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Progress and prospects of ergot alkaloid research.

Ergot alkaloids, produced by the plant parasitic fungi Claviceps purpurea are important pharmaceuticals. The chemistry, biosynthesis, bioconversions, physiological controls, and biochemistry have been extensively reviewed by earlier authors. We present here the research done on the organic synthesis of the ergot alkaloids during the past two decades. Our aim is to apply this knowledge to the synthesis of novel synthons and thus obtain new molecules by directed biosynthesis. The synthesis of clavine alkaloids, lysergic acid derivatives, the use of tryptophan as the starting material, the chemistry of 1,3,4,5-tetrahydrobenzo[cd]indoles, and the structure activity relationships for ergot alkaloids have been discussed. Recent advances in the molecular biology and enzymology of the fungus are also mentioned. Application of oxygen vectors and mathematical modeling in the large scale production of the alkaloids are also discussed. Finally, the review gives an overview of the use of modern analytical methods such as capillary electrophoresis and two-dimensional fluorescence spectroscopy.

Animals↗

Production of alkaloids and differentiation in a submerged culture of Claviceps purpurea (Fr.) Tul.

The submerged culture of the Claviceps purpurea strain studied was polymorphous. In the process of alkaloid synthesis, cytodifferentiation preceded biochemical differentiation. The onset of the alkaloid phase was characterized by: predominance of chlamydospores in the culture, the presence of vegetative cells with reduced or arrested proliferation, maximum acetylCoA carboxylase activity, the maximum amount of total fatty acids, an over-average cell pool tryptophan level and minimum tryptophan synthetase activity. Intracellular ricinoleic acid was an indicator of differentiation of the culture towards alkaloid formation and also of alkaloid synthesis. The cytodifferentiation period in the initial phases of fermentation, when the cell has several alternative possibilities of development, is regarded as the most sensitive sector of the regulatory mechanisms of alkaloid formation.

Acetyl-CoA Carboxylase↗

Saprophytic production of clavine alkaloids and activity of hydroxymethylglutaryl-CoA reductase.

In submerged Claviceps cultures the activity of hydroxymethylglutaryl-CoA reductase preceded the increase of alkaloid production and of sterol content. During the first alkaloid phase, cell mevalonate was involved in the biosynthesis of both alkaloids and steroids. In the second production phase, it was predominantly used for alkaloid synthesis. Hydroxymethylglutaryl-CoA reductase appears to be a suitable target for physiological manipulation to increase clavine alkaloid yields.

Claviceps↗

Alkaloidal constituents from Aconitum jaluense.

Aconitum jaluense Komar. (Ranunculaceae) is one of the Aconitum plants growing in Korean peninsula. An investigation of the alkaloidal constituents of this species led to the isolation of seven C19-norditerpenoid and a C20-diterpenoid alkaloid. Three of them have been identified as neoline, mesaconitine, and hypaconitine, which were isolated from this plant collected from Mt. Bultasan in the north part. The other five alkaloids were determined as lipomesaconitine, lipohypaconitine, 15alpha-hydroxyneoline, hokbusine A, and napelline, which have not been found in this plant. Structures of those alkaloids were determined on the basis of their spectral data. It is of interest to note that a comparison of the present work and the previous report showed some differences in the alkaloidal contents.

Aconitine↗

Biotechnology and genetics of ergot alkaloids.

Ergot alkaloids, i.e. ergoline-derived toxic metabolites, are produced by a wide range of fungi, predominantly by members of the grass-parasitizing family of the Clavicipitaceae. Naturally occurring alkaloids like the D-lysergic acid amides, produced by the "ergot fungus" Claviceps purpurea, have been used as medicinal agents for a long time. The pharmacological effects of the various ergot alkaloids and their derivatives are due to the structural similarity of the tetracyclic ring system to neurotransmitters such as noradrenaline, dopamine or serotonin. In addition to "classical" indications, e.g. migraine or blood pressure regulation, there is a wide spectrum of potential new applications of this interesting group of compounds. The biotechnology of ergot alkaloids has a long tradition, and efficient parasitic and submerse production processes have been developed; the biochemistry of the pathway and the physiology of production have been worked out in detail. The recent identification of a cluster of genes involved in ergot alkaloid biosynthesis in C. purpurea and the availability of molecular genetic techniques allow the development of strategies for rational drug design of ergoline-related drugs by enzyme engineering and by biocombinatorial approaches.

