Biosynthetic pathways with labeled precursor

The next example is used to demonstrate how different pathways could produce the same pheromone component. Helicoverpa zea and Helicoverpa assulta are closely related species that use aldehydes as the major pheromone. Helicoverpa zea uses a blend of components with Z11-16 Aid as the major component, and minor components include 16 Ald, Z9-16 Aid, and Z7-16 Aid (Klun et al., 1980). H. assulta uses Z9-16 Ald as the major component and Z11-16 Aid as a minor component (Cork et al., 1992 Sugie et al., 1991). The biosynthesis of Zll-16 Aid occurs by Al 1 desaturation of 16 CoA to produce Z1 l-16 CoA, which is reduced to the aldehyde. This probably occurs in both species, but Z9-16 Ald could be produced by the action of a A9 desaturase using 16 CoA as a substrate or by the Al 1 desaturation of 18 CoA to produce Zll-18 CoA that is then chain shortened to Z9-16 CoA. To determine between these two pathways, deuterium-labeled precursors were applied topically to the glands in dimethyl sulfoxide and females injected with PBAN 1 h later the glands were extracted and analyzed for incorporation using GC/MS (Choi et al., 2002). Figure 3.4 shows the data and biosynthetic pathways. 

Although the general biosynthetic pathway to 3-prenylquinoline alkaloids and their relatives is apparent from the feeding experiments with labeled precursors discussed above (Scheme 28), many details remain to be resolved. Theories are based on chemotaxonomic and stereochemical evidence and have been reviewed for isoprenoid quinoline alkaloids and coumarins (38) some aspects will be discussed here. 

The biosynthesis of the furan moiety was investigated for skimmianine, a dictamnine-related alkaloid, using Fagara coco (Rutaceae). Thus, when C-4- or C-5-labeled 3R-mevalonic acid was used in the biosynthetic pathway, C-labeled skimmianine was observed in both cases. When the labeled skimmianine was degraded, radioactive skimmianic acid was obtained from C-4-labeled 3R-mevalonic acid, but radioinactive skimmianic acid was obtained when C-5-labeled 3R-mevalonic acid was fed. Consequently, it was concluded that the C-T and C-2 carbons (furan moiety) of skimmianine were derived from the C-5 and C-4 carbons of mevalonic acid, respectively [4].The biosynthetic precursor of dictamnine is therefore a quinoline alkaloid with a prenyl moiety, derived from dimethylallylpyrophosphate (DMAPP), at the C-3 position, such as platy-desmine [5]. It was also shown that the methyl carbon of the methoxyl moiety at the C-4 position of skimmianine was derived from methionine. 

Chiral oxazaborolidines are also efficient catalysts for the enantioselective reduction of aldehydes labeled with isotopic hydrogen at the carbonyl function. Treatment wifli cate-cholborane in the presence of (/ )- or (S)-B-n-butyloxazaborolidine (3 d) affords Cl-deuteriated or tritiated primary alcohols with enantiomeric excesses generally exceeding 90%. Use of catecholborane is essential, since for BH3 THF the achiral uncatalyzed process is competitive with the chiral catalyst-mediated pathway, thereby reducing the enantiomeric purity of products. Enantiomerically enriched [ 1 - H]- and [ 1- H] alcohols have been extensively used in the study of enzymatic mechanisms and biosynthetic pathways , and as precursors for chiral [ H, H, H]acetic acid and [ H, H]fluoroacetic acid . Equivalent results are obtained when BusSnH is employed in the presence of BITIP (58, prepared in situ from (R)- or (5)-l,l -bi-2-naphthol and Ti(0-/-Pr)4 2 1) ° (Figure 11.23). 

The proposed pathway for the biosynthesis of the avermectins (Fig. 3) has been described in a review (23). Some of the details are yet to be elucidated, although the steps, in general, are based on firm evidence from four types of studies incorporation of labeled precursors, conversion of putative intermediates by producing strains and blocked mutants, in vitro measurement of biosynthetic enzymes, and studies with enzyme inhibitors. The biosynthesis of the oleandrose units was elucidated from studies using and labeled glucose, which indicated a direct conversion of glucose to... 

