Derivatives of methanopterin, a coenzyme involved in methanogenesis

Degradational studies of methanopterin, a coenzyme involved in methanogenesis, are reported. The results of these studies are in full accordance with the proposed structure of methanopterin as N-[1'-(2'-amino-4'-hydroxy-7' -methyl-6'-pteridinyl)ethyl]-4-[2', 3', 4', 5'-tetrahydroxypent-1'-yl(5'-1' )O-alpha-ribofuranosyl-5'-phosphoric acid] aniline in which the phosphate group is esterified with alpha-hydroxyglutaric acid. Acid hydrolysis of methanopterin cleaved the 5'----1' glycosidic bond and yielded a 'hydrolytic product' which was identified as N-[1'-(2'-amino-4'-hydroxy-7' -methyl-6'-pteridinyl)ethyl]-4-[2', 3', 4', 5'-tetrahydroxypent-1'-yl]aniline. Alkaline permanganate oxidation of methanopterin yielded 7-methylpterin-6-carboxylic acid. Catalytic (or enzymatic) hydrogenation of methanopterin gave a mixture of 6-ethyl-7-methyl-7,8-dihydropterin, 6-ethyl-7-methylpterin and a third compound, named methaniline which was identified as 4-[2', 3', 4', 5'-tetrahydroxypent-1'-yl(5'----1')O-alpha -ribofuranosyl-5'-phosphoric acid]aniline, in which the phosphate group is esterified with alpha-hydroxyglutaric acid. Methanosarcina barkeri contains a closely related coenzyme called sarcinapterin, which was identified as a L-glutamyl derivative of methanopterin, where the glutamate moiety is attached to the alpha-carboxylic acid group of the alpha-hydroxyglutaric acid moiety of methanopterin via an amide linkage.     

Elucidation of the structure of methanopterin, a coenzyme from Methanobacterium thermoautotrophicum, using two-dimensional nuclear-magnetic-resonance techniques

Methanopterin is a coenzyme involved in methanogenesis. From 2 kg wet cells of Methanobacterium thermoautotrophicum about 35 mumol methanopterin were isolated. The structure of this compound was elucidated by various two-dimensional nuclear-magnetic-resonance techniques. Methanopterin was identified as N-[1'-(2"-amino-4"-hydroxy-7" - methyl-6"- pteridinyl) ethyl]-4-[2',3',4',5'- tetrahydroxypent-1'- yl (5' leads to 1") O-alpha-ribofuranosyl-5"-phosphoric acid] aniline, in which the phosphate group is esterified with alpha-hydroxyglutaric acid. The molecular formula of the sodium salt of methanopterin at pH 7.0 is C30H38O16N6PNa3 X chiH2O (chi is about 4). The anhydrous sodium salt of methanopterin has a molecular mass of 838.60 Da and the molar absorption coefficient at 342 nm is 7.4 mM-1 cm-1 at pH 7.0.     

Biosynthesis of methanopterin

The biosynthetic pathway for the generation of the methylated pterin in methanopterins was determined for the methanogenic bacteria Methanococcus volta and Methanobacterium formicicum. Extracts of M. volta were found to readily cleave L-7,8-dihydroneopterin to 7,8-dihydro-6-(hydroxymethyl)pterin, which was confirmed to be a precursor of the pterin portion of the methanopterin. [methylene-2H]-6-(Hydroxymethyl)pterin was incorporated into methanopterin by growing cells of M. volta to an extent of 30%. Both the C-11 and C-12 methyl groups of methanopterin originate from [methyl-2H3]methionine, as confirmed by the incorporation of two C2H3 groups into 6-ethyl-7-methylpterin, a pterin-containing fragment derived from methanopterin. Cells grown in the presence of [methylene-2H]-6-(hydroxymethyl)pterin, [ethyl-2H4]-6-[1 (RS)-hydroxyethyl]pterin, [methyl-2H3]-6- (hydroxymethyl)-7-methylpterin, [ethyl-2H4, methyl-2H3]-6-[1 (RS)-hydroxyethyl]-7-methylpterin, and [1-ethyl-3H]-6-[1 (RS)-hydroxyethyl]-7-methylpterin showed that only the non-7-methylated pterins were incorporated into methanopterin. Cells extracts of M. formicicum readily condensed synthetic [methylene-3H]-7,8-H2-6-(hydroxymethyl)pterin-PP with methaniline to generate demethylated methanopterin, which is then methylated to methanopterin by the cell extract in the presence of S-adenosylmethionine. These observations indicate that the pterin portion of methanopterin is biosynthetically derived from 7,8-H2-6-(hydroxymethyl)pterin, which is coupled to methaniline by a pathway analogous to the biosynthesis of folic acid. This pathway for the biosynthesis of methanopterin represents the first example of the modification of the specificity of a coenzyme through a methylation reaction.     

