Tritium-labelled compounds Acetate

The introduction of tritium into molecules is most commonly achieved by reductive methods, including catalytic reduction by tritium gas, PH2], of olefins, catalytic reductive replacement of halogen (Cl, Br, or I) by H2, and metal pH] hydride reduction of carbonyl compounds, eg, ketones (qv) and some esters, to tritium-labeled alcohols (5). The use of tritium-labeled building blocks, eg, pH] methyl iodide and pH]-acetic anhydride, is an alternative route to the preparation of high specific activity, tritium-labeled compounds. The use of these techniques for the synthesis of radiolabeled receptor ligands, ie, dmgs and dmg analogues, has been described ia detail ia the Hterature (6,7). 

Tritium-labeled (GlcNAc)2-Ep was obtained from (VII) by the procedure described in Method A, starting with the synthesis of the glycosyl chloride (VIII). The specific activity of the [ H] (GlcNAc)2-Ep was 1.1 X 10 cpm/mmole. [ C] (GlcNAc)2-Ep was prepared by the same method, using [ C] acetic anhydride instead of the corresponding tritium-labeled compound. 

Two important classes of reactions use labelled tin compounds to prepare labelled compounds for mechanistic and analytical purposes. In the first type of reaction, labelled trialkyl- or triaryl tin hydrides (stannanes) are used to reduce (replace) several different groups such as halogen, —N02, —N=C, —N=C=Se, —COOR, —SR or an acetal group with a deuterium or a tritium atom. 

In the case of labeled compounds, entry is normally without special designation in the formula, but name entries under such formulas show the labeled nature of the compound C4H8O2, isobutyric-I-C acid, indicates that the number one carbon atom is labeled. Deuterium and tritium compounds show labeling in the formula index, as CH3DO, methanol-d, or C2H2D2O, acet-d2-aldehyde. 

The reduction of a halogen compound by a metal catalyst such as zinc in the presence of tritiated water or tritium-labelled acetic acid will obviously produce a compound with a tritium atom in place of the original halogen. Reduction of this kind, although sometimes used for labelling, requires an excess of tritium in the water, acetic acid or other solvent. 

In our studies, we have administered tritium-labeled vitamin A in one of its two physiological plasma transport vehicles (associated with either retinol-binding protein or chylomicrons) so that tracer data can be extrapolated to the vitamin A compounds of interest (retinol, retinyl esters, and metabolites). To prepare pH]retinol in its plasma transport complex (Green and Green, 1990b), vitamin A-depleted rats are used as donors to maximize hepatic secretion of the labeled vitamin on acciunulated liver apoRBP. pH]Retinol or pH]retinyl acetate in an emulsion with Tween 40 is administered intravenously to donor rats and blood is harvested 100 min later when plasma radioactivity is maximal. Plasma is isolated and stored under a nitrogen atmosphere at 4°C plasma is used for in vivo studies within 23 days. 

A 98% conversion efficiency has been achieved at 1.5 atmospheric pressure of CO2 and 10% excess of water, while the yield was 85% only when stoichiometric amounts of water and carbon dioxide were used. No isotopic fractionation has been noted when 5 to 10% excess of water has been used in the reaction in equation 5. Doubly labelled methane has been used in simultaneous analysis of and both in the atmosphere and in environmental carbon dioxide as well as in analysis of and H-labelled organic compounds. Proportional counters filled with methane possess very good counting properties and there is no need to add any inactive counting gas. and tritium-labelled methanes have also been produced" by pyrolysis of sodium acetate in molten sodium hydroxide (equation 6) ... 

C-Labelled dotriacontane has been used to study the retention of tobacco smoke in the respiratory system of smoke-exposed laboratory animals. High-resolution autoradiography studies within the respiratory tract needed tritium-labelled dotriacontane (34) with a specific activity of 0.5 Cimmol" This compound has been prepared by synthesizing the diacetylene 35 and by its reduction to dotriacontane-15,15,16,16,17,17,18,18- H, 34 (equation 48). Sodium acetylide with myristyl bromide yielded hexadec-l-yne 36. Oxidative coupling of 36 using cupric acetate gave the diyne 35 which, upon reduction with tritium gas in the presence of Adams catalyst, yielded 34, with the specific activity of 0.50 Ci mmol as required. 

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. 

In principle any except tetrasubstituted olefins could be labeled by catalytic exchange, but in practice it is difficult to find experimental conditions under which significant tritium incorporation can be catalyzed without reducing the double bond. However, if the olefinic starting material is separable from the reduced product, it is sometimes possible to find reaction conditions that disfavour complete reduction, then isolate the unreduced olefin which has become labeled. Two examples with which this has been accomplished are pleuromutilin (71) and cyclosporin A (72) " °. Treatment with a deficiency of tritium gas for 60-90 min in the presence of 10% Pd/C in ethyl acetate (71) or DMF (72 followed by removal of reduced product by HPLC, provided the compounds, labeled as indicated, at specific activities of 10 and 19Ci/mmol, respectively. Unfortunately, it proved to be difficult to separate the cis/trans mixture formed with 72, which stimulated the development of a tritide labeling approach (see Chapter 4, Section 4.3.1). 

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