Phytochemistry (v.68, #2)

Quaternary protoberberine alkaloids by Lenka Grycová; Jiří Dostál; Radek Marek (150-175).
This contribution reviews some general aspects of the quaternary iminium protoberberine alkaloids, including their plant sources, isolation procedures, analytical methods, spectral data, and reactivity. Examples of pharmacological effects and interactions with biological targets are described.This contribution reviews some general aspects of the quaternary iminium protoberberine alkaloids. The alkaloids represent a very extensive group of secondary metabolites with diverse structures, distribution in nature, and biological effects. The quaternary protoberberine alkaloids (QPA), derived from the 5,6-dihydrodibenzo[a,g]quinolizinium system, belong to a large class of isoquinoline alkaloids.Following a general introduction, the plant sources of QPA, their biosynthesis, and procedures for their isolation are discussed. Analytical methods and spectral data are summarized with emphasis on NMR spectroscopy. The reactivity of QPA is characterized by the sensitivity of the iminium bond C=N+ to nucleophilic attack. The addition of various nucleophiles to the protoberberine skeleton is discussed. An extended discussion of the principal chemical reactivity is included since this governs interactions with biological targets.Quaternary protoberberine alkaloids and some related compounds exhibit considerable biological activities. Recently reported structural studies indicate that the QPA interact with nucleic acids predominantly as intercalators or minor groove binders. Currently, investigations in many laboratories worldwide are focused on the antibacterial and antimalarial activity, cytotoxicity, and potential genotoxicity of QPA.
Keywords: Berberidaceae; Quaternary protoberberine alkaloid; Plant source; Nucleophilic addition; NMR spectroscopy; X-ray diffraction; Cytotoxicity; DNA binding;

Unusual features of a recombinant apple α-farnesene synthase by Sol Green; Ellen N. Friel; Adam Matich; Lesley L. Beuning; Janine M. Cooney; Daryl D. Rowan; Elspeth MacRae (176-188).
Functional and kinetic analysis of a recombinant α-farnesene synthase from apple skin identified a prenyltransferase activity allowing biosynthesis of α-farnesene directly from geranyl diphosphate and isopentenyl diphosphate. Mutagenesis of the DDXXD active site motif abolished α-farnesene and monoterpene synthase, and prenyltransferase activities of this enzyme.A recombinant α-farnesene synthase from apple (Malus  ×  domestica), expressed in Escherichia coli, showed features not previously reported. Activity was enhanced 5-fold by K+ and all four isomers of α-farnesene, as well as β-farnesene, were produced from an isomeric mixture of farnesyl diphosphate (FDP). Monoterpenes, linalool, (Z)- and (E)-β-ocimene and β-myrcene, were synthesised from geranyl diphosphate (GDP), but at 18% of the optimised rate for α-farnesene synthesis from FDP. Addition of K+ reduced monoterpene synthase activity. The enzyme also produced α-farnesene by a reaction involving coupling of GDP and isoprenyl diphosphate but at <1% of the rate with FDP. Mutagenesis of active site aspartate residues removed sesquiterpene, monoterpene and prenyltransferase activities suggesting catalysis through the same active site. Phylogenetic analysis clusters this enzyme with isoprene synthases rather than with other sesquiterpene synthases, suggesting that it has evolved differently from other plant sesquiterpene synthases. This is the first demonstration of a sesquiterpene synthase possessing prenyltransferase activity.
Keywords: α-Farnesene; Prenyltransferase; Sesquiterpene; Terpene synthase; Farnesyl diphosphate; Malus domestica; Apple;

