A. Ian Scott

Biogenetic-type synthesis of the indole alkaloids

Abstract A review of the rationale and methodology for the laboratory construction of the major classes of indole alkaloid based on their known or presumed biosynthesis is presented. The experimental results obtained provide analogies for the principal condensation and rearrangement reactions uncovered in biochemical investigations in this field and suggest new incorporation experiments. The prediction and discovery of new classes of alkaloid based on the observed transformations have been made and, in turn, new theories have been developed to explain some of the hitherto unsolved problems of biotransformation and structure in this series. Finally, the suggestion is made that many of the complex structures encountered in the various families may arise through rather unspecific processes, some indeed of a nonenzymic nature.

Biosynthesis of polyketides. Purification and inhibition studies of 6-methylsalicylic acid synthase

Abstract 6-MSA 3 synthase has been purified 190-fold with 33% yield. The purification was found to be dependent on the presence of glycerol. The acetylenic inhibitors 3-pentynoyl- and 2-hexynoyl-NAC completely inhibit 6-MSA production at concentrations in which fatty acid synthesis, TAL production as well as NADPH oxidation are only partially affected. These results confirm earlier studies on the specificity of inhibition by acetylenic inhibitors and support a mechanism wherein the NADPH-mediated reduction step occurs on a 6-carbon rather than on an 8-carbon intermediate.

Indole alkaloid biosynthesis: Partial purification of “ajmalicine synthetase” from Catharanthus roseus

Abstract The “ajmalicine synthetase” system of Catharanthus roseus has been partially purified from callus, seedlings and mature plants. On gel filtration of the cell-free extract, four β-D-glucosidase isozymes were observed in seedlings and plants. Only two were present in the callus. A protein peak at 55,000 daltons in all three materials was capable of synthesizing ajmalicine from tryptamine and secologanin in the presence of NADPH. This “ajmalicine synthetase” rapidly lost its ability to synthsize ajmalicine, but retained the β-glucosidase activity.

Mechanisms of Indole Alkaloid Biosynthesis. Recognition of Intermediacy and Sequence by Short-Term Incubation

Abstract Autoradiography and radiochemical analysis of two-dimensional chromatograms of the alkaloids of Vinca rosea seedlings have been used to determine the activity of various postulated intermediates after administration of 2- 14 C- dl -tryptophan. The technique holds promise as a method for (a) distinction between “dynamic” and “static” metabolites, (b) determination of the sequence of occurrence of alkaloids from 5 min to 8 days, (c) the discovery of hitherto unknown intermediates, and (d) the attainment of specific incorporations of up to 30%.

Biosynthesis of Natural Products

The experimental verification of a proposed biosynthetic pathway for a given natural product is often difficult to obtain with the use of the whole organism (permeability factors) or, in the case of higher plants, a cell-free system. Until the purified enzyme for each step of biosynthesis is available, biosynthetic studies can, however, be carried out, albeit with modest incorporation values, by means of either hydroponic or injection methodology. Where viable seed sources are available it is suggested that improvements of several orders of magnitude in incorporation can be achieved by a short-term incubation (small pool size of precursors; trapping of reactive intermediates) or a long-term feeding (equilibration of precursor with the compartmentalized or induced synthetases). In bacterial, fungal, mammalian, and plant systems, where incorporation efficiencies provide the opportunity to study 13C enrichment (at least equal to natural abundance of the isotope), we can expect a rapid expansion since the method removes the tedium of carbon-by-carbon degradation. For the next few years, however, the prognosis would seem to favor parallel studies of 13C and 2H, and of 14C/3H ratio techniques since the last-mentioned method provides more information concerning the stereoselectivity of labeling processes on the microgram scale.

Regio- and stereospecific models for the biosynthesis of the indole alkaloids—II : Biogenetic type synthesis of Aspidosperma and Iboga alkaloids from a corynanthe precursor☆

Abstract The thermal reactivity of dihydro-stemmadenine acetate ( 14 ) has been investigated as a model for the biochemical conversion of Corynanthe to Iboga alkaloids in which the highly reactive achiral ester dehydrosecodine A( 17 ) is invoked to rationalize the observed products pseudocatharanthine ( 7 ) and its 15, 20 dihydro derivative ( 8 ). Interception of the reactive intermediate from the alcoholysis of dihydropreakuammicine acetate ( 15 ) has provided further insight into the mechanism of this and related rearrangements. Generation of the isomeric ester dehydrosecodine B( 24 ) from stemmadenine ( 1 ) has been demonstrated in a biogenetic-type conversion to the Aspidosperma alkaloids (±)-tabersonine ( 9 ) and (±) vincadifformine ( 27 ) by two different processes. These reactions constitute a separation of the mechanisms operative in the vigorous treatment of stemmadenine acetate on a silica surface which was shown previously to yield a mixture of Aspidosperma and Iboga alkaloids and also serve to explain the occurrence of racemic and antipodal versions of complex indole alkaloids in Nature.