Biotechnology↗

Identification of genes induced in alkaloid-producing cultures of Claviceps sp.

In order to identify genes which are expressed during alkaloid synthesis in an axenic culture of Claviceps sp. (strain ATCC 26245), a cDNA library from a producing culture was differentially screened with cDNA from producing (cDNA+) and non-producing (cDNA-) cultures, respectively. Altogether, ten cDNA clones were obtained, the alkaloid-synthesis-correlated expression of which was confirmed by Northern analyses. Evaluation of their nucleotide and derived amino-acid sequences identified one gene unequivocally, coding for dimethylallyltryptophan-synthase (DMAT-S), the initial enzyme of the specific alkaloid pathway. For two other genes significant homologies to known fungal genes were detected: one clone showed homology to the Neurospora crassa ccg1 gene, coding for a clock-regulated putative general stress protein; seven cDNA clones, derived from the same gene, which is highly expressed under these conditions, contained typical hydrophobin domains and long stretches of asparagine/glycine repeats (like QID3 from Trichoderma harzianum), thus probably representing a cell-wall constituent. These data show that this is not only a successful approach to clone genes specific for the alkaloid-pathway of C. purpurea, but also of genes which might be involved in the differentiation of sclerotial hyphae, the prerequisite for alkaloid synthesis.

Alkaloids↗

LC-MS determination of Taxus alkaloids in biological specimens.

A semi-quantitative LC-MS method was developed for the detection of the pseudo alkaloids of Taxus baccata (yew) from human body fluids and tissue samples. This method was used to examine the cause of death of a 43-year-old man who died several hours after he drank a decoction of taxus leaves. Autopsy and histology demonstrated early signs of myocardial hypoxia. Since investigation of the stomach content did not yield evidence of taxus ingestion, the taxus alkaloids were determined in blood, stomach content and tissue samples of the deceased by LC-MS. The samples were prepared by solid phase extraction on RP-18 columns. Chromatographic separation was achieved by HPLC on a RP-8 column, coupled to an ion trap mass spectrometer (Finnigan LCQ). An atmospheric pressure electrospray ionisation was performed. Spectra of the alkaloids were recorded in the single MS mode and in the MS-MS mode and compared with reference spectra obtained from an extract of yew leaves. In the stomach content, the kidneys, the liver and a heart blood sample of the deceased, alkaloids of Taxus baccata, predominantly taxine B and iso-taxine B, were identified. The semi-quantitative evaluation of the heart blood revealed a taxine concentration of 11 micro g taxine/g. As far as we know this is the first case in which a semi-quantitative analysis of taxine alkaloids has been performed.

Adult↗

Isolation and partial characterization of norcoclaurine synthase, the first committed step in benzylisoquinoline alkaloid biosynthesis, from opium poppy.

Norcoclaurine synthase (NCS) catalyzes the condensation of dopamine and 4-hydroxyphenylacetaldehyde (4-HPAA) to yield norcoclaurine, the common precursor to all benzylisoquinoline alkaloids produced in plants. In opium poppy (Papaver somniferum L.), NCS activity was detected in germinating seeds, young seedlings, and all mature plant organs, especially stems and roots. However, the highest levels of activity were found in cell-suspension cultures treated with a fungal elicitor. NCS activity was induced more than 20-fold over an 80-h period in response to elicitor treatment. Compared to opium poppy. basal NCS activity was 3-and 5-fold higher in benzylisoquinoline alkaloid-producing cell cultures of Eschscholzia californica and Thalictrum flavum ssp. glaucum, respectively. In contrast, NCS activity was not detected in cultured cells of Nicotiana tabacum and Catharanthus roseus, which do not produce benzylisoquinoline alkaloids. NCS displayed maximum activity between pH 6.5 and 7.0, and a broad temperature optimum between 42 and 55 degrees C. Enzyme activity was not affected by Ca2+ or Mg2+, and was not inhibited by a variety of benzylisoquinoline alkaloids. NCS showed hyperbolic saturation kinetics for 4-HPAA, with an apparent Km of 1.0 mM. However, the enzyme exhibited sigmoidal saturation kinetics for dopamine with a Hill coefficient of 1.84. NCS enzymes from E. californica and T. flavum displayed similar properties. These data indicate that NCS exhibits positive cooperativity between substrate-binding sites. Enzymes of this type catalyze regulatory, or rate-limiting, steps in metabolism, suggesting that NCS plays a role in controlling the rate of pathway flux in benzylisoquinoline alkaloid biosynthesis.

