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An array of analogues of the wasp toxin philanthotoxin-433, in which the asymmetric polyamine moiety was exchanged for spermine and the headgroup replaced with a variety of structurally diverse moieties, was prepared using parallel solid-phase synthesis approaches. In three analogues, the spermine moiety was extended with an amino acid tail, six compounds contained an N-acylated cyclohexylalanine, and four analogues were based on a novel diamino acid design with systematically changed spacer length between N-cyclohexylcarbonyl and N-phenylacetyl substituents. The analogues were studied using two-electrode voltage-clamp electrophysiology employing Xenopus laevis oocytes expressing GluA1(i) AMPA or GluN1/2A NMDA receptors. Several of the analogues showed significantly increased inhibition of the GluN1/2A NMDA receptor. Thus, an analogue containing N-(1-naphtyl)acetyl group showed an IC(50) value of 47 nM. For the diamino acid-based analogues, the optimal spacer length between two N-acyl groups was determined, resulting in an analogue with an IC(50) value of 106 nM.

Introduction – Rhodiola rosea is a broadly used medicinal plant with largely unexplored natural variability in secondary metabolite levels. Objective – The aim of this work was to develop a non-target procedure for 1H NMR spectroscopic fingerprinting of rhizome extracts for pattern recognition analysis and identification of secondary metabolites responsible for differences in sample composition. To achieve this, plants from three different geographic areas (Swiss Alps, Finland, and Altai region in Siberia) were investigated. Results – A sample preparation procedure was developed in order to remove polymeric polyphenols as the 1H NMR analysis of low-molecular-weight metabolites was hampered by the presence of tannins. Principal component analysis disclosed tight clustering of samples according to population. PCA models based on the aromatic region of the spectra showed that the first two components reflected changes in the content of salidroside and rosavin, respectively, the rosavin content being negatively correlated to that of rhodiocyanoside A and minor aromatics. Score plots and non-parametric variance tests demonstrated population-dependent changes according to harvest time. Data consistency was assessed using score plots and box-and-whisker graphs. In addition, a procedure for presenting loadings of PCA models based on bucketed data as high-resolution plots, which are reminiscent of real 1H NMR spectra and help to identify latent biomarkers, is presented. Conclusion – This study demonstrated the usefulness of the established procedure for multivariate non-target 1H NMR metabolic profiling of Rhodiola rosea. Copyright © 2010 John Wiley & Sons, Ltd.

1H NMR metabolic profiles of urine from sheep exposed to prenatal nutritional restriction (n = 19) and a control group with normal prenatal nutritional requirements (n = 19), followed by either conventional (n = 10 + 10) or high carbohydrate high fat postnatal diet (n = 9 + 9), were studied. Urine was sampled from 2, 6, 19 and 24-month-old animals receiving differential dietary treatments during the first 6 months and the same normal diet later. Principal component analysis of 1H NMR spectra (n = 164) showed a V-shaped metabolic trajectory as a function of age and diet, starting with urines with a high amount of glucose, indicative of monogastric-like metabolism, and exhibiting concomitant increase of metabolites related to rumen microflora (mainly glycine conjugates of benzoic and phenylacetic acid) as the ruminal metabolism developed. Urines from young (2-month-old) animals exposed to prenatal undernutrition followed by normal postnatal diet showed metabolic patters that are ahead in time on the metabolic trajectory relative to the prenatal control group. No long-term effects of fetal undernutrition, alone or in combination with postnatal hypernutrition were observed.

The aim of this study was to explore feasibility of 1H NMR metabolic fingerprinting for discrimination of authenticity of saffron using principal component analysis (PCA) modeling. Authentic reference Iranian saffron (n = 31) and commercial samples (n = 32) were used. Cross-validated PCA models based on 1H NMR spectra of solutions prepared by direct extraction of grinded saffron with methanol-d4 distinguished reference Iranian saffron samples from commercial samples that formed several distinct clusters, some of which represent falsified samples as confirmed by microscopic analysis. The production sites and drying conditions of the authentic reference Iranian samples were not reflected in the current dataset. Picrocrocin and glycosyl esters of crocetin emerged as the most important 1H NMR markers of authentic saffron by using statistical correlation spectroscopy. In conclusion, 1H NMR spectra of saffron extracts combined with pattern recognition by PCA provide immediate means of unsupervised classification of saffron samples.

