Analogs of 1-phosphonooxy-2,2-dihydroxy-3-oxo-5-(methylthio)pentane, an acyclic intermediate in the methionine salvage pathway: a new preparation and characterization of activity with E1 enolase/phosphatase from Klebsiella oxytoca

The methionine salvage pathway allows the in vivo recovery of the methylthio moiety of methionine upon the formation of methylthioadenosine (MTA) from S-adenosylmethionine (SAM). The Fe(II)-containing form of acireductone dioxygenase (ARD) catalyzes the penultimate step in the pathway in Klebsiella oxytoca, the oxidative cleavage of the acireductone 1,2-dihydroxy-3-oxo-5-(methylthio)pent-1-ene (2) by dioxygen to give formate and 2-oxo-4-(methylthio)butyrate (3). The Ni(II)-bound form (Ni-ARD) catalyzes an off-pathway shunt, forming 3-(methylthio)propionate (4), carbon monoxide, and formate. Acireductone 2 is formed by the action of another enzyme, E1 enolase/phosphatase, on precursor 1-phosphonooxy-2,2-dihydroxy-3-oxo-5-methylthiopentane (1). Simple syntheses of several analogs of 1 are described, and their activity as substrates for E1 enolase/phosphatase characterized. A new bacterial overexpression system and purification procedure for E1, a member of the haloacid dehalogenase (HAD) superfamily, is described, and further characterization of the enzyme presented.     

A new synthesis of certain 7-(beta-D-ribofuranosyl) and 7-(2-deoxy-beta-D-ribofuranosyl) derivatives of 3-deazaguanine via the sodium salt glycosylation procedure.

A facile synthesis of 7-beta-D-ribofuranosyl-3-deazaguanine (1) and certain 8-substituted derivatives of 1 via the sodium salt glycosylation method has been developed. Glycosylation of the sodium salt of methyl 2-chloro(or methylthio)-4(5)-cyanomethylimidazole-5(4)-carboxylate (5 and 13b) with 2,3,5-tri-O-benzoyl-D-ribofuranosyl bromide (6) gave exclusively methyl 2-chloro(or methylthio)-4-cyanomethyl-1-(2,3, 5-tri-O-benzoyl-beta-D-ribofuranosyl)imidazole-5-carboxylate (7 and 14a), respectively. Ammonolysis of 7 and 14a provided 6-amino-2-chloro(or methylthio)-3-beta-D-ribofuranosylimidazo-[4,5-c]pyridin-4(5H)-one (11 and 17), which on subsequent dehalogenation (or dethiation) gave 1. Similarly, reaction of the sodium salt of 5 and 13b with 1-chloro-2-deoxy-3,5-di-O-p-toluoyl-alpha-D-erythro-pentofuranose (8), and ammonolysis of the glycosylated imidazole precursors (9 and 16) gave 6-amino-2-chloro(or methylthio)-3-(2-deoxy-beta-D-erythro-pentofuranosyl) imidazo[4,5-c]-pyridin-4(5H)-one (10a and 15), respectively. Dehalogenation of 10a or dethiation of 15 gave 2'-deoxy-7-beta-D-ribofuranosyl-3-deazaguanine (10b). This procedure provided a direct method of obtaining 10b without the contaminating 9-glycosyl isomer 4.     

Syntheses, crystal structures and antimicrobial activities of thioether ligands containing quinoline and pyridine terminal groups and their transition metal complexes

Eleven transition metal complexes of three asymmetrical tridentate thioether ligands, 8-((pyridin-2-yl)methylthio) quinoline (TQMP2), 8-((pyridin-3-yl)methylthio) quinoline (TQMP3), 8-((pyridin-4-yl)methylthio) quinoline (TQMP4) and one symmetrical pentadentate ligand 2,6-bis (8-quinolinylthiomethyl) pyridine (DTQMP) were prepared. The structures of all these complexes were identified by means of elemental analysis (EA), infrared spectra (IR) and single-crystal diffraction, providing three different kinds of basic conformations, (1) discrete mononuclear structures, (2) dinuclear rings and (3) 1D polymer chains. The antibacterial, antifungal and pesticide activities of the four ligands and 11 complexes were also studied.     

