Tabersonine

Abstract: Described is a concise, highly stereocontrolled strategy to the Aspidosperma family of indole alkaloids, one that is readily adapted to the asymmetric synthesis of these compounds. The strategy is demonstrated by the total synthesis of (±)-tabersonine (rac-1), proceeding through a 12-step sequence. The basis for this approach was provided by a highly regio- and stereoselective [4 + 2] cycloaddition of 2-ethylacrolein with 1-amino-3-siloxydiene developed in our laboratory. Subsequent elaboration of the initial adduct into the hexahydroquinoline DE ring system was accomplished efficiently by a ring-closing olefin metathesis reaction. A novel ortho nitrophenylation of an enol silyl ether with (o-nitrophenyl)phenyliodonium fluoride was developed to achieve an efficient, regioselective introduction of the requisite indole moiety. The final high-yielding conversion of the ABDE tetracycle into pentacyclic target rac-1 relied on intramolecular indole alkylation and regioselective C-carbomethoxylation. Our approac...

Abstract: Antitumor substances related to vinblastine and vincristine are exclusively found in the Catharanthus roseus (Madagascar periwinkle), a member of the Apocynaceae plant family, and continue to be extensively used in cancer chemotherapy. Although in high demand, these valuable compounds only accumulate in trace amounts in C. roseus leaves. Vinblastine and vincristine are condensed from the monoterpenoid indole alkaloid (MIA) precursors catharanthine and vindoline. Although catharanthine biosynthesis remains poorly characterized, the biosynthesis of vindoline from the MIA precursor tabersonine is well understood at the molecular and biochemical levels. This study uses virus-induced gene silencing (VIGS) to identify a cytochrome P450 [CYP71D1V2; tabersonine 3-oxygenase (T3O)] and an alcohol dehydrogenase [ADHL1; tabersonine 3-reductase (T3R)] as candidate genes involved in the conversion of tabersonine or 16-methoxytabersonine to 3-hydroxy-2,3-dihydrotabersonine or 3-hydroxy-16-methoxy-2,3-dihydrotabersonine, which are intermediates in the vindorosine and vindoline pathways, respectively. Biochemical assays with recombinant enzymes confirm that product formation is only possible by the coupled action of T3O and T3R, as the reaction product of T3O is an epoxide that is not used as a substrate by T3R. The T3O and T3R transcripts were identified in a C. roseus database representing genes preferentially expressed in leaf epidermis and suggest that the subsequent reaction products are transported from the leaf epidermis to specialized leaf mesophyll idioblast and laticifer cells to complete the biosynthesis of these MIAs. With these two genes, the complete seven-gene pathway was engineered in yeast to produce vindoline from tabersonine.

Abstract: Cells of Catharanthus roseus (L.) G. Don were genetically engineered to over-express the enzymes strictosidine synthase (STR; EC 4.3.3.2) and tryptophan decarboxylase (TDC; EC 4.1.1.28), which catalyze key steps in the biosynthesis of terpenoid indole alkaloids (TIAs). The cultures established after Agrobacterium-mediated transformation showed wide phenotypic diversity, reflecting the complexity of the biosynthetic pathway. Cultures transgenic for Str consistently showed tenfold higher STR activity than wild-type cultures, which favored biosynthetic activity through the pathway. Two such lines accumulated over 200 mg · L−1 of the glucoalkaloid strictosidine and/or strictosidine-derived TIAs, including ajmalicine, catharanthine, serpentine, and tabersonine, while maintaining wild-type levels of TDC activity. Alkaloid accumulation by highly productive transgenic lines showed considerable instability and was strongly influenced by culture conditions, such as the hormonal composition of the medium and the availability of precursors. High transgene-encoded TDC activity was not only unnecessary for increased productivity, but also detrimental to the normal growth of the cultures. In contrast, high STR activity was tolerated by the cultures and appeared to be necessary, albeit not sufficient, to sustain high rates of alkaloid biosynthesis. We conclude that constitutive over-expression of Str is highly desirable for increased TIA production. However, given its complexity, limited intervention in the TIA pathway will yield positive results only in the presence of a favorable epigenetic environment.

Abstract: Late exponential phase hairy root cultures of Catharanthus roseus were elicited with pectinase and jasmonic acid. The effects of elicitor concentration and exposure time on growth and levels of several compounds in the indole alkaloid biosynthetic pathway were monitored. Pectinase decreased the fresh weight to dry weight ratio of the roots, while addition of jasmonic acid had no significant effect. Selective effects on indole alkaloid yields were observed upon addition of elicitors. An increase of 150% in tabersonine specific yield was observed upon addition of 72 units of pectinase. Transient studies at the same level demonstrated possible catabolism as serpentine, tabersonine, and lochnericine levels decreased immediately after elicitation. The levels of these compounds recovered back to control levels or were higher than the control levels after some time. Jasmonic acid was found to be a unique elicitor leading to an enhancement in flux to several branches in the indole alkaloid pathway. Jasmonic acid addition caused an increase in the specific yields of ajmalicine (80%), serpentine (60%), lochnericine (150%), and horhammericine (500%) in dosage studies. Tabersonine, the likely precursor of lochnericine and horhammericine, decreased at lower levels of jasmonic acid and then increased with increasing jasmonic acid concentration. Transient studies showed that lochnericine and tabersonine levels go through a maxima, then decrease back to control levels and reduce below control levels, respectively. The yields of ajmalicine, serpentine, and horhammericine increased continuously after the addition of jasmonic acid. The methods described in this article could generally be used in devising strategies for enhancement in productivity of secondary metabolites and for probing and studying the complex secondary metabolite pathways in plant tissue cultures.