Positional enrichment, carbon labeling

A more recent experimental technique employs C as the isotopic label Instead of locating the position of a label by a laborious degradation procedure the NMR spectrum of the natural product is recorded The signals for the carbons that are enriched m are far more intense than those corresponding to carbons m which IS present only at the natural abundance level... 

To understand the evaluation of a CLE, we need to introduce some terms The word isotopomer is a combination of the terms isotope, and isomer. An isotopomer is one of the different labeling states in which a particular metabolite can be encountered [248] that is, a molecule with n carbon atoms has 2" isotopomers. These are usually either depicted using outlined and filled circles for unlabeled and labeled atoms, respectively (see Fig. 14), or are described in text format for example, C 010 would be the isotopomer of a three-carbon molecule labeled at the second position. An isotopomer fraction is the percentage of molecules in this specific labeling state. The positional enrichment is the sum of all isotopomer fractions in which a specific carbon atom in a specific metabolite is labeled [248]. Consequently, the usage of isotopomers enables to account for more information While a molecule with n carbon atoms will yield n positional enrichments, there are 2 — 1 isotopomer fractions (the 2"th measurement is redundant as, by definition, isotopomer fractions must sum up to unity) [260],... 

The measurements of the labeled metabolites may be performed with GC- or LC-MS, or by NMR. Because it is the most commonly used method, we will only consider GC-MS based approaches here. Obviously and unfortunately, it is not possible to directly measure the isotopomer enrichments by GC-MS, because the apparatus only yields total masses of molecules or fractions thereof, but not directly the position of a label. Each MS peak is produced by all isotopomers with the same molecular weight that is, the same number of labeled carbon positions. Sometimes this concept is also called mass isotopomers [264]. In a so-called retrobiosynthetic approach, it has been shown that the labeling state of many intracellular pools can be determined indirectly by measuring the labels in macromolecular biomass components at steady state for example, the labeling state of alanine from hydrolyzed protein reflects the label of pyruvate [265]. Using this approach, it is possible to quantify fluxes into storage components. 

A specific variant of El MS is isotope ratio (IR) MS [46]. It is based on electron impact ionization with maximized ionization probability. IR MS is limited to the analysis of gases of high volatility and low reactivity such as CO2, N2 or SO2. The analytes of interest thus have to be transformed into one of these gases before introduction into the IR MS. Information on the position of C labelings in the analyte can be only obtained, if all carbons are isolated position specific and subsequently combusted. In this context Corso and Brenna [47] showed position specific analysis by IR MS for methylpalmitate through pyrolytic fragmentation. IR MS exhibits an extremely high precision of 0.00001 % for the isotope ratio measurement and is optimal to quantify low label enrichments [48]. This is especially important for in vivo studies with ani-... 

The JC NMR spectrum of [13CH3]methionine enriched dicyanocobalamin also revealed labelling of the six methyl carbons at the C-l, -2, -5, -7, -15 and -17 positions of the molecule, showing that the C-l methyl is not derived from the extruded C-20 methylene of uroporphyrinogen III. In accord with this, vitamin B 2 derived from [5-uC]ALA showed enrichment of seven rather than eight of the nuclear carbon atoms (Scheme 24) (72PNA2585, 78ACR29). 

Eight methyl AT-(l/f-benzimidazol-2-yl)carbamates 78 with various 5-substituents have been labelled with 13C at carbon-2, and at the carbonyl and methoxy carbons according to the synthetic scheme of equation 25 using commercially available 13C-enriched (91-92 atom%) carbon tetrachloride, methanol and thiourea63. The three labelled positions indicated with asterisks are at or near the site involved in binding to tubulin (which performs a variety of vital functions in the cell). A correlation has been found between anthelmintic potency of the benzimidazole carbamate group of anthelmintic drugs widely used to control nematode parasites in sheep and their ability to bind tubulin. 

A compound that is especially easy to observe is glutamate. This amino acid, most of which is found in the cytoplasm, is nevertheless in relatively rapid equilibrium with 2-oxoglutarate of the citric acid cycle in the mitochondria. The accompanying scheme shows where isotopic carbon from certain compounds will be located when it first enters the citric acid cycle and traces some of the labels into glutamate. For example, uniformly enriched fatty acids will introduce label into the two atoms of the pro-S arm of citrate and into 4- and 5-positions of glutamate whereas [2- C]acetate will introduce label only into the C4 position as marked by in the scheme. In the NMR spectrum a singlet resonance at 32.4 ppm will be observed. However, as successive turns of the citric acid cycle occur the isotope will appear in increasing amounts in the adjacent... 

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