Catharanthus roseus alkaloid production

The two alkaloids vinblastine and vincristine found in Catharanthus roseus have been recent targets of total synthesis because of their potency in cancer chemotherapy. The reduced tree diagram for the Fukuyama plan to vincristine is shown in Figure 4.66. There are three points of convergence, four branches leading to the target product and four tiers of reaction yields. 

The isolation of the antitumor agents vincaleukoblastine (1) and leuro-cristine (2) from Catharanthus roseus (L.) G. Don has proved to be one of the most important developments in both natural product chemistry and the clinical treatment of cancer during the 1960s to 1980s. More alkaloids (over 90) have been isolated from C. roseus than from any other plant, and because of the complexity of the alkaloid mixture this work has required the most advanced isolation and structure determination techniques. The exceptional interest in the broad spectrum of antitumor activity of these compounds has resulted in numerous achievements in the pharmaceutical, clinical pharmacologic, and therapeutical sciences. Simultaneously, strenuous efforts have been made in three areas of the natural product chemistry (i) elaboration of a practical semisynthesis of... 

VLB and VCR are said to be present in only minute amounts in Catharanthus roseus (L.) G. Don 0.00025% of leaf dry weight), and their separation from other monomeric or binary alkaloids involves a complicated and tedious procedure based on their differential basicity followed by chromatography. Industrial production of VLB and VCR has therefore been a serious problem, and consequently these drugs are among the most expensive on the pharmaceutical market. 

The discovery of medicinal alkaloids from Catharanthus roseus G. Don (Vinca rosea L.) represents one of the most important introductions of plant products into the cancer chemotherapeutic armamentarium. The relatively unique effects and toxicities of these agents have allowed the design of multiagent chemotherapy programs that have demonstrated sufficient effectiveness to achieve cures even of advanced tumors in many instances. This great accomplishment is possible only because of the inclusion of many different drugs, including the binary Vinca alkaloids. 

Nef, C., Rio, B. and Chrestin, H. 1991. Induction of catharanthine synthesis and stimulation of major indole alkaloid production by Catharanthus roseus cells under non-growth-altering treatment with Pythium vexans extracts. Plant Cell Reports, 10 26-29. 

Cathenamine (100) has been identified as an early intermediate in terpenoid indole alkaloid biosynthesis (cf. Vol. 8, p. 27). It has also been isolated from Guettarda eximia. Another alkaloid, 4,21-dehydrogeissoschizine (99), has now been isolated from this plant it is readily converted into (100) in alkaline solution.29 On incubation with an enzyme preparation from Catharanthus roseus cell cultures in the presence of NADPH at pH 7, (99) was converted into ajmalicine (102), 19-ep/-ajmalicine (103), and tetrahydroalstonine (104), which are the normal products with this enzyme preparation. In the absence of NADPH, cathenamine (100) accumulated.30 The reaction to give (100) proceeded linearly with time, and was dependent on the concentration of protein and substrate. No conversion occurred in the absence of enzyme. 

Within the natural products field, the investigation of complete biosynthetic pathways at the enzyme level has been especially successful for plant alkaloids of the monoterpenoid indole alkaloid family generated from the monoterpene gluco-side secologanin and decarboxylation product of tryptophan, tryptamine [3-5]. The most comprehensive enzymatic data are now available for the alkaloids ajmalicine (raubasine) from Catharanthus roseus, and for ajmaline from Indian Rauvolfia serpentina [6] the latter alkaloid with a six-membered ring system bearing nine chiral carbon atoms. Entymatic data exsist also for vindoline, the vincaleucoblastin (VLB) precursor for which some studies on enzymatic coupling of vindoline and catharanthine exist in order to get the so-called dimeric Catharanthus indole-alkaloids VLB or vincristine [7-9] with pronounced anti-cancer activity [10, 11]. 

MERILLON, J.-M., OUELHAZI, L., DOIREAU, P., CHENIEUX, J.-C., RIDEAU, M., Metabolic changes and alkaloid production in habituated and non-habitualed cells of Catharanthus roseus grown in hormone-free medium. Comparing hormone-deprived non-habituated cells with habituated cells. J. Plant Physiol., 1989,134, 54-60. 

