Synthesis and biological activities of 2-Pyrimidinone Nucleosides. III.1,2 5-Alkynyl-2-Pyrimidinone 2′-Deoxyribosides

Abstract

The syntheses of 5-ethynyl-, 5-phenylethynyl- and 5-propynyl-2(1H)-pyrimidinone 2′-deoxyribosides are described. Two of the new nucleosides, 14 a and 14 c, showed significant antiviral activities against HSV-1 and HSV-2 in tissue culture.     

Biosynthesis of riboflavin. Single turnover kinetic analysis of GTP cyclohydrolase II.

GTP cyclohydrolase II catalyzes the conversion of GTP into a mixture of 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate (Compound 2), formate, and pyrophosphate. Moreover, GMP was recently shown to be formed as a minor product. The major product (Compound 2) serves as the first committed intermediate in the biosynthesis of the vitamin, riboflavin. Numerous pathogenic microorganisms are absolutely dependent on endogenous synthesis of riboflavin. The enzymes of this pathway are therefore potential drug targets, and mechanistic studies appear relevant for development of bactericidal inhibitors. Pre-steady state quenched flow analysis of GTP cyclohydrolase II shows the rate-determining step to be located at the beginning of the reaction sequence catalyzed by the enzyme. Thus, GTP is consumed at a rate constant of 0.064 s(-1), and the reaction product, Compound 2, is formed at an apparent rate constant of 0.062 s(-1). Stopped flow experiments monitored by multiwavelength photometry are well in line with these data. 2-Amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone triphosphate can serve as substrate for GTP cyclohydrolase II but does not fulfill the criteria for a kinetically competent intermediate. A hypothetical reaction mechanism involves the slow formation of a phosphoguanosyl derivative of the enzyme under release of pyrophosphate. The covalently bound phosphoguanosyl moiety is proposed to undergo rapid hydrolytic release of formate from the imidazole ring and/or hydrolytic cleavage of the phosphodiester bond.     

Location of polar substituents and fatty acyl chains on lipid A precursors from a 3-deoxy-D-manno-octulosonic acid-deficient mutant of Salmonella typhimurium. Studies by 1H, 13C, and 31P nuclear magnetic resonance

Eight anionic disaccharide precursors of lipid A accumulate at 42 degrees C in 3-deoxy-D-manno-octulosonic acid-deficient temperature-sensitive mutants of Salmonella typhimurium. These compounds comprise a series of lipids based on the minimal structure, O-[2-amino-2-deoxy-N2,O3-bis(3-hydroxytetradecanoyl)-beta-D-glucopyranos yl] -(1----6)-2-amino-2-deoxy-N2, O3-bis(3-hydroxytetradecanoyl)-alpha-D-glucopyranose 1,4'- bisphosphate (designated lipid IVA) that differ from each other by the presence of an additional phosphoethanolamine moiety (IIIA), or an aminodeoxypentose moiety (IIA), or both (IA). A homologous set of metabolites is further derivatized with a palmitoyl function; these are designated IVB, IIIB, IIB, and IB (Raetz, C. R. H., Purcell, S., Meyer, M. V., Qureshi, N., and Takayama, K. (1985) J. Biol. Chem. 260, 16080-16088). The attachment of the palmitoyl moiety, known to be on the reducing terminal GlcN residue by mass spectrometry, was determined to be O-beta of the N2-linked beta-hydroxymyristoyl group of that residue of IVB by 13C NMR and two-dimensional 1H chemical shift correlation spectroscopy experiments. 31P NMR indicated the presence of diphosphodiester moieties in IIIA, IIIB, and IA and monophosphodiester moieties in IIA and IA. Selective 1H decoupling of the 31P spectrum of IIIA demonstrated that the O-diphosphoethanolamine moiety is attached to the O4' position in IIIA. On the basis of the observed 31P chemical shifts it was concluded that the aminodeoxypentose is located at position 1 in IIA and IA, while diphosphoethanolamine is most likely located at O-4' in IA and IIIB, as in IIIA.     

Synthesis of 5-chloroformycin A, 5-chloro-2''-deoxyformycin A and certain related 5,7-disubstituted 3-beta-D-ribofuranosylpyrazolo[4,3-d] pyrimidines from formycin A.