The aim of this study was to investigate structures and acetylcholinesterase inhibitory activities of lycopodane-type alkaloids isolated from an Icelandic collection of Lycopodium annotinum ssp. alpestre. Ten alkaloids were isolated, including annotinine, annotine, lycodoline, lycoposerramine M, anhydrolycodoline, gnidioidine, lycofoline, lannotinidine D, and acrifoline, as well as a previously unknown N-oxide of annotine. 1H and 13C NMR data of several of the alkaloids were provided for the first time. Solvent-dependent equilibrium constants between ketone and hemiketal form of acrifoline were determined. Conformation of acrifoline was characterized using NOESY spectroscopy and molecular modelling. The isolated alkaloids were evaluated for their in vitro inhibitory activity against acetylcholinesterase and butyrylcholinesterase. Ligand docking studies based on mutated 3D structure of Torpedo californica acetylcholinesterase provided rationale for low inhibitory activity of the isolated alkaloids as compared to huperzine A or B, which are potent acetylcholinesterase inhibitors belonging to the lycodine class. Based on the modelling studies the lycopodane-type alkaloids seem to fit well into the active site gorge of the enzyme but the position of their functional groups is not compatible with establishing strong hydrogen bonding interactions with the amino acid residues that line the binding site. The docking studies indicate possibilities of additional functionalization of the lycopodane skeleton to render potentially more active analogues.

In order to clarify the interaction of gossypol with proteins, the pure diastereoisomeric Schiff bases from L-tryptophan methyl ester and both gossypol enantiomers were prepared. Their c.d. and n.m.r. spectra demonstrate that the interaction between gossypol and tryptophan, previously reported to involve a weakly associated complex, consists in Schiff base formation. Recent studies on enzyme inhibition by gossypol are discussed; it is suggested that nonspecific covalent binding of gossypol to proteins may be responsible for a significant proportion of the in vitro effects of gossypol.

article i nfo Article history: Novel peptidomimetic backbone designs with stability towards proteases are of interest for several pharmaceutical applications including intracellular delivery. The present study concerns the cellular uptake and membrane-destabilising effects of various cationic chimeras comprised of alternating N-alkylated β- alanine and α-amino acid residues. For comparison, homomeric peptides displaying octacationic functionalities as well as the Tat47-57 sequence were included as reference compounds. Cellular uptake studies with fluorescently labelled compounds showed that guanidinylated chimeras were taken up four times more efficiently than Tat47-57. After internalisation, the chimeras were localised primarily in vesicular compartments and diffusively in the cytoplasm. In murine NIH3T3 fibroblasts, the chimeras showed immediate plasma membrane permeabilising properties, which proved highly dependent on the chimera chain length, and were remarkably different from the effects induced by Tat47-57. Finally, biophysical studies on model membranes showed that the chimeras in general increase the permeability of fluid phase and gel phase phosphatidylcholine (PC) vesicles without affecting membrane acyl chain packing, which suggests that they restrict lateral diffusion of the membrane lipids by interaction with phospholipid head groups. The α- peptide/β-peptoid chimeras described herein exhibit promising cellular uptake properties, and thus represent proteolytically stable alternatives to currently known cell-penetrating peptides.

The Letter describes an improved, rapid and mild strategy for the loading of primary alcohols onto a polystyrene trityl resin via a highly reactive trityl bromide linker. This protocol facilitates an efficient resin loading even of acid-sensitive or heat-labile alcohols, which otherwise require expensive or non-commercial resin types. Secondary alcohols were only attached in moderate to low yields, while attempts to load a tertiary alcohol expectedly failed. Importantly, selective attachment of diols via a primary alcohol group in the presence of more hindered alcohol groups proved possible. The effects of activation time and reagent excess as well as alcohol structure were investigated. This improved method provides a convenient access to O-linked resin-bound N -Fmoc-protected amino alcohols that may be employed in SPS of peptides with C-terminal alcohol functionalities. In the case of a sensitive alcohol containing an activated aziridine functionality, the use of the trityl bromide linker proved superior to a recently described silver triflate-assisted trityl chloride resin-based procedure.

The hyphenated technique HPLC–SPE–NMR is proving to be a useful analytical tool for structure elucidation of mixture components, particularly for mass-limited samples where traditional isolation procedures are either time consuming or challenging. In this work, we investigated SPE trapping performance of 25 model natural products within a format corresponding to that of HPLC–SPE–NMR hyphenation. Six different silica-based bonded phases and two polymeric phases were evaluated. The trapping efficiency of polystyrene/divinylbenzene polymers was generally superior compared to silica bonded phases, which showed variable results and performed well only with hydrophobic analytes. Acetonitrile concentration in the loading solvent was critical for trapping on polymeric phase (Resin GP), as small changes of the organic solvent concentration (±3%) could alter the trapping efficiency significantly. Flow rate changes of the loading solvent within 0.8–5.0 mL/min did not affect trapping kinetics. Simulation of multiple trapping showed excellent performance of this approach for hydrophobic analytes, and moderate gain for more polar analytes that do not trap quantitatively in a single trapping step. Determination of 50% breakthrough levels by frontal chromatography analysis showed feasibility of accumulation of analyte amounts corresponding to about 0.5 μmol (10 mm × 2 mm i.d. Resin GP cartridge).

