Isotope Exchange of Azidothymidine with Tritium

The following modes of isotope exchange of azidothymidine (3'-azido-3'-deoxythymidine) with tritium were studied: solid- and liquid-phase isotope exchange with gaseous tritium and isotope exchange in solution with tritium water. Catalytic reactions of azidothymidine with gaseous tritium in solution result in virtually complete reduction of the azido group to amino group. This reduction also occurs in the course of solid-phase catalytic hydrogenation; the yield of 3'-amino-3'-deoxythymidine ranges from 20 to 70%. The molar radioactivity of tritium-labeled azidothymidine prepared by solid-phase catalytic isotope exchange with gaseous tritium and by isotope exchange in solution with tritium water does not exceed 0.5 Ci mmol^{- 1}.     

Synthesis of High-Molar-Activity Tritium-Labeled Biologically Active Compounds Containing Aromatic and Heterocyclic Fragments

Procedures are examined for tritium labeling of biologically active compounds. By isotope exchange with tritium water, it is possible to prepare products with the molar radioactivity of about 1 PBq mol^{- 1}. The molar radioactivities of compounds prepared by solid-phase isotope exchange with gaseous tritium at 180-220°C reached 5-6 PBq mol^{- 1}. The degree of labeling varied by a factor of more than 100 depending on the physicochemical properties of the substrate. Selective hydrogenation of a heterocyclic fragment of an organic compound, leaving intact the aromatic fragment, was performed for the first time by solid-phase tritiation.     

Synthesis of tritium-labeled 5-fluorouracil and 5-fluorocytosine

The effect of various catalysts and temperature on the solid-phase isotope exchange of 5-fluorouracil and 5-fluorocytosine with tritium was studied. The isotope exchange yielding the desired compounds is accompanied by dehalogenation and hydrogenation of the 5,6-double bond of the pyrimidine ring. Performing the reaction at a temperature below 160°C allowed the process to be carried out selectively, i.e., with the preservation of the functional groups and double bond in the starting compound. The yields of various products formed in the reactions of tritium with the above compounds were estimated. Synthesis conditions were found, and tritium-labeled 5-fluorouracil and 5-fluorocytosine were prepared with the molar radioactivity of 0.45 Ci mmol⁻¹ (16.7 TBq mol⁻¹) and 4.4 Ci mmol⁻¹ (0.16 PBq mol⁻¹), respectively, and with the purity exceeding 98%.     

Solid-Phase Catalytic Hydrogenation of Orotic Acid and 5-Bromouracil with Tritium on a Copper Catalyst

The performance of a copper-based catalyst in solid-phase catalytic hydrogenation of orotic acid and 5-bromouracil with gaseous tritium was studied. Hydrogen isotope exchange in the carboxy group of orotic acid was combined with decarboxylation in a one-pot process. The catalyst performance was judged from the molar radioactivity of [6-³H]uracil and [5-³H]uracil formed by catalytic hydrogenation with gaseous tritium of orotic acid and 5-bromouracil, respectively. In solid-phase catalytic dehalogenation, the performance of the copper-based catalyst is comparable with that of the palladium catalyst, but this level is attained at a higher temperature. To evaluate the performance of the copper catalyst in isotope exchange reactions, additional studies with a wider range of substrates are required.     

Solid-Phase Catalytic Reactions of Tritium with Carbohydrates: 1. Influence of Temperature,Catalysts, and Solid Phase Composition on Solid-Phase Catalytic Hydrogenation of <Emphasis Type="Italic">D</Emphasis>-Ribose with Tritium

Solid-phase catalytic hydrogenation of D-ribose with tritium was studied in relation to the temperature (varied in the range 90–130°C), platinum-group catalysts, and solid phase composition. Based on the results obtained, general approaches to synthesis of tritium-labeled ribose were formulated.     

Selective Tritium Labeling by a Solid-Phase Procedure

Ways were considered how substrates that contain such reactive centers as halogen atoms and double bonds can be labeled with tritium by solid-phase isotope exchange. The feasibility was demonstrated for tritium labeling by selective solid-phase dehalogenation, hydrogenation, and isotope exchange. Highly labeled vitamin K₁, dihydrofusicoccin, and thyroxine and its derivatives were prepared.     

Synthesis of [3H]Sulfobromophthalein and Related Problems of Tritium Labeling by Solid-Phase Isotope Exchange

Tritium-labeled sulfobromophthalein with a molar radioactivity of 0.5-0.6 PBq mol^-1 was prepared. Various aspects of tritium labeling of organic compounds by solid-phase catalytic isotope exchange are considered. A number of arguments are given in favor of the hypothesis that the degree of isotope exchange mainly depends on the efficiency of tritium spillover in the bulk of the organic substances applied onto the catalyst surface. At present, it can be considered as a reliably proved fact that at temperatures up to 180-200°C the solid-phase isotope exchange mainly occurs via reaction with tritium cations. Apparently, the contribution of the reactions with atomic tritium to labeling is significant only if there is no spillover of tritium cations to the bulk of the organic compound and the substrate withstands heating to 280-300°C.     