A facile synthesis of 7-amino-5-chloro-3-beta-D-ribofuranosylpyrazolo [4,3-d]pyrimidine (5-chloroformycin A, 6), 7-amino-5-chloro-3-(2-deoxy-beta-D-erythro-pentofuranosyl) pyrazolo [4,3-d]-pyrimidine (5-chloro-2'-deoxyformycin A, 13) and certain related 5,7-disubstituted pyrazolo[4,3-d]pyrimidine ribonucleosides is described starting with formycin A. Thiation of tri-O-acetyloxoformycin B (4b) with phosphorus pentasulfide, followed 3-beta-D-ribofuranosyl-7-thioxopyrazolo[4,3-d] pyrimidin-5(1H,4H,6H)-one (3b) in excellent yield. Chlorination of 4b with either phosphorus oxychloride or phenyl phosphonicdichloride furnished the key intermediate 5,7-dichloro-3-(2,3, 5-tri-O-acetyl-beta-D-ribofuranosyl)pyrazolo[4,3-d]pyrimidine (5a), which on deacetylation afforded 5,7-dichloro-3-beta-D-ribofuranosylpyrazolo [4,3-d]pyrimidine (5b). Ammonolysis of 5a with liquid ammonia gave 6, whereas with MeOH/NH3, a mixture of 6 and 7-methoxy-5-chloro-3-beta-D-ribofuranosylpyrazolo[4,3-d]pyrimidine (7) was obtained. Reaction of 6 with lithium azide and subsequent hydrogenation afforded 5-aminoformycin A (10). Treatment of 5a with thiourea gave 5-chloro-3-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl) pyrazolo[4,3-d]pyrimidine-7(1H,6H)-thione (8a), which on further reaction with sodium hydrosulfide furnished 3-beta-D-ribofuranosylpyrazolo [4,3-d]pyrimidine-5,7(1H,4H,6H)-dithione (11). The four-step deoxygenation procedure using phenoxythiocarbonylation of the 2'-hydroxy group of the 3', 5'-protected 6 gave 5-chloro-2'-deoxyformycin A (13).     

Arylglycerol-gamma-Formyl Ester as an Aromatic Ring Cleavage Product of Nonphenolic beta-O-4 Lignin Substructure Model Compounds Degraded by Coriolus versicolor

4-Ethoxy-3-methoxyphenylglycerol-gamma-formyl ester (compound IV) was identified as a degradation product of both 4-ethoxy-3-methoxyphenylglycerol-beta-syringaldehyde ether (compound I) and 4-ethoxy-3-methoxyphenylglycerol-beta-2,6-dimethoxyphenyl ether (compound II) by a ligninolytic culture of Coriolus versicolor. An isotopic experiment with a C-labeled compound (compound II') indicated that the formyl group of compound IV was derived from the beta-phenoxyl group of beta-O-4 dimer as an aromatic ring cleavage fragment. However, compound IV was not formed from 4-ethoxy-3-methoxyphenylglycerol-beta-guaiacyl ether (compound III). gamma-Formyl arylglycerol (compound IV) could be a precursor of 4-ethoxy-3-methoxyphenylglycerol (compound VI), because 3-(4-ethoxy-3-methoxyphenyl)-1-formyloxy propane (compound VII) was cleaved to give 3-(4-ethoxy-3-methoxyphenyl)-1-propanol (compound VIII) by C. versicolor. 4-Ethoxy-3-methoxyphenylglycerol-beta,gamma-cyclic carbonate (compound V), previously found as a degradation product of compound III by Phanerochaete chrysosporium (T. Umezawa, and T. Higuchi, FEBS Lett., 25:123-126, 1985), was also identified from the cultures with compound I, II, and III and degraded to give the arylglycerol (compound VI). An isotopic experiment with C-labeled compounds II' and III' indicated that the carbonate carbon of compound V was derived from the beta-phenoxyl groups of beta-O-4 substructure.     

Synthesis of phosphono-pyrimidine and -purine ribonucleosides

The reaction of the lithiated species of 2', 3', 5'-tri-O-protected uridines and 6-chloropurine ribonucleoside with diethyl chlorophosphate provided the corresponding diethyl phosphonate derivatives (III, IV and VIII). The Arbuzov-reaction of 2', 3', 5'-tri-O-protected 4-chloro-2(1H)-pyrimidinone ribonucleoside with triethyl phosphite afforded the diethyl phosphonate derivative (VI).     