Microbial transformation of deoxyandrographolide and their inhibitory activity on LPS-induced NO production in RAW 264.7 macrophages

A series of analogues of deoxyandrographolide (1) transformed by Cunninghamella blakesleana AS 3.2004 were isolated and identified by spectral methods including 2D NMR. Among them, 3-oxo-17,19-dihydroxy-7,13-ent-labdadien-15,16-olide (9), 3-oxo-19-hydroxy-1,13-ent-labdadien-15,16-olide (16), 3-oxo-1β-hydroxy-14-deoxy-andrographolide (17) and 3-oxo-2β-hydroxy-14-deoxyandrographolide (18) are new compounds. And their structure-activity relationships (SAR) of inhibitory activity on LPS-induced NO production in RAW 264.7 macrophage cells were also discussed.     

Synthesis of double headed imidazoline acyclo C-nucleosides: 1,4-bis[1-amino-5-oxo-4-substituted(imidazolin-2-yl)]galacto-tetritols

Condensation of 2,3,4,5-tetra-O-acetyl-galactaroyl dichloride (1) with two equivalents of the alpha-amino esters 2a-c gave the corresponding 2,3,4,5-tetra-O-acetyl-galactaric acid diamides 3a-c. Heterocyclization of 3a-c by heating with hydrazine hydrate took place with concomitant de-O-acetylation of the polyacetoxyalkyl chain to give 1,4-bis[1-amino-5-oxo-4-substituted(imidazolin-2-yl)] galacto-tetritols (5a-c) and not the theoretically possible 1,2,4-triazinones 4 as indicated by spectral data. Compounds 5a-c readily reacted with p-nitrobenzaldehyde to give the corresponding p-nitrobenzylideneamino derivatives 6a-c. Acetylation of 5a-c afforded the 2,3,4,5-tetra-O-acetyl-1,4-bis[1-acetamido-5-oxo-4-substituted(imidazolin-2-yl)]galacto-tetritols (7a,b,d). De-O-acetylation of 7a,b,d gave 1,4-bis[1-acetamido-5-oxo-4-substituted (imidazolin-2-yl)]galacto-tetritols (8a-c).     

Two new 9,11-secosterols from the marine sponge Spongia officinalis. Synthesis of 9,11-seco-3 beta,6 alpha,11-trihydroxy-5 alpha-cholest-7-en-9-one.

Two new 9,11-secosterol, 9,11-seco-3 beta,6 alpha,11-trihydroxy-5 alpha-cholest-7-en-9-one (2) and 9,11-seco-3 beta,6 alpha,11-trihydroxy-24- methylene-5 alpha-cholest-7-en-9-one (3), have been isolated from the marine sponge Spongia officinalis and their structures elucidated by analysis of spectral data including 1H nuclear magnetic resonance correlation spectroscopy (COSY) experiments. Partial synthesis of 2 starting from 3 beta,6 alpha-dihydroxy-9-oxo-9,11-seco-5 alpha-cholest-7-en-11- al (1) confirmed the structure assignment.     

Studies on Nucleosides: Part XXVIII1. Synthesis of 4-Amino (or Hydroxy)-6-Methylthio-1-(3′-Deoxy-β- D-ribofuranosyl)-1-H-pyrazolo[3, 4-d]Pyrimidines

Abstract

4-Amino-6-methylthio-1-(3′-deoxy-β-D-ribofuranosyl)-1H-pyrazolo-[3, 4-d]pyrimidine (11) and 6-methylthio-4(5H)-oxo-1-(3′-deoxy-β-D-ribofuranosyl)-1H-pyrazolo[3, 4-d]pyrimidine (12) have been synthesized from 1, 2-di-O-acetyl-5-O-benzoyl-3-deoxyribofuranose (5) and 4, 6-bis (methylthio)-1H-pyrazolo-[3, 4-d]pyrimidine (6). in a convergent fashion. Structural proofs are based on MS, IR, ¹H NMR, ¹³C NMR and elemental analyses.     