Acetaldehyde↗

Determination and in-depth chromatographic analyses of alkaloids in South American and greenhouse-cultivated coca leaves.

Methodology is described for the detection and/or determination of cocaine and minor alkaloids in South American coca as well as in greenhouse- and tropical-cultivated field coca of known taxonomy. Coca leaf from Bolivia, Peru, Ecuador and Colombia were subjected to the determination of cocaine, cis- and trans-cinnamoylcocaine, tropacocaine, hygrine, cuscohygrine and the isomeric truxillines. The greenhouse samples were cocaine-bearing leaves of the genus Erythroxylum and included E. coca var. coca, E. novogranatense var. novogranatense and E. novogranatense var. truxillense, and the alkaloids determined were cocaine, ecgonine methyl ester, cuscohygrine, tropacocaine and the cinnamoylcocaines. The tropical-cultivated coca were E. novogranatense var. novogranatense and E. coca var. coca. Cocaine and minor alkaloids were isolated from basified powdered leaf samples using a toluene extractant, followed by acid-Celite column chromatography. The isolated alkaloids were determined by capillary gas chromatography with flame ionization or electron-capture detection. Methodology is also presented for the isolation and mass spectral analysis of numerous trace-level coca alkaloids of unknown structure.

Alkaloids↗

Monoclonal antibodies to antitumor Vinca alkaloids: thermodynamics and kinetics.

Spleen cells from a mouse and a rat immunized with vinblastine coupled to bovine serum albumin were fused in two independent experiments with P3 X 63-Ag8 (non-secreting variant) mouse myeloma cells. Three mouse X mouse (Vinca 1-3) and two rat X mouse (Vinca 4 and 5) hybrids were selected for production of Vinca alkaloid binding monoclonal antibodies. Each antibody had characteristic cross-reactivities with alkaloids structurally related to vinblastine: Vinca 1 reacted preferentially with deacetylated alkaloids (deacetyl vinblastine and vindesine) and Vinca 2 had a higher affinity for vinblastine and vincristine. Vinca 3-5 recognized equally vinblastine, vincristine and vindesine but differed with respect to their affinities for other analogues. No significant cross-reactivity of the monomeric alkaloids vindoline or catharanthine was observed with any antibody, and dimeric alkaloids modified in the catharanthine moiety had reduced immunoreactivity. Mouse monoclonal antibodies (Vinca 1 and 3) showed moderate affinity (2.2 X 10(-7) and 5.8 X 10(-9) M) for their respective best ligands and fast kinetics (dissociation rate constants greater than 3 X 10(-3) sec-1). Vinca 4 and 5, derived from the rat X mouse hybrids, had much higher affinities (1.5 X 10(-11) and 1.1 X 10(-11) M) and slower kinetics (dissociation rate constants: 2.4 X 10(-5) and 7.2 X 10(-6) sec-1). The major difference between these two antibodies was that Vinca 4 binds and releases the antigen more rapidly than Vinca 5 does. Somatic hybridization techniques thus generated monoclonal antibodies recognizing a given class of low mol. wt antigens with variable specificity, affinity and kinetic behavior, allowing the selection of reagents most appropriate for particular immunochemical applications.

Animals↗

Chromosomal aberrations and mutation in cultured mammalian cells induced by pyrrolizidine alkaloids.

4 pyrrolizidine alkaloids, heliotrine, lasiocarpine, petasitenine, and senkirkine, were tested for their effects of V79 Chinese hamster cells. All the alkaloids produced cytoplasmic vacuolization, and caused cellular and nuclear enlargement. Chromosomal aberrations were induced in cells treated with the alkaloids. Heliotrine and petasitenine induced interchromosomal exchanges, and lasiocarpine and senkirkine caused chromatid gaps. All the alkaloids induced an 8-azaguanine-resistant mutation in V79 cells by direct treatment for 48 h. Mutation was also induced by treatment with the alkaloids for 1 h in the presence of a metabolic activation system.

Animals↗

Effect of culture process on alkaloid production by Catharanthus roseus cells. I. Suspension cultures.

