Deacetylvindoline

Salutaridinol 7-0-acetyltransferase catalyzes the conversion of the phenanthrene alkaloid salutaridinol to salutaridinol-7-Oacetate, the immediate precursor of thebaine along the morphine biosynthetic pathway in P. somniferum (Fig. 10.7).26 Acetyl CoA-dependent acetyltransferases have an important role in plant alkaloid metabolism. They are involved in the synthesis of monoterpenoid indole alkaloids in medicinal plant species such as Rauwolfia serpentina. In this plant, the enzyme vinorine synthase transfers an acetyl group from acetyl CoA to 16-epi-vellosimine to form vinorine. This acetyl transfer is accompanied by a concomitant skeletal rearrangement from the sarpagan- to the ajmalan-type (reviewed in2). An acetyl CoA-dependent acetyltransferase also participates in vindoline biosynthesis in Catharanthus roseus, the source of the chemotherapeutic dimeric indole alkaloid vinblastine (reviewed in2). Acetyl CoA deacetylvindoline 4-O-acetyltransferase catalyzes the last step in vindoline biosynthesis. A cDNA encoding acetyl CoA deacetylvindoline 4-0-acetyltransferase was recently successfully isolated.27... 

The translation of the sequence of the cDNA encoding deacetylvindoline 4-O-acetyltransferase compared to other putative plant acetyltransferases revealed a conserved region near the carboxy terminus of the proteins. This sequence was used to design a degenerate antisense oligodeoxynucleotide primer for PCR. The sense primer was based upon an internal peptide sequence of salutaridinol 7-0-acetyltransferase. This approach finally yielded a partial cDNA that encoded salutaridinol 7-O-acetyltransferase. The full-length clone was obtained by RACE-PCR and was functionally expressed in S. frugiperda Sf9 cells.28 The amino acid sequence of salutaridinol 7-O-acetyltransferase is most similar (37% identity) to that of deacetylvindoline acetyltransferase of C. roseus.27... 

Earlier biotransformation studies with vindoline were reported from the Eli Lilly Laboratories (166-168), and microbial transformation products included N-demethylvindoline (48), deacetylvindoline (49), and a structurally novel com-... 

Recent work in our laboratories has confirmed the existence of a similar pathway in the oxidation of vindoline in mammals (777). The availability of compounds such as 59 as analytical standards, along with published mass spectral and NMR spectral properties of this compound, served to facilitate identification of metabolites formed in mammalian liver microsome incubations. Two compounds are produced during incubations with mouse liver microsome preparations 17-deacetylvindoline, and the dihydrovindoline ether dimer 59. Both compounds were isolated and completely characterized by spectral comparison to authentic standards. This work emphasizes the prospective value of microbial and enzymatic transformation studies in predicting pathways of metabolism in mammalian systems. This work would also suggest the involvement of cytochrome P-450 enzyme system(s) in the oxidation process. Whether the first steps involve direct introduction of molecular oxygen at position 3 of vindoline or an initial abstraction of electrons, as in Scheme 15, remains unknown. The establishment of a metabolic pathway in mammals, identical to those found in Strep-tomycetes, with copper oxidases and peroxidases again confirms the prospective value of the microbial models of mammalian metabolism concept. 

DeLuca and co-workers have reached somewhat different conclusions. In support of their pathway they have isolated an A-methyltransferase which catalyzes the N -methylation of 16-methoxy-2,3-dihydro-3-hy-droxytabersonine (104) to 4-deacetoxyvindoline (43) (2/6). The Af-methyl-transferase activity was found to co-chromatograph with chlorophyll on Sephadex 100, but subsequent centrifugation gave a supernatant which possessed the A-methyltransferase activity and a pellet which contained the chlorophyll. Highest substrate specificity was shown by 105 (0.07 pkat/100 (jlI enzyme), followed by 2,3-dihydrotabersonine (106). 4-0-Deacetyl-Aa-demethylvindoline (98) was not a substrate, but, unfortunately, 16-0,Aa-bisdemethyl-4-0-deacetylvindoline (107), which is a key intermediate in Stockigt s proposed pathway, was not tested (2/6). 

Derivatives with five- and six-membered oxygen-containing rings have been isolated when vindoline and deacetylvindoline were treated under superacid conditions.686 Formation of the unexpected oxolane derivatives (R = Ac 4%, R = H 18%) was accounted for by rearrangement through ethyl and hydride shifts (Scheme 5.66). 