Poppy alkaloid profiling by electrospray tandem mass spectrometry and electrospray FT-ICR mass spectrometry after [ring-13C6]-tyramine feeding by Jürgen Schmidt; Chotima Boettcher; Christine Kuhnt; Toni M. Kutchan; Meinhart H. Zenk (189-202).
[ring-13C6]-Tyramine as a biogenetic precursor of Papaver alkaloids was fed to Papaver somniferum seedlings. The alkaloid pattern was elucidated both by direct infusion high-resolution ESI-FT-ICR mass spectrometry and liquid chromatography/electrospray tandem mass spectrometry. The structure of about 20 alkaloids displaying an incorporation of the [ring-13C6]-labeled tyramine could be elucidated. These alkaloids belong to morphinans, benzylisoquinolines, protoberberines, benzo[c]phenanthridines, phthalide isoquinolines and protopines. The information gained from the alkaloid profile demonstrates that the combination of these two mass spectrometric methods represents a powerful tool for evaluating biochemical pathways and facilitates the study of the flux of distant precursors into these natural products. Papaver alkaloids play a major role in medicine and pharmacy. In this study, [ring-13C6]-tyramine as a biogenetic precursor of these alkaloids was fed to Papaver somniferum seedlings. The alkaloid pattern was elucidated both by direct infusion high-resolution ESI-FT-ICR mass spectrometry and liquid chromatography/electrospray tandem mass spectrometry. Thus, based on this procedure, the structure of about 20 alkaloids displaying an incorporation of the labeled tyramine could be elucidated. These alkaloids belong to different classes, e.g. morphinan, benzylisoquinoline, protoberberine, benzo[c]phenanthridine, phthalide isoquinoline and protopine. The valuable information gained from the alkaloid profile demonstrates that the combination of these two spectrometric methods represents a powerful tool for evaluating biochemical pathways and facilitates the study of the flux of distant precursors into these natural products.
Keywords: Papaver somniferum; Electrospray; MS/MS; Poppy alkaloids; 13C-Labelling; FT-ICR mass spectrometry;

Synergism and redundancy in a plant volatile blend attracting grapevine moth females by Marco Tasin; Anna-Carin Bäckman; Miryan Coracini; Daniel Casado; Claudio Ioriatti; Peter Witzgall (203-209).
Grapevine moth females flew upwind to a blend of β-caryophyllene, (E)-β-farnesene and (E)-4,8-dimethyl-1,3,7-nonatriene. Leaving out any compound from this blend almost abolished attraction. Substituting these three compounds with other grape volatiles, which are perceived by the female antenna, partly restored attraction.A flight tunnel study was done to decipher the behavioral effect of grape odor in grapevine moth Lobesia botrana. A blend of 10 volatile compounds, which all elicit a strong antennal response, attracts mated grapevine moth females from a distance, by upwind orientation flight. These 10 grape volatiles are in part behaviorally redundant, since attraction to a 3-component blend of β-caryophyllene, (E)-β-farnesene and (E)-4,8-dimethyl-1,3,7-nonatriene was not significantly different from the 10-component blend. Blending these three compounds had a strong synergistic effect on female attraction, and omission of any one compound from this 3-component blend almost abolished attraction. It was nonetheless possible to substitute the three compounds with the other grape volatiles which are perceived by the female antenna, to partly restore attraction. Several blends, of varying composition, elicited significant attraction. The observed behavioral plasticity in response to grape volatile blends probably reflects the variation of the natural plant signal, since females oviposit on different grape varieties, in different phenological stages.
Keywords: Vitis vinifera; Vitaceae; Grapevine; Attraction bioassay; Wind tunnel; Plant volatiles; Kairomone; Odor signal; Host finding; Lobesia botrana;

