A-3 ,4’-Anhydrovinblastine

There are, however, a few examples in which the exclusive participation of peroxidase in the synthesis of natural bioactive alkaloids has been reported. These mainly concern the biosynthesis of some monoterpenoid indole alkaloids, especially the synthesis of a-3 ,4 -anhydrovinblastine (XLVII) [76],... 

Strictosidine is the common precursor of ajmalicine (XLVni), on the one hand, and of vindoline (LIH) and catharanthine (LIV), on the other, these last two being the precursors of a-3 ,4 -anhydrovinblastine (XLVn), vinblastine (XLIX R = CH3) and vincristine (XLIX R = CHO), in that order [76],... 

C. roseus is, therefore, an amazing chemical factory, which produces more than 100 different monoterpenoid indole alkaloids, many of them possessing notable pharmacological activity [158-159]. The main alkaloids present in C. roseus plants are catharanthine, vindoline and a-3 ,4 -anhydrovinblastine. Ajmalicine and serpentine are also present in significant amounts in the plant [160-165]. Class III plant peroxidases have been involved in the synthesis of many of these monoterpenoid indole alkaloids. 

Catharanthine (LIV) and vindoline (Lin) are regarded as the monomeric precursors of the dimeric alkaloids vinblastine and vincristine, via a-3 ,4 -anhydrovinblastine. C. roseus peroxidase catalyzes the coupling reaction of catharanthine and vindoline (Scheme XXVI) to lead to a-3 ,4 -anhydrovinblastine (XLVH) or, more properly, to an iminium intermediate (LVI) from which a-3 ,4 -anhydrovinblastine is directly derivated [52,74,166]. a-3 ,4 -Anhydrovinblastine is then converted to vinblastine (XLIX, R = CH3) and vincristine (XLIX, R = CHO) in C. roseus plants [167-169], a-3 ,4 -Anhydrovinblastine (XLVn), or the unstable iminium intermediate (LVI) formed during the coupling reaction, is then assumed to be the precursor of all dimeric alkaloids in C. roseus. 

In 1975, Potier and collaborators proposed that, in planta, the dimeric vinblastine type alkaloids resulted from the coupling of catharanthine and vindoline and, in light of this hypothesis, they reported for the first time the chemical synthesis of a dimer with the natural configuration through a modified Polonovski reaction [18, 19]. This reaction resulted in the formation of an iminium dimer which, after reduction with NaBH4, yielded a-3 ,4 -anhydrovinblastine, Fig. (2), later proved to be the first dimeric biosynthetic precursor of vinblastine in the plant. The group of Potier investigated possible modifications of anhydrovinblastine and produced vinorelbine, Fig. (1), which was the first active derivative with an altered cleavamine (catharanthine) moiety [20, 21]. 

Sottomayor M, Ros Barcelo A (2003) Peroxidase from Catharanthus roseus (L.) G. Don and the biosynthesis of a-3, 4 -anhydrovinblastine a specific role for a multifunctional enzyme. Protoplasma 222 97-105. doi 10.1007/s00709-003-0003-9... 

Additionally, there had, for many years, been a vast synthetic effort underway aimed at the synthesis of the two monomeric units, where it was anticipated that the two units could be joined to form the vinblastine-type bisindole alkaloids. Coincidentally, as it transpired, 20 years of effort in the areas of synthesis and biosynthesis converged, at almost the same time, on the compound 3, 4 -anhydrovinblastine (8). 

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. 

The chemical reactivity of N-6 (or N ), directed entirely by the basicity of this atom, is controlled by the nature and stereochemistry of the substituents at C-4 (vide supra). Oxidation of N-6 occurs under mild conditions in several naturally occurring bisindole alkaloids. Thus, treatment of a dichloromethane solution of leurosine (4) with m-chloroperben-zoic acid at -20°C for 4 hr gives the N -oxide (15) in greater than 90% yield after preparative reversed-phase chromatography (46). Leurosine A/ -oxide has also been isolated from Catharanthus roseus and should therefore be considered a naturally occurring bisindole (50). The analogous conversion of vinblastine (1) to its A/ -oxide (16) proceeds under similar conditions but requires longer exposure to the peraeid (24 hr) (5/) 3, 4 -anhydrovinblastine is converted to its N -oxide (17) in 10 min at 0°C... 

SOTTOMAYOR, M., LOPEZ-SERRANO, M., DICOSMO, F., ROS-BARCELO, A., Purification and characterization of alpha-3 4 -anhydrovinblastine synthase (peroxidase-like) from Catharanthus roseus (L.) G. Don. FEBS Lett., 1998, 428, 299-303. 

Furthermore, the same cell-free extracts will also couple vindoline and catha-ranthine to yield the dimeric 3, 4 -anhydrovinblastine, which forms fhe natural dimeric alkaloids, leurosine, Catharine and vinblastine (Fig. 2.11) (Kutney, 1987). The enzyme, which apparently brings about the coupling appears to be a POD (Endo et al, 1986 Goodbody et al, 1988). A commercial method for production of vincrisfine depends on fhe efficienf conversion of 3, 4 -anhydrovinblasfine fo vinblastine, which is yet to be achieved. 

Endo, T., Goodbody, A., Vukovic, J. and Misawa, M. (1986) Enzymes from Catharanthus roseus cell suspension cultures that couple vindoUne and catharanthine to form 3, 4 -anhydrovinblastine. Phytochemistry, 27,2147-9. 

