Strictosidine

The in vivo transformation of [6-14C]strictosidine (19) to gelsemine in Gelsemium sempervirens was claimed with an incorporation of 0.47% (33). This provides another experimental support to the proposal that strictosidine appears to be the original precursor in the biosynthesis of monoterpenoid indole alkaloids, although the detailed pathway of this biosynthetic process still remains obscure. 

The alkaloidal glucoside strictosidine was recognized in 1968 as the biosynthetic precursor of monoterpenoid indole alkaloids.3 The enzyme that... 

Fig. 10.1 Reaction catalyzed by strictosidine synthase (Str) in monoterpenoid indole alkaloid formation.

KUTCHAN, T.M., HAMPP, N., LOTTSPEICH, F., BEYREUTHER, K., ZENK, M.H., The cDNA clone for strictosidine synthase from Rauvolfia serpentina - DNA sequence determination and expression in Escherichia col., FEBSLett., 1988, 237,40-44. 

SMITH, G.N., Strictosidine A key intermediate in the biogenesis of indole alkaloids, Chem. Commun. 1968, 912-914. 

PFITZNER, U., ZENK, M.H., Immobilization of strictosidine synthase from Catharanthus cell cultures and preparative synthesis of strictosidine, Planta Med., 1982, 46,10-14. 

HAMPP, N., ZENK, M.H., Homogeneous strictosidine synthase from cell suspension cultures of Rauvolfia serpentina, Phytochemistry, 1988,27, 3811-3815. 

KUTCHAN, T.M., Expression of enzymatically active cloned strictosidine synthase from the higher plant Rauvolfia serpentina in Escherichia coli, FEBS Lett., 1989, 257, 127-130. 

KUTCHAN, T.M., BOCK, A., DITTRICH, H., Heterologous expression of strictosidine synthase and berberine bridge enzyme in insect cell culture, Phytochemistry, 1994, 35, 353-360. 

Scheme 22. The enzymatic synthesis of strictosidine (86) by immobilized strictosidine synthase.

Since the last major review of the biosynthesis of the monoterpenoid indole alkaloids (97), there have been several full and partial 98-104) reviews of various aspects of the work that has been conducted since 1974. Two major developments have dominated the field in this period, namely, the demonstrations that (i) strictosidine (33) is the universal precursor of the monoterpenoid indole alkaloids and (ii) selected cell-free systems of C. roseus have the ability to produce the full range of alkaloid structure types, including the bisindoles. This section traces some aspects of these developments, paying particular attention to work been carried out with C. roseus, and omitting work, important though it may be, on other monoterpenoid indole alkaloid-producing plants, e.g., Rauwolfia, Campto-theca, and Cinchona. 

The 200GW line proved to be quite different, and of particular interest was the discovery that this line produced catharanthine (4) at levels about three times that of the intact plant (0.005%) (155,159,160). Curiously, the predominant alkaloid (60.48%) was strictosidine lactam (41), which is not normally seen in extracts of intact plants. Variation of the pH and added phytohormones did not significantly alter the pattern of alkaloids produced by this cell line (160). Further cell line studies (161) afforded one line (176G) which produced mainly ajmalicine (39) and lochnericine (73) and one (299Y) which apparently contained relatively inactive p-glucosi-dases, since the major alkaloids produced were strictosidine (33) (83%) and strictosidine lactam (41) (Table XIII). 

In vivo feeding experiments with singly and doubly labeled strictosidine (33) in C. roseus shoots afforded labeled ajmalicine (39), serpentine (40), vindoline (3), and catharanthine (4). Vincoside (85, page 37) was not incorporated into the alkaloids, suggesting that it was biologically inert 188). Brown and co-workers 190) conducted somewhat parallel studies examining the precursor relationship of strictosidine in C. roseus. Incorporation into tetrahydroalstonine (75), ajmalicine (39), catharanthine (4), and vindoline (3) was observed. 

