Labelling with tritium

Eor products having relatively low specific activity, such as some compounds labeled with and which are synthesized on the scale of several millimoles, classical organic chemical separation methods may be utilized, including extraction, precipitation, and crystallization. Eor separation of complex mixtures and for products having high specific activity, such as those labeled with tritium, etc, chromatographic methods utilizing paper, thin... 

This reaction was studied by use of tritium. The phenylalanine was labeled with tritium at the 4-position of the phenyl ring. When the product, tyrosine, was isolated, it retained much of the original radioactivity, even though the 4-position was now substituted by a... 

Binding assays for the saxitoxins were conducted with homogenized rabbit brain and saxitoxin exchange-labelled with tritium at C-11 (92, 93). If the various saxitoxins were available with suitably intense radiolabels, then the equilibrium dissociation constant, K, could be measured directly for each. Since only saxitoxin is currently available with the necessary label, the binding experiments instead measure the ability of a compound to compete with radiolabelled saxitoxin for the binding site. The value obtained, Kj, corresponds to the uilibrium dissociation constant, K, that would be observed for the compound if it were measured directly. Affinity is defined for this assay as the reciprocal of Kj. The affinities of several of the saxitoxins (94) are summarized in Figure 11, expressed relative to saxitoxin and plotted on a logarithmic scale. 

Synthesis of simple seven-membered ring compounds labelled with tritium... 

Reduction of aldonolactones and their derivatives with isotopically modified reducing agents leads to sugars labeled at the anomeric center. Glycosides substituted with deuterium or labeled with tritium are widely employed for kinetic isotope-effect measurements, mechanistic studies, isotope-tracing experiments, and so on. 

More experimental data where the secondary KIE was larger than the EIE were subsequently published by Subramanian and Saunders (1984). The 2-arylethyl system was employed in these studies because other relevant data, such as the primary deuterium KIE, were available for this reaction. Special techniques were developed to determine the primary and the secondary tritium KIEs for this system. Three isotopically distinct elimination reactions (49-51) are possible for a 2-arylethyl derivative tracer labelled with tritium at the 2-position. 

Nucleotide Uptake. When uracil labelled with tritium was supplied to 15-day-old ozone-treated cotton leaves, a 2-fold increase in uracil incorporation into the RNA fraction was observed (Table IV). In this one particular case, there appeared to be a greater percentage of uracil incorporation into RNA in treated tissue than in control tissue. 

Oxidation of Initiated Habilitate Pakrritate uniformly labeled with tritium (3H) to a specific activity of 2.48 X 10 counts per minute (cpm) per micromole of palmitate is added to a mitochondrial preparation that oxidizes it to acetyl-CoA. The acetyl-CoA is isolated and hydrolyzed to acetate. The specific activity of the isolated acetate is 1.00 X 107 cpm//xmol. Is this result consistent with the /1-oxidation pathway Explain. What is the final fate of the removed tritium ... 

FIGURE 25-3 Visualization of bidirectional DNA replication. Replication of a circular chromosome produces a structure resembling the Greek letter theta (0). (a) Labeling with tritium (3H) shows that both strands are replicated at the same time (new strands shown in red). The electron micrographs illustrate the replication of a circular E. coli plasmid as visualized by autoradiography, (b) Addition of 3H for a... 

As for any other organic molecules containing CH bonds, heteroaromatics can be labeled with tritium by reaction with energetic tritium atoms from neutron irradiation. 

The borohydride reduction of 2 - and 3 -ketocytidine derivatives having or lacking an N4-acetyl group provided a facile route to cytosine nucleosides labeled with tritium at specific positions of the sugar.28 Reduction of either 20a or 20c with sodium borohydride in ethanol-benzene afforded the arabino and the ribo derivatives in the ratio of 4 1. Treatment of the reduction mixture with methanolic ammonium hydroxide gave the deacetylation products, 82b and 19b, isolated in crystalline form by preparative, thin-layer chromatography. 

The stilbene was labelled with tritium since its low concentrations and chemical similarity with styrene precluded normal chemical or physical analysis. From this study it was concluded that head to head structures are not weak links. 

Compounds may be labeled with tritium by several methods. The classic synthetic methods utilizing labeled intermediates have the advantage of yielding products that have predictable specific activities, are specifically labeled, and have a minimum of aged by-products. Among the methods of tritium labeling are ... 

