Indole alkaloids from suspension cultures

Indole alkaloids from suspension cultures. of Tabernaemontana species. 

R. van der Heijden, A. Hermans-Lokkerbol, R. Verpoorte and A. Baerheim Svendsen, Pharmacognostical studies on Tabernaemontana XX. Ion-pair droplet counter-current chromatography of indole alkaloids from suspension cultures,... 

Pawelka and Stockigt (5S5) identified eight different indole alkaloids from cell suspension cultures of O. elliptica. The pattern of the alkaloids found was quite different from the above-mentioned results reported by Kuoadio and co-workers (779). The alkaloids identified were tetrahydroal-stonine, cathenamine, pleiocarpamine and two other methoxy-substituted heteroyohimbine alkaloids Corynanthe/heteroyohimbine type), norfluo-rocurarine Strychnos type), and apparicine and epchrosine Aspi-dosperma type). The last mentioned alkaloid was a new compound (729) not known from intact plants. 

Arens H, Borbe HO, Ulbrich B, Stockigt J 1982 Detection of pericine, a new CNS-active indole alkaloid from Picralima nitida cell suspension culture by opiate receptor binding studies. Planta Med 46 210-214... 

The work by Scott and Lee 165) on the isolation of a crude enzyme system from a callus tissue culture of C. roseus was followed by studies of Zenk et al. on an enzyme preparation from a cell suspension system which produced indole alkaloids 166). The cell-free preparation was incubated with tryptamine and secologanin (34) in the presence of NADPH to afford ajmalicine (39), 19-epiajmalicine (92), and tetrahydroalstonine (55) in the ratio 1 2 0.5. No geissoschizine (35) was detected. In the absence of NADPH, an intermediate accumulated which could be reduced with a crude homogenate of C. roseus cells in the presence of NADPH to ajmalicine (39). Thus, the reaction for the formation of ajmalicine is critically dependent on the availability of a reduced pyridine nucleotide. 

The conversion of anhydrovinblastine (8) to vinblastine (1) has been examined by several different groups, using intact plants, cell suspension systems, and cell-free preparations. From the studies discussed above it was clear that 3, 4 -anhydrovinblastine (8) was probably the initially formed intermediate in the condensation of vindoline (3) and catharanthine (4) prior to vinblastine (1). Kutney and co-workers have reported (225,226) on the biotransformation of 3, 4 -anhydrovinblastine (8) using cell suspension cultures of the 916 cell line from C. roseus a line which did not produce the normal spectrum of indole alkaloids. After 24 hr the major alkaloid products were leurosine (11) and Catharine (10) in 31 and 9% yields, respectively, with about 40% of the starting alkaloid consumed. 

Biosynthetic research relating to the isoquinoline family was extremely successful, with such important members as morphine [3, 14], codeine [3, 15] or berberine [3, 14,16-18]. Extensive efforts have provided details pertaining to multiple sets of enzymes participating in the biosynthesis of the alkaloids above, in many cases with the help of plant cell suspension culture techniques. Since 1988, when the breakthrough in cloning of cDNA from alkaloid biosynthesis occurred [19, 20], a significant number of enzymes known from the indoles and isoquinolines biosynthesis have been isolated, their biochemical properties described and the majority of their corresponding cDNAs cloned and functionally over-expressed in non-plant hosts such as Escherichia coli, yeast or insect cells. 

DE LUCA, V., BALSEVICH, J., TYLER, R.T., KURZ, W.G.W, Characterization of a novel V-methyltransferase (NMT) from Catharanthus roseus plants. Detection of NMT and other enzymes of the indole alkaloid biosynthetic pathway in different cell suspension culture systems. Plant Cell Rep., 1987,6,458-461. 

The enzyme STR1 that was first characterized in Catharanthus roseus cell suspension cultures produces the central indole alkaloid intermediate H-3-a-(S)-strictosidine from tryptamine and secologanin (Fig. 8.9). It is well known that strictosidine represents the central intermediate precursor for several thousand indole alkaloids found in Nature. STR1 was the first gene to be cloned from R serpentina that involved a committed step in alkaloid biosynthesis.31 This was soon followed by the identification and isolation of an STR clone from Catharanthus roseus32 whose sequence was 80 % identical to the same gene from R serpentina.31... 