Fig. 3. Biosynthesis of TIAs in C. roseus. Solid arrows indicate single enzymatic conversions, whereas dashed arrows indicate multiple enzymatic conversions. AS Anthranilate synthase, DXS D-l-deoxyxylulose 5-phosphate synthase G10H geraniol 10-hydroxylase CPR cytochrome P450 reductase TDC tryptophan decarboxylase STR strictosidine synthase SGD strictosidine /1-D-glucosidase D4H desacetoxyvindoline 4-hydroxylase DAT acetyl-CoA 4-O-deacetylvindoline 4-O-acetyl transferase. Genes regulated by ORCA3 are underlined. Reprinted with permission from [91]. Copyright (2000) American Association for the Advancement of Science...

DE LUCA, V., BALSEVICH, J., KURZ, W.G.W., Acetylcoenzyme A deacetylvindoline O-acetyltransferase, a novel enzyme from Catharanthus. J. Plant Physiol., 1985,121,417-428. 

FAHN, W LAUSSERMAIR, E DEUS-NEUMANN, B., STOCKIGT, J, Late enzymes in vindoline biosynthesis. S-adenosyl-L-methionine ll-0-demethyl-17-O-deacetylvindoline 11-0-methyltransferase and an unspecific acetylesterase. Plant Cell Rep., 1985, 4,337-340. 

Deacetylvindoline ll-O- Methyltransferase and unspecific acetylesterase., Plant Cell Reports., 1985,4,337-340. 

The structures of cleavamine and velbanamine are considered here since they are probably derived from precursors with the iboga skeleton. Cleavamine (XLVII), mp 109°-113°, [a]D +56° (CHCI3), was obtained along with deacetylvindoline when leurosine (structure unknown but closely related to XLI) was refluxed with concentrated hydrochloric acid, stannous chloride, and tin (42). Velbanamine (XLVIII), mp 139°-141°, [a]D +56° (CHCI3), was the indolic product when vincaleuko-blastine (XLI R = Me) or leurocristine (XLI R = CHO) were treated in the same way (42). Aside from the question of the mode of fission of the dimers (XLI), the production of the new tetracyclic systems in XLVII and XLVIII can be regarded as proceeding via a reverse Mannich... 

Figure 2.11 Biosynthesis of vindoline, catharanthine and the dimeric alkaloids vinblastine and vincristine. T16H, tabersonine-16-hydroxylase OMT, 5-adenosylmethionine 16-hydroxy-tabersonine-O-methyltransferase NMT, 5-adenosylmethionine 16-methoxy-2,3-dihydro-3-hydroxymethyltabersonine-/ /-methyltransferase D4H, desacetoxy-vindoline-4-dioxygenase DAT, acetylcoenzyme A 4-0-deacetylvindoline-4-0-acetyltransferase.

The final step in the formation of vindoline is the acetylation of 4-0-deacetylvindoline by a 4-0-acetyltransferase (DAT). This enzyme has been purified to homogeneity from C. roseus leaves (De Luca, 1993 Facchini and De Luca, 2008). 

Figure 2.12 A hypothetical view of compartmentation of indole alkaloid biosynthesis in Catharanthus roseus. Enzymes located with dashed arrows are hypothetical and circles indicate membrane associated enzymes (after Meijer et at, 1 993b). Cl OH, geraniol-1 0-hydroxylase NMT, 5-adenosyl-L-methionine 11 -methoxy 2,16-dihydro-16-hydroxytabersonine N-methyltransferase DAT, acetylcoenzyme A deacetylvindoline 1 7-0-acetyltransferase OHT, 2-oxyglutarate-dependent dioxygenase SSpC, strictosidine-((3)-glucosidase SSS, strictosidine synthase.

Fahn, W. and Stockigt, J. (1990) Purification of acetyl-CoA 17-O-deacetylvindoline 17-O-acetyltransferase from Catharanthus roseus leaves. Plant Cell Rep., 8, 613-6. 

The total synthesis of vindoline (205) from the pentacyclic ketone (206), whose formation was discussed in last year s Report, has now been completed (Scheme 27), essentially according to the route adopted earlier in the synthesis of vin-dorosine. The reduction of the C-17 ketone function in the penultimate stage with normal hydride reducing agents was not stereospecific. However, this was circumvented by addition of aluminium chloride, which presumably formed a complex involving the C-16 hydroxy-group and N reduction by a bulky complex hydride then ensured preferential a-attack with formation of deacetylvindoline. ... 

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