Ajmalicine formation

From literature it becomes clear that light reduces ajmalicine formation while stimulating serpentine and catharanthine formation. It should be kept in mind, however, that exposure of plant cell cultures to light in large-scale culture vessels might be problematic or at least expensive. The applicability of light in a large-scale production process should therefore be doubted. 

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. 

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-... 

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). 

Zenk, M. H., H. El-Shagi, H. Arens, J. StoV ckigt, E. W. Weiler, and B. Deus, "Formation of the Indole Alkaloids Serpentine and Ajmalicine in Cell Suspension Cultures of Catharanthus roseus," in Plant Tissue Culture and Its Biotechnological Application, Eds. W. Barz, E. Reinhard, M. H. Zenk, New York Springer-Verlag, 1977, pp. 27-43. 

Biomimetic conversions reported51" on 7Vb,21-dehydrogeissoschizine include the formation of 17-hydroxydihydrocathenamine (64) on treatment with 2% aqueous hydrochloric acid and of isovallesiachotamine (65) in buffered solution at pH 4. Reduction (by NaBH4) of (64), followed by dehydration, affords 19-epi-ajmalicine (66) hence the configuration at C-19 is as depicted in (64). 19-epi-Ajmalicine (66) can also be obtained by the reaction of (61) with alumina, followed by reduction this may involve the intermediate formation of the fugitive dienamine (67), which can equilibrate with the (Z)-isomer of (61) before cyclization to the (19R) product (64) (Scheme 8).52... 

An alternative mechanism (shown in Scheme 10) for the formation of (109) from demethylcorynantheine (102) postulates the prior formation of a hemiacetal (110) followed by an irreversible attack on the mercurinium ion by the hydroxy-group to give an intermediate of structure (111). The inherent plausibility of such a mechanism led Goutarel et al.75 to study the mercuration-demercuration of cory-nantheine which, in an aqueous medium, can in principle give rise to the same hemiacetal (110), and thence the acetal (111). In fact this reaction gave a mixture of the acetals (104), (109), and their C-16 epimers which, on treatment with polyphosphoric acid, gave a mixture of ajmalicine (85a) and 19-epiajmalicine (85b) in a ratio of ca. 45 55. 

The mass spectra of ajmalicine (IXa) and its deuterated derivatives IXb-IXd exhibit a prominent ion at M-l (IXa) or M-2 (IXb-d), which is presumably due to formation of the corresponding 3-dehydro derivative XLI (R2, R j = H or D, as appropriate) by loss of the C-3 hydrogen atom. Other, but much less intense, ions at m/e 337 and 321 are formed by loss of a methyl or methoxyl group from ring E. 

The same conclusions concerning the stereochemistry of tetrahydroalstonine were reached from a comparison of the dissociation constants and rate of methiodide formation of tetrahydroalstonine and its stereoisomers (64). Both the reduced basicity of tetrahydroalstonine, pK 5.83 (cf. ajmalicine, pK 6.31) and its reduced rate of methiodide formation when compared with ajmalicine are in consonance with its formulation as a cis D/E isomer, in which Nb is to some extent sterically hindered... 

Fifty per cent of the M-l peak is found at M-2 in the spectra of 3-deu-terioyohimbine (CCCVII, prepared by NaBD4 reduction of 3,4-dehydro-yohimbine perchlorate) and of 3,5,6-trideuterioajmalicine (CCCIX, prepared by NaBD4 reduction of serpentine hydrochloride), so that one-half of the hydrogen lost in the formation of this peak comes from C-3 or, in the case of ajmalicine, from C-3 and C-6. 

Figure 2.9 Enzymic formation of ajmalicine. TDC, tryptophan decarboxylase STS, strictosidine synthase STG, strictosidine glucosidase POD, peroxidase.

Isolation of the stereospecific strictosidine s)mthase (STS) and formation of strictosidine with the 3a-(S) configuration proved conclusively that this was the natural precursor of the terpenoid indole alkaloids. Strictosidine occurs naturally in Rhazya stricta and the synthase has been isolated from a number of other species Amsonia salicifolia, A. tabemaemontam, Catharanthus pusillus, C. roseus, Rauvolfia verticillata, R. vomitoria, R. serpentina, Rhazya orientalis and Voacanga africana. The enzyme has been purified to homogeneity from R. serpentina (Hampp and Zenk, 1988). A comparison of the activity of STS from C. roseus roots, the only portion of the plant to contain ajmalicine, with that present in plant cell cultures producing the same alkaloid demonstrated that the plant cell cultures are far more metabolically active (Ziegler and Facchini, 2008). 

Schmidt, D. and Stockigt, J. (1995) Enzymic formation of the sarpagan-bridge a key step in the biosynthesis of sarpagine-ajmalicine-type alkaloids. Planta Med., 61, 254-8. 

In continuation of his biomimetic syntheses of heteroyohimbine alkaloids Brown" has succeeded in converting secologanin tetra-acetate (117) into elenolic acid (118), and in completing the synthesis of ajmalicine (80) essentially by the route published" earlier (Scheme 14). Contrary to the previous workers, who apparently isolated only ajmalicine. Brown eta/." obtained ajmalicine (80), 19-epiajmalicine (119) and tetrahydroalstonine (120). Since one of the lactams (121) afforded only 19-epiajmalicine (119) and in the general reaction sequence the proportion of (80), (119), and (120) in the final product depended on the length of time allowed for Schiff base formation rather than on the ratio of isomers present in the methyl elenolate [ester of (118)], it is apparent that interconversion of the imines (122) via the equilibria shown in Scheme 14 is responsible for the non-stereospecificity of the synthesis. 

ITie intermediates leading to ajmalicine have extensively been studied by Zenk, StOckigt, and co-workers (209-211). Cathenamine (210-213) is believed to be a major intermediate in this pathway. Moreover, evidence was presented for the intermediacy of 4,21-dehydrocorynantheine aldehyde (214) and 4,21-dehydrogeissoschizine (215) (Fig. 14). The formation of... 

The formation of ajmalicine from the carbinolamine or cathenamine requires a reduction. Hemscheidt (217) and Stdckigt et al. (222) described an enzyme cathenamine reductase (CR), which used cathenamine as substrate and NADPH as cofactor, yielding ajmalicine and 19-epi-ajmalicine. Hemscheidt and Zenk (223) reported partial purification of an NADPH-dependent tetrahydroalstonine synthase (THAS) from C. roseus cell cultures. This enzyme only yields tetrahydroalstonine, and the substrate was the iminium form of cathenamine. The Km for this substrate is 62 /nAf. The molecular mass of the enzyme was estimated to be 81 kDa. 

An important disadvantage which is frequently mentioned in the literature is the low degree of cell differentiation in suspension cultures of plant cells. As product formation in plants largely occurs in differentiated tissue, it seems reasonable that the undifferentiated state might not favor the formation of secondary products. In some cases, for example, the production of ajmalicine in cultures of Catharanthus roseus, it is indeed shown that the product, originally formed in the roots of the plant, is better produced in hairy root cultures than in cell suspension cultures. 

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