Iridoid glucosides and flavones from the aerial parts of Avicennia marina

Two new iridoid glucosides, namely, 2'-O-[(2E,4E)-5-phenylpenta-2,4-dienoyl]mussaenosidic acid (1; mussaenosidic acid = [1S-(1alpha,4aalpha,7alpha,7aalpha)]-1-(beta-D-glucopyranosyloxy)-1,4a,5,6,7,7a-hexahydro-7-hydroxy-7-methylcyclopenta[c]pyran-4-carboxylic acid) and 2'-O-(4-methoxycinnamoyl)mussaenosidic acid (2), were isolated from the aerial parts of the mangrove plant Avicennia marina. Beside that, one known iridoid glucoside, 2'-O-coumaroylmussaenosidic acid (3) and four known flavones (flavone = 2-phenyl-4H-1-benzopyran-4-one) including 4',5-dihydroxy-3',7-dimethoxyflavone (4), 4',5-dihydroxy-3',5',7-trimethoxyflavone (5), 4',5,7-trihydroxyflavone (6), and 3',4',5-trihydroxy-7-methoxyflavone (7) were also isolated and identified. The structures of these compounds were elucidated by NMR spectroscopy and by low- and high-resolution mass spectrometry. The chemotaxonomic significance of these findings was discussed. In addition, each isolated compound was evaluated for the ability of alpha,alpha-diphenyl-beta-picrylhydrazyl (DPPH) radical-scavenging activity.     

The structure of a molybdopterin precursor. Characterization of a stable, oxidized derivative

An oxidized pterin species, termed compound Z, has been isolated from molybdenum cofactor-deficient mutants of Escherichia coli and shown to be the direct product of oxidation of a molybdopterin precursor which accumulates in these mutants. The complete structural characterization of compound Z has been accomplished. A carbonyl function at C-1' of the 6-alkyl side chain can be reacted with 2,4-dinitrophenylhydrazine to yield a phenylhydrazone and can be reduced with borohydride, producing a mixture of two enantiomers, each with a hydroxyl group on C-1'. Compound Z contains one phosphate/pterin and no sulfur. The phosphate group is insensitive to alkaline phosphatase and to a number of phosphodiesterases but is quantitatively released as inorganic phosphate by mild acid hydrolysis. From 31P and 1H NMR of compound Z it was inferred that the phosphate is bound to C-2' and C-4' of a 4-carbon side chain, forming a 6-membered cyclic structure. Mass spectral analysis showed an MH+ ion with an exact mass of 344.0401 corresponding to the molecular formula C10H11N5O7P, confirming the proposed structure.     

A facile synthesis and anti-avian influenza virus (H5N1) screening of some novel pyrazolopyrimidine nucleoside derivatives

Treatment of 5-amino-1-(9-methyl-5,6-dihydronaphtho[1',2':4,5]thieno[2,3-d]pyrimidin-11-yl)-1H-pyrazole-4-carbonitrile (1) with formic acid afforded pyrazolo[3,4-d]pyrimidin-4-one derivative 2. The sodium salt of the latter compound (generated in situ) was treated with some alkyl halides to afford the corresponding N-substituted compounds 3-7. The siloxy derivative 8 (generated also in situ from 2) was ribosylated and glycosylated to yield compounds 9 and 11, respectively. Deprotection of compounds 9 and 11 in methanolic ammonia produced the free nucleosides 10 and 12, respectively. Moreover, the prepared compounds were tested for antiviral activity against H5N1 virus [A/chicken/Egypt/1/2006] and some of them revealed moderate results compared with the other tested compounds.     

Biosynthesis of the 7-methylated pterin of methanopterin.

The incorporation of [15N]glycine and [U-methyl-2H]methionine into methanopterin by growing cells of a methanogenic bacterium was measured to establish the biosynthetic route of the methylated pterin in the structure. The tetrahydromethanopterin produced by the cells was oxidatively cleaved to produce 7-methylpterin, and the amount of label incorporated into this pterin was measured by gas chromatography-mass spectrometry of the ditrimethylsilyl derivative of this compound. Approximately 27% of the 7-methylpterin and the guanine present in the cell was derived from the fed [15N]glycine. [U-methyl-2H]methionine was incorporated with the initial retention of all three deuteriums. These results are consistent with the biosynthesis of the pterin of methanopterin originating from GTP and its 7-methyl group arising from the methyl group of methionine.     

Microbiological degradation of bile acids, further degradation of a cholic acid metabolite containing the hexahydroindane nucleus by Corynebacterium equi.

