Labeled precursors

Miscellaneous Alkaloids. Stukimic acid (57) is a precursor of anthranihc acid (28) and, in yeasts and Escherichia coli (a bacterium), anthranHic acid (o-aminobenzoic acid) is known to serve as a precursor of tryptophan (26). A similar but yet unknown path is presumed to operate in higher plants. Nonetheless, anthranHic acid itself is recognized as a precursor to a number of alkaloids. Thus damascenine [483-64-7] (134), C qH NO, from the seed coats of JSHgella damascena has been shown (95) to incorporate labeled anthranHic acid when unripe seeds of the plant are incubated with labeled precursor. 

Microbiological procedures which exploit the ability of bacteria and photosynthetic algae to incorporate exogenous labeled precursors such as 14CO2, SO%, and 32pQ3- [ can be used to label complex molecules in cells such as proteins (qv) and nucleic acids (qv), which are then processed to give labeled constituents such as uniformly labeled C-amino acids, C-nucleotides, C-fipids, LS-amino acids, etc (8). 

Even higher organisms can be used for the production of labeled compounds. Plants, tobacco, or Canna indica for example, when grown in an exclusive atmosphere of radioactive carbon dioxide, [ 002], utilize the labeled precursor as the sole source of carbon for photosynthesis. After a suitable period of growth, almost every carbon atom in the plant is radioactive. Thus, plants can serve as an available source of C-labeled carbohydrates (9). 

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... 

Since ivermectin (= 22,23-dihydroavermectin B ) is obtained by catalytic reduction of avermectin B, the same procedure using tritium gas convenientiy affords tritiated ivermectin (22,23- [JT]-22,23-dihydroavermectin B ). The preparation of a tritiated derivative containing a 22,23-double bond starts with the readily available 5-ketone, which is reduced with [JT]-sodium borohydride stereospecificaHy to a 5- [JT]-derivative (40). Carbon-14 labeled avermectins can be obtained by a biosynthetic process using sodium (l- C)propionate as labeled precursor (48). 

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. 

A characteristic of all the above reactions is that the yield of the aldoliza-tion product depends on the pH of the reaction mixture (324), the maximum yield usually occurring near pH 7. Such reactions have been carried out in vitro in dilute aqueous buffer under so-called physiological conditions, i.e., conditions attainable in the living cell. Although this oversimplified technique for the study of alkaloid biogenesis is now being abandoned in favor of experiments in vivo with labeled precursors, such reactions are still of interest to organic chemists. 

Further experiments with labeled precursors were necessary to shed a little more light on this puzzling observation. Pyramine, biosynthesized from AIRs labeled with, 4C on C-l on the ribose part, exhibited only marginal radioactivity. This result rules out C-l of ribose in AIRs as a precursor of pyramine. This conclusion was confirmed with a precursor labeled at the C-l position with the stable l3C isotope. The mass spectrum of the ethylthio derivative of pyramine was identical with that of an unlabeled sample (Scheme 9). 

Solvolysis of the deuterium-labeled precursor, 54, indicates that the intermediate ion 51 arises from a symmetrical precursor, as the product ketone 55 has an equal distribution of deuterium in the 3 and 4 positions. 

The turnover rate of a transmitter can be calculated from measurement of either the rate at which it is synthesised or the rate at which it is lost from the endogenous store. Transmitter synthesis can be monitored by administering [ H]- or [ " C]-labelled precursors in vivo these are eventually taken up by neurons and converted into radiolabelled product (the transmitter). The rate of accumulation of the radiolabelled transmitter can be used to estimate its synthesis rate. Obviously, the choice of precursor is determined by the rate-limiting step in the synthetic pathway for instance, when measuring catecholamine turnover, tyrosine must be used instead of /-DOPA which bypasses the rate-limiting enzyme, tyrosine hydroxylase. 

Localisation of specific NT terminals. After its injection a labelled precursor should be taken up and detected in appropriate nerve terminals (and possibly cell bodies) so that the intensity of emission reflects the density of nerve terminals and the innervation. Using this procedure it has been possible to show that very little [ F] fluorodopa is concentrated in the striatum of Parkinsonian patients, compared with normals (Fig. 14.1). Whether the label remains on dopa or is transferred to dopamine will not greatly affect the result since both will label DA neurons although some will occur in noradrenergic nerve terminals. 