2,5-diamino-6-ribitylamino-4(3H)-pyrimidinone 5'-phosphate synthases of fungi and archaea

The pathway of riboflavin (vitamin B2) biosynthesis is significantly different in archaea, eubacteria, fungi and plants. Specifically, the first committed intermediate, 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate, can either undergo hydrolytic cleavage of the position 2 amino group by a deaminase (in plants and most eubacteria) or reduction of the ribose side chain by a reductase (in fungi and archaea). We compare 2,5-diamino-6-ribitylamino-4(3H)-pyrimidinone 5'-phosphate synthases from the yeast Candida glabrata, the archaeaon Methanocaldococcus jannaschii and the eubacterium Aquifex aeolicus. All three enzymes convert 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate into 2,5-diamino-6-ribitylamino-4(3H)-pyrimidinone 5'-phosphate, as shown by 13C-NMR spectroscopy using [2,1',2',3',4',5'-13C6]2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate as substrate. The beta anomer was found to be the authentic substrate, and the alpha anomer could serve as substrate subsequent to spontaneous anomerisation. The M. jannaschii and C. glabrata enzymes were shown to be A-type reductases catalysing the transfer of deuterium from the 4(R) position of NADPH to the 1' (S) position of the substrate. These results are in agreement with the known three-dimensional structure of the M. jannaschii enzyme.     

Synthesis, properties and reactions of 3 beta-benzoyloxy-7 alpha-15 beta-dichloro-5 alpha-cholest-8(14)-ene.

Treatment of 3 beta-benzoyloxy-14 alpha,15 alpha-epoxy-5 alpha-cholest-7-ene (I) with gaseous HCl in chloroform at -40 degrees C gave, in 87% yield, 3 beta-benzoyloxy-7 alpha,15 beta-dichloro-5 alpha cholest-8(14)-ene (III). Reduction of the latter compound with lithium aluminum hydride in ether at room temperature for 20 min gave, in 86% yield, 7 alpha-15 beta-dichloro-5 alpha-cholest-8(14)-en-3 beta-ol (IV). The latter compound was fully characterized and assignments of the individual carbon peaks in the 13C nuclear magnetic resonance spectra of this sterol have been completed. Reduction of III with excess lithium aluminum hydride in refluxing ether for 4 days gave, in 74% yield, 5 alpha-cholesta-7,14-dien-3 beta-ol (VI). Reduction of the dichloro-steryl benzoate III with lithium triethylborohydride in tetrahydrofuran gave, in 88% yield, 5 alpha-cholest-8(14)-en-3 beta-ol (VII). A similar reduction using lithium triethylborodeuteride led to the formation of [7 beta, 15 xi-2 H2]-VIIa. Treatment of III with concentrated HCl in a mixture of chloroform and methanol gave, in 79% yield, 3 beta-benzoyloxy-5 alpha-cholest-8(14)-en-15-one (II) which was characterized as such and as the corresponding free sterol.     

Separation and characterization of six (1 leads to 3)-beta-glucanases from Saccharomyces cerevisiae.

Using a system of chromatography through columns of DEAE-Bio-Gel, HTP-Bio-Gel, and CM-Bio-Gel, we isolated and characterized six different (1 leads to 3)-beta-glucanases from cell wall autolysates and cell extracts of Saccharomyces cerevisiae haploid strain 2180B. These enzymes were designated glucanases I, II, IIIA, IIIB, IV, and V. The haploid mating type S. cerevisiae strain 2180A and the diploid strains S. cerevisiae 2180D and S. cerevisiae 595 contained the same complex of glucanases. Glucanases II and IIIA were exoenzymes, and glucanases I, IIIB, IV, and V were endoenzymes. The enzymes exhibited different molecular weights, kinetic properties, and activities on isolated yeast cell walls. The products of substrate (laminarin) hydrolysis were quantified by using high-pressure liquid chromatography and were significantly different for the four endoglucanases.     

Inhibitors of sterol synthesis. A highly efficient and specific side-chain oxidation of 3 beta-acetoxy-5 alpha-cholest-8(14)-en-15-one for construction of metabolites and analogs of the 15-ketosterol.