Ferric ion-induced coupling of catharanthine (135) and vindoline (140) in aqueous acidic media to produce 3,4 -anhydrovinblastine has been proposed to occur via the formation of a cation radical (136) of the tertiary amine of catharanthine (Scheme 30). Rearrangement and subsequent fragmentation between C16 and C21 leads to ring opening. A second oxidation followed by nucleophilic attack of the diiminium (137) by vindolene (140) results in the formation of iminium (139), which on borohydride reduction yields 3,4 -anhydrovinblastine (77 %) [238]. 

Goodbody, A. E., T. Endo, J. Vukovic, J. P. Kutney, L. S. L. Choi, and M. Misawa, Enzymic coupling of catharanthine and vindoline to form 3, 4 -anhydrovinblastine by horseradish peroxidase, Planta Medica, 54, 136-140 (1988). 

Finally, the monomeric precursors vindoline and catharanthine are coupled in a reaction thought to be catalyzed by a class III Prx, namely, CrPrxl, to yield the dimeric alkaloid 3, 4 -anhydrovinblastine, which is converted into vinblastine and then into vincristine (Scheme 4.7) [35, 36]. 

More recently, Kutney and co-workers (220) have investigated whether the same dihydropyridinium intermediate 109 is involved in the enzymatic conversion of catharanthine (4) and vindoline (3) to anhydrovinblastine (8) as is involved in the chemical conversion. Use of a cell-free preparation from a 5-day culture of the AC3 cell line gave 18% of the bisindole alkaloids leurosine (11), Catharine (10), vinamidine (25), and hydroxy-vinamidine (110), with 10 predominating. When the incubations were carried out for only 5-10 min, the dihydropyridinium intermediate was detected followed by conversion to the other bisindole alkaloids, with FAD and MnClj required as cofactors. Clearly a multienzyme complex is present in the supernatant, but further purification led to substantial loss of enzymatic activity. The chemical formation of anhydrovinblastine (3) is carried out with catharanthine A-oxide (107), but when this compound was used in the enzyme preparation described, no condensation with vindoline (3) occurred to give bisindole alkaloids. This has led Kutney and co-workers to suggest (220) that the A-oxide 108 is not an intermediate in the biosynthetic pathway, but rather that a 7-hydroperoxyindolenine... 

When 3, 4 -[ao - H]anhydrovinblastine (8) was incubated with a cell-free preparation at pH 6.3 for 50 hr, leurosine (11) and Catharine (10) were labeled to the extent of 8.15 and 15.15%, respectively, and vinblastine (1) was labeled to 1.84% (776,227). Approximately the same level of incorporation was obtained by Scott s group, using 3, 4 -[21 - H]anhydrovinblas-tine (8) and isolating vinblastine (1) from cell-free extracts of C. roseus (228). Scott s failure (87) to observe incorporation of the same precursor into vinblastine (1) in whole plants was explained by the established (82) instability of anhydrovinblastine (8). 

It would thus appear that the presence of an a-acetoxy-group at C-15 severely inhibits the fission of the 16,21-bond in the coupling reaction, since the isovinblastine O-acetate (258) was obtained in yields of only 6 and 4%, respectively, from (257) and (260). The effect of a /3 -acetoxy-group is less well defined Honma and Ban " report the formation of anhydrovinblastine (255), but only as the minor product of the reaction, whereas Kutney and Worth report the formation of (253) and (254), but in unspecified yield. For the synthesis of vinblastine derivatives the absence of a C-15 substituent, as in catharanthine and dihydro-catharanthine, seems preferable for example, catharanthine N-oxide was... 

Goodbody and co-workers (7/9) have examined the production of alkaloids in root and shoot cultures induced from seedlings of C. roseus. The pattern of alkaloids in the root cultures was similar to that of the roots from intact plants. Thus ajmalicine (39) and catharanthine (4) were produced, but no vindoline (3), a major leaf alkaloid, and no bisindole alkaloids. Similarly, the pattern of the alkaloid content of the shoot cultures was like that of the leaves of the intact plant, showing the presence of vindoline (3), catharanthine (4), and ajmalicine (39), with 3 predominating. A search for the bisindole alkaloids in the cultures indicated the presence of anhydrovinblastine (8) and leurosine (11) in the shoot cultures (2.6 and 0.3 xg/g fresh weight, respectively), but no vinblastine (1) or vincristine (2). 

The enzyme-catalyzed formation of anhydrovinblastine (8) from catharanthine (4) and vindoline (3) was first examined by Kutney and co-workers (170,219) using a cell-free preparation. [ao f- H]Catharanthine (4) and [acety/- C]vindoline (3) were incubated for 3-8 hr, both separately and jointly with a preparation from C. roseus, which led to the isolation of labeled anhydrovinblastine (8) and leurosine (11) incorporations were of the order of 0.54 and 0.36%, respectively. On this basis, anhydrovinblastine (8) was proposed as the key biosynthetic intermediate en route to vinblastine (1) and vincristine (2). 

Amino nitroxides, 383 5-Aminotetrazoles, 336, 337 N-Amino-2,4,6-triphenylpyridinium perchlorate, 409 Ammonium acetate, 11 Ammonium cerium(IV) sulfate, 56 Ammonium metavanadate, 418 5a-Androstane-3(3.17 3-diol, 6 5a-Androstane-3d-ol-17-one, 6 A -Androstene-3,17-dione, 167 Angustmycin, 407 2,S -Anhydronucleosides, 88 Anhydrovinblastine, 389 Anion exchange resins, 69 Annelation, 71, 90, 148 Anthraquinone, 29 Anthrasteroids, 167 Antimony(V) chloride, 12 Apomorphine, 236... 

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