The processes for production of indole alkaloids in shake flask suspension cultures of Catharanthus roseus cells using Zenk's alkaloid production medium (APM) were evaluated. The 1-stage process consisted of inoculating APM and incubating for 15 days. The 2-stage process involved 6 d of cultivation in growth medium followed by 15 d of incubation in APM. Growth, main nutrient consumption and alkaloid production were monitored. Both culture processes produced approximately 20 g dw per 1 biomass. However, 2-stage cultures yielded an inorganic nutrient richer and more active plant cell biomass, richer in inorganic nutrients, as indicated by higher (greater than 70%) nutrient availability and consumption. Total and individual indole alkaloid production were 10 times higher (740 mg l-1 and 25 to 4000 micrograms per g dw, respectively) for 2-stage than for 1-stage cultures. For both processes, highest alkaloid productivity coincided with complete extracellular consumption of major inorganic nutrients, especially nitrate, by the cells. Complete carbohydrate consumption in 2-stage cultures resulted in a 40% decline in production. Small but significant (approximately 10%) product release was observed for both culture regimes, which seemed not to be related to cell lysis.

Alkaloids↗

Instability of pleomorphic tubulin paracrystals artificially induced by Vinca alkaloids in tissue-cultured cells.

The processes of tubulin paracrystal induction in Chinese hamster ovary cells treated with a Vinca alkaloid, ie, vinblastine or vincristine, and treated simultaneously with one of the Vinca alkaloids and colcemid or colchicine were followed by four different microscopic techniques, in particular by tubulin-immunofluorescence. Vinca alkaloid alone, in lower concentrations, induced basically tactoid or needle-shaped (N-shaped) paracrystals. However, the formation of crystalloid was greatly enhanced by increasing the concentration of Vinca alkaloid. Square or barrel-shaped (S-shaped) and hexagonal paracrystals were also commonly induced by simultaneous treatment with a Vinca alkaloid and colcemid or colchicine. Large rectangular paracrystals often displayed fibrillar or lamellar fine structures which ran perpendicular to the long axis but tended to cleave into fragments by spontaneous splitting. Electron micrographs revealed the fine structure of crystalloids to be aggregates of numerous filaments. The growth of paracrystals, particularly N-shaped crystals, was markedly inhibited when cells were exposed to drug(s) at a low temperature (4 degrees C). We confirmed that both N- and S-shaped paracrystals dissociated rapidly after the culture medium was replaced with fresh, drug-free medium. Glutaraldehyde-fixed paracrystals treated with RNase solution were stained with acridine orange, showing a weak orange color. Possible factors involved in the assembly and disassembly of tubulin paracrystals are discussed.

Animals↗

The recovery of alkaloids after their location on thin layer chromatograms.

A method was developed for the recovery of 29 alkaloids from TLC plates after they had been complexed with either potassium iodoplatinate (for tertiary amines) or carbon disulphide (for primary and secondary amines). A comparison was made between the percentage recoveries of these alkaloids from 5 different types of TLC plate. In addition, the stability of these alkaloids on TLC plates over a 7 day period was studied. Assays were, where possible, carried out by UV spectrophotometry but gas chromatography was used for alkaloids which possessed low UV absorbtivity values. All the alkaloids extracted from TLC plates were examined by low resolution mass spectrometry.

Alkaloids↗

Gas chromatographic-mass spectrometric method for determination of anabasine, anatabine and other tobacco alkaloids in urine of smokers and smokeless tobacco users.

A selected ion monitoring method for determination of the tobacco alkaloids anabasine, anatabine, nornicotine, metanicotine, dihydrometanicotine, and 2,3'-bipyridyl in urine of smokers and smokeless tobacco users is described. The method involves conversion of the secondary amine alkaloids to tertiary amine derivatives by reductive alkylation using an aldehyde and sodium borohydride, and chromatography on a 5% phenylmethylsilicone capillary column. These derivatives have good chromatographic properties, allowing determination of concentrations as low as 1 ng/ml. The alkaloid 2,3'-bipyridyl is unaffected by the derivatization procedure and may be determined simultaneously with the other alkaloids. The structural analogues 2-(3-pyridyl)hexahydroazepine, 5-methyldihydrometanicotine, and 6-methyl-2,3'-bipyridyl were synthesized for use as internal standards. Using the method, concentrations and 24 h excretion of anabasine, anatabine, and nornicotine in urine of twenty-two smokers, eight chewing tobacco users, and six oral snuff users were determined and compared with concentrations and excretion of nicotine and its metabolite cotinine. Excretion of nicotine and cotinine was similar in all tobacco users, but excretion of anabasine, anatabine and nornicotine was substantially greater in urine of smokeless tobacco users, presumably due to absence of pyrolysis of these alkaloids in smokeless tobacco products.