Four prenylated acetophenones 2,6-dihydroxy-4-geranyloxyacetophenone (1), 4-geranyloxy-2,6,β-trihydroxyacetophenone (2), 2,6-dihydroxy-4-geranyloxy-3-prenylacetophenone (3), and 4-geranyloxy-3-prenyl-2,6,β-trihydroxyacetophenone (4) have for the first time been isolated from Melicope obscura (1 and 2) and Melicope obtusifolia ssp. obtusifolia var. arborea (3 and 4). The distribution of prenylated acetophenones in Rutaceae is reviewed and the results, including the new records, indicate that prenylated acetophenones are valuable as chemotaxonomic markers for the subfamily Rutoideae, tribe Xanthoxyleae sensu Engler.

N-alkyl-beta-alanine oligomers (beta-peptoids) with alpha-chiral side chains [(R)- or (S)-1-(phenylethyl)amino groups] were synthesized and analyzed by CD spectroscopy. These chiral beta-peptoid homomers exhibited chain-length-dependent and solvent-dependent ellipticity, strongly indicating the presence of a secondary structure in solution. The CD behaviour was only slightly temperature-dependent upon heating, as also previously observed for stable alpha-peptoid helices containing the same type of side chains. Thus, the data presented here comprise the first evidence for a chain length-dependent secondary folding of compounds with this novel peptidomimetic backbone design. In addition, applicability of a novel hyphenated technique, HPLC-SPE-NMR/MS, for analysis of crude SPPS reaction products was demonstrated.

We describe the synthesis and characterization of the first generation of oligomers consisting of alternating repeats of α-amino acids and chiral N-alkyl-β-alanine (β-peptoid) residues. These chimeras are stable toward proteolysis, non-hemolytic, and possess antibacterial activity comparable to well-known antimicrobial agents. Moreover, the chimeras exhibit length-dependent, concentration-dependent, solvent-dependent, and ion-strength-dependent ellipticity, indicating the presence of a secondary structure in solution. Thus, α-peptide/β-peptoid oligomers represent a promising novel peptidomimetic backbone construct for biologically active ligands.

Polyamine toxins isolated from the venoms of spiders and wasps and their synthetic analogues are uncompetitive antagonists of ligand-gated ionotropic receptors in the central- and peripheral nervous systems, and have proved valuable as tools for the investigation of receptor structure and function. In the present letter we describe the efficient solid-phase synthesis (SPS) of novel hybrid toxins using a BAL resin. This strategy enables the bidirectional construction of toxin molecules and has a potential in SPS of chemically diverse libraries of toxin analogues for structure–activity relationship (SAR) studies.

Biotransformation of deoxypodophyllotoxin to epipodophyllotoxin by three major human hepatic enzymes, CYP1A2, CYP2C9 and CYP3A4, heterologously expressed in E coli DH5 alpha, was investigated. It was shown that CYP3A4 catalysed the hydroxylation of deoxypodophyllotoxin into epipodophyllotoxin in yields up to 90%. The structure of the metabolite was determined using HPLC-MS and HPLC-SPE-NMR. techniques. There was no detectable production of epipodophyllotoxin or podophyllotoxin by CYP1A2 and CYP2C9 enzymes. The CYP3A4 enzyme shows a distinctly different reactivity to deoxypodophyllotoxin compared to the semi-synthetic derivatives etoposide and teniposide, which are degraded by 3-O-demethylation. These findings demonstrate a novel system for the production of 2,7'-cyclolignans, starting from the easily accessible deoxypodophyllotoxin. (c) 2006 Elsevier B.V. All rights reserved.

1. Subject and source Vincetoxicum pumilum Decne. [syn. Alexitoxicon pumilum (Decne.) Pobed., Antitoxicum pumilum (Decne.) Pobed., Cynanchum pumilum (Decne.) Bornm.] (ApocynaceaeeAsclepiadoideae) is a perennial herb endemic to Central Asia (Jalili and Jamzad, 1999). The plant (roots and aerial parts) was collected in Deh-Ghaibi near Mashhad, Iran. A voucher specimen (accession number 35007) was deposited in herbarium FUMH (Ferdowsi University Mashhad Herbarium).

The Fukuyama–Mitsunobu amination strategy has emerged as an efficient means of N-alkylation of peptides and sulfonamides, as well as a method for synthesis of polyamines on solid phase. Here, an array of reagent combinations for solid-phase alkylation with secondary alcohols was examined in various solvents. The classical reagents DEAD–PPh 3 as well as DEAD–PEt 3 proved applicable for a single alkylation step. Sharply dropping yields in successive alkylation steps were identified as the most serious limitation of the use of Fukuyama–Mitsunobu reaction in SPS of polyamines using primary and in particular secondary alcohols.

