Synthesis and analysis of selenomethionine metabolites.

This paper describes the enzymatic synthesis of selenomethionine metabolites of the transmethylation and polyamine synthesis pathways: adenosylselenomethionine, adenosylselenohomocysteine, decarboxylated adenosylselenomethionine, and methylselenoadenosine. These compounds and the corresponding methionine metabolites were simultaneously separated by a single HPLC run. The sensitivity of the HPLC method is about 20 pmol per compound. The method may be used for direct analysis of the metabolite levels in tissues or cells treated with selenomethionine and it provides an assay method for the pulse-chase type of analysis of relative flows for both selenium- and sulfur-containing compounds in transmethylation and polyamine pathways.

Oxidative deamination of Se-(1-carboxyethyl)-,Se-(1-carboxypropyl)- and Se-(2-carboxyethyl)-selenocysteine by snake venom L-aminoacid oxidase.

Details are reported for the synthesis of Se-(1-carboxyethyl)-selenocysteine (1-CESeC), Se-(1-carboxypropyl)-selenocysteine (1-CPSeC) and Se-(2-carboxyethyl)-selenocysteine (2-CESeC). They can be obtained in pure cristalline form with good yield. Some chromatographic properties, useful for their identification, are described. The three aminoacids are good substrates for snake venom L-aminoacid oxidase, giving the corresponding alpha-ketoacids as reaction products.

Fast chemical synthesis of [75Se]L-selenomethionine.

A fast chemical synthesis of high specific activity [75Se]L-selenomethionine (10 Ci/mmol--370 GBq/mmol) is described with a view to 73Se labeling and PET studies. The overall radiochemical yield of the preparation is better than 80%. The purification method uses commercially available reverse phase HPLC columns and 9% NaCl as mobile phase. The final labeled compound is obtained in less than 3 h and the chemical, radiochemical and optical purities of the L-isomer are higher than 99.0%.

Synthesis and characterization of a selenium-containing substrate of alpha-chymotrypsin. Selenium-77 nuclear magnetic resonance observation of an acyl-alpha-chymotrypsin intermediate.

The selenium-containing ester p-nitrophenyl (phenylselenyl)acetate, C6H5SeCH2C(O)-OC6H4-p-(NO2), has been synthesized, characterized as a substrate for alpha-chymotrypsin (k2/KM = 15.2 X 10(3) M-1 s-1, KMapp = 5.16 X 10(-6) M, pH 7.77, 33% CH3CN, 25 degrees C), and shown to be an active-site titrant for the enzyme. A synthesis of the selenium-77 enriched p-nitrophenyl (phenylselenyl)acetate in 53% yield from 94.4% elemental selenium-77, followed by its reaction with alpha-chymotrypsin (pH 5.0, 0-3 degrees C), permitted the observation of the (phenylselenyl)acetyl-alpha-chymotrypsin reaction intermediate by selenium-77 NMR spectroscopy. This acyl-enzyme species had a chemical shift of 275.1 ppm relative to dimethyl selenide. Accompanying this resonance was a lower intensity, pH-dependent resonance that is assigned to (phenylselenyl)acetate on the basis of a pH titration of the model compound. Deacylation in the presence of hydrazine sulfate produced a resonance at 332.3 ppm in addition to the 302.2 ppm resonance of (phenylselenyl)acetate at pH 7.85. Denaturation of the acyl-enzyme resulted in a shift of the 275.1 ppm resonance to 334.6 ppm at pH 4.90, in good agreement with the selenium-77 chemical shift of the model compound, methyl (phenylselenyl)acetate, in CDCl3 (333.3 ppm). The large shielding observed for the native acyl-enzyme in comparison to the denatured species can be attributed to a resonance-perturbed ester linkage and/or steric compression at a nonbonding orbital of the selenium nucleus.

Synthesis of L-selenodjenkolate and its degradation with methionine gamma-lyase.