Following Zenk s demonstration that strictosidine (33) is the key precursor of the indole alkaloids in a variety of plants in the family Apocyna-ceae, as well as several plants in other families, Battersby et al. 193) repeated experiments with both the 3a and 3p isomers of the primordial glucoside. The earlier results were not confirmed, for it was the 3a isomer which was specifically incorporated. Thus, the cycle of experiments is now complete for the involvement of strictosidine as the key intermediate in both enzyme systems and intact plants. 

The enzyme responsible for the stereospecific condensation of trypt-amine and secologanin 34) was called strictosidine synthase, and its presence was demonstrated by Treimer and Zenk 194) in a number of indole alkaloid-producing plants, including Amsonia salicifolia, Catharanthus roseus, Ochrosia elliptica, Rauwolfia vomitoria, Rhazya orientalis, Stem-madenia tomentosa. Vinca minor, and Voacanga africana. Enzyme activity as high as 1698 pkat/mg protein was observed for O. elliptica. No... 

Scott s group has also reported on the isolation of strictosidine syn-... 

Although ajmalicine (39) is not on the pathway to the bisindole alkaloids, it is a compound of substantial commercial interest, and several of the intermediates in its formation are probable intermediates in the extended biosynthetic pathway. This work is therefore reviewed for the purpose of completeness of studies on C. roseus. Considerable progress has been made on the biosynthesis of ajmalicine (39), and the studies on the formation of strictosidine (33) and cathenamine (76) have already been described. One of the preparations described by Scott and Lee was a supernatant from a suspension of young seedlings of C. roseus which af-... 

The next area for study was the pathway between strictosidine (33) and cathenamine (76), where the initial step is viewed as hydrolysis by p-glucosidase and opening of the hemiacetal to a dialdehyde 93. Attempts to trap this intermediate 200) have thus far failed, and it appears that recyclization and dehydration occur too rapidly, giving rise to 4,21-dehy-drocorynantheine aldehyde (94). Thus, an enzyme preparation was incubated at pH 7.0 in the presence of KBH4 to afford the C-16 epimers 69 and 95, thereby impuning the existence of 94. 

The glucosidases involved in the biosynthetic pathway have been studied in detail by Hemscheidt and Zenk 203), and two of the isolated enzymes were specific for the hydrolysis of strictosidine (33) and were purified 120-fold. The enzyme was isolated from a number of indole alkaloid producing plants, including C. roseus, C.pusilus, and C. trichophyllus. The pH optimum was 6.5, and the values were 0.2 mM for enzyme I and 0.1 mM for enzyme II. Molecular weights were estimated at 230,000 for enzyme I and 450,000 for enzyme II. Unlike the case of the enzyme system of Scott et al. 198,199), tryptamine did not activate either of the two enzymes. The enzymes were highly substrate specific vincoside (85)... 

Incubation of geissoschizine (35) with a cell-free extract from C. roseus 210) in the presence of NADPH caused the accumulation of an isomer of isositsrikine whose structure was established chemically to be the (167 ) isomer 58. None of the 16-epi isomer 95 was detected in the cell-free incubations or in feeding experiments with intact plants. Additionally, Stdck-igt has reviewed enzymatic studies on the formation of strictosidine (33) and cathenamine (76) (277), and Zenk has provided a very elegant summary of the enzymatic synthesis of ajmalicine (39) (272). 

Vincamine, vinblastine and vincristine are very important clinic alkaloids. They are produced naturally by plants vincamine by Vinca minor, and vinblascine and vincristine by Madagascar periwinkle Catharanthus roseus). The vindoline synthesis pathway starts with strictosidine and, via dehydrogeissoschizine, preakuammicine, stemmadenine and tabersonine, is converted to vindoline and vincristine (Figure 42). Conversion from vindoline to vinblastine is based on the NADH enzyme activity. Vinblastine and vincristine are very similar alkaloids. The difference is that vincristine has CHO connected to N, whereas vinblastine in the same situation has only CO3. This synthetic structural differences influence their activity. Vinblastine is used to treat Hodgkin s disease (a form of lymphoid cancer), while vincristine is used clinically in the treatment of children s leukaemia. Vincristine is more neurotoxic than vinblastine. 

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