As part of a program directed towards the chemical syntheses of potential metabolites and analogs of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (I), a potent regulator of cholesterol metabolism, several routes have been explored for the preparation of 3 beta-hydroxy-15-keto-5 alpha-chol-8(14)-en-24-oic acid (IV). These investigations led to a remarkably specific and efficient side-chain oxidation of I. For example, treatment of the acetate of I with a mixture of trifluoroacetic anhydride, hydrogen peroxide, and sulfuric acid for 3.5 h at -2 degrees C gave a crude product consisting of 3 beta-acetoxy-24-trifluoroacetoxy-5 alpha-chol-8(14)-en-15-one (XI), 3 beta-acetoxy-24-hydroxy-5 alpha-chol-8(14)-en-15-one (XII), and 3 beta, 24-diacetoxy-5 alpha-chol-8(14)-en-15-one (XIII) in yields of 58%, 8%, and 3%, respectively, by HPLC analysis. XI was readily hydrolyzed to XII upon treatment with triethylamine in methanol at room temperature. Oxidation of XII with Jones reagent gave 3 beta-acetoxy-15-keto-5 alpha-chol-8(14)-en-24-oic acid (XVIII) from which its methyl ester (IX) was prepared by treatment with diazomethane. Mild alkaline hydrolysis of XVIII gave the 3 beta-hydroxy-delta 8(14)-15-keto C24 acid (IV). Hydrolysis of the crude product of the side-chain oxidation with K2CO3 in methanol gave 3 beta,24-dihydroxy-5 alpha-chol-8(14)-en-15-one (XIV) which was oxidized with Jones reagent to yield 3,15-diketo-5 alpha-chol-8(14)-en-24-oic acid (XV). Treatment of XV with diazomethane gave its methyl ester (XVI) which, upon controlled reduction with NaBH4, yielded methyl 3 beta-hydroxy-15-keto-5 alpha-chol-8(14)-en-24-oate (XVII). Compound IX was also prepared by an independent route. Full 1H and 13C NMR assignments are presented for 12 new compounds. IV caused a approximately 56% reduction of the level of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells at a concentration of 2.5 microM. In contrast, the corresponding 3,15-diketo acid XV had no detectable effect on reductase activity under the same conditions.     

Arylglycerol-γ-Formyl Ester as an Aromatic Ring Cleavage Product of Nonphenolic β-O-4 Lignin Substructure Model Compounds Degraded by Coriolus versicolor

4-Ethoxy-3-methoxyphenylglycerol-γ-formyl ester (compound IV) was identified as a degradation product of both 4-ethoxy-3-methoxyphenylglycerol-β-syringaldehyde ether (compound I) and 4-ethoxy-3-methoxyphenylglycerol-β-2,6-dimethoxyphenyl ether (compound II) by a ligninolytic culture of Coriolus versicolor. An isotopic experiment with a ¹³C-labeled compound (compound II′) indicated that the formyl group of compound IV was derived from the β-phenoxyl group of β-O-4 dimer as an aromatic ring cleavage fragment. However, compound IV was not formed from 4-ethoxy-3-methoxyphenylglycerol-β-guaiacyl ether (compound III). γ-Formyl arylglycerol (compound IV) could be a precursor of 4-ethoxy-3-methoxyphenylglycerol (compound VI), because 3-(4-ethoxy-3-methoxyphenyl)-1-formyloxy propane (compound VII) was cleaved to give 3-(4-ethoxy-3-methoxyphenyl)-1-propanol (compound VIII) by C. versicolor. 4-Ethoxy-3-methoxyphenylglycerol-β,γ-cyclic carbonate (compound V), previously found as a degradation product of compound III by Phanerochaete chrysosporium (T. Umezawa, and T. Higuchi, FEBS Lett., 25:123-126, 1985), was also identified from the cultures with compound I, II, and III and degraded to give the arylglycerol (compound VI). An isotopic experiment with ¹³C-labeled compounds II′ and III′ indicated that the carbonate carbon of compound V was derived from the β-phenoxyl groups of β-O-4 substructure.     

The synthesis of some branched-chain-sugar nucleoside analogues.