Separation of urinary oestrogen sulphates from oestrogen glucosiduronates on diethylaminoethyl-Sephadex columns

1. Gradient elution (sodium chloride concentration gradient) on DEAE-Sephadex columns is used to separate urinary oestrogen conjugates of pregnancy urine. Changes in the shape of the gradient alter the chromatograms in a predictable manner so that the dispersion of peaks can be modified at will. 2. Suitable choice of the gradient results in the separation from each other of oestrogen sulphates, oestrogen 16(or 17)-glucosiduronates, oestrogen 3-glucosiduronates and free oestrogens. 3. Evidence for the presence of oestriol 3-sulphate, oestrone 3-sulphate, 17β-oestradiol 3-sulphate, 16-oxo-17β-oestradiol 3-sulphate and oestriol 16(or 17)-sulphate in the sulphate fraction of DEAE-Sephadex chromatograms of pregnancy urine is provided.     

Oleanane-type triterpenes of Embelia schimperi leaves

An investigation of an ethyl acetate extract of Embelia schimperi leaves has led to the isolation of 10 oleanane-type triterpenes characterized as 3beta,16alpha-di-O-acetyl-13beta, 28-epoxyoleanane (1), 3beta-acetyl-16-oxo-13beta, 28-epoxyoleanane (2), 3beta-acetyl-16alpha-hydroxy-13beta, 28-epoxyoleanane (3), 3beta-acetyl-16alpha-hydroxyoleanane-13beta, 28-olide (4), 3beta-acetyl-28-hydroxy-16-oxo-12-oleanene (5), 3beta, 28-di-O-acetyl-16alpha-hydroxy-12-oleanene (6), 3beta-acetyl-11alpha, 28-dihydroxy-16-oxo-12-oleanene (7), 3beta, 11alpha, 16alpha, 28-tetrahydroxy-12-oleanene (8), 3beta-acetyl-16alpha, 28alpha-dihydroxy-13beta, 28-oxydooleanane (9) and 3beta, 28alpha-dihydroxy-16-oxo-13beta, 28-oxydooleanane (10). The known compounds isolated from the same extract included 3beta, 16alpha-dihydroxy-13beta, 28-epoxyoleanane (protoprimulagenin A) (11), 3beta-hydroxy-16-oxo-13beta, 28-epoxyoxyoleanane (aegicerin) (12), 3, 16-dioxo-13beta, 28-epoxyoleanane (embilionone) (13), 3beta, 28-dihydroxy-16-oxo-12-oleanene (schimperinone) (14), taraxerone (15), taraxerol (16) and stigmasterol (17). Structure elucidations were carried out spectroscopically.     

Versatile nuclearity in copper complexes with ortho functionalized 1,3-bis(aryl)triazenido ligands

The synthesis, characterization and crystal structures of three new copper complexes derived from 1,3-bis(aryl)triazenido ligands bearing either a methoxycarbonyl, methylthio or a hydroxymethyl group in the ortho position of one of the aromatic rings are reported. In addition to the coordination of the triazenido fragment, the Lewis basic groups coordinate to the copper centers to form complexes with different nuclearity: {1-[2-(methoxycarbonyl)phenyl]-3-[4-methylphenyl]}triazene and {1-[2-(methylthio)phenyl]-3-[4-methylphenyl]}triazene form stable dinuclear and tetranuclear Cu(I) complexes, respectively. Reaction of {1-[2-(hydroxymethyl)phenyl]-3-[4-methylphenyl]}triazene with either Cu(I) or Cu(II) results in a novel Cu(II) hexanuclear macrocyclic complex.     

