Synthesis of tritium-labeled N-acetyl PGE2 carboxamide (CP-27,987)

The preparation of N-acetyl PGE2 carboxamide (CP-27,987) regioselectively labeled at C-18 and C-19 with tritium is described. The overall radiochemical yield was 8.0% at a specific activity of 0.49 Ci/mmole. The synthesis employed is applicable to the preparation of tritium labeled natural prostaglandins and a variety of analogs.     

Stereochemistry of the β-cyanoalanine synthetase and S-alkylcysteine lyase reactions

The stereochemistry of the replacement of the SH-group of cysteine by CN catalyzed by β-cyanoalanine synthetase was studied using cysteine stereospecifically tritiated at C-3. Analysis of the resulting β-cyanoalanine by conversion into fumarate via aspartate and malate showed that the reaction had occurred with retention of configuration at C-3. Using cystine stereospecifically labeled at C-3 with tritium or with tritium and deuterium, it was found that the α,β-elimination reaction catalyzed by S-alkylcysteine lyase involves stereo-specific replacement of the β-substituent of the substrate by a hydrogen derived from the solvent, D₂O or H₂O, with retention of configuration to give pyruvate containing a chiral methyl group. The results are discussed, particularly in the light of mechanistic proposals by Braunstem and co-workers.     

Distribution of tritium in D-glucose and starch labeled by tritium-atom bombardment

Samples of D-glucose and starch were labeled by tritium-atom bombardment. Up to 51% incorporation into D-glucose as non-labile tritium was achieved for crystalline, anhydrous D-glucose and 41% for the amylose-butyl alcohol complex. Distribution of tritium in the carbon skeleton of D-glucose was calculated by comparing the specific molar activity of D-glucose with that of its derivatives. Derivatives prepared were D-gluconic acid, D-arabino-hexulose phenylosotriazole, 4-formyl-2-phenyltriazole, 2-phenyltriazole-4-carboxylic acid, D-arabino-hexulose phenylflavazole, 3-formyl-1-phenylflavazole, and formaldehyde dimedone. The tritium distribution showed definite structural effects. Generally, the products from films of D-glucose and the amylose-butyl alcohol complex had nearly uniform distribution of tritium in D-glucose. The product from crystalline α-D-glucose monohydrate had zero tritium at C-2 and twice the expected amount of tritium at C-5, and that from starch granules had zero or near zero tritium at C-3 and close to twice the expected amount of tritium at C-2.     

Absence of reactive intermediates in the formation of catechol estrogens by rat liver microsomes

Release of 3H2O from regiospecifically labeled estradiol was measured during 2-hydroxylation of this estrogen by rat liver microsomes. The amount of tritium remaining in the isolated catechol estrogen was also determined. Virtually all the tritium was removed from C-2 during the reaction confirming the absence of an NIH shift. About 20% of the tritium at C-1 was also lost without any such change occurring at C-4 or C-6,7 of the steroid molecule. These findings provide evidence for the formation of an arene oxide or o-semiquinone intermediate during the conversion of estradiol to 2-hydroxyestradiol. No indication of adduct formation at either C-1 or C-4 during this biotransformation was obtained although the 2-hydroxylated product was able to react with a nucleophile such as glutathione. The different regiospecificity of tritium loss in the generation of catechol estrogens and in their subsequent reaction leads to the important conclusion that the reactive intermediates in the two processes must be different. The possible role of catechol estrogens in neoplastic transformation is discussed.     

Evidence for a C-2→C-1 intramolecular hydrogen-transfer during the acid-catalyzed isomerization of D-glucose to D-fructose ag]

In mechanistic studies by isotope-exchange tecniques of the conversion of D-fructose and D-glucose into 2-(hydroxyacetyl)furan, it was shown that both sugars are converted in acidified, tritiated water into the furan containing essentially no carbon-bound tritium. As the hydroxymethyl carbon atom of the furan corresponds to C-1 of the hexose, this result suggests that one of the hydrogen atoms in this group, when it is produced from D-glucose, must arise intramolecularly. This hypothesis was verified by synthesizing D-glucose-2-³H and converting it into the furan in acidified water. The 2-(hydroxyacetyl)furan obtained was labeled exclusively on the hydroxymethyl carbon atom, thus showing that intramolecular hydrogen-transfer occurs, during the conversion, from C-2 of D-glucose to the carbon atom corresponding to C-1. The specific activities of the product and reactant permitted calculation of the tritium isotope-effect (k_h/k_t=4.4) for the reaction. The precise step for the transfer from C-2 of the aldose to the carbon atom corresponding to C-1 was found to be during the isomerization of D-glucose to D-fructose, as evidenced by the conversion of D-glucose-2-³H into D-fructose-1-³H in acidified water.     

