Tritium-labeled compounds

Isotopically Labeled Compounds. The hydrogen isotopes are given special names H (protium), H or D (deuterium), and H or T (tritium). The superscript designation is preferred because D and T disturb the alphabetical ordering in formulas. 

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). 

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... 

Other methods of sensitive detection of radiotracers have been developed more recently. Eourier transform nmr can be used to detect (nuclear spin 1/2), which has an efficiency of detection - 20% greater than that of H. This technique is useful for ascertaining the position and distribution of tritium in the labeled compound (14). Eield-desorption mass spectrometry (fdms) and other mass spectral techniques can be appHed to detection of nanogram quantities of radiolabeled tracers, and are weU suited for determining the specific activity of these compounds (15). 

When specifically labelled compounds are required, direct chemical synthesis may be necessary. The standard techniques of preparative chemistry are used, suitably modified for small-scale work with radioactive materials. The starting material is tritium gas which can be obtained at greater than 98% isotopic abundance. Tritiated water can be made either by catalytic oxidation over palladium or by reduction of a metal oxide ... 

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. 

The incorporation of 14C into compounds at a suitable site often requires extensive and complicated syntheses, and thus a relatively long time. This usually means that 14C-labeled compounds are unsuitable for studies to be carried out during discovery. There are however, very rapid methods for incorporating 3 H into compounds. The newer methods, generally involving metal-catalyzed exchange reactions [15-18], in our experience, mean that suitable labels can often be prepared in 2 or 3 weeks. These time scales make the approach viable for discovery support. Additionally, and importantly, these methods can lead to specific incorporation of tritium. 

For mechanistic studies => to test some hypothesis about a reaction mechanism or about how a certain organism metabolizes a compound => often need to synthesize a particularly labeled compound (with deuterium, tritium, or C). 

This tritiated water can become incorporated into the normal bases of DNA and may contribute significantly to the overall apparent binding levels measured in DNA (4). Such problems are circumvented in the case of 14C labeled hydrocarbons. Tritium labeled compounds can be prepared at sufficiently high specific activities (often 20-80 Ci/mmole) that fentomole amounts of adduct may be detected. 

In the preparation of tritium-labeled compounds there are four stages ... 

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

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. 

The comprehensive dedicated research ultimately made it possible to decode the patterns of labelling in almost any type of tritium labelled compound at low isotopic abundance (e.g., 3 x 10 4 to 3 x 10 2 per cent. 3H per site) with the aid of 3H-NMR directly, rapidly, reliably and non-destructive analytical means. Since, 1971, the 3H-NMR spectroscopy, utilizing only millicurie (mCi) quantities of radioactivity, emerged as a most useful analytical tool for the study of tritium labelled compounds. 

Useful labelled compounds containing deuterium or tritium normally require the isotopic hydrogen to be attached to carbon, so the acid-base equilibrium will require cleavage of a C-H bond, where acid... 

Nagarajan, S. Kellogg, M. S. DuBois, G. E. Williams, D. S. Gresk, C. J. Markos, C. S. Understanding the Mechanism of Sweet Taste Synthesis of Tritium Labeled Guanidineacetic Acid, J. Labelled Compounds and Radio-pharmaceut. 1992, 31, 599-607. 

H transfer. Tritium is sometimes transferred from substrates to solvents or proteins. For this reason and more importantly for that above, the use of 14C is preferable to 3H, although the higher specific activities and relative cheapness of 3H-labeled compounds often more than compensate for the disadvantages. 

The problem of radiochemical purity with respect to the chemical state of aged tritium or 14C-labeled compounds is still more acute. Because of the short range... 

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. 

In many situations, the experimenter will prefer to buy labeled compounds from commercial suppliers rather than attempt to synthesize them. The radiochemical purity of such purchased compounds cannot be assumed. Radiation-induced selfdecomposition (radiolysis) can result in the formation of a variety of labeled degradation products, which must be removed before experimental use of the compounds. The extent of radiolysis depends on the nature of the labeled compound, how long it has been stored, and the manner of storage. Radiolysis is most significant with low-energy (3 emitters (especially tritium) since the decay energy is dissipated almost entirely with the compound itself. Furthermore, impurities involving other radionuclides may be present. 

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

Carbon-14 has been introduced at the Cqj position of the pentene group of drug 172153,154 starting with Ba14C03 in 28% yield and tritium has been introduced into the position at Cp) of the chain in the final reaction step by reduction of the ketone group with NaBTzt155 (equation 81). The double-labelled compound 172 is applied in pharmacokinetic studies in animals155. 

Syntheses and uses of isotopically labelled compounds 3. Synthesis of carbon-14 and tritium labelled analogues of manoalide... 

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

---