Antibacterials and modulators of bacterial resistance from the immature cones of Chamaecyparis lawsoniana by Eileen C.J. Smith; Elizabeth M. Williamson; Neale Wareham; Glenn W. Kaatz; Simon Gibbons (210-217).
Several anti-staphylococcal diterpenes were isolated from the immature cones of the conifer Chamaecyparis lawsoniana. The most active, ferruginol, caused an 80-fold potentiation of oxacillin activity against EMRSA-15, with more modest potentiation of antibiotic activity against effluxing clinical isolates of Staphylococcus aureus. The results of an efflux inhibition assay suggest that ferruginol is a weak efflux pump inhibitor.As part of an on-going project to characterize compounds from immature conifer cones with antibacterial or modulatory activity against multidrug-resistant (MDR) strains of Staphylococcus aureus, eight compounds were isolated from the cones of Chamaecyparis lawsoniana. The active compounds were mainly diterpenes, with minimum inhibitory concentrations ranging from 4 to 128 μg/ml against MDR effluxing S. aureus strains and two epidemic methicillin-resistant (EMRSA) clinical isolates. The compounds extracted were the diterpenes ferruginol, pisiferol and its epimer 5-epipisiferol, formosanoxide, trans-communic acid and torulosal, the sesquiterpene oplopanonyl acetate and the germacrane 4β-hydroxygermacra-1(10)-5-diene. Some of these compounds also exhibited modulatory activity in potentiating antibiotic activity against effluxing strains and ferruginol, used at a sub-inhibitory concentration, resulted in an 80-fold potentiation of oxacillin activity against strain EMRSA-15. An efflux inhibition assay using an S. aureus strain possessing the MDR NorA efflux pump resulted in 40% inhibition of ethidium bromide efflux at 10 μM ferruginol (2.86 μg/ml). We report the 1H and 13C NMR data for the cis A/B ring junction epimer of pisiferol which we have named 5-epipisiferol. We also unambiguously assign all 1H and 13C NMR resonances for trans-communic acid.
Keywords: Chamaecyparis lawsoniana; Diterpene; MRSA; Antibacterial; Multidrug-resistant; Efflux pump; Modulator; Ferruginol;

Antiproliferative activity is predominantly associated with ellagitannins in raspberry extracts by Heather A. Ross; Gordon J. McDougall; Derek Stewart (218-228).
Raspberry extracts enriched in ellagitannins were more effective in inhibiting the in vitro growth of cancer cells than extracts enriched in anthocyanins.Raspberry extracts enriched in polyphenols, but devoid of organic acids, sugars and vitamin C, were prepared by sorption to C18 solid phase extraction matrices and tested for their ability to inhibit the proliferation of human cervical cancer (HeLa) cells in vitro. The raspberry extract reduced proliferation in a dose-dependent manner whether this was judged by cell number or measurements of cell viability. However, measurements based on cell viability were more accurate and gave an EC50 value of 17.5 μg/ml gallic acid equivalents (GAE) at day 4 of culture.Raspberry extracts were fractionated by sorption to Sephadex LH-20 into an unbound fraction, which was obviously enriched in anthocyanins, and a bound fraction. The unbound anthocyanin-enriched fraction was much less effective in reducing proliferation then the original extract and gave an EC50 value estimated at 67 μg/ml. The LH-20 bound fraction was more effective than the original raspberry extract (EC50  = 13 μg/ml) suggesting that the main anti-proliferative agents were retained in the bound fraction. Analysis of the original extract, the unbound and the LH20 bound fractions by LC–MS confirmed that the unbound fraction was enriched in anthocyanins and the bound fraction primarily contained ellagitannins. The ellagitannin-rich bound fraction had the highest antioxidant capacity as measured by the ferric reducing antioxidant potential (FRAP) assay. The mechanism by which the ellagitannins inhibit proliferation of cancer cells is discussed.
Keywords: Anthocyanins; Antioxidant; Antiproliferation; Cancer; Cell viability; Ellagic acid; Ellagitannins; Growth inhibition; Raspberry;