Structure elucidation of new compounds from acidic treatment of the progestins gestodene and drospirenone

Gestodene acidic treatment afforded a single rearrangement product, namely 13-beta-ethyl-18,19-dinorpregna-4,14,16-trien-3,20-dione 3, which was originated through HCl-catalyzed Rupe rearrangement. Drospirenone acidic treatment yielded two epimeric lactones by addition of HCl to the 6beta,7beta-cyclopropane ring, namely 7beta-(chloromethyl)-15beta,16beta-methylene-3-oxo-17beta-pregn-4-ene-21,17-carbolactone 4 and 7beta-(chloromethyl)-15beta,16beta-methylene-3-oxo-17alpha-pregn-4-ene-21,17-carbolactone 5. The structure of the compounds was assessed by spectroscopic and crystallographic methods.     

A short efficient synthesis of 16-oxygenated estratriene 3-sulfates

A novel synthesis of sodium 17-oxo-16 alpha-hydroxy-1,3,5(10)-estratrien-3-yl sulfate (4), sodium 16 alpha, 16 beta-dihydroxy-1,3,5(10)-estratrien-3-yl sulfate (5) and sodium 16-oxo-17 beta-hydroxy-1,3,5(10)-estratrien-3-yl sulfate (6) is described. 16 alpha-Bromo-3-hydroxy-1,3,5(10)-estratrien-17-one (1) was efficiently synthesized in one step with 70-97% yield by bromination of 3-hydroxy-1,3,5(10)-estratrien-17-one with cupric bromide. 3,16 alpha-Dihydroxy-1,3,5(10)-estratrien-17-one (3) was quantitatively obtained by controlled stereospecific hydrolysis of the bromoketone 1 with sodium hydroxide in aqueous pyridine. The bromoketone 1 was converted to the 16 alpha-hydroxy-17-ketone 3-sulfate 4 by sulfation with chlorosulfonic acid in pyridine and a subsequent controlled hydrolysis in a high yield without formation of the other ketols. Treatment of the sulfate 4 with sodium borohydride have the triol sulfate 5. The sulfate 4 was also rearranged to the 17 beta-hydroxy-16-ketone 6 with sodium hydroxide in water in a quantitative yield.     

Quantitative analysis of a model opioid peptide and its cyclic prodrugs in rat plasma using high-performance liquid chromatography with fluorescence and tandem mass spectrometric detection

An isocratic reversed-phase HPLC method for the simultaneous quantitation of alpha-lipoic acid and five of its metabolites in human plasma as well as in human urine employing solid-phase extraction and pulsed amperometric detection was developed and validated. The method was found to be sufficiently precise and accurate for the measurement of alpha-lipoic acid and its metabolites 6,8-bis(methylthio)octanoic acid, 4,6-bis(methylthio)hexanoic acid and 2,4-bis(methylthio)butanoic acid in plasma and urine samples, obtained from patients suffering from diabetic neuropathy as well as from healthy volunteers following daily oral administration of 600 mg alpha-lipoic acid. The quantitation of the metabolite bisnorlipoic acid was often impaired by interferences caused by an unidentified metabolite. However, bisnorlipoic acid was detected in few test samples and the concentrations were consistently low. Despite the poor recovery of the metabolite tetranorlipoic acid from plasma, reproducibility and accuracy were found to be from acceptable magnitude to assess concentration time courses. Thus, the obtained analytical results are considered as reliable and well suited for pharmacokinetic studies of alpha-lipoic acid and its metabolites.     