Extract of Naravelia zeylanica (Ranunculaceae) yielded three simple benzamides, 3,4-methylenedioxybenzamide, 4-methoxybenzamide and 4-hydroxy-3-methoxybenzamide. These simple C6C1 metabolites have been encountered as natural products for the first time. The compounds were identified by direct comparison of their spectral (1H- and 13C-NMR) and chromatographic (GCMS) data with those of authentic samples. Authentic 4-hydroxy-3-methoxybenzamide was synthesized in one step by treatment of 4-hydroxy-3-methoxybenzonitrile with sodium perborate. Authentic 3,4-methylenedioxybenzamide was synthesized from the corresponding acid.

[reaction: see text] A mild resin-immobilization strategy employing a readily prepared trityl bromide resin for anchoring building blocks via a phenol group has been developed. With N(alpha)-Fmoc-Tyr-OPfp as a starter building block, it was possible to prepare asymmetrically substituted hybrids of spider- and wasp-type polyamine toxins using solid-phase peptide synthesis conditions.

A new natural product, named selagoline, and two known alkaloids, huperzine A and serratidine, were isolated from Huperzia selago (Lycopodiaceae) collected in Iceland. Their structures were determined using 600 and 800MHz one- and two-dimensional NMR methods supported by Fourier-transform mass spectrometry. Possible role of selagoline as a precursor of 5,15-oxidolycopodane, a component of the classical alkaloids L28 and L31, is discussed.

[reaction: see text] An expedient solid-phase synthetic approach to secondary and tertiary amines was developed. The protocol employs conversion of resin-bound amino alcohols to the corresponding iodides, followed by iodide displacement with primary or secondary amines or with unprotected amino alcohols. This two-step procedure, affording products in good to excellent yields, is suitable for solid-phase synthesis of polyamines.

The natural triterpene betulinic acid and its analogues (betulinic aldehyde, lupeol, betulin, methyl betulinate and betulinic acid amide) caused concentration-dependent alterations of erythrocyte membrane shape towards stomatocytes or echinocytes according to their hydrogen bonding properties. Thus, the analogues with a functional group having a capacity of donating a hydrogen bond (COOH, CH2OH, CONH2) caused formation of echinocytes, whereas those lacking this ability (CH3, CHO, COOCH3) induced formation of stomatocytes. Both kinds of erythrocyte alterations were prohibitive with respect to Plasmodium falciparum invasion and growth; all compounds were inhibitory with IC50 values in the range 7–28 μM, and the growth inhibition correlated well with the extent of membrane curvature changes assessed by transmission electron microscopy. Erythrocytes pre-loaded with betulinic acid or its analogues and extensively washed in order to remove excess of the chemicals could not serve as hosts for P. falciparum parasites. Betulinic acid and congeners can be responsible for in vitro antiplasmodial activity of plant extracts, as shown for Zataria multiflora Boiss. (Labiatae) and Zizyphus vulgaris Lam. (Rhamnaceae). The activity is evidently due to the incorporation of the compounds into the lipid bilayer of erythrocytes, and may be caused by modifications of cholesterol-rich membrane rafts, recently shown to play an important role in parasite vacuolization. The established link between erythrocyte membrane modifications and antiplasmodial activity may provide a novel target for potential antimalarial drugs.

The first phytochemical investigation of the recently discovered East African liana Ancistrocladus tanzaniensis is described, resulting in the isolation and structural elucidation of two new naphthylisoquinoline alkaloids, ancistrotanzanines A (5) and B (6), and the known compound ancistrotectoriline A (7). Ancistrotazanine A (5) represents a hitherto unprecedented 5,3'-coupling type between the naphthalene and isoquinoline portions, while 6 and 7 are 5,8'-coupled. The structures of the compounds were determined by spectroscopic, chemical, and chiroptical methods. Compounds 5 and 6 showed good activities against the pathogens of leishmaniasis and Chagas' disease, Leishmania donovani and Trypanosoma cruzi, while 5-7 displayed moderately potent antiplasmodial activities against Plasmodium falciparum parasites.

Leaves of Melicope borbonica (Bory) T.G. Hartley (syn. Euodia borbonica var. borbonica (Bory) Engl.) were collected in July 1998 at Plaine d’Affouches, Reunion Island. Leaves of Melicope obscura (Cordem.) T.G. Hartley (syn. Euodia obscura Cordem.) were collected in February 2000 at Plaine de Palmiste, Reunion Island. Voucher specimens (DFHAA3 and DFHAA4) were deposited in Herbarium C (Botanical Museum, University of Copenhagen, Copenhagen, Denmark).