A cheap and time sparing solid phase biosynthesis of the octadecanoid cis(+)-12-oxo-phyodienoic acid is provided to the “jasmonate community”. With this method high amounts of enantioselective oxylipin metabolites can by synthesized for physiological studies and octadecanoid-metabolomics.The pathway of jasmonic acid (JA) biosynthesis was established in the 1980s by Vick and Zimmerman but, until now, the preparative biosynthesis of the jasmonic acid precursors 12-oxo-phytodienoic acid (OPDA) and 3-oxo-2-[2′-pentenyl]-cyclopentan-1-octanoic acid (OPC-8:0) in their endogenous and biologically relevant cis(+)-configuration was only possible in small amounts and had to put up with high costs. This was mainly due to the lack of high amounts of pure and enzymatically active allene oxide cyclase (AOC), which is a key enzyme in the biosynthesis of jasmonates in that it releases, in a coupled reaction with allene oxide synthase (AOS), the first cyclic and biological active metabolite – OPDA. We describe here the expression and purification of AOS and AOC and their subsequent coupling to solid matrices to produce an enantioselective, reusable bioreactor for octadecanoid production. With the method described here it is possible to produce optically pure enantiomers of octadecanoids in high amounts in a cost- and time-efficient manner. Furthermore, it could be demonstrated that a physical interaction of AOS and AOC, hitherto postulated to be required for substrate channeling from AOS to AOC, is not necessary for the in vitro cyclization of the unstable epoxide generated by the AOS reaction.
Keywords: Allene oxide cyclase; Allene oxide synthase; Jasmonates; Jasmonic acid; 12-Oxo-phytodienoic acid; Octadecanoid pathway; Oxylipins; Plant hormones;

Unrevealed structural requirements for auxin-like molecules by theoretical and experimental evidences by Noel Ferro; Patrick Bultinck; Ana Gallegos; Hans-Jörg Jacobsen; Ramon Carbo-Dorca; Thomas Reinard (237-250).
Quantum chemical methods and biostatistical analysis have detected flexible structure–activity requirements of auxin-like molecules linked to hardness (η, HOMO–LUMO gap). A decarboxylated organobromine compound (2,6-dibromophenol) shows auxin like effects.An computational-biostatistical approach, supported by ab initio optimizations of auxin-like molecules, was used to find biologically meaningful relationships between quantum chemical variables and fresh bioassay’s data. It is proven that the auxin-like recognition requires different molecular assembling states. We suggest that the carboxyl group is not the determining factor in explaining the biological auxin-like conduct. The biological effects depends essentially on the chemical condition of the ring system. The aim to find active molecules (quantum objects) via statistical grouping-analysis of molecular quantum similarity measures was verified by bioactivity assays. Next, this approach led to the discovery of a non-carboxylated active auxin-like molecule (2,6-dibromo-phenol). This is the first publication on structure activity relationship of auxin-like molecules, which relies on highly standardized bioassays of different auxins screened in parallel as well as analysed by multi-dimensional scaling.
Keywords: Auxin; Structure–activity relationship; Molecular quantum similarity measures; Plant growth regulation;

Minor betalains in fruits of Hylocereus species by Sławomir Wybraniec; Barbara Nowak-Wydra; Katarzyna Mitka; Piotr Kowalski; Yosef Mizrahi (251-259).
Betalains of fruit peel and flesh of Hylocereus cacti are presented. The sinapoyl moiety was found in the structure of betacyanins from fruit peel and the migration of the phyllocactin malonyl group was noticed for the first time.Betacyanins in peel and flesh of fruits of different Hylocereus species were identified by means of GC/MS, electrospray MS/MS, HPLC as well as 1H and 13C NMR techniques. As hitherto unknown pigments: betanidin 5-O-(2′-O-β-d-apiofuranosyl)-β-d-glucopyranoside, betanidin 5-O-(4′-O-malonyl)-β-d-glucopyranoside and betanidin 5-O-[(5″-O-E-sinapoyl)-2′-O-β-d-apiofuranosyl]-β-d-glucopyranoside were elucidated. The sinapoyl moiety attachment position in the structure of betacyanins was established for the first time. The peel contained a more complex pattern of betacyanins with apiofuranosyl moiety. Other recently identified pigments were also present in the samples and their 1H or 13C NMR spectra were recorded. In the case of phyllocactin and its 4′-isomer the migration of the malonyl group was noticed.
Keywords: Hylocereus polyrhizus; Hylocereus ocamponis; Hylocereus undatus; Cactaceae; Acylated betacyanins; Betalains; Betanin; Phyllocactin; Hylocerenin; Sinapic acid; Acyl migration;