Synthesis of 2-bromomethyl-3-hydroxy-2-hydroxymethyl-propyl pyrimidine and theophylline nucleosides under microwave irradiation. Evaluation of their activity against hepatitis B virus

Alkylation of 2-methylthiopyrimidin-4(1H)-one (1a) and its 5(6)-alkyl derivatives 1b-d as well as theophylline (7) with 2,2-bis(bromomethyl)-1,3-diacetoxypropane (2) under microwave irradiation gave the corresponding acyclonucleosides 1-[(3-acetoxy-2-acetoxymethyl-2-bromomethyl)prop-1-yl]-2-methyl-thio pyrmidin-4(1H)-ones 3a-d and 7-[(3-acetoxy-2-acetoxymethyl-2-bromomethyl)prop-1-yl]theophylline (8), which upon further irradiation gave the double-headed acyclonucleosides 1,1 '-[(2,2-diacetoxymethyl)-1,3-propylidene]-bis[(2-(methylthio)-pyrimidin-4(1H)-ones] 4a-c, and 7,7 '-[(2,2-diacetoxymethyl)-1,3-propylidene]-bis(theophylline) (9). The deacetylated derivatives were obtained by the action of sodium methoxide. The activity of deacetylated nucleosides against Hepatitis B virus was evaluated. Compound 5b showed moderate inhibition activity against HBV with mild cytotoxicity.     

Discovery of 5-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-7-phenyl-(E)-2,3,6,7-tetrahydro-1,4-thiazepines as a new series of apoptosis inducers using a cell- and caspase-based HTS assay

We report the discovery of 5-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-7-(4-methylphenyl)-(E)-2,3,6,7-tetrahydro-1,4-thiazepine (2a) as an inducer of apoptosis using our proprietary cell- and caspase-based HTS assay. Through structure activity relationship (SAR) studies, 5-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-7-(2-methoxy-4-(methylthio)phenyl)-(E)-2,3,6,7-tetrahydro-1,4-thiazepine (5d) was identified as a potent apoptosis inducer with an EC(50) value of 0.08 microM in T47D cells, which was >15-fold more potent than screening hit 2a. Compound 5d also was found to be highly active in a growth inhibition assay with a GI(50) value of 0.05 microM in T47D cells and to function as an inhibitor of tubulin polymerization.     

Ametryne and Prometryne as Sulfur Sources for Bacteria

Bacteria were isolated that could utilize quantitatively the s-triazine herbicide prometryne [N,N′ -bis(1-methylethyl)-6-(methylthio)-1,3,5-triazine-2,4-diamine] or ametryne [N-ethyl-N′-(1-methylethyl)-6-(methylthio)-1,3,5-triazine- 2,4-diamine], or both, as a sole source of sulfur for growth. The success of enrichments depended on previous exposure of the soil inoculum to s-triazine herbicides. Deaminoethylametryne [4-(1-methylethyl)amino-6-(methylthio)-1,3,5-triazine-2-(1H)-one], methylsulfonic acid, and sodium sulfate could also be used as sulfur sources. Utilization of a compound was quantified as the growth yield per mole of sulfur supplied. Yields were about 6 kg of protein per mol of sulfur. The product of the desulfuration of an s-triazine was identified as the corresponding hydroxy-derivative. This is the first substantiated report of the utilization of these s-triazines as sulfur sources by bacteria.     

The X-ray investigation of 16 alpha,17 alpha-thiazolidine derivatives of delta 4- and delta 5-pregnanes and conformational analysis of their oxathiolane analog

An X-ray study of 3,20-dioxo-4-pregnene-[16 alpha,17 alpha-d]--2',2'-dimethylthiazolidine (I) and 3 beta-hydroxy-20-oxo-5--pregnene-[16 alpha,17 alpha-d]-2',2'- dimethylthiazolidine (II) has been carried out. Two independent molecules in crystal II have significantly different conformations of the D and E rings, although according to the atom-atom potential calculations the energy of interaction of these molecules with their neighbors in crystal is the same. The calculation of conformational energy of 3,20-dioxo-4-pregnene-[17 alpha,16 alpha-d]-2',2'--dimethyloxathiolane (III) by the molecular mechanics method (MMM) indicates a possibility of existence of two similar conformers also for this molecule. The MMM calculation shows also that the conformation of molecule III (as well as progesterone) with the 17 beta-acetyl group torsion angle C(16)C(17)C(20)0(20) close to -120 degrees is possible.     