Voltage-dependent, non-competitive inhibition by philanthotoxin-343 (PhTX-343) analogues, with reduced charge or length, of nicotinic acetylcholine receptors (nAChR) of TE671 cells and ionotropic glutamate receptors (N-methyl-D-aspartate receptors (NMDAR) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR)) expressed in Xenopus oocytes from rat brain RNA was investigated. At nAChR, analogues with single amine-to-methylene or amine-to-ether substitutions had similar potencies to PhTX-343 (IC(50)=16.6 microM at -100 mV) whereas PhTX-(12), in which both secondary amino groups of PhTX-343 were replaced by methylenes, was more potent than PhTX-343 (IC(50)=0.93 microM at -100 mV). Truncated analogues of PhTX-343 were less potent. Inhibition by all analogues was voltage-dependent. PhTX-343 (IC(50)=2.01 microM at -80 mV) was the most potent inhibitor of NMDAR. At AMPAR, most analogues were equipotent with PhTX-343 (IC(50)=0.46 microM at -80 mV), apart from PhTX-83, which was more potent (IC(50)=0.032 microM at -80 mV), and PhTX-(12) and 4,9-dioxa-PhTX-(12), which were less potent (IC(50)s>300 microM at -80 mV). These studies show that PhTX-(12) is a selective nAChR inhibitor and PhTX-83 is a selective AMPAR antagonist.

Nineteen species of Passiflora (Passifloraceae) were examined for the presence of cyanogenic glycosides. Passibiflorin, a bisglycoside containing the 6-deoxy-β- d -gulopyranosyl residue, was isolated from P. apetala , P. biflora , P. cuneata , P. indecora , P. murucuja and P. perfoliata . In some cases this glycoside co-occurs with simple β- d -glucopyranosides: tetraphyllin A, deidaclin, tetraphyllin B, volkenin, epivolkenin and taraktophyllin. P. citrina contains passicapsin, a rare glycoside with the 2,6-dideoxy-β- d - xylo -hexopyranosyl moiety, while P. herbertiana contains tetraphyllin A, deidaclin, epivolkenin and taraktophyllin, P. discophora tetraphyllin B and volkenin, and P. × violacea tetraphyllin B sulfate. The remaining species were noncyanogenic. The glycosides were identified by 1 H and 13 C NMR spectroscopy following isolation by reversed-phase preparative HPLC. From P. guatemalensis , a new glucoside named passiguatemalin was isolated and identified as a 1-(β- d -glucopyranosyloxy)-2,3-dihydroxycyclopentane-1-carbonitrile. An isomeric glycoside was prepared by catalytic hydrogenation of gynocardin. α-Hydroxyamides corresponding to the cyanogenic glycosides were isolated from several Passiflora species. These α-hydroxyamides, presumably formed during processing of the plant material, behave as cyanogenic compounds when treated with commercial Helix pomatia crude enzyme preparation. Thus, the enzyme preparation appears to contain an amide dehydratase, which converts α-hydroxyamides to cyanohydrins that liberate cyanide; this finding is of interest in connection with analysis of plant tissues and extracts using Helix pomatia enzymes.

Philanthotoxin?433 (PhTX?433) is a known potent inhibitor of ionotropic glutamate receptors, and analogues have been synthesised to identify more potent and selective antagonists. Herein we report the synthesis of four PhTXs with a cyclopropane moiety introduced into their polyamine chain, and their inhibition of an ??amino?3?hydroxy?5?methyl?4?isoxazolepropionic acid (AMPA) receptor subtype by using two?electrode voltage?clamp assays on Xenopus oocytes expressing the GluA1flop subunit. All analogues were found to be more potent than PhTX?343, with trans?cyclopropyl?PhTX?343 being the most potent (?28?fold) and cis?cyclopropyl?PhTX?343 least potent (?4?fold). Both cis? and trans?cyclopropyl?PhTX?444 had intermediate potency (both ?12?fold). Molecular modelling indicates that a cyclopropane moiety confers a favourable steric constraint to the polyamine part, but this is compromised by a cis conformation due to enhanced intramolecular folding. Elongated PhTX?444 analogues alleviate this to some extent, but optimal positioning of the amines is not permitted.

The protonation states of a series of piperidinedicarboxylic acids (PDAs), which are conformationally constrained acidic alpha-amino acids, have been studied by (13)C NMR titration in water. The resulting data have been correlated with theoretical results obtained by HF/6-31+G calculations using the polarizable continuum model (PCM) for the description of water. The PDAs are highly ionizable and contain one or two possible internal hydrogen bonds. In the present study, we show that the PCM model is able to reproduce the relative stabilities of the different protonation states of the PDAs. Furthermore, our results show that prediction of relative pK(a) values for two different types of ionizable functional groups covering a pK(a) range from 1.6 to 12.1 is possible with a high degree of accuracy.