CANEL, C., LOPES-CARDOSO, M.I., WH1TMER, S VAN DER FITS, L., PASQUALI, G., VAN DER HEIJDEN, R HOGE, J.H., VERPOORTE, R., Effects of over-expression of strictosidine synthase and tryptophan decarboxylase on alkaloid production by cell cultures of Catharanthus roseus. Planta, 1998,205, 414-419. 

A large amount of structural information is available that describes the variety of indole alkaloids produced in plants. This has recently been followed by significant increases in our knowledge of the biosynthetic pathways that lead to their production and of the genes involved. Several reviews have appeared recently that describe the chemistry, biochemistry, cell and molecular biology of alkaloid biosynthesis.1 3 This chapter will selectively review recently characterized genes that appear to be responsible for the diversity and complexity of monoterpenoid indole alkaloids produced by plants. A particular focus will be on the reactions leading to the biosynthesis of vindoline (Fig. 8.1) in Catharanthus roseus. 

Extensive studies to quantitate the production of indole alkaloids in Catharanthus roseus hairy root cultures have revealed that they accumulate several compounds including ajmalicine, serpentine, catharanthine, tabersonine, horhammericine, and lochnericine.27, 28 The presence of tabersonine in hairy roots has raised speculations that this intermediate in vindoline biosynthesis, together with catharanthine, is transported from this potential site of biosynthesis through the vasculature to the stem and to the leaves where tabersonine is further elaborated into vindoline within laticifers and/or idioblasts.26 However, oxidized derivatives of tabersonine, such as horhammericine and lochnericine, are present at 5 to 15 times the levels of tabersonine in hairy roots,27 and presumably this prevents their transport and/or use for vindoline biosynthesis. In this context, it would be interesting to... 

Quaternary ammonium compounds are important as osmoprotectants in times of water deficiency and cold stress, as well as being constituents of membrane phospholipids, p-Alanine and phosphoethanolamine are each methylated three times on their nitrogens to produce the products P-alanine betaine and phosphocholine, respectively (Fig. ll.l).24"27 An example of O-methylation and N-methylation occurring on the same molecule can be found in the indole alkaloid vindoline from Catharanthus roseus (Fig. 11.1) 28,29... 

The terpenoid indole alkaloids have a variety of chemical structures and a wealth of biologic activities (Fig. 2a) (59, 60). Terpenoid indole alkaloids are used as anticancer, antimalarial, and antiarrhythmic agents. Although many biosynthetic genes from this pathway remain unidentified, recent studies have correlated terpenoid indole alkaloid production with the transcript profiles of Catharanthus roseus cell cultures (61). 

The antitumor alkaloids vinblastine (1) and vincristine (2) were the first natural products to be used on a large scale as anticancer agents, and they thus blazed the trail for others that came afterward. Vinblastine (as vincaleukoblastine) was isolated from Catharanthus roseus (L.) G. Don, which was formerly known... 

The presence or absence of phosphate ions plays an important role in the expression and accumulation of some secondary products. Zenk et al. (47) have demonstrated a 50% increase in anthraquinone accumulation in cell cultures of Morinda citrofolia when phosphate was increased to a concentration of 5g7h In suspension cultures of Catharanthus roseus, the overall accumulation of secondary metabolites like tryptamine and indole alkaloids has been shown to occur rapidly when cells were shifted to a medium devoid of phosphate (48,49). A study on the uptake of phosphate and its effect on phenylalanine ammonia lyase and the subsequent accumulation of cinnamoyl putrescine in cell suspension cultures of Nicotiana tabacum demonstrated marked sensitivity to phosphate concentration (5DX Enhanced phenylalanine ammonia lyase activity and increased production of cinnamoyl putrescine was induced by subculture onto phosphate-free medium while suppression of these effects and stimulation of growth was observed with phosphate concentrations of 0.02-0.5uM. Interestingly, phenylalanine ammonia lyase activity is stimulated by increasing phosphate concentrations in cell suspension of Catharanthus roseus (51). 

