Isotopic labelling with stable isotopes

Syntheses of nucleosides labeled with stable isotopes and their application to structural biology 99YZ299. 

For quantitative work, it is necessary to estimate the concentration of 5-amino-l-(P-D-ribofuranosyl)imidazole in aqueous solution. It seems that the only available method is the Bratton-Marshall assay, which was originally developed for the estimation of arylamines in biological fluids. The principle of the method is the spectrometric estimation of a salmon-pink colored dyestuff obtained by diazotation in situ, followed by coupling with /V-( 1 -naphthyl)ethyl-enediamine.65 The only remaining problem then is to know the molar extinction of this dye because pure samples of AIRs are not available. A value of 16800 at 520 nM was obtained for the dyes prepared from a model compound, 5-amino-l-cyclohexylimidazole-4-carboxylic acid (54), which is crystalline. A comparable molar extinction can be expected for the dye prepared from imidazole 55, if the carboxyl group does not exert too much influence on the chromophore. Actually, its influence is perceptible even with the naked eye, the dyestuff prepared from 53 having a somewhat different, wine-red color, with max>520 nM. The molar extinction for 55 is 17400 at 500 nM. When the decarboxylation of 54 was conducted under mild acidic conditions (pH 4.8, 50°C, 1 hour), estimation of 5-aminoimidazole 55 by the Bratton-Marshall method led to the conclusion that the reaction was almost quantitative.66 Similar conditions for the final decarboxylation were adopted in the preparation of samples of AIRs labeled with stable isotopes.58... 

Thiamine was biosynthesized by resting cells of S. typhimurium strain thilO/T-ath-383, which can synthesize thiamine from exogenous glucose, AIRs, and thiazole.54 Derepression was achieved by conventional means. The organism was cultivated in the presence of a suboptimal amount of thiamine (20 nM), the washed cells were resuspended in a minimal medium containing glucose (10 mM), thiazole (1-2 mM), and labeled AIRs (10 p,M). During the incubation (1.5 hours 37°C), the level of thiamine diphosphate in the cells had risen from about 0.04 to 0.5 nmol/mg. In work with molecules labeled with stable isotopes, thiamine was extracted and cleaved by ethanethiol to 4-amino-5-(ethyl-... 

Escherichia coli Adenine and adenosine are inhibitory74 and the synthesis of thiamine can be derepressed by culture in their presence.13,75 adth- Mutants are known.76 [l4C]Formate incorporates at C-2 of pyramine without dilution of molar activity. Glycine labeled with stable isotopes was fed to E. coli and the pyramine was analyzed by mass spectrometry. The two carbon atoms of glycine separated during the biosynthesis. The carboxyl was found12 at C-4, and the C-N fragment was the precursor of C-6-N-1. In conclusion, it is beyond doubt that pyramine synthesis follows the AIR pathway in E. coli. 

For human studies, the choice of stable isotopes is limited because radioisotopes are associated with ionization radiation and thus with some potential harmful effects for humans. Studying the bioavailability of compounds labeled with stable isotopes requires complex techniques such as gas chromatography coupled with mass spectrometry (GC-MS), liquid chromatography coupled with MS (LC-MS), and atmo-... 

The results obtained with the ICAT labeling strategy are similar, in principle, to those obtained by in vivo labeling with stable isotopes in that the relative ratios of proteins from different samples are obtained (Fig. 3.2). The important difference between the methods is that the ICAT-labeling procedure is performed in vitro on protein lysates. Therefore, the ICAT-labeling strategy can be applied to samples that cannot be labeled in vivo. Because the ICAT-labeling method can be used with virtually any sample, it... 

Membrane-integrated proteins were always hard to express in cell-based systems in sufficient quantity for structural analysis. In cell-free systems, they can be produced on a milligrams per milliliter scale, which, combined with labeling with stable isotopes, is also very amenable forNMR spectroscopy [157-161]. Possible applications of in vitro expression systems also include incorporation of selenomethionine (Se-Met) into proteins for multiwavelength anomalous diffraction phasing of protein crystal structures [162], Se-Met-containing proteins are usually toxic for cellular systems [163]. Consequently, rational design of more efficient biocatalysts is facilitated by quick access to structural information about the enzyme. 

An alternative approach uses tracers, isotopically enriched materials, which are added to the system studied. Labeled compounds are available with the heavy isotope enriched to a level of 99 atom%. These are more difficult to produce than radioisotopes, making them relatively expensive. Materials labeled with stable isotopes are non-radioactive and pose no hazard to human health or the environment (Barrie Prosser, 2000). 

New derivatives of 4-amino and 2,4-diaminopteridines have been synthesized and their capability to inhibit neuronal nitric oxide synthase evaluated <99JMC4108>. The synthesis of folic acid multiply labeled with stable isotopes, for bioavailability studies in human nutrition, has been reported <99JCS(P1)1311>, Synthesis and antiviral evaluation of several 6-(methylenecarbomethoxy)pteridine-4,7-diones have been described <99JHC435>. Synthesis and biochemical evaluation of bis(6,7-dimethyl-8-D-ribityllumazines) as potential bisubstrate analog inhibitors of riboflavin synthase have been reported <99JOC4635>. Synthesis and cyclization of novel lumazine-enediyne chimeras have been reported <99H13>. 

II. SYNTHESES AND USES OF COMPOUNDS CONTAINING C=C, C=0 OR C=N GROUPS LABELLED WITH STABLE ISOTOPES... 

No signals for protons at C(2) have been found in the H NMR spectrum of 30. This indicates complete deuteriation at this position and that no significant deuterium exchange took place under the conditions of the synthesis. Compounds labelled with stable isotopes are finding increasing applications in biosynthetic studies31. Labelled HMVA are required for metabolic studies in insects29. 

Bjostad L. B. and Roelofs W. L. (1986) Sex pheromone biosynthesis in the red banded leafroller moth studied by mass-labeling with stable isotopes and analysis with mass spectrometry. J. Chem. Ecol. 12, 431-450. 

Gaflhey, T. E., Hammar, C. G., Holmstedt, B., and McMahon, R. E. (1971). Ion specific detection of internal standards labeled with stable isotopes. Anal. Chem. 43, 307-310. 

Techniques applying test compounds labeled with stable isotopes are applied as well (Browne et al. 1993). However, they have not found broad acceptance in substituting radioisotopes in this field due to their restrictions in quantification of unknown metabolites. 

Gas chromatography-selected ion monitoring (GC-SIM) is often used for the quantification of GAs by GC-MS, an internal standard labeled with stable isotopes (usually with deuterium) being used as the most reliable and sensitive method. A mass chromatogram reconstructed from full-scan GC-MS is also used for semiquantification. The amounts of GAs are determined by measuring the peak areas of ions characteristic for each GA and comparing these areas to those of authentic samples. When an internal standard labeled with a stable isotope is used, the ratio of the area of an ion peak characteristic for a sample and the area of the corresponding peak of the labeled internal standard is used for quantification. The analysis of GAs by GC-MS has been discussed in numerous publications and review articles.254-256... 

The internal standard should show physical and chemical properties that are as close as possible to those of the molecule that has to be measured. It must be pure, absent from the sample and, of course, inert towards the compounds in the sample. The internal standards can be classified into three categories structural analogues that are labelled with stable isotopes, structural homologues and compounds from the same chemical family. These various types of internal standards are classified here in descending order according to their usefulness and their price. In fact, the starting material for labelled compounds is fairly... 

A. Synthesis of Bismuth Compounds Labelled with Stable isotopes. . . 580... 

B. Synthesis and Applications of Arsenic Compounds Labelled with Stable Isotopes... 

---