The AMPA receptor agonist Thio-AMPA, the 3-isothiazolol analogue of AMPA was converted into the selective NMDA antagonist, 2, in which a 3-isothiazolone unit is a bioisosteric analogue of the peptide bond of the NMDA antagonist, γ-(R)-Glu-Gly. The isomeric 3-oxygenated isothiazole amino acid, 3, and the corresponding isothiazole phosphono amino acid 4 were also synthesized, and were shown to be selective AMPA receptor antagonists. Compound 1, in which the peptide bond of γ-(R)-Glu-Gly is replaced by an ester group, was synthesized and shown to be unstable in the test buffer system.

Passiflora morifolia, which under natural conditions contains cyanohydrin glucosides linamarin, lotaustralin and epilotaustralin, converted cyclopentanecarbonitrile, 2-cyclopentenecarbonitrile and 3-methylbutanenitrile into the corresponding cyanohydrin glucosides. Turnera angustifolia, which normally produces glucosides of cyclopentenone cyanohydrin, converted cyclopentanecarbonitrile, 2-methylpropanenitrile and 2-methylbutanenitrile, but not 3-methylbutanenitrile, into the corresponding cyanohydrin glucosides. Mixtures of epimers were produced when these glucosides contained chiral cyanohydrin carbon atoms. Feeding with cyclopentanecarbonitrile resulted in formation of 1-(beta-D-glucopyranosyloxy)cyclopentanecarbonitrile, a saturated analogue of deidaclin and tetraphyllin A. Neither plant utilized cyclopropanecarbonitrile as substrate. The experiments demonstrate broad substrate specificity of nitrile hydroxylases present in these plants. A novel glycoside, 2-[6-O-(beta-D-xylopyranosyl)-beta-D-glucopyranosyloxy]propane (isopropyl primeveroside), was isolated from P. morifolia. The compound represents a rare example of natural isopropyl glycoside; its characterization included assignment of all 1H and 13C NMR signals of the primeverosyl group using two-dimensional NMR methods. Biosynthesis of the isopropyl moiety of the primeveroside is unclear, but the formation of alcohols corresponding to natural cyanohydrins may be a previously unrecognized extension of the cyanohydrin biosynthesis pathway in higher plants.

The effects of gossypol on glucose metabolism, ATP levels and intracellular sodium levels in murine TM3 Leydig and TM4 Sertoli cell lines were investigated, and their response compared. Relative ATP levels and sodium ion levels in the two cell lines were determined by 31P and 23Na NMR spectroscopy. Short-term effect of gossypol on phosphate metabolism in immobilized and perfused cells was apparent in 31P NMR spectra only with relatively high concentrations of gossypol, corresponding to about 40 times IC50. However, incubation with low gossypol concentrations markedly affected the energetic status of the cells, especially of the TM3 cells. Although inhibition of proliferation by gossypol was stronger with the TM4 cells, the decrease of intracellular ATP level and increase of sodium ion concentration were more pronounced in the TM3 cells. The growth-inhibitory effect of (-)-gossypol was stronger than that of (+)-gossypol, with the eudesmic ratio of 2-2.5. The enantioselectivity of the effect of gossypol on the energy metabolism of TM3 and TM4 cells was low, in contrast to the in vivo antispermatogenic effect, which was reported to be solely associated with (-)-gossypol. Inhibition of energy production in somatic testicular tissue is thus unlikely to be major cause of the antispermatogenic effect of gossypol.

Bioactivity-guided fractionation of extracts of roots and leaves of Cajanus cajan afforded 8 compounds: betulinic acid, biochanin A, cajanol, genistein and 2'-hydroxygenistein, longistylin A and C, and pinostrobin. The two stilbenes, longistylin A and C, and betulinic acid showed a moderately high in vitro activity against the chloroquine-sensitive Plasmodium falciparum strain 3D7.

[reaction: see text]. Polyamines containing piperidine and piperazine moieties have been synthesized on solid support using SN2 alkylation of resin-bound secondary amines with 2-nitrobenzenesulfonates (nosylates). The effect of solvent on this alkylation was investigated. The methodology was employed for the total synthesis of novel analogues of wasp polyamine toxins (philanthotoxins).

