Labeled substrate

Nucleophilic substitution in cyclohexyl systems is quite slow and is often accompanied by extensive elimination. The stereochemistry of substitution has been determined with the use of a deuterium-labeled substrate (entry 6). In the example shown, the substitution process occurs with complete inversion of configuration. By NMR amdysis, it can be determined that there is about 15% of rearrangement by hydride shift accon any-ing solvolysis in acetic acid. This increases to 35% in formic acid and 75% in trifiuoroacetic acid. The extent of rearrangement increases with decreasing solvent... 

The occurrence of syn elimination in 5-decyl systems has been demonstrated with the use of diastereomeric deuterium-labeled substrates. Stereospecifically labeled 5-substituted decane derivatives were prepared and subjected to appropriate elimination conditions. By comparison of the amount of deuterium in the E and Z isomers of the product, it was... 

Compound 5 can be trapped through a Diels-Alder reaction with maleic anhydride and thus be shown to be an intermediate. Further evidence for a mechanism involving two subsequent allyl conversions has been provided by experiments with " C-labeled substrates. 

As another example, studies with deuterium-labeled substrates have shown that the reaction of ethanol with the coenzyme NAD+ catalyzed by yeast alcohol dehydrogenase occurs with exclusive removal of the pro-R hydrogen from ethanol and with addition only to the Re face of NAD+. 

Experiments with 14C-labelled substrates also demonstrated conclusively the intramolecularity of the rearrangement. The generally accepted scheme involves the formation of the dienone LXXXV which can lose a hydrogen atom if R = H, to form the ortho product or if R H further rearrangement to LXXXVI occurs with subsequent formation of the para product, viz. 

Cyclobutyl substrates also solvolyze abnormally rapidly and give similar products. Furthermore, when the reactions are carried out with labeled substrates, considerable, though not complete, scrambling is observed. For these reasons, it has been suggested that a common intermediate (some kind of nonclassical intermediate, e.g., 25, p. 408) is present in these cases. This common intermediate could then be obtained by three routes ... 

The metabolic activity of other white-rot fungi including Phanerochaete chrysosporium and Pleurotus ostreacus has been discussed in the context of polycyclic aromatic hydrocarbons. For example, the mineralization potential of the manganese peroxide system fmmNematolomafrowardii for a number of substrates has been demonstrated (Hofrichter et al. 1998) the formation of CO2 from labeled substrates ranged from 7% (pyrene) to 36% (pentachlorophenol), 42% (2-amino-4, 6-dinitrotoluene), and 49% (catechol). 

The microbial degradation of contaminants under anaerobic conditions using humic acids as electron acceptors has been demonstrated. These included the oxidations (a) chloroethene and 1,2-dichloroethene to CO2 that was confirmed using C-labeled substrates (Bradley et al. 1998) and (b) toluene to CO2 with AQDS or humic acid as electron acceptors (Cervantes et al. 2001). The transformation of l,3,5-trinitro-l,3,5-triazine was accomplished using Geobacter metallireducens and humic material with AQDS as electron shuttle (Kwon and Finneran 2006). 

Illustrative examples of the application of C-labeled substrates to biodegradation include the following ... 

Although quite extensive use of has been made in studies on the degradation of alkyl sulfonates (Hales et al. 1986), C1 has achieved only limited application on account of technical difficulties resulting from the low specific activities and the synthetic inaccessibility of appropriately labeled substrates. One of the few examples of its application to the degradation of xenobiotics is provided by a study of the anaerobic dechlorination of hexachlorocyclohexane isomers (Jagnow et al. 1977), the results of which are discussed in Chapter 7, Part 3. 

Although the radioactive isotope H has been extensively used for studies on the uptake of xenobiotics into whole cells, the intrusion of exchange reactions and the large isotope effect renders this isotope rather less straightforward for metabolic studies. Both deuterium H-labeled substrates, and oxygen and OH2 have, however, been extensively used in metabolic studies, since essentially pure labeled compounds are readily available and mass spectrometer facilities have become an essential part of structural determination. 

The use of H-labeled substrates has been used to determine details of the dehydrogenation of ciT-dihydrodiols produced by dioxygenases from aromatic substrates (Morawski et al. 1997), and it was possible to demonstrate the specificity of hydrogen transfer from the dihydrodiol substrates to NAD. 

H-labeled substrates have been used to determine the dissipation and degradation of aromatic hydrocarbons in a contaminated aquifer plume (Thierrin et al. 1995). Its application was particularly appropriate since the site was already contaminated with the substrates. With suitable precautions, this procedure seems capable of extension to determining the presence—though not the complete structure—of metabolites, provided that the possibility of exchange reactions were taken into account. 

Hunkeler D (2002) Quantification of isotope fractionation in experiments with deuterium-labeled substrate. 

The degradation of phenylacetate has remained enigmatic for several years, and details of the pathway used by Escherichia coli were elucidated using C-labeled substrate. Using the full complement of NMR technology, the structures of critical intermediates were determined and provided details of an unusual pathway (Ismail et al. 2003). 

Apart from its use in studies of nitrogen fixation, N-labeled substrates have been used to assess the association of contaminants with organic matter in soil, and in metabolism. 

Padmanabhan P, S Padmanabhan, C DeRito, A Gray, D Gannon, JR Snape, DC Tsai, W Park, C Jeon, EL Madsen (2003) Respiration of C-labeled substrates added to soil in the field and subsequent 16S rRNA gene analysis of C-labeled soil DNA. Appl Environ Microbiol 69 1614-1622. 

It is appropriate at this stage to evaluate procedures that will be used to assess the effectiveness of bioremediation, which have been discussed in Chapter 13. These may include (a) use of radiolabeled substrates (although these will not generally be permitted in field operations) and the application of C-labeled substrates, (b) evaluation of the occurrence of metabolites, and (c) evaluation of markers such as specific enzymes. 

Both H-thymidine incorporation and radiolabeled leucine incorporation techniques have been recently used to determine bacterial activity and growth in the rhizosphere of barley seedling (28), Bacteria were initially released from the rhizosphere using homogenization and centrifugation before adding the labeled substrates. The cell incorporation rate was twice as high in the rhizosphere than in bulk soil. In addition, both the leucine and thymidine incorporation rates increased with the distances from the root tip (28). 

Actually, all deuterium labeled substrates are enantiomerically enriched but not 100%... 

Matsuoka, K., Nishimura, S. I. and Lee, Y. C. (1995). A bi-fluorescence-labeled substrate for ceramide glycanase based on fluorescence energy-transfer. Carbohydr. Res. 276, 31-42. 

Using labelled tin compounds to produce labelled substrates via reduction reactions... 

The reaction mechanism was considered to be oxidative cyclization, and pal-ladacyclopentene 32 was formed. Reductive elimination then occurs to give cyclobutene 33, whose bond isomerization occurs to give diene 28. The insertion of alkyne (DMAD) into the carbon palladium bond of 32 followed by reductive elimination occurs to give [2+2+2] cocyclization product 27. Although the results of the reactions of E- and Z-isomers of 29 with palladium catalyst 26a were accommodated by this pathway, Trost considered the possibility of migration of substituents. Therefore, 13C-labeled substrate 25 13C was used for this reaction. 

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