Evidence for a C-2→C-1 intramolecular hydrogen-transfer during the acid-catalyzed isomerization of D-glucose to D-fructose ag]

In mechanistic studies by isotope-exchange tecniques of the conversion of D-fructose and D-glucose into 2-(hydroxyacetyl)furan, it was shown that both sugars are converted in acidified, tritiated water into the furan containing essentially no carbon-bound tritium. As the hydroxymethyl carbon atom of the furan corresponds to C-1 of the hexose, this result suggests that one of the hydrogen atoms in this group, when it is produced from D-glucose, must arise intramolecularly. This hypothesis was verified by synthesizing D-glucose-2-³H and converting it into the furan in acidified water. The 2-(hydroxyacetyl)furan obtained was labeled exclusively on the hydroxymethyl carbon atom, thus showing that intramolecular hydrogen-transfer occurs, during the conversion, from C-2 of D-glucose to the carbon atom corresponding to C-1. The specific activities of the product and reactant permitted calculation of the tritium isotope-effect (k_h/k_t4.4) for the reaction. The precise step for the transfer from C-2 of the aldose to the carbon atom corresponding to C-1 was found to be during the isomerization of D-glucose to D-fructose, as evidenced by the conversion of D-glucose-2-³H into D-fructose-1-³H in acidified water.     

Stereochemistry of 1,2-hydrogen loss during aromatization in the brain

The stereochemistry of hydrogen loss from C-1 and C-2 during aromatization in rat brain was studied using androstenedione containing a known distribution of isotopic label. Comparison of the tritium content of the estrone obtained from the aromatization of androstenedione labeled predominantly in the 1 alpha,2 alpha positions with that in estrone obtained from a parallel incubation using substrate with label in the 1 beta,2 beta orientation gave an estrone alpha/beta ratio of 3.6. This ratio compares with a calculated value of 4.3 for an aromatization mechanism involving loss of the 1 beta,2 beta-hydrogens. The distortion from the predicted value is due to the loss of tritium from the alpha-substrate which is unrelated to aromatization. The ratio determined experimentally is compatible with 2 beta-tritium loss since random or alpha-elimination from C-2 would yield alpha/beta ratios of 2.2 and 1.3 respectively. In an analogous manner the stereochemistry of tritium loss at C-1 was determined using [1 alpha-3H] and [1 beta-3H]androstenedione. The alpha/beta ratio of the isolated estrone was 3.6 which is in good agreement with the calculated value of 3.3 for 1 beta-tritium elimination. Our results therefore show that estrogen formation in the brain occurs with the same stereospecificity of hydrogen loss at C-1 and C-2 as in placental microsomes.     

Stereochemistry and kinetic isotope effects in the bovine plasma amine oxidase catalyzed oxidation of dopamine.

The stereochemistry of the bovine plasma amine oxidase catalyzed oxidation of 2-(3,4-dihydroxyphenyl)-ethylamine (domapine) has been investigated by comparing 3H/14C ratios of 3,4-dibenzyloxyphenethyl alcohols, derived from 3,4-dihydroxyphenylacetaldehydes, to starting dopamines chirally labeled at C-1 and C-2. The oxidation of [2RS-3H]-, [2R-3H]-, and [2S-3H]dopamine leads to products which have retained 53, 59, and 47% of their tritium. Similarly, oxidation of [1RS-3H]-, [1R-3H]-, and [1S-3H]dopamine leads to an 80, 80, and 92% retention of tritium. The configurational purity of tritium at C-2 of dopamine and C-1 of the dopamine precursor 3-methoxy-4-hydroxyphenethylamine has been confirmed employing dopamine-beta-hydroxylase (specific for the pro-R hydrogen at C-2) and pea seedling amine oxidase (specific for the pro-S hydrogen at C-1). In addition, chromatographically resolved isozymes of bovine plasma amine oxidase have been demonstrated to lead to the same stereochemical result as pooled enzyme fractions. We have been able to rule out carbon interchange and tritium transfer in the ethylamine side chain of dopamine as the source of the apparent nonstereospecificity. Estimated primary tritium isotope effects are 1 for [2-3H]dopamines and 5--6 and 26--34 for [1R-3H]- and [1S-3H]dopamine, respectively. We propose the presence of alternate dopamine binding modes, characterized by absolute but opposing stereochemistries and differential primary tritium isotope effects at C-1.     

Hydroxylation of testosterone and 5alpha-dihydrotestosterone--isolation of 2alpha-hydroxytestosterone (author's transl)]

The hydroxylation at C-2 of testosterone and 5alpha-dihydrotestosterone is measured by isolation of HTO after incubating "metabolically labile" tritium labeled steroids with the enzymes. After hydroxylation of testosterone in rat liver 2alpha-hydroxytestosterone was isolated and identified by gaschromatography and mass spectra.     