Application of NMR spectroscopy to heterocyclic chemistry has developed very rapidly during the past 15 years, and the technique is now used almost as routinely as H NMR spectroscopy. There are four main areas of application of interest to the heterocyclic chemist (i) elucidation of structure, where the method can be particularly valuable for complex natural products such as alkaloids and carbohydrate antibiotics (ii) stereochemical studies, especially conformational analysis of saturated heterocyclic systems (iii) the correlation of various theoretical aspects of structure and electronic distribution with chemical shifts, coupling constants and other NMR derived parameters and (iv) the unravelling of biosynthetic pathways to natural products, where, in contrast to related studies with " C-labelled precursors, stepwise degradation of the secondary metabolite is usually unnecessary. 

Evidence for de novo synthesis of pheromone components was obtained by showing that labeled acetate and mevalonate were incorporated into ipsdienol by male Ips pini [103,104]. Similarly, labeled acetate and other labeled intermediates were shown to be incorporated into frontalin in a number of Dendroctonus species [105]. Possible precursors to frontalin include 6-methyl-6-hep-ten-2-one, which was incorporated into frontalin by D. ruffipennis [106]. The precursor 6-methyl-6-hepten-2-one also was shown to be converted to bre-vicomin in the bark beetle, Dendroctonus ponderosae [107]. In addition, the expression patterns of HMG-CoA reductase and HMG-CoA synthase are tightly correlated with frontalin production in Dendroctonus jeffreyi [108, 109]. A geranyl diphosphate synthase cDNA from I. pini was also isolated, functionally expressed, and modeled [110]. These data indicate that the de novo isoprenoid biosynthetic pathway is present in bark beetles. A variety of other monoterpene alcohols such as myrcenol, pityol, and sulcitol are probably synthesized through similar pathways [111]... 

In suberizing potato tuber disks, labeled oleic acid was incorporated into co-hy-droxyoleic acid and the corresponding dicarboxylic acid, the two major aliphatic components of potato suberin [73]. Exogenous labeled acetate was also incorporated into all of the aliphatic components of suberin, including the very long chain acids and alcohols in the wound-healing potato slices. The time-course of incorporation of the labeled precursors into the suberin components was consistent with the time-course of suberization. The biosynthetic pathway for the major aliphatic components of suberin is shown in Fig. 8a. 

A non-ribosomal biosynthetic pathway is clearly indicated for cyclosporin A, considering the uncommon structural elements MeBmt, L-a-aminobutyric acid and D-alanine as well as the plethora of isolated congeners [20,21]. Non-ribosomal biosynthesis directed by multienzyme thiotemplates have been reported for other small peptides of microbial origin, for example, gramicidin S [22] and enniatin [23]. Experimental data for cyclosporin A were obtained by feeding appropriate labelled precursors to cultures of T. inflation strains. The distribution profile of the labelled atoms in cyclosporin A was determined by 3H- or 13C-NMR spectroscopy. In preliminary trials with several tritium and carbon-14 labelled precursors, [met/y>/-3H]methionine proved to be the most suitable marker for the biosynthetic preparation of radiolabelled cyclosporin A for pharmacokinetic and metabolic studies [24],... 

The biosynthetic pathway to both nicotine (5) and the tropane alkaloids includes A-methylputrescine (4) as a probable intermediate. New results6 obtained for nicotine (5) and scopolamine (6) with 1 l-13C,14C wef/zy/awmo-15Nl-A-methyl putrescine 1(4) labels as shown nicely confirm this. The specific incorporation of both stable isotopes was closely similar to that of the 14C label in both alkaloids, indicating intact incorporation of the precursor. The labelling patterns deduced are illustrated ( = 13C, = 15N), and they are in accord with earlier results that were... 

Biosynthetic pathways are usually worked out by isotopic labelling of potential precursors and we shall mark the label with a coloured blob. If ornithine is labelled with l4C and fed to the plant, labelled hygrine is isolated. 

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