By Reduction of Unsaturated Precursors The method of choice for labeling with tritium is the reduction of a suitable unsaturated precursor (containing a double bond, carbonyl group, etc.) with carrier-free tritium gas or tritiated metal hydrides. The major limitation of this method is the availability of a suitable unsaturated precursor of the desired compound. It is essential to carry out the synthesis in a non-hydroxylic solvent (dioxane, ethyl acetate, etc.). Reductions carried out in alcohol or water will lead to almost complete exchange of the tritium gas with the solvent. 

Although random labeling with tritium usually involves some element of uncertainty as to the identity of the labeled molecules with the original sample, this labeling method frequently offers the only practical means of preparing the necessary amounts of tagged materials for field studies. Carbon- 14-labeled gibberellic acid has been prepared, but the yields have been too low to make its use practicable (8). 

The classical histamine H3 antagonist thioperamide has been labelled and evaluated as radioligand for the H3 receptor by Alves-Rodrigues et al [27], [3H]thioperamide was synthesised from [3H]4-(lH-imidazol-4-yl)piperidine, labelled with tritium in the 2 or 5 position of the imidazole and at the 4 position of the piperidine, and cyclohexylisothiocyanate (scheme 10) [28], It was purified with a semipreparative HPLC method and isolated with a specific activity of 6 Ci/mmol. 

III. SYNTHESIS AND USES OF COMPOUNDS CONTAINING C=C, C=0 OR CN GROUPS LABELLED WITH TRITIUM... 

The synthesis of pressor amines in the body may convincingly be considered to begin with the essential amino acid, phenylalanine as illustrated in Table I. Gurin and Delluva (86) have reported that phenylalanine labeled with tritium, or containing C14 located both in the carboxyl group and in the position adjacent thereto, was converted in the rat to radioactive adrenaline. Only one C14 atom was present in the side chain and it was in the terminal group bearing the secondary amine (86). However the intermediate steps in the synthesis are by no means so certain, nor does this necessarily preclude other paths of synthesis. 

Hydrogen exchange reactions in liquid ammonia can be used to prepare many organic compounds labelled with tritium or deuterium. 

In contrast to the oxidative reactions discussed above, the only reported biotransformations of reserpine (21) and rescinnamine (23) (42-44) appear to involve hydrolytic processes. Reserpine is readily metabolized by liver homogenates from the mouse (43), rat, guinea pig, dog, and cat (44) to yield methyl reserpate (22) and 3,4,5-trimethoxybenzoic acid in yields of up to 70% (43). The use of reserpine labeled with tritium in the 2 and 6 positions of the trimethoxybenzoate residue indicated that no significant metabolism of reserpine by another route occurred before hydrolysis, reserpine and 3,4,5-trimethoxybenzoic acid being the only detectable radioactive components of the incubation mixture at the conclusion of the reaction (44). An... 

The appearance of triamcinolone (labelled with tritium) in the blood of rabbits following intramuscular adminstration has been evaluated for microsphere systems prepared using the heat denaturation method (Figure 5). A characteristic change in the release characteristics of the drug was achieved. 

The present work was undertaken in an attempt to shed light on the mechanism of hydroxyproline formation in collagen biosynthesis and on the site of action of ascorbate. It was recognized that these phenomena were very likely to be closely interrelated. Proline labeled with tritium was used because it was anticipated that tritium released from proline during hydroxylation would appear in the tissue water as tritiated water. The use of tritiated proline in such a system might then provide a new parameter for following the hydroxylation reaction, provided that the formation of tritiated water was stoichiometrically related to the formation of hydroxyproline. Such an approach might conceivably permit the demonstration of hydroxylation and peptide bond formation as separate chemical steps. 

An ingenious experiment showed that this cyclization is not as simple as it seems. If the starting material is labelled with tritium (radioactive 3H) next to the ring, the product shows exactly 50% of the label where it is expected and 50% where it is not. 

When the hydrogen transferred as hydride to the cofactor is retransferred to the same carbon atom in the product, the movement is far more difficult to detect. The conversion of D-glucose 6-phosphate (58) into lL-mt/o-inositol 1-phosphate (61) occurs by cyclization of the carbon skeleton, with formation of a new bond between C-l and C-6. When each carbon atom in turn was specifically labeled with tritium, there was complete retention of tritium, even in the presence of added NADH, although there was an apparent, small isotope-effect with D-glucose-5-t 6-phosphate.19 The mechanism proposed for the cyclization19 was an initial oxidation at C-5 to give NADH and xylo-hexos-5-ulose 6-phosphate (59), followed by an aldol reaction causing cyclization to lL-myo-inosose-2 1-phosphate (60), which is then... 

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