Alkaloids↗

Biological activity of the alkaloids of Erythroxylum coca and Erythroxylum novogranatense.

The cultivated Erythroxylum varieties E. coca var. coca, E. coca var. ipadu, E. novogranatense var. novogranatense and E. novogranatense var. truxillense contain 18 alkaloids, identified so far, belonging to the tropanes, pyrrolidines and pyridines, with cocaine as the main alkaloid. The biological activity of the following alkaloids has been reported in the literature: cocaine, cinnamoylcocaine, benzoylecgonine, methylecgonine, pseudotropine, benzoyltropine, tropacocaine, alpha- and beta-truxilline, hygrine, cuscohygrine and nicotine. The biological activity of cocaine and nicotine is not reviewed here, because it is discussed elsewhere in the literature. Hardly anything is known about the biological activity of the other alkaloids present in the four varieties mentioned. The biosynthesis of the coca alkaloids has been outlined.

Alkaloids↗

Stem bark alkaloids of Rauvolfia caffra.

Thirty two alkaloids were isolated from the stem bark of Rauvolfia caffra and 28 were identified. The alkaloids represented corynane (3), strictamine (1), sarpagan (4), akuammicine (2), pleiocarpamine (1), indolenine (1), dihydroindole (6), peraksine (3), heteroyohimbine (2), hydroxyheteroyohimbine (2), oxindole (1), 2-acyl-indole (1), suaveoline (3) and yohimbine (2) types. The anhydronium base serpentine was detected but not isolated. The principal alkaloids were ajmaline and norajmaline (dihydroindoles), ajmalicinine and ajmalicine (heteroyohimbines), geissoschizol (E-seco indole) and pleiocarpamine and the heteroyohimbine derived alkaloids were predominantly normal configuration compounds. The biosynthetic and ethnopharmacological significance of the alkaloids is discussed.

Humans↗

Nicotine-related alkaloids and metabolites as inhibitors of human cytochrome P-450 2A6.

S-(-)-Nicotine and 13 of the most prevalent nicotine-related alkaloids and metabolites (i.e., S-(-)-nornicotine, myosmine, beta-nicotyrine, S-cotinine, S-norcotinine, S-(-)-nicotine N-1'-oxide, S-(-)-nicotine Delta1'-5'-iminium ion, S-(-)-anabasine, S-(-)-N-methylanabasine, anabaseine, S-(-)-anatabine, nicotelline, and 2,3'-bipyridyl) were evaluated as inhibitors of human cDNA-expressed cytochrome P-450 2A6 (CYP2A6) mediated coumarin 7-hydroxylation. Tobacco alkaloids myosmine, S-(-)-nornicotine, S-cotinine, S-norcotinine, S-(-)-nicotine N-1'-oxide, S-(-)-nicotine Delta1'-5'-iminium ion, S-(-)-N-methylanabasine, anabaseine, and nicotelline had Ki values for inhibition of coumarin 7-hydroxylation ranging from 20 microM to more than 300 microM whereas nicotine and S-(-)-anatabine were much more potent (i.e. 4.4 and 3.8 microM, respectively). The tobacco alkaloids 2,3'-bipyridyl (7.7 microM) and S-(-)-anabasine (5.4 microM), were somewhat less potent compared with S-(-)-nicotine or S-(-)-anatabine in inhibition of human CYP2A6. beta-Nicotyrine, in which the N-methylpyrrolidino moiety of nicotine was replaced by the aromatic N-methylpyrrole ring, was shown to inhibit human CYP2A6 with much greater potency (Ki=0.37 microM) compared with S-(-)-nicotine. Among the compounds examined, only nicotine and beta-nicotyrine were mechanism-based inhibitors of human CYP2A6. The potency of the mechanism-based CYP2A6 inhibitors suggests that, for smokers, modulation of CYP2A6 may be greater than that predicted on the basis of serum concentration of these alkaloids. Our results indicate that the prominent nicotine-related alkaloid beta-nicotyrine present after smoking potently inhibits human CYP2A6.

Alkaloids↗