The metabolism of cycloartenol,lanosterol, 24-methylenecholesterol and fucosterol in Chlorella ellipsoidea

Lanosterol and cycloartenol labelled with tritium at C-2, and 24-methylenecholesterol and fucosterol labelled with tritium at C-2 and C-4 were fed to actively growing cultures of Chlorella ellipsoidea. Lanosterol and cycloartenol were converted to each of the five desmethyl sterols of C. ellipsoidea. Lanosterol was more efficiently incorporated than cycloartenol.Although there was some evidence for the reduction of the 24-methylene group, it was apparent that 24-methylene-cholesterol was converted primarily to the C₂₉ sterols, clionasterol and poriferasterol. Labelled fucosterol was reduced at the 24(28) double bond, producing clionasterol.     

Synthesis of 3H and 13C labeled mRNA cap dinucleotides--useful tools for NMR, biochemical, and biological studies

For deeper understanding the roles of the mRNA cap structure in cellular processes isotopically labeled dinucleotide cap analogues have been synthesized as tools for NMR and in vivo studies. Tritium or carbon C-13 labeled methyl iodide was used as a source of the isotope material. In order to minimize the number of steps during the radioisotopic synthesis the methylation with tritium labeled methyl iodide was performed with Gp(3)G as a substrate. The C-13 isotope was introduced into the cap dinucleotide by methylation of GDP with C-13 Methyl iodide, followed by coupling the product with guanosine 5'-phosphorimidazolide in DMF with zinc chloride as a catalyst.     

Fragments formed by the side chain cleavage of a 20-aryl analog of 20alpha-hydroxycholesterol by adrenal mitochondria.

An analog of 20alpha-hydroxycholesterol, (20R)-20-phenyl-5-pregnene-3beta,20-diol, which is completely substituted at C-22 was prepared with radioisotopes at various positions. The analog labeled with 3H at C-M and 14C at C-4 and C-IU was converted into radioactive pregnenolone by an enzyme preparation derived from adrenal mitochondria. Cleavage of the phenyl analog labeled with 3H in the aromatic ring by the same enzyme preparation led to the formation of [3H]phenol. Using the substrate doubly labeled with 14C at C-4 and 3H in the aromatic ring, it appeared that the products of the reactions, pregnenolone and phenol, were formed in equal amounts. During incubation of the side chain labeled substrate, another labeled fragment was formed. It was identified as acetophenone, a product resulting from cleavage of the C17,20 bond. The steroidal fragment corresponding to this C8 ketone was traced using nuclear label analog. From its nonpolar chromatographic properties it appears to be a C-17-deoxy-C19 steroid.     

Trichodiene biosynthesis and the stereochemistry of the enzymatic cyclization of farnesyl pyrophosphate

Cyclization of trans,trans-[1-³H₂,12,13-¹⁴C]farnesyl pyrophosphate (2a) by a preparation of trichodiene synthetase isolated from the fungus, Trichothecium roseum, gave trichodiene (5a), which was shown by chemical degradation to retain both tritium atoms of the precursor at C-11. Incubation of 1S-[1-³H,12,13-¹⁴C]farnesyl pyrophosphate (2b) and 1R-[1-³H,12,13-¹⁴C]farnesyl pyrophosphate (2c) with trichodiene synthetase and degradation of the resulting labeled trichodienes, 5b and 5c, established that the displacement of the pyrophosphate moiety from C-1 of the precursor and formation of the new C-C bond in the formation of trichodiene takes place with net retention of configuration. These results are accounted for by an isomerization-cyclization mechanism involving the intermediacy of nerolidyl pyrophosphate (4).     

Synthesis of [3H]- and -[14C]-labelled sulprostone (CP-34,089/ZK-57,671)

The synthesis of N-methanesulfonyl 16-phenoxy-ω-tetranor PGE₂ carboxamide (sulprostone — CP-34,089/ZK-57,671) labeled with tritium and carbon-14 is described. Sulprostone labeled with tritium in the phenoxy moiety by means of catalytic hydrogenolysis was obtained in a 17% radiochemical yield with a specific activity of 1.0 Ci/mmol. The methanesulfonamide-¹⁴C derivative of sulprostone was prepared from methyl-¹⁴C iodide in an 11.8% radiochemical yield having a specific activity of 18.8 mCi/mmol.     

