Carbon double labeling

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). 

In order to obtain more insights into the biosynthetic mechanism, [Me- C-Me-d ] double-labeled methionine was fed to the organism. The C-NMR spectrum of resulting neosaxitoxin showed a clean triplet for C-13 beside the natural abundance singlet. The result indicated that only one deuterium was left on the methylene carbon. 

In another experiment, [l,2- C2-2-dJ double-labeled acetate was fed. First we observed a complete loss of deuterium atoms. In a short incubation, however, we obtained neosaxitoxin partially retaining a deuterium atom (40% equivalent of incorporated acetate molecule). The location of the deuterium atom was on C-5, which was originally the carboxyl carbon of acetate, suggesting that it migrated from the adjacent methyl-derived carbon C-6. 

Recurrent is the lack of adequate techniques to assess carbon flows through the plants and microbes into soil organic matter (151). Most important is the development of techniques and protocols to separate rhizosphere from nonrhizosphere soil as well as possibly to facilitate analyses of soil carbon dynamics. The use of carbon isotopes, and, where possible, application of double labeling with C and C, seems inevitable in order to separate the contribution of different substrates to the formation of the soil organic matter pool and to get to an understanding of the ecological advantage of exudates and rhizodeposits. 

Remote double labelling techniques have been used successfully in the determination of enzyme KIEs (Kiick, 1991). A variant of this technique was applied to a nonenzymatic reaction by Matsson and co-workers (Axelsson et al., 1990). They determined the primary carbon and secondary deuterium KIEs for the SN2 reaction between methyl iodide and hydroxide ion in 50% dioxane-water at 25°C. The a-carbon KIE was determined by the UC method (Axelsson et al. 1987,1991). In this method, a mixture of substrate molecules labelled with UC (tm = 20.4 min) and 14C is used. The reactants and products... 

The most frequently used combination of radioisotopes in double labeling experiments is tritium and 14C. Ideally the ratio of the activities in the corresponding channels (Fig. 7.16) should to be high enough to insure the carbon counts in the tritium channel are negligible compared to tritium counts. Usually this is not achieved and a correction must be applied. 

The reaction of alkenes with ozone constitutes an important method of cleaving carbon-carbon double bonds.138 Application of low-temperature spectroscopic techniques has provided information about the rather unstable species that are intermediates in the ozonolysis process. These studies, along with isotope labeling results, have provided an understanding of the reaction mechanism.139 The two key intermediates in ozonolysis are the 1,2,3-trioxolane, or initial ozonide, and the 1,2,4-trioxolane, or ozonide. The first step of the reaction is a cycloaddition to give the 1,2,3-trioxolane. This is followed by a fragmentation and recombination to give the isomeric 1,2,4-trioxolane. The first step is a... 

Reaction of Atomic Carbon with Pyrroie. Cocondensation of arc generated C with pyrrole 71 gives pyridine 72. This clean reaction is useful to insure that the system is functioning properly when setting up a new carbon arc reactor. Double labeling experiments with atoms and produce 72 with both the... 

The metathesis reaction consists of a movement of double bonds between different molecules, as shown in Figure 1.4. Thus, the metathesis reaction can be addressed as a transalkylideneation reaction. The cleavage of the carbon-carbon double bonds was established using isotopic labelled compounds that were subjected to ozonolysis after reaction (9). 

Of greater relevance catalytically is that the combined use of l3C enrichment and 13C nutation NMR spectroscopy can distinguish between proposed rival mechanisms for the Ziegler-Natta catalyzed polymerization of acetylene. In the four-center insertion mechanism the enriched acetylene (HC =C H) is incorporated as shown in Scheme 6. It is to be noted that the, 3C—13C bond label is here incorporated into a carbon-carbon double bond, the length of which is significantly smaller than that of a carbon-carbon single bond, which is how the enriched acetylene would be incorporated in the two-center mechanism shown in Scheme 7. The results of nutation experiments leave little doubt that the Ziegler-Natta polymerization of acetylene proceeds by a four-center mechanism. 

Carbon-14 has been introduced at the Cqj position of the pentene group of drug 172153,154 starting with Ba14C03 in 28% yield and tritium has been introduced into the position at Cp) of the chain in the final reaction step by reduction of the ketone group with NaBTzt155 (equation 81). The double-labelled compound 172 is applied in pharmacokinetic studies in animals155. 

Fig. 2.4 Double labeling experiments to assign the Met carbonyl carbon resonances of SSI. A) [M]SSI ...

Kainosho M, Tsuji T, Assignment of the three methionyl carbonyl carbon resonances in Streptomyces subtilisin inhibitor by a carbon-13 and nitrogen-15 double-labeling technique. A new strategy for structural studies of proteins in solution, Biochemistry, 21 6273-6279, 1982. 

Small isotope-effects can be detected by double-labeling techniques, in which the carbon skeleton is labeled with 14C, and the ratio of 14C to tritium is measured both in the substrate and the product. Care must be taken in the observation and interpretation of isotope effects determined from the hydrogen-isotope content of the product. Just as in non-enzymic reactions (see p. 154), discrimination against the substrate containing deuterium or tritium leads to an increase in the isotopic content of the substrate, and this decreases the apparent isotope-effect towards the end of the reaction. 

This mechanism predicts that the hydrogen atom and cyanide group of HCN will be added across the same side of the carbon-carbon double bond and, consistent with this prediction, the addition of DCN see Isotopes Isotope Labeling) to cyclohexadiene (equation 9) has been shown to occur stereospecifically cis see Stereochemistry)) ... 

MSA does not contain any chiral carbon centers. Before the aromatization of the six-membered ring occurs, two prochiral carbons (C-2 and C-4 in the six-carbon intermediate) evolve, each of which loses a hydrogen in the process of the dehydratization/aromatization steps. In addition, C-3 of the six-carbon intermediate forms a chiral center when the ketone is reduced to a hydroxyl by a ketoreductase activity (Fig. 5). The chirality of this hydroxyl carbon is unclear since the intermediate has not been isolated. It is also unknown if this carbon retains its chirality in an eight-carbon intermediate or whether the hydroxyl is eliminated by dehydration prior to the third condensation reaction. The stereospecificity at the prochiral C-2 and C-4 carbons in the reaction intermediates was addressed using chemically synthesized (] )- and (S)-[1- C, 2- H]malonate precursors which were enzymatically converted into CoA derivatives via succinyl CoA transferase [127,128]. Thus, the prochiral methylene in malonyl CoA was replaced by chiral, double-labeled (S)- or (J )-[1- C, 2- H]malonyl CoA substrates in the reaction mixture with 6-MSAS. The condensation is expected to occur with inversion of configuration and the intact methylene... 

Androsterone and 5)3-androsterone, which are the major metabolites, were thought to be uniquely derived from the plasma androstenedione pool. Korenman et al. [170,388] demonstrated by the double isotope tracer method using carbon-14-labeled testosterone and tritium-labeled androstenedione that neither androsterone nor 5 -androsterone is a unique metabolite of a plasma androstenedione pool. A unique steroid metabolite has been defined by Dorfman [326] as a steroid persisting or formed during metabolism which can be related to one and only one tissue steroid. Korenman et al. obtained different tritium/carbon-14 ratios for androsterone and 5/3-androsterone, suggesting that other pathways of testosterone and androstenedione metabolism also exist. These other pathways, possibly metabolism of testosterone and androstenedione by peripheral tissue, may be responsible for the relative enrichment of either the 5a- or the 5/8-isomer of the urinary metabolites [305]. 

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