A convenient method for the synthesis of a new selenium-containing amino acid, L-selenodjenkolate (3,3'-methylenediselenobis(2-aminopropionic acid)), is described. The starting material, selenocystine, was found to be prepared easily from commercially available beta-chloro-L-alanine, elemental selenium, and sodium borohydride. These synthetic procedures are useful for the preparation of the isotope-labeled compounds. The physiochemical properties of selenodjenkolate thus prepared are reported. This amino acid undergoes alpha, beta-elimination to produce pyruvate, formaldehyde, ammonia, and selenium by bacterial methionine gamma-lyase under aerobic conditions: the Vmax and Km values were determined to be 0.5 mumol/min/mg and 2.3 mM, respectively.

Ribavirin, tiazofurin, and selenazofurin: mononucleotides and nicotinamide adenine dinucleotide analogues. Synthesis, structure, and interactions with IMP dehydrogenase.

A series of dinucleotides, analogous to nicotinamide adenine dinucleotide but containing the five-membered base nucleosides tiazofurin (1a), selenazofurin (1b), ribavirin (2), and AICAR (3) in place of nicotinamide ribonucleoside, were prepared in greater than 50% yield by reacting the corresponding nucleotide imidazolidates (6a-d) with adenosine 5'-monophosphate (AMP). The symmetric dinucleotides of tiazofurin (TTD, 8e) and selenazofurin (SSD, 8f) were also prepared by a similar methodology. These dinucleotides were characterized by 1H NMR and fast atom bombardment MS and were evaluated for their inhibitory potency against a partially purified preparation of tumoral inosine monophosphate dehydrogenase (IMPD) from P388 cells. The order of potency found was SAD (8b) greater than TAD (8a) much greater than SSD (8f) congruent to TTD (8e) congruent to RAD (8c) much much greater than ZAD (8d). On kinetic analysis none of the dinucleotides produced competitive inhibition of IMPD with NAD as a variable substrate. In addition to their superior IMPD inhibitory activity, SAD and TAD appear to be the only dinucleotides, besides NAD, that are formed naturally by the NAD pyrophosphorylase from P388 lymphoblasts.

Identification and synthesis of a naturally occurring selenonucleoside in bacterial tRNAs: 5-[(methylamino)methyl]-2-selenouridine.

Escherichia coli, Clostridium sticklandii, and Methanococcus vannielii synthesize 75Se-labeled amino acid transfer ribonucleic acids [( 75Se]tRNAs) when grown with low levels (approximately equal to 1 microM) of 75SeO32-. When E. coli [75Se]tRNA was digested to nucleosides and analyzed by reversed-phase high-performance liquid chromatography, a single selenonucleoside accounted for 70-90% of the 75Se label in the bulk tRNA. This nucleoside was shown to be indistinguishable in a number of its properties from authentic 5-[(methylamino)methyl]-2-selenouridine. Preparation of the authentic selenonucleoside was accomplished and the synthetic compound characterized by its UV and 1H NMR spectral properties. The new selenonucleoside also accounted for 40-60% of the 75Se found in [75Se]tRNA from C. sticklandii or M. vannielii. Each of these anaerobic bacteria contains one additional selenonucleoside in their tRNA populations distinct from 5-[(methylamino)methyl]-2-selenouridine. Pure seleno-tRNAGlu isolated from C. sticklandii contains one 5-[(methylamino)methyl]-2-selenouridine and one 4-thiouridine per tRNA molecule.

Investigation of two fluorinated reagents for the analysis of selenium by gas chromatography.

As potential reagents for the determination of traces of selenium by gas chromatography, the compounds 4-fluoro-o-phenylenediamine and 4-trifluoromethyl-o-phenylenediamine have been examined and compared with seven o-diamines previously reported for analytical purposes. Retention times of the fluoro-piaselenols are shorter than all other compounds but, with the electron-capture detector, their responses differ widely. However, the detection limit of the trifluoromethylpiaselenol compares favourably with the commonly used derivative, 5-nitropiaselenol. The analytical requirements of 4-trifluoro-o-phenylenediamine as a reagent, and the results of its application to the determination of selenium in various biological matrices, are presented.

Synthesis and antitumor activity of some aromatic seleno lactones.