The synthesis of [3H]gibberellin A3 and [3H]gibberellin A1 by the palladium-catalyzed actions of carrier-free tritium on gibberellin A3

Reaction of gibberellin A₃ (GA₃) with carrier-free tritium gas and 5% palladium on calcium carbonate as catalyst gave a complex mixture of products, several of which were isolated and identified. Three of the purified products are the radioactive forms of naturally occurring gibberellins: [³H]GA₃ (1), [³H]GA₁ (2) and [³H]tetrahydro GA₃ (4). Another substance was isolated and tentatively identified as [³H]16,17-dihydro GA₃ (3). GLC was used to determine the specific activities of 1 and 2. [³H]GA₃ likely arises from palladium catalysed nonspecific exchange of GA₃ alkane hydrogen atoms with tritium. [³H]GA₁ is also exchange labeled but most of its radioactivity is due to tritium addition to the C-1,2 olefinic bond of GA₃.     

Trichothecene Biosynthesis in Fusarium sporotrichioides: Origin of the Oxygen Atoms of T-2 Toxin

Mass spectral analysis of T-2 toxin formed during the growth of Fusarium sporotrichioides (ATCC 24043) in the presence of H₂¹⁸O showed incorporation of up to three ¹⁸O atoms per toxin molecule. The carbonyl oxygens of the acetates at C-4 and C-15 and of the isovalerate at C-8 were derived from H₂O. Toxin formed in the presence of ¹⁸O molecular oxygen incorporated up to six ¹⁸O atoms per toxin molecule. The overall incorporation was 78 and 92% of toxin molecules labeled for H₂¹⁸O and ¹⁸O₂ labeled samples, respectively. The oxygens of position 1, the 12,13-epoxide, and the hydroxyl groups at C-3, C-4, C-8, and C-15 were all derived from molecular oxygen.     

Synthesis of [H]- and -[C]-labelled sulprostone (CP-34,089/ZK-57,671)

The synthesis of N-methanesulfonyl 16-phenoxy-ω-tetranor PGE₂ carboxamide (sulprostone — CP-34,089/ZK-57,671) labeled with tritium and carbon-14 is described. Sulprostone labeled with tritium in the phenoxy moiety by means of catalytic hydrogenolysis was obtained in a 17% radiochemical yield with a specific activity of 1.0 Ci/mmol. The methanesulfonamide-¹⁴C derivative of sulprostone was prepared from methyl-¹⁴C iodide in an 11.8% radiochemical yield having a specific activity of 18.8 mCi/mmol.     

Stereochemistry of C-4 demethylation during conversion of obtusifoliol into poriferasterol by Ochromonas malhamensis

Obtusifoliol-[2,2,4-³H₃] was synthesised and incubated with the chrysophyte alga Ochromonas malhamensis which converted it into poriferasterol. A reaction sequence applied to poriferasterol showed that the tritium retained at C-4 occupied the 4α-position. This demonstrates that biological C-4 demethylation of a 4α-methylsterol precursor by O. malhamensis results in the axial 4β-hydrogen being inverted into the equatorial 4α-position of the 4-desmethyl sterol product.     

Precursors of ricinine in the castor bean plant

Isotopic tracer experiments confirmed that glycerol and succinic acid are good precursors of the pyridine ring of ricinine in castor bean plants. Tritium from C-2 was lost from tritiated glycerol while tritium from C-1 was retained. Thus a derivative of dihydroxyacetone is likely to be intermediate. By simultaneous feeding of glycerol-1-(3)-[³H] and succinic acid-2(3)-[¹⁴C], it was hoped to find precursors of ricinine containing both labels, but none could be found. There was no evidence for the appearance of labeled quinolinic acid, which is presumed to be a precursor of ricinine.     

ANALYSIS OF TRITIUM INCORPORATION INTO INDIVIDUAL CELLS BY AUTORADIOGRAPHY OF SQUASH PREPARATIONS

The relation between tritium content of individual cells and grain count obtained in autoradiographs of squashed cells was investigated. The tissues used were root meristems of Tradescantia paludosa and intestinal epithelium of the mouse. The relation between grain count and tritium content is affected by self-absorption which depends on the thickness of the labeled cell. Therefore, squashed preparations were sectioned to determine the uniformity of thickness of nuclei. In a preparation of mouse cells, thicknesses were 1.18 ± 0.35 µ, and in a preparation of Tradescantia cells, 2.97 ± 0.35 µ. The effects of similar and larger variations in thickness upon grain count were studied in material squashed with different pressures; no marked correlation was found. The lack of correlation is explained by the geometric relation between labeled nuclei and the emulsion. By counting grains and directly measuring tritium content in a glass proportional counting tube in the same preparation, the yield of grains per disintegration was measured in Tradescantia cells and found to be 1 grain for 10.9 disintegrations with AR 10 autoradiographic film and 1 grain for 19.3 disintegrations for NTB nuclear track liquid emulsion. Latent image fading may pose a problem with long exposures; the conditions of its occurrence are as yet not well known.