The internal storage capacity is limited by the number of storage cells. Excreted alkaloids which are dissolved in the medium are exposed to catalytic activities in the medium (e.g., peroxidases) and can thus be degraded as was demonstrated for quinolizidine alkaloids in lupine cell cultures (92). A role for peroxidases in product degradation has been recently demonstrated for indole alkaloids produced by suspension cultures of Tabemaemontana. Reduction of peroxidase activity by removal of Ca ions from the medium resulted in the formation of the alkaloid... 

A number of terpenoid indole alkaloids have pharmaceutical interest. These alkaloids are isolated from plants belonging to the families Apocy-naceae, Loganiaceae, and Rubiaceae. For the production of alkaloids by means of plant cell cultures, plants of the latter two families have proved to be rather recalcitrant (e.g., see Cinchona alkaloids). On the other hand, it has been reported by Pawelka and Stockigt that all apocynaceous cell suspensions they studied did produce terpenoid indole alkaloids 588). Here we confine ourselves to alkaloids which have direct commercial interest the production of new, potentially interesting, compounds is not reviewed here. For this we refer the reader to reviews by Balsevich (589), van der Heijden et al. (tribe Tabernaemontaneae) (590), and Omar (Rhazya stricta) (591). 

Schallenberg and Berlin (27) selected several 5-methyltryptophan-resistant cell lines from wild-type cells of different C. roseus cell suspension cultures. This resulted in increased tryptophan synthesis in all cell lines, and up to 30 times the normal levels of free tryptophan were detected. Increased tryptophan production did not, however, result in higher levels of tryptamine nor indole alkaloids. 

The enzyme strictosidine synthase (EC 4.3.3.2) is responsible for the stereospecific coupling of tryptamine and secologanin, yielding strictosidine (Fig. 12). This glucoalkaloid is the precursor for all terpenoid indole and related alkaloids, including among others the Cinchona quinoline alkaloids. Hampp and Zenk (707) isolated and purified this enzyme to homogeneity from a cell suspension culture of R. serpentina. The enzyme could successfully be immobilized on CNBr-activated Sepharose 4B, as was reported for this enzyme isolated from Catharanthus roseus (102,708). It proved to be more stable than the C. roseus enzyme the half-life of the immobilized enzyme was 100 days at a temperature of 37°C. 

C21H22N2O3, Mr 350.42, amorphous, [a]D -52° (CHCI3). 0 unstable, reactive alkaloid, existing as /d -immonium salts, isolated from Guettarda exi-mia (Rubiaceae) and synthesized enzymatically with enzymes from Catharanthus roseus cell suspension cultures C. plays a key role in the biosynthesis of monoterpenoid indole alkaloids, e.g., the hetero-yohimbine alkaloids such as ajmalicine, 19-epi-aj-malicine, and tetrahydroalstonine (see Alstonia alkaloids). 

Other cell suspension cultures of Catharanthus roseus synthesize and accumulate substantial amounts of both serpentine (8) and ajmalicine (7). Serpentine (8), the principal alkaloid of some cell cultures of Catharanthus roseus, is derived from ajmalicine (7) in contrast to some other indole alkaloids, serpentine is located inside the vacuole (Deus-Neumann and Zenk, 1984 Zenk et al 1985). Serpentine... 

The alkaloid productivity and the storage capacity in cultured plant cells can be influenced by the pH gradient between the medium and the accumulation sites inside the plant cell (vacuoles). A shift in the medium pH from low to high value was used to release the intracellularly stored alkaloids into the culture medium in cell suspension culture of C. roseus [42]. Similarly, transient modifications in the medium pH value led to the release of indole alkaloids in culture medium during immobilized cell cultivation of C. roseus [43]. 

Yoshikawa T, Furuya T (1983) Regeneration and in vitro flowering of plants derived from callus cultures of opium poppy Papaver somniferum). Experientia 39 1031-1033 Zenk MH, El-Shagi H, Arens H, Stockigt J, Weiler EW, Deus B (1977) Formation of the indole alkaloids serpentine and ajmalicine in cell suspension cultures of Catharanthus roseus. In Barz W, Reinhard E, Zenk MH (eds) Plant tissue culture and its bio-technological application. Springer, Berlin Heidelberg New York, pp 27-43... 

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