Regio- and stereospecific models for the biosynthesis of the indole alkaloids—I : Development of strategic approaches and preliminary experiments

Abstract Potential models for both proven and presumed rearrangement processes of indole alkaloid biosynthesis are presented which impute a key role to the reactive dihydropyridine-acrylic ester, dehydrosecodine (19). Plausible mechanisms are considered for the generation of 19 from the Corynanthe-Strychnos alkaloids which lead to several important consequences for both regio- and stereochemical control of the cyclisation of 19 and its close relatives, including the prediction of some hitherto undiscovered classes of indole alkaloid. Preliminary experimental observations are described which indicate the feasibility of generating and utilising dehydrosecodines (as 19) as models for several rearrangement processes of the biosynthetic pathway. A recent criticism of these experiments is analyzed.

Biosynthesis of patulin. Dehydrogenase and dioxygenase enzymes of penicillium patulum

Abstract Two aromatic dehydrogenases catalyzing the reversible conversions of gentisyl alcohol and m -hydroxybenzyl alcohol to their corresponding aldehydes have been partially purified. These partially purified dehydrogenases were shown to require NADPH. In the case of the gentisyl alcohol-gentisaldehyde interconversion, a 46-fold purification was achieved using POLYCLAR AT and DEAE-cellulose chromatography. A cell-free system capable of converting gentisaldehyde to patulin was prepared with a pH optimum of 8.0. The system was dependent on O 2 and NADPH, was stimulated by ATP and inhibited by the Fe 2+ chelators, α, α-dipyridyl and o -phenanthroline. These results suggest a dioxygenase mechanism for patulin synthesis from gentisaldehyde.

Regio- and stereospecific models for the biosynthesis of the indole alkaloids—III : The Aspidosperma-Iboga-secodine relationship

Abstract In vitro transformation of the Aspidosperma alkaloid (−) tabersonine ( 1 ) to (±)-pseudocatharanthine ( 7 ) via (+) allocatharanthine ( 6 ) and dehydrosecodine A ( 3 ) is described as a model for the biochemical interconversion of Aspidosperma and Iboga alkaloids. Facile conversion of 1 , 2 and 7 in xylene solution to the carbazole ( 9 ) suggests the intermediacy of dehydrosecodines (as 8 ) in these reactions. In methanol solution the racemic salt ( 12 ) is formed in 50% yield from catharanthine at 175°. Further pyrolysis of the salt yields the carbazole ( 9 ). Reduction of the salt ( 12 ) with NaBH 4 affords (±) dihydrosecodine ( 16 ) identical with the natural alkaloid from Rhazya stricta .

Biosynthesis of polyketides the synthesis of 6-methylsalicylic acid and triacetic acid lactone in Penicillium patulum

Abstract An enzyme fraction from P. patulum has been prepared which will synthesize 6-MSA 5 and fatty acids from acetyl CoA, malonyl CoA and NADPH. The 6-MSA synthetase was purified threefold by ammonium sulphate fractionation and catalyses the formation of 0.86 nmoles of 6-MSA per minute per mg of protein. Chemical degradation of 14C-6-MSA biosynthesized from 1,3-14C-malonyl CoA, acetyl CoA and NADPH gave a ratio of 2.39 1 for the radioactivity in the aromatic ring to that in the terminal carboxyl group. This is consistent with the acetatepolymalonate origin of 6-MSA; the deviation from the theoretical value of 2 1 is probably due to malonyl CoA decarboxylase activity leading to the formation of 1-14C-acetyl CoA. The rate of synthesis of 6-MSA was generally about ten times faster than the rate of fatty acid synthesis, but this ratio varied with the preparation. The 6-MSA synthetase is inhibited by isodoacetamide and N-ethymaleimide, which suggests that the synthetase activity contains sulphydryl groups in the active sites. The 6-MSA synthetase activity is inhibited by the following acetylenic thioesters: 3-pentynoyl-NAC, 3-hexynoyl-NAC and 2-hexynoyl-NAC. The activity is not inhibited by 3-hexynoic acid. The synthesis of 6-MSA can be abolished at a concentration of 10−4 M 3-hexynoyl-NAC without affecting the synthesis of free palmitic and stearic acid. The synthesis of 6-MSA by P. patulum under in vivo conditions is also inhibited by 3-hexynoyl-NAC. The syntheses of 6-MSA and TAL (in the presence of NADPH) are inhibited to comparable extents by 5 × 10−5 M 3-hexynoyl-NAC while the synthesis of fatty acids is not inhibited below 10−4 M. The mechanism of 6-MSA synthesis is discussed in relation to the formation of TAL and to the possible mode of action of the acetylenic thioester inhibitors.