Beale, S.I. and Castelfranco, P.A., The biosynthesis of 5-aminolevulinic acid in plants. II. Formation of C-5-Aminolevulinic acid from labeled precursors in greening plant tissues, Plant Physiol, 53, 297, 1974. 

When [ H]-labeled precursors are employed the resulting compounds can be used as internal standards for analysis, especially by utilization of mass spectrometric methods. Appropriate deuterated standards are shown in Fig. 7. The introduction of deuterium into the A9-THC precursors can be done with Grignard reagents such as C[ H3]MgI or reducing substances such as LiAl[ H4]. The general procedures for the synthesis with these [ Hj-labeled precursors are the same as described above for the unlabeled compounds [76,78]. 

A common feature of the reactions described in (1) is that the hydrogen migration can be traced directly from appropriate mass shifts in the spectra of suitably 2H labelled precursor ions. 

The site of pheromone production is varied amongst the insects just as there are variable structures in the different orders. Several reviews are available detailing the ultrastructure of these glands [9-11]. Evidence that pheromone biosynthesis occurs in these cells and tissues requires that the isolated tissue be shown to incorporate labeled precursors into pheromone components. In the more studied model insects this criteria has been met. 

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. 

Use of encapsulated labeled precursors in lipid vesicles enabled the Hawaiian group to conduct the biosynthetic studies - with the exception of workup of the sponge - entirely in the field. Incorporation of doubly labeled [13C, 15N]cyanide into a Ciocalypta sp. and an Acanthella sp. produced labeled 9-isocyanoneopupukeanane (77) and kalihinol-F (112) respectively [71]. Detection of incorporation was followed by 13C NMR experiments. 

In more recent studies the use of HPLC allowed isolation and counting of individual sterols after administration of labelled precursors. The sterols isolated from mantles and viscera of the nudibranch Doris verrucosa were identified as cholestanol, cholesterol, 24-dehydrocholesterol and 7-dehydrocholesterol [103]. After injection of dl-[2-14C]-mevalonic acid DBED salt, cholesterol (57) and 7-dehydrocholesterol (58) were isolated as the acetates by reversed phase HPLC. Both sterols were found significantly labelled specific radioactivity associated with 7-dehydrocholesterol was higher by one order of magnitude than that associated with cholesterol. This fact would indicate either that the reduction of the A1 double bond of 7-dehydrocholesterol to afford cholesterol occurs at a low rate, or that the cholesterol found in D. verrucosa comes partly from a dietary source. 

The linear structure of 93 was derived from experiments with labeled precursor molecules and by correlation of vibrational frequencies calculated from estimated force constants with the recorded IR absorptions. The three fundamentals were observed as well as the UV/VIS spectrum,131 which was resolved and analyzed by gas phase measurements.132 The predicted triplet ground state was confirmed by recording the ESR spectrum of 93 isolated in various matrices.131... 

Genetic analysis indicates that two of the 10 sad mutants of A. strigosa that we isolated represent different mutant alleles at the Sadi locus.6 These mutants accumulate radiolabelled 2,3-oxidosqualene but not p-amyrin when the roots are fed with 14C-labelled precursor mevalonic acid, suggesting that the triterpenoid pathway is blocked between 2,3-oxidosqualene and P-amyrin.34 The roots of these mutants also lack detectable P-amyrin synthase activity, but, like the wild type and the other mutants, are unimpaired in cycloartenol synthase (CS) activity and sterol biosynthesis.34 The transcript levels for AsbASl are substantially reduced in roots of sadl mutants, while AsCSl transcript levels are unaffected,35 suggesting that the sadl mutants are either mutated in the AsbASl gene itself or in a gene involved in its regulation. 

Mechanistic insight into this process was obtained by administration of labeled trideca- or undeca-3,6,9-trienoic acid instead of the natural C12 precursor (Figure 2). In this case, the artificial 2H metabolites can be analyzed by mass spectrometry without interference from the plants own 1H metabolites, since a homo- or norectocarpene is formed. The sequence of the oxidative decarboxylation/cyclization reaction proceeds without loss of 2H atoms from the double bonds but with loss of a single 2H atom from certain methylene groups of the precursor acids (Figure 3). If C(l) and a 2H atom from C(5) of the labeled precursor is lost, finavarrene is the product of the reaction channel. If the methylene group... 

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