1-(2,3-Epoxy-5-O-trityl-beta-D-lyxofuranosyl)uracil was treated with a number of carbon nucleophiles. Ethynyl lithium gave 3'-deoxy-3'-ethynyl-5'-O-trityl-ara-uridine, which was reduced to the corresponding 3'-ethenyl compound. Sodium cyanide gave 3'-cyano-3'-deoxy-5'-O-trityl-ara-uridine which upon alkaline hydrolysis gave the corresponding 3'-carboxamido compound. 1,3-Dithian-2-yl lithium gave 3'-deoxy-3'-(1,3-dithian-2-yl)-5'-O-trityl-ara-uridine. The trityl group was removed from each of these compounds by mild acidic hydrolysis. Treatment of 2 with 0.1M H2sO4 and mercury (II) acetate afforded 3'-acetyl-3'-deoxy-ara-uridine which upon reduction with NaBH4 gave 3'-deoxy-3'-(1-hydroxyethan-1-yl)-ara-uridine. Acetylation of 6 yielded 5'-O-acetyl-3'-acetyl-2',3'-didehydro-2',3'-dideoxyuridine which upon reduction with NaBH4 produced a mixture of 5'-O-acetyl-2',3'-didehydro-2',3'-dideoxy-3'-(1-hydroxyethan -1-yl)uridine and 1-(R)[5-(S)-acetoxymethyl-4-(1-hydroxyethan-1-yl)-tetrahydrofuran- 2-yl]- uracil. Reduction of 14 with Raney nickel followed by removal of the trityl group gave 3'-deoxy-3'-methyl-ara-uridine.     

Variation of (1 leads to 3)-beta-glucanases in Saccharomyces cerevisiae during vegetative growth, conjugation, and sporulation.

The total (1 leads to 3)-beta-glucanase activities associated with cell extracts and cell walls of Saccharomyces cerevisiae were measured during vegetative growth, conjugation, and sporulation. Using a system of column chromatography, we resolved (1 leads to 3)-beta-glucanase activity into six different enzymes (namely, glucanases I, II, IIIA, IIIB, IV, and V). The contributions of the individual enzymes to the total activity at the different stages of the life cycle were determined. Total glucanase activity increased during exponential growth and decreased in stationary resting-phase cells. Glucanase IIIA was the predominant enzyme in stationary resting-phase cells. Glucanases I, II, IIIB, and IV were either absent or present at low levels in stationary phase cells, but their individual activities (in particular, glucanase IIIB activity) increased substantially during exponential growth. Total (1 leads to 3)-beta-glucanase activity did not change significantly during conjugation of two haploid mating strains, S. cerevisiae 2180A and 2180B, and no notable changes were detected in the activities of the individual enzymes. Sporulation was accompanied by a rapid increase and then a decrease in total glucanase activity. Most of the increase was due to a dramatic rise in the activity of glucanase V, which appeared to be a sporulation-specific enzyme. Glucanase activity was not derepressed by lowering the glucose concentration in the growth medium.     

Studies on bovine adrenal estrogen sulfotransferase. III. Facile synthesis of 3'-phospho- and 2'-phosphoadenosine 5'-phosphosulfate.

A practical synthesis of 3'-phosphoadenosine 5'-phosphosulfate (IV) in yields of 68-72% from adenosine 2',3'-cyclic phosphate 5'-phosphate (II) is described. Reaction of II with triethylamine-N-sulfonic acid affords adenosine 2',3'-cyclic phosphate 5'-phosphosulfate (III) which, on treatment with ribonuclease-T2, provides IV. Spleen phosphodiesterase, on the other hand, converts III to 2'-phosphoadenosine 5'-phosphosulfate (V). The biological activity of IV, measured by sulfate transfer to [6,7-3H2]estrone as mediated by bovine adrenal estrone sulfotransferase (3'-phosphoadenylyl-sulfate:estrone 3-sulfotransferase, EC 2.8.2.4), is identical with that obtained with a sample of IV prepared by an established biochemical procedure. By contrast, V exhibits approximately one-third the activity of the natural isomer.     