Deus-Neumann, B. and Zenk, M. H., Instability of indole alkaloid production in Catharanthus roseus cells in suspension cultures, Planta Med., 50 427 31 (1984)... 

Since the yields of active dimeric alkaloids from Catharanthus roseus is the lowest of any medicinally useful compounds (yield of VCR == 3 x 10 " %), material for studies of structure-activity relations has been scarce. Nevertheless, such studies were performed with VLB and it has been claimed that some of the products of molecular modification are more active in animal tests than VLB itself. 

Tissue or cell cultures of Catharanthus roseus continue to be investigated, in the search for improved methods of production of pharmacologically important alkaloids. Three such investigations have recently been reported, and although several alkaloids were obtained, including some heteroyohimbine and yohimbine alkaloids, no new ones were encountered, and none of the valuable bisindole alkaloids was obtained. However, pleiocarpamine, one of the alkaloids isolated, had not previously been 6 2... 

Higher plants have evolved an extraordinary variety of secondary metabolic pathways, the resulting products of which have been put to use by man providing pharmaceuticals for drug use, insecticides and various allelochemicals for pest control, and extracts for the flavor and fragrances industries. In spite of advances in synthetic organic chemistry, plants remain a major source of natural products, particularly in the specialty chemicals industry. Compounds, such as the insecticide derived from Azadiraohta indioa or the antitumor alkaloids vinblastine and vincristine found in periwinkle (Catharanthus roseus) (1 ), have complicated structures which preclude at the present time the development of an economical chemical synthesis (Figure 1). In the case of... 

Induction and production media are used in a two-stage process in the first stage biomass is produced, and the second stage allows alkaloid production. Morris, however, selected conditions for a Catharanthus roseus culture in which high alkaloid accumulation could be combined with high biomass accumulation (37). 

The initial pH of culture media is generally adjusted to 5.5-6. So far, little is known about the influence of the initial pH of the culture medium on productivity. For Catharanthus roseus cultures contradictory results have been reported no effect at an initial pH of 5.5, 6.0, or 6.5 (55) but higher productions at pH 5.5 (39) and at pH 7.0 (40), with the results seeming to be cell line specific. In cultures of Hyoscyamus muticus a 7-fold increase in alkaloid production was found on media with an initial pH of 3.5 (41). More alkaloid was released by Mcotiana rustica hairy roots when grown on media with lower initial pH values (i.e., pH 5-5.5) buffering of the media with 50 mM MES decreased growth and total alkaloid production (42). 

Increased alkaloid production induced by increased osmotic strength of the culture media was detected in Catharanthus roseus suspension cul-... 

The release of sanguinarine from the cells into the medium after elicitation was used as the basis for the design of an industrial process for production of the alkaloid 72,99,487,492,493). As the cells of P. somniferum remain viable after a 72-hr exposure to a fungal elicitor, it is possible to recycle the cells after induction of alkaloid biosynthesis. Because about 40-60% of the alkaloid is released to the medium, a simple change of medium is sufficient to collect the alkaloid and to recover viable cells. By a repeating sequence of elicitation and medium replenishment, a semicontinuous production process was obtained. Such a process was also developed for alkaloid production in Catharanthus roseus, Ruta grave-olens, and P. somniferum cell cultures using different elicitors (99). 

Several important alkaloids have been isolated from Catharanthus roseus plants ajmalicine (34) and serpentine (35) from the root, and the dimeric alkaloids vinblastine (36) and vincristine (37) from the leaves. Therefore, much research has been done on production of these alkaloids by means of plant cell culture. Catharanthus roseus is, in fact, one of the most widely studied plants for the production of secondary metabolites in cell culture systems. We here discuss the two types of alkaloids separately. Table XXIX summarizes patents concerning the production of alkaloids by means of cell cultures of C. roseus. 

Influence of Plant Growth Regulators on Alkaloid Production in Catharanthus roseus Cell Cultures... 

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