Synthesis of coumarin-polyamine-based molecular probe for the detection of hydroxyl radicals generated by gamma radiation

To develop a molecular probe for detection of hydroxyl radicals in the vicinity of DNA, the coumarin-polyamine complexes, N(1),N(12)-bis[2-oxo-2H-chromene-3-carbonyl]-1,12-diamine-4,9-diazadodecane (5) and tris[2-(2-oxo-2H-chromene-3-carboxamido)ethyl]amine (7), and their hydroxylated derivatives, N(1),N(12)-bis[7-hydroxy-2-oxo-2H-chromene-3-carbonyl]-1,12-diamine-4,9-diazadodecane (6) and tris[2-(7-hydroxy-2-oxo-2H-chromene-3-carboxamido)ethyl]amine (8), have been synthesized. Using computer-generated molecular modeling, the derivatives have been docked onto DNA dodecamer d(CGCGAATTCGCG)(2), the ligand-DNA complexes have been minimized, and the free binding energies (DeltaG(binding)) and inhibition constants (K(i)) have been calculated. Compound 7 is not water-soluble at the concentrations required for the project. When aqueous solutions of 5 are irradiated with gamma rays, the relationship between induced fluorescence and dose is linear in the range of 0 to 10 Gy. The fluorescence emission spectrum of irradiated 5 is similar to that of its dihydroxy derivative 6, indicating conversion of 5 to 6, and induction of fluorescence records formation of hydroxyl radicals in aqueous solution. The dicoumarin-polyamine 5, a novel compound for the detection of hydroxyl radicals close to DNA, is a sensitive and quantitative probe with potential for applications in biological systems.     

Biliary metabolites of estriol in the rat

Following the subcutaneous administration of estriol-6,7-³H to rats, biliary metabolites were identified and quantitated. Approximately 70% of the metabolites were excreted in the form of “glucosiduronate” conjugates. 3, 17β-Dihydroxy-2-methoxy-1,3,5(10)-estratrien-16-one was the major metabolite in this conjugate fraction. Significant amounts of 3,17β-dihydroxy-1,3,5(10)-estratrien-16-one and 2,3,17β-trihydroxy-1,3,5(10)-estratrien-16-one, as well as smaller quantities of 1,3,5(10)-estratriene-2,3,16α,17β-tetrol and 2-methoxy-1,3,5(10)-estratriene-3,16α, 17β-triol, were also found. In 17α-ethinylestradiol - treated animals, the rate of excretion of radioactivity and the proportion of 16-oxo-17β-ol metabolites found in the “glucosiduronate” fraction were reduced.     

Pathways that produce volatile sulphur compounds from methionine in Oenococcus oeni

Aims:  Determination of pathways involved in synthesis of volatile sulphur compounds (VSC) from methionine by Oenococcus oeni isolated from wine.
Methods and Results:  Production of VSC by O. oeni from methionine was investigated during bacterial cultures and in assays performed in the presence of resting cells or protein fractions. Cells of O. oeni grown in a medium supplemented with methionine produced methanethiol, dimethyl disulphide, methionol and 3-(methylthio)propionic acid. Methional was also detected, but only transiently during the exponential growth phase. It was converted to methionol and 3-(methylthio) propionic acid in assays. Although this acid could be produced alternatively from 2-oxo-4-(methylthio) butyric acid (KMBA) by oxidative decarboxylation. In addition, KMBA was a precursor for methanethiol and dimethyl disulphide synthesis. Interestingly, assays with resting cells and protein fractions suggested that a specific enzyme could be involved in this conversion in O. oeni .
Conclusion:  This work shows that methional and KMBA are the key intermediates for VSC synthesis from methionine in O. oeni . Putative enzymatic and chemical pathways responsible for the production of these VSC are discussed.
Significance and impact of the study:  This work confirms the capacity of O. oeni to metabolize methionine and describes the involvement of potential enzymatic pathways.