Preparation and characterization of NADP derivatives alkylated at 2'-phosphate and 6-amino groups

Reaction of NADP with 3-propiolactone at pH 6 gave new NADP derivatives carboxyethylated at the 2'-phosphate or 6-amino group, or both: 2'-O-(2-carboxyethyl)phosphono-NAD (I), N6-(2-carboxyethyl)-NADP (II), and 2'-O-(2-carboxyethyl)phosphono-N6-(2-carboxyethyl)-NAD (III). Their structures were assigned on the basis of ultraviolet, 1H-NMR and 31P-NMR spectra, and also treatment with nucleotide pyrophosphatase or alkaline phosphatase. Carbodiimide-promoted reaction of derivative I with 1,2-diaminoethane gave 2'-O-[N-(2-aminoethyl)carbamoylethyl]phosphono-NAD (IV); derivative III gave 2'-O-[N-(2-aminoethyl)carbamoylethyl]phosphono-N6-[N-(2-aminoethyl ) carbamoylethyl]-NAD (IV). The same reaction of derivative II, on the other hand, gave a mixture of N6-[N-(2-aminoethyl)carbamoylethyl]-NADP (Va) and its 3'-phosphate isomer (Vb). The mixture was converted to Va via the 2',3'-cyclic derivative (Vc). Their structures were assigned on the basis of ultraviolet and 1H-NMR spectra, and also treatment with alkaline phosphatase or 3'-nucleotidase. All the NADP derivatives obtained in this work could be reduced with yeast glucose-6-phosphate dehydrogenase.     

Ring opening is not rate-limiting in the GTP cyclohydrolase I reaction

GTP cyclohydrolase I catalyzes a mechanistically complex ring expansion affording dihydroneopterin triphosphate and formate from GTP. Single turnover quenched flow experiments were performed with the recombinant enzyme from Escherichia coli. The consumption of GTP and the formation of 5-formylamino-6-ribosylamino-2-amino-4(3H)-pyrimidinone triphosphate, formate, and dihydroneopterin triphosphate were determined by high pressure liquid chromatography analysis. A kinetic model comprising three consecutive unimolecular steps was used for interpretations where the first intermediate, 5-formylamino-6-ribosylamino-2-amino-4(3H)-pyrimidinone 5'-triphosphate, was formed in a reversible reaction. The rate constant k(1) for the reversible opening of the imidazole ring of GTP was 0.9 s(-1), the rate constant k(3) for the release of formate from 5-formylamino-6-ribosylamino-2-amino-4(3H)-pyrimidinone triphosphate was 2.0 s(-1), and the rate constant k(4) for the formation of dihydroneopterin triphosphate was 0.03 s(-1). Thus, the hydrolytic opening of the imidazole ring of GTP is rapid by comparison with the overall reaction.     

Inhibitors of sterol synthesis. Chemical synthesis and spectral properties of 3 beta-hydroxy-5 alpha-cholesta-8(14),24-dien-15-one, 3 beta,25-dihydroxy-5 alpha-cholest-8(14)-en-15-one, and 3 beta,24-dihydroxy-5 alpha-cholest-8(14)-en-15-one and their effects on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells.

Side-chain functionalized delta 8(14)-15-ketosterols have been synthesized from 3 beta-acetoxy-24-hydroxy-5 alpha-chol-8(14)-en-15-one (VI) as part of a program to prepare potential metabolites and analogs of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (I), a potent regulator of cholesterol metabolism. Oxidation of VI to the 24-aldehyde VII, followed by Wittig olefination with isopropyltriphenylphosphonium iodide gave 3 beta-acetoxy-5 alpha-cholesta-8(14),24-dien-15-one (VIII), which was hydrolyzed to the free sterol IX. Oxymercuration of VIII followed by hydrolysis of the 3 beta-acetate gave 3 beta,25-dihydroxy-5 alpha-cholest-8(14)-en-15-one (IV). Hydroboration-oxidation of VIII followed by hydrolysis of the 3 beta-acetate gave 3 beta,24-dihydroxy-5 alpha-cholest-8(14)-en-15-one (V) as a 5:4 mixture of the 24R and 24S epimers. 1H and 13C nuclear magnetic resonance (NMR) assignments and mass spectral fragmentation patterns, supported by high-resolution measurements, are presented for IV and its 3 beta-acetate, V, VII, VIII, and IX. Characterization of IV by NMR and of trimethylsilyl ethers of IV and V by gas chromatography-mass spectrometry was compatible with spectral data for samples of IV and V isolated previously after incubation of I with rat liver mitochondria in the presence of NADPH. Sterols IV, V, and IX were very potent in lowering of the level of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in Chinese hamster ovary cells; their potency was comparable to that of I.     