The hyphenated technique HPLC-SPE-NMR is an important tool for rapid dereplication of complex mixtures of in particular small molecules and has been successfully employed in natural product research. However, positively charged alkaloids at low pH are often poorly trapped on the generally used SPE cartridge limiting the general application of the procedure. In this work, two new approaches for efficient SPE trapping of alkaloids and elution efficiencies were evaluated using 24 model alkaloids. Use of a 0.1 M NaOH solution as the post-column dilution greatly enhanced trapping of alkaloids on the commonly used cartridge containing divinylbenzene polymer (GP resin). This procedure, however, was unsuitable for trapping phenolic alkaloids. Severe line broadening and immiscibility with water made chloroform-d(1) unsuited as eluent. None of these problems occurred when methanol-d(4) was used as eluent. Previously, mixed mode cation exchange sorbents have not been used in HPLC-SPE-NMR analysis of natural products. In contrast to GP resin this material showed good retention and elution characteristics for retention and elution of alkaloids. As well the use of methanol-d(4) containing 1% aqueous NaOD (40%) as methanol-d(4) containing 5% aqueous NH(4)OH (30%) as eluents were successful, even though elution of alkaloids with pK(a) of the corresponding acid above 10 proved difficult. Alkaloid extracts of Huperzia selago containing complex aliphatic alkaloids and Triclisia patens containing bisbenzylisoquinoline alkaloids were used for validation of the protocols for analysis of a diverse collection of alkaloids. Mixed mode cation exchange sorbent was efficient for trapping and elution of both types of alkaloids as evidenced by acquisition of 2D NMR data for all trapped compounds. In contrast, GP resin proved only viable for all the H. selago alkaloids whereas trapping and elution of bisbenzylisoquinoline alkaloids were dubious.

Dehydroabietinol isolated from Hyptis suaveolens (L.) Poit. was found to inhibit growth of chloroquine-sensitive as well as chloroquine-resistant strains of Plasmodium falciparum cultivated in erythrocytes in vitro (IC 50 26-27 microM). However, erythrocytes exposed to dehydroabietinol were transformed in a dose-dependent manner towards spherostomatocytic forms with concomitant formation of endovesicles, as disclosed by transmission electron microscopy. The erythrocyte shape alterations caused by dehydroabietinol correlated well with its apparent IC 50 value. Thus, dehydroabietinol incorporates into the erythrocyte membrane, and since invasion and survival of Plasmodium parasites is known to depend on the function of the erythrocyte membrane, the observed antiplasmodial effect of dehydroabietinol is presumably an indirect effect on the host cell. Because of these findings, microscopic investigations should be generally used to support claims of antimalarial effects of apolar natural products.

[structure: see text] Leaves of the edible passion fruit plant, Passiflora edulis, contain benzylic beta-D-allopyranosides 1 and 2, representatives of a rare class of natural glycosides with D-allose as the only sugar constituent. The glycoside 1 is the first known cyanogenic glycoside containing a sugar different from D-glucose attached directly to the cyanohydrin center. Asymmetric perturbation of the (1)L(b) transition of the benzene chromophore was shown to be useful for determination of absolute configuration of the cyanohydrin center of aromatic cyanogenic glycosides.

1H NMR spectroscopy-based metabolic phenotyping was used to identify biomarkers in the plasma of patients with rheumatoid arthritis (RA). Forty-seven patients with RA (23 with active disease at baseline and 24 in remission) and 51 healthy subjects were evaluated during a one-year follow-up with assessments of disease activity (DAS-28) and 1H NMR spectroscopy of plasma samples. Discriminant analysis provided evidence that the metabolic profiles predicted disease severity. Cholesterol, lactate, acetylated glycoprotein, and lipid signatures were found to be candidate biomarkers for disease severity. The results also supported the link between RA and coronary artery disease. Repeated assessment using mixed linear models showed that the predictors obtained from metabolic profiles of plasma at baseline from patients with active RA were significantly different from those of patients in remission (P=0.0007). However, after 31 days of optimized therapy, the two patient groups were not significantly different (P=0.91). The metabolic profiles of both groups of RA patients were different from the healthy subjects. 1H NMR-based metabolic phenotyping of plasma samples in patients with RA is well suited for discovery of biomarkers and may be a potential approach for disease monitoring and personalized medication for RA therapy.

The biosynthesis of deidaclin in Turnera angustifolia and of linamarin in Passiflora morifolia were investigated using intact plant tissues. Radiolabelled precursors, 2-(2′-cyclopentenyl)[2- 14 C]glycine and l -[U- 14 C]-valine were fed to freshly harvested shoots either alone or together with the presumed nitrile intermediates, 2-cyclopentenecarbonitrile and 2-methylpropanenitrile. The cyanohydrin glucosides were isolated and purified, and the incorporation of the radioactive labels was determined after enzymatic degradation of the glucosides to cyanide. The labels from the amino acid precursors were incorporated into the nitrile group of their corresponding cyanohydrin glucosides, and the incorporation was in each case strongly inhibited by simultaneous feeding with either of the two nitriles. Turnera angustifolia was able to synthesize linamarin when fed with 2-methylpropanenitrile, even though linamarin could not be demonstrated to be present in this plant naturally.