Synthesis of tritium-labeled Ganciclovir

The influence of temperature on the solid-phase isotope exchange of Ganciclovir with tritium was studied. Synthesis conditions were found, and tritium-labeled Ganciclovir with the molar radioactivity of 25 Ci mmol⁻¹ (0.925 PBq mol⁻¹) and purity higher than 98% was prepared.     

Efficiency of isotope exchange between sodium 4-phenylbenzoate and activated tritium

The efficiency of the protium–tritium isotope exchange in the sodium 4-phenylbenzoate (PBNa) molecule on activating the reaction on a tungsten filament at 1940 K (target temperature 77 and 295 K) and on heating the substrate supported on 5% Pd/C in the presence of gaseous tritium is compared. It is shown that the reaction mechanism is laregly determined by the properties of the material on which this reaction occurs and not only by the method of generation of activated tritium species. In the reaction of tritium atom with PBNa deposited on glass walls of the reaction vessel, the isotope substitution of tritium for protium occurred by the radical mechanism, leading to the formation of [³H]PBNa and hydrogenation products. It is assumed that the spillover of tritium atom over the support (carbon) surface is accompanied by polarization of the electronic shell and formation of the cluster (^{3 +})(\(\bar e\)), which leads to changes in the composition of the reaction products. The combined treatment of PBNa on 5% Pd/C allows estimation of the concentration of clusters on the carbon surface, which reaches 10.9 particles per 100 nm² (9.2 nm² per cluster).     

Tritium labeling of bioorganic compounds by isotope exchange

Procedures for preparing labeled compounds by liquid- and solid-phase methods using gaseous tritium and by isotope exchange with tritium water are considered as different manifestations of a common complex of processes occurring in the presence of tritium, a substrate, and a catalyst. The studies performed allow purposeful optimization of the conditions of tritium labeling of practically any biologically active substance, which makes possible more detailed investigation of the functioning of living objects.     

Nonequilibrium processes in reactions of hot tritium atoms with cooled solid targets. Influence of the atomizer temperature on formation of labeled substances

A system consisting of a cold target and “hot” atoms generated by dissociation of tritium on a tungsten wire was studied with the aim to determine conditions for preparing tritium-labeled organic compounds with the maximal radiochemical yield. The influence of the atomizer temperature on the result of the reaction of tritium atoms with amino acids and tetraalkylammonium bromides was studied; homological series of the substrates were examined with the aim to evaluate the contributions of functional groups and hydrocarbon tail to the processes occurring in the target. The dependence of the yield of the labeled parent compound on the atomizer temperature varied in the range 1600–2000 K was determined. The rates of decarboxylation and deamination sharply grew with increasing temperature of the tungsten wire. The highest yield of labeled amino acids was attained at an atomizer temperature of 1800–1900 K, and at higher temperature their yield decreased. The difference between the activation energies of the elimination of the carboxy and amino groups and of the isotope exchange of hydrogen for tritium in the C-H bond appeared to be 93 and 59 kJ mol^?1, respectively. For alkyltrimethylammonium bromides with the alkyl radicals C₁₂H₂₅, C₁₄H₂₉, and C₁₆H₃₃, the yield of the labeled parent compound reached 80–90% and was virtually independent of the atomizer temperature. The capability of tritium atoms to penetrate into the targets was evaluated. For the exponential model of the attenuation of the flow of tritium atoms inside the target, the attenuation factor for freeze-dried amino acids and alkyltrimethylammonium bromides as targets was 1.8 nm^?1.     

A Comparative Study of the Reactions of Thermally Activated Tritium with Sugars and Diazines and of Solid-Phase Catalytic Hydrogenation of These Compounds with Tritium

The feasibility of the labeling procedure involving thermal activation (TA) of tritium was examined with the substrates that are commonly labeled by solid-phase catalytic hydrogenation (SCH) with tritium. Comparative characteristics of SCH and TA as procedures for tritium labeling of sugars and diazines were obtained. These two methods ensure comparable rates of tritium incorporation into purine and pyrimidine bases. The SCH allows preparation of tritium-labeled compounds with the maximum possible molar radioactivity. The molar radioactivity of the same compounds labeled using TA did not exceed 37 TBq mol⁻¹, because only a small fraction of the substrate could react with atomic tritium. Longer reaction times and increased amounts of tritium taken into the reaction resulted in stronger degradation of the substrates. On the assumption that the reactive tritium atoms penetrate into the target to a depth of 0.5 nm, the actual specific radioactivity of the labeled compound in the zone accessible for atomic tritium reaches 0.2–2 PBq mol⁻¹. Ways are suggested to increase the molar radioactivity of compounds labeled using thermal activation of tritium.__________Translated from Radiokhimiya, Vol. 47, No. 3, 2005, pp. 284–288.Original Russian Text Copyright © 2005 by Sidorov, Badun, Baitova, Baitov, Platoshina, Myasoedov, Fedoseev.     