The use of iodotrimethylsilane in nucleosidation procedure.

1-O-Acetyl-2,3,5-tri-O-benzoyl-beta-D-ribofuranose (I) was reacted with iodotrimethylsilane (II) and the product, the glycosyl iodide, was coupled with silylated uracil to afford 1-(2,3,5-tri-O-benzoyl-beta-D-ribofuranosyl)uracil (III; 89%), with silyated cytosine to afford, on subsequent acetylation, 1-(2,3,5-tri-O-benzoyl-beta-0D-ribofuranosyl)-4-acetamido-2-(1H)-pyrimidinone (IVb; 81%), and with chloromercuri-N-benzoyl-adenine to afford Va and on subsequent debenzoylation, adenosine (Vb; 49%).     

Studies on the mechanism and regulation of C-4 demethylation in cholesterol biosynthesis. The role of adenosine 3′:5′-cyclic monophosphate

1. An assay for demethylation has been developed based on the release of tritium from 4,4-dimethyl[3alpha-(3)H]cholest-7-en-3beta-ol (II). 2. The maximum release of (3)H from 3alpha-(3)H-labelled compound (II) in a rat liver microsomal preparation occurs in the presence of NADPH and NAD(+) under aerobic conditions. 3. Incubation of 3alpha-(3)H-labelled compound (II) with NADPH under aerobic conditions leads to the formation of a 3alpha-(3)H-labelled C-4 carboxylic acid. This compound undergoes dehydrogenation on subsequent anaerobic incubation with NAD(+). 4. The (3)H released from the steroid was located in [4-(3)H]nicotinamide and the medium. Incubation with synthetic [4-(3)H(2)]NADH gave a similar result. 5. In the presence of glutamate dehydrogenase and alpha-oxoglutarate part of the (3)H released from the steroid was transferred to glutamate. 6. A series of 3-oxo steroids were reduced equally well by [4-(3)H(2)]NADH and [4-(3)H(2)]NADPH. The reduction of 5alpha-cholest-7-en-3-one was shown to use the 4B H atom from the nucleotide. 7. 3':5'-Cyclic AMP was shown to be a competitive inhibitor of the 3beta-hydroxy dehydrogenase enzyme in the demethylation reaction.     

Degradation mechanisms of phenolic beta-1 lignin substructure model compounds by laccase of Coriolus versicolor

Phenolic beta-1 lignin substructure model compounds, 1-(3,5-dimethoxy-4-hydroxy-phenyl)-2-(3,5-dimethoxy-4-ethoxyphenyl)propa ne-1, 3-diol (I) and 1-(3,5-dimethoxy-4-ethoxyphenyl)-2-(3, 5-dimethoxy-4-hydroxyphenyl)propane-1,3-diol (II) were degraded by laccase of Coriolus versicolor. Substrate I was converted to 1-(3,5-dimethoxy-4-hydroxyphenyl)-2-(3,5-dimethoxy-4-ethoxyphenyl)-3- hydroxypropanone (III), 1-(3,5-dimethoxy-4-ethoxyphenyl)-2-hydroxyethanone (IV), syringaldehyde (V), 1-(3,5-dimethoxy-4-ethoxyphenyl)-3-hydroxypropanal (VI), 2,6-dimethoxy-p-hydroquinone (VII), and 2,6-dimethoxy-p-benzoquinone (VIII). Furthermore, incorporations of 18O of 18O2 into ethanone (IV) and 18O of H218O into hydroquinone (VII) and benzoquinone (VIII) were confirmed. Substrate II gave 1-(3,5-dimethoxy-4-hydroxyphenyl)ethane-1, 2-diol (IX), 1-(3,5-dimethoxy-4-hydroxyphenyl)-2-hydroxyethanone (X), and 3,5-dimethoxy-4-ethoxybenzaldehyde (XI). Also 18O of H218O was incorporated into glycol (IX) and ethanone (X). Based on the structures of the degradation products and the isotopic experiments, it was established that three types of reactions occurred via phenoxy radicals of substrates caused by laccase: (i) C alpha-C beta cleavage (between C1 and C2 carbons); (ii) alkyl-aryl cleavage (between C1 carbon and aryl group); and (iii) C alpha (C1) oxidation.