PAY, the following phase I metabolites could be identified: 9-O-demethyl PAY, 16-carboxy PAY, 9-O-demethyl-16-carboxy PAY, 17-O-demethyl PAY, 17-O-demethyl-16,17-dihydro PAY, 9,17-O-bisdemethyl PAY, 9,17-O-bisdemethyl-16,17-dihydro PAY, 17-carboxy-16,17-dihydro PAY, and 9-O-demethyl-17-carboxy-16,17-dihydro PAY. These metabolites indicated that PAY was metabolized via the same pathways as MG. Several metabolites were excreted as glucuronides or sulfates. The metabolism studies in rats showed that PAY and its metabolites corresponded to the MG-related dehydro compounds detected in urine of the Kratom users. In conclusion, PAY and its metabolites may be further markers for a Kratom abuse in addition of MG and its metabolites. FigureIsolation of paynantheine from kratom leaves; high-resolution mass spectrum of the glucuronide of its 9-O-demethyl metabolite, and paynantheine structure with marked sides of biotransformation.

Physiological changes and UV protection in the aquatic liverwort Jungermannia exsertifolia subsp. cordifolia along an altitudinal gradient of UV-B radiation

Here we report the effects of a natural altitudinal gradient of UV-B radiation, from 1140 to 1816 m altitude, on the physiology of the aquatic liverwort Jungermannia exsertifolia Steph. subsp. cordifolia (Dumort.) Váña collected in mountain streams. Photosynthetic pigments, net photosynthesis and dark respiration rates, chlorophyll fluorescence, protein concentration, sclerophylly, and UV-absorbing compounds [both global UV absorbance of methanol-extractable UV-absorbing compounds (MEUVAC) and concentrations of five individual compounds] were measured. Two new caffeic acid derivatives were discovered: 5″-(7″,8″-dihydroxycoumaroyl)-2-caffeoylmalic acid and 5″-(7″,8″-dihydroxy-7-O-β-glucosyl-coumaroyl)-2-caffeoylmalic acid, whereas three additional compounds were already known in other species: p-coumaroylmalic acid, phaselic acid (both compounds in their cis- and trans- forms) and feruloylmalic acid. Most physiological variables changed considerably along the altitudinal gradient, but only six showed significant linear relationships with altitude: MEUVAC levels, the concentrations of the two new secondary compounds, the maximal apparent electron transport rate through PSII (ETRmax) and the maximal non-photochemical quenching (NPQmax) increased with altitude, whereas photoinhibition percentage decreased. A principal components analysis (PCA) was conducted to rank the values of the physiological and ecological variables obtained along the altitudinal transect, showing that those variables correlated with altitude were responsible for the ordination of the sampling points. The liverwort was not adversely affected by the changing conditions along the altitudinal gradient and, in particular, by the increasing UV-B irradiance, probably because the characteristics shown by high-altitude populations may confer tolerance to high UV-B levels. The response to UV-B of the two new compounds suggests that they could be used as indicators of the spatial changes in UV-B radiation.

Lignan derivatives from the liverwort Bazzania trilobata

Eight lignan derivatives trilobatin D-K, as well as jamesopyrone were isolated from the liverwort Bazzania trilobata. Their structures have been elucidated based on extensive NMR spectral evidence.

Chlorinated macrocyclic bisbibenzyls from the liverwort Bazzania trilobata

Abstract Bazzanines A-J, 10 cyclic bis bibenzyl derivatives with two biphenyl linkages and substituted with 1–6 chlorine atoms, as well as Bazzanin K, a new dichlorinated macrocycle consisting of a phenanthrene and a bibenzyl moiety also connected with two biphenyl linkages, have been isolated from the liverwort Bazzania trilobata . The structures have been elucidated based on extensive NMR spectral evidence and by mass spectrometry.