Synthesis of tritium-labeled 2′,3′-dideoxy-2′,3′-didehydrothymidine and 3′-azidothymidine-5′-phosphamide

Tritium-labeled 2′,3′-dideoxy-2′,3′-didehydrothymidine and 3′-azidothymidine-5′-phosphamide were prepared by isotope exchange with highly enriched tritium water. Tritium water was prepared by oxidation of high-percentage tritium on PdO. The isotope exchange was performed at 100°C in the dioxane-triethylamine mixed solvent (9: 1 by volume). The molar radioactivities (GBq mol^?1) and yields (%) of the products were, respectively, as follows: 2′,3′-dideoxy-2′,3′-didehydrothymidine, 82, 44; 3′-azidothymidine-5′-phosphamide, 200, 71.     

Solid-phase catalytic reactions of tritium with carbohydrates: 4. Mechanism of isomerization of <Emphasis Type="Italic">D</Emphasis>-glucose in the course of solid-phase catalytic hydrogenation with tritium

The influence exerted on solid-phase catalytic hydrogenation (SCH) of D-glucose with tritium by the temperature varied in the range 90–140°C, platinum group catalysts, solid phase composition, reaction time, and surface area of the support was examined. Fructose and mannose were identified in the reaction products along with labeled glucose. The mechanism of the isomerization of glucose into fructose and mannose in the solid phase under the action of hydrogen spillover was suggested. The glucose isomerization occurs by a complex mechanism analogous to acid-catalyzed keto-enol tautomerization of epimeric sugars in solution, and the active species in SCH of D-glucose with tritium is spillover hydrogen in the form of proton.     

Heterogeneous Catalytic Synthesis of Organic Compounds Labeled with Hydrogen Isotopes without Using Solvents

Solid-phase procedures for hydrogenolysis, hydrogenation, and isotope exchange are described. The possibilities of the solid-phase method for deuterium or tritium labeling of organic compounds are demonstrated. The influence of the reaction conditions on the yield of the labeled products and on the degree of hydrogen isotope incorporation into them is considered. An attempt is made to rationalize the data obtained by processes occurring in the hydrogen isotope–catalyst–support–substrate system.     

Influence of Tritium Spillover on the Efficiency of Labeling of Organic Compounds

Various mechanisms of hydrogen spillover are discussed. A number of evidences are given that tritium incorporation by isotope exchange occurs with the participation of both tritium cations and atomic tritium. The molar radioactivity of the labeled products prepared at temperatures of up to 180°C is, apparently, mainly determined by reactions of the organic compound with tritium cations, and the degree of isotope exchange depends chiefly on the efficiency of tritium spillover to the bulk of the organic substance supported on the catalyst.     

Effect of Various Additives on the Yield and Molar Radioactivity of Labeled Products in Solid-Phase Isotope Exchange

A series of tritium-labeled amino acids were prepared by high-temperature solid-phase isotope exchange in the presence of various additives, namely, of 4-dimethylaminopyridine and rhodium trichloride. These agents strongly affect the degree of isotope exchange. 4-Dimethylaminopyridine provides successful labeling of high-melting compounds; it presumably increases the probability of the reaction of active tritium species with the substrate. The positive effect of rhodium salts may be due to an increase in the thermal stability of amino acids, i.e., in the presence of rhodium trichloride the same yield can be attained at higher temperatures. Therefore, despite modification of the catalyst occurring when rhodium trichloride is reduced, the molar radioactivity of the labeled product appears to be higher than without rhodium salts. Thus, in the presence of rhodium salts, the decrease in the yield of labeled amino acids with increasing temperature is less pronounced than the increase in the molar radioactivity of these substrates, which allows preparation of highly labeled products in reasonable yields.     

Solid-phase catalytic reactions of tritium with carbohydrates: 5. Mechanism of isomerization of epimeric disaccharides in the course of solid-phase catalytic hydrogenation with tritium

The influence exerted on the solid-phase catalytic hydrogenation of lactulose with tritium by temperature in the range 130–160°C, platinum group catalysts, solid phase composition, and support surface area was studied. Lactose was identified in the reaction products along with labeled lactulose. The mechanism of isomerization of sugars in the solid phase under the action of hydrogen spillover was suggested. Isomerization of sugars occurs by a complex mechanism similar to acid-catalyzed keto-enol tautomerization of epimeric sugars in solution, and the active species in SCH of sugars with tritium is spillover hydrogen in the form of proton.     

Solid-Phase Catalytic Hydrogenation of Uracil with Tritium: Synthesis of Tritium-Labeled β-Alanine

Solid-phase catalytic hydrogenation of uracil with gaseous tritium was studied. Isotope exchange of protons at C⁵ and C⁶ is accompanied by hydrogenation of the 5,6-double bond in the pyrimidine core; as a result, a mixture of tritium-labeled uracil and 5,6-dihydrouracil is formed. The influence of the reaction temperature and time on the yield and molar radioactivity of these compounds was examined. The best conditions for cleavage of 5,6-dihydrouracil into -alanine were found. [5,6-³H₂]Uracil (49 Ci mmol^{- 1}), [5,5,6,6-³H₄]5,6-dihydrouracil (100 Ci mmol^{- 1}), and [2,2,3,3-³H₄]-alanine (100 Ci mmol^{- 1}) were prepared.