Direct hyphenation of analytical-scale high-performance liquid chromatography, photo-diode array detection, mass spectrometry, solid-phase extraction and nuclear magnetic resonance spectroscopy (HPLC-PDA-MS-SPE-NMR) has been used for accelerated dereplication of crude extract of Haplophyllum acutifolium (syn. Haplophyllum perforatum). This technique allowed fast on-line identification of six quinolinone alkaloids, named haplacutine A-F, as well as of acutine, haplamine, eudesmine, and 2-nonylquinolin-4(1H)-one. Acutine and haplacutine E, isolated by preparative-scale HPLC, showed moderate antiplasmodial activity with IC(50) values of 2.17+/-0.22 microM and 3.79+/-0.24 microM, respectively (chloroquine-sensitive Plasmodium falciparum 3D7 strain).

Biotransformation of deoxypodophyllotoxin to epipodophyllotoxin (ePTOX) by three major human hepatic enzymes, CYP1A2, CYP2C9 and CYP3A4, heterologously expressed in E. coli DH5α, was investigated. It was shown that CYP3A4 catalysed the hydroxylation of deoxypodophyllotoxin into epipodo-phyllotoxin in yields up to 90%. The structure of the metabolite was determined using HPLC-MS and HPLC-SPE-NMR techniques [1]. There was no detectable production of epipodophyllotoxin or podophyllotoxin by CYP1A2 and CYP2C9 enzymes. The CYP3A4 enzyme shows a distinctly different reactivity to deoxypodophyllotoxin compared to the semisynthetic derivatives etoposide and teniposide, which are degraded by 3-O-demethylation. These findings demonstrate a novel system for the production of 2,7'-cyclolignans, starting from the easily accessible deoxypodophyllotoxin. Further kinetic analysis revealed that the Michaelis–Menten Km and Vmax for hydroxylation of deoxypodophyllotoxin by CYP3A4at C-7 position were 1.93µM and 1.48 nmol/min/nmol, respectively. Deoxypodophyllotoxin was subjected to automated docking analysis in order to get better knowledge of the interaction between the CYP 3A4 enzyme and the substrate, using the PatchDock algorithm with distance constraints. Automated docking showed that the β-hydrogen atom at C-7 position is in the most appropriate binding orientation at the site of oxidation. The docking results are consistent with the experimental data for the bioconversion of deoxypodophyllotoxin into epipodophyllotoxin by CYP 3A4 [2]. In addition, the effects of five lignans, deoxypodophyllotoxin, epipodophyllotoxin, podophyllotoxin, demethylenedeoxyodophyllotoxin, and demethylenepodo-phyllotoxin, on CYP3A4 were compared in order to investigate the influence of the methylenedioxy group on the biotransformation process. The docking method hypothesized inhibition by epipodophyllotoxin when the methylenedioxy moiety is involved and the substrate is oriented towards the iron atom of the heme group of CYP 3A4.

Cyanohydrin glucosides from Piriqueta taubatensis, P viscosa ssp. australis, Turnera angustifolia, T. caerulea, T. chamaedryfolia, T. hassleriana and Wormskioldia longipedunculata were isolated and identified. Members of the Turneraceae produce cyclopentanoid cyanohydrin glucosides as weil as linamarin. This is the first report of linamarin from the Turneraceae. Both types of 1,4-dioxygenated cyclopentanes ( cis and trans ) were represented. The relationship of the Turneraceae to the Passifloraceae and Flacourtiaceae is discussed briefly.

Epidermal growth factor (EGF) has an in vitro inhibitory effect on tumor cells which exhibit a high number of EGF receptors (EGFR). Studies were performed in order to delineate the effects of EGF on glucose metabolism of MDA-468 human breast cancer cells, which have a large number of EGFR. Glucose consumption and lactate production were found to be substantially increased in MDA-468 cells following EGF exposure, while no such effects were detected in MCF-7 breast cancer cells, which have a very low number of EGFR. When glucose levels in the growth medium were increased, the toxicity of EGF was diminished. The energetic status of MDA-468 cells perfused with growth medium containing EGF was monitored by 31P magnetic resonance spectroscopy, and no signs of compromised metabolic state or viability were noted for up to 36 h. The rate of glucose transport and phosphorylation was quantitated by 13C magnetic resonance spectroscopy, utilizing [6-13C]2-deoxyglucose, and a 97% increase was found in MDA-468 cells following EGF administration. The profound effects of EGF on glucose metabolism in cells with very high numbers of EGFR and the lack of toxicity in the perfused system may indicate that the growth-inhibitory effect is confined to the in vitro cultured cells.