Strictosidine synthesis

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

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

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. 

The power of engineered enzymes in the synthesis of novel alkaloids, to generate structural diversity and establish new alkaloid libraries, is best represented by the enzyme strictosidine synthase (STR1). 

Although previous studies suggested that SG is highly glycosylated, it appears to contain very little carbohydrate, since its expected Mr of 63 kDa is very close to the size obtained by SDS PAGE.36, 39 In addition, the enzyme appears to contain a C-terminal sequence KKXKX that is a putative retention signal for type I transmembrane ER proteins. However additional detailed studies must be done to provide evidence for the localization of SG in relation to the site of synthesis of strictosidine. 

The basic stmcture of monoterpenoid indole alkaloids includes an indole nucleus derived from tryptophan via tryptamine (L) and a versatile C9 or CIO unit arising from the monoterpenoid secologanin (LI). Strictosidine synthase catalyzes the synthesis of strictosidine (LII) from tryptamine and secologanin (Scheme XXIV) [76],... 

In analogy with the synthesis of strictosidine and vincoside from tryptamine, a mixture of 5a-carboxystrictosidine (33a) (major) and 5a-carboxyvincoside (33d) could be obtained by condensation of L-tryptophan with secologanin. Again in... 

The cell-free synthesis of strictosidine (79) and cathenamine (82) has been further explored, and the conditions under which these key compounds are formed have been optimized. Strains from C. roseus suspension cultures that were resistant to inhibition of their growth by various tryptophan analogues have been selected. The free tryptophan level in cells of these strains could be 30—40 times higher than in normal cells. Tryptophan at this level did not induce tryptophan decarboxylase, nor the production of alkaloids. It is to be noted, however, that stimulation of alkaloid production by tryptophan and tryptamine ° in cultures of normal cells has been reported. In the case of tryptamine the two most prominent metabolites were A/ -acetyltryptamine and JVN-dimethyltrypt-amine. 

The three arguments mentioned above, i.e., indole orientation, relationship of the amine side chain with residue Glu309 and aldehyde 44, may influence the reaction catalyzed by Pictet-Spenglerase STRl. All these structural interactions in the catalytic pocket most likely explain the comparatively lower activity of STRl against novel substrates for the synthesis of novel strictosidine analogues. 

Strategy I STR-mediated synthesis of novel strictosidines by recruiting newly discovered substrate analogues. 

Strategy II Synthesis of modified strictosidines using rational reengineered STR mutants. 

Strategy III Chemo-enzymatic synthesis of alkaloids from strictosidine (2) and azastrictosidine (87). 

---