Several aromatic seleno lactones have been synthesized and shown to possess significant inhibitory activity against human colon tumor-8r cells in culture at concentrations lower than 1 mM. Although all of the compounds tested were found to be active, 5-hydroxy-3-[(phenylseleno)methyl]hydrocoumarinoctanoate (3d) and 5-hydroxy-3-[(phenylseleno)methyl]hydrocoumarindecanoate (3e) were found to be the most effective in inhibiting cell growth. In situ formation of the corresponding alpha-methylene lactones is postulated to account for the cytotoxic activity in this class of compounds.

Synthesis of [75Se]trimethylselenonium iodide from [75Se]selenocystine.

The synthesis of [75Se]trimethylselenonium iodide from [75Se]selenocystine is described. The starting compound is reduced to [75Se]selenocysteine with borohydride and reacted with methyl iodide to form [75Se]Se-methyl-selenocysteine, then treated with methyl iodide in formic acid solution to form Se-dimethyl-selenocysteine selenonium iodide. Over a period of days, the selenonium intermediate undergoes spontaneous elimination to form alanine and dimethyl selenide, which reacts with methyl iodide to give the trimethylselenonium product in over 90% yield.

Synthesis and evaluation of 24-(isopropyl[75Se]seleno)chol-5-en-3 beta-ol.

Selenium-75-labeled 24-(isopropylseleno)chol-5-en-3 beta-ol (4) has been prepared by reaction of sodium isopropyl-[75Se]selenol [( 75Se]2) with 3 beta-acetoxy-24-bromochol-5-ene (3). This new 75Se-labeled adrenal imaging agent shows pronounced adrenal uptake in rats. The concentration of radioactivity in rat adrenals increased steadily from 1 to 24 h after injection and then decreased slowly over the 21-day period. After 3 days the adrenal/blood and adrenal/liver ratios were 85:1 and 32:1, respectively, which are sufficient for adrenal imaging by single photon techniques. After 6 h the adrenal/blood ratio was 17:1 and the adrenal/liver ratio was 7:1. We propose that these ratios are sufficiently high for positron emission tomography of the adrenals. The absorbed radiation dose values to human organs have been estimated for the 75Se- and 73Se-labeled agent.

New selenium-75 labeled radiopharmaceuticals: selenonium analogues of dopamine.

Selenium-75 labeled selenonium analogues of dopamine, [2-(3,4-dimethoxyphenyl)ethyl]dimethylselenonium iodide (4) and its dihydroxy analogue (7), were prepared by reducing [75Se]selenious acid with sodium borohydride at pH 6.0 and reacting the NaSeH produced with 1-(3,4-dimethoxyphenyl)-2-(p-toluenesulfonyloxy)ethane. Tissue distribution studies in rats given the 75Se-labeled selenonium agents intravenously demonstrated high initial heart uptake (2.38% dose/g at 5 min). Prolonged adrenal retention (t1/2 = 10 h) and high adrenal to blood ratio of compound 4 (21/1 at 4 h after injection) were observed. The high uptake and adrenal to blood ratio suggest the potential use of compound 4 as a radiopharmaceutical for the adrenal gland.

Antioxidants in purification, stabilization, and formulation of the antineoplastic agent 6-selenoguanosine.

6-Selenoguanosine (NSC 137679) was stablized and formulated as a lyophilized parenteral paroduct using ascorbic acid as an antioxidant. In addition to preventing the oxidation of 6-selenoguanosine to the corresponding diselenide in aqueous solution, ascorbic acid reduced the diselenide already in the bulk drug. Dithioerythritol and sodium bisulfite also were evaluated as antioxidants. Dithioerythritol had effects similar to ascorbic acid, while sodium bisulfite reacted rapidly with 6-selenoguanosine.

Synthesis and chromatographic properties of Se-carboxymethyl-selenohomocysteamine (2-carboxymethyl, 3-aminopropyl selenide).

Details are reported for the synthesis of Se-carboxymethylselenohomocysteamine from selenohomocysteamine and monochloroacetic acid. Data on its behaviour on paper and ion-exchange chromatography are also reported, which allow its identification.