1. The further degradation of a cholic acid (I) metabolite, (4R)-4-[4alpha-(2-carboxyethyl)-3aalpha-hexahydro-7abeta-methyl-5-oxoindan-1beta-yl]valeric acid (IIa), by Corynebacterium equi was investigated. This organism effected ring-opening and gave (4R)-4-[2alpha-(2-carboxyethyl)-3beta-(3-carboxypropionyl)-2beta-methylcyclopent-1beta-yl]valeric acid (VI). The new metabolite was isolated as its trimethyl ester and identified by partical synthesis. It was not utilized by C. equi. 2. (4R)-4[4alpha-(2-Carboxyethyl)-3aalpha-decahydro-8abeta-methyl5-oxa-6-oxoazulen-1beta-yl]valeric acid (IVa), which is a hypothetical initial oxidation product in the above degradation, was not converted by C. equi into the expected metabolite (VI), but into 3 - [2beta - [(2S) - tetrahydro - 5 - oxofur - 2 - yl] - 1beta - methyl - 5 - oxocyclopent - 1alpha - yl]-propionic acid (VIII), the structure of which was established by partial synthesis. 3. Both the possible precursors of the metabolite (VI), an isomer of the epsilon-lactone (IVa), the gamma-lactone (XIa), and the open form of these lactones, the hydroxytricarboxylic acid (V), were also not utilized by C. equi. 4. Under some incubation conditions, C. equi also converted compound (IIa) and 3-(3aalpha-hexahydro-7abeta-methyl-1,5-dioxoindan-4alpha-yl)propionic acid (IIb) into 5-methyl-4-oxo-octane-1,8-dioic acid (III), (4R)-4-(2,3,4,6,6abeta,7,8,9,9aalpha,9bbeta-decahydro-6abeta-methyl-3-oxo-1H-cyclopenta[f]quinolin-7beta-yl)valeric acid (VII) and probably a monohydroxy derivative of compound (IIa) and compound (III), respectively. 5. The possibility that an initial step in the degradation of compound (IIa) by C. equi is oxygenation of the Baeyer-Villiger type, yielding compound (IVa), is discussed. Metabolic pathways of compound (IIa) to compounds (III), (VI), (VII) and (VIII) are also considered.     

Synthesis of some newer derivatives of substituted quinazolinonyl-2-oxo/thiobarbituric acid as potent anticonvulsant agents

5-[1'-[3"-Aminoacetyl-2"-methyl-6",8"-dihalosubstitutedquinazolin-4"(3"H)-onyl]-thiosemicarbazido]-2-oxo/thiobarbituric acids 3a-3h and 5-[2'-amino-5'-[3"-aminomethylene-2"-methyl-6",8"-dihalosubstitutedquinazolin-4"(3"H)-onyl]-1',3',4'-thiadiazol-2'-yl]-2-oxo/thiobarbituric acid 5a-5h were prepared by incorporating 1-[3'-aminoacetyl-2'-methyl-6",8"-dihalosubstituted-quinazolin-4'(3'H)-onyl]-thiosemicarbazides 2a-2d and 2-amino-5-[3'-aminomethylene-2'-methyl-6',8'-dihalosubstituted-quinazolin-4'(3'H)-onyl]-1,3,4-thiadiazoles 4a-4 h respectively at 5(th) position of 2-oxo/thiobarbituric acids (via Mannich reaction). All the newly synthesized compounds were screened for their anti-convulsant activity in MES and PTZ models and were compared with standard drugs phenytoin sodium and sodium valproate. Interestingly, these compounds were found to be devoid of sedative and hypnotic activities when tested. Out of the compounds studied, the most active compound 5h, that is 5-[2'-amino-5'-[3"-aminomethylene-2"-methyl-6",8"-dibromoquinazolin-4"(3"H)-onyl]-1',3',4'-thiadiazol-2'-yl]-2-thiobarbituric acid showed activity (90%) more potent than the standard drug.     

The structure of the molybdenum cofactor. Characterization of di-(carboxamidomethyl)molybdopterin from sulfite oxidase and xanthine oxidase

A di-(carboxamidomethyl) derivative of molybdopterin, the organic component of the molybdenum cofactor, has been prepared under conditions favoring retention of all of the structural features of the molecule. The specific radioactivity of [1-14C]iodoacetamide incorporated relative to the amount of phosphate indicated two alkylation sites per pterin. Energy-dispersive x-ray analysis of the derivative showed the presence of 2 sulfurs in the derivative. An exact mass corresponding to the molecular formula C14H18N7O5S2 was obtained for the MH+ ion of the alkylated, dephosphorylated compound by fast atom bombardment mass spectroscopy. 1H NMR spectra of the phosphorylated and dephosphorylated forms of alkylated molybdopterin, in conjunction with the other data, have provided strong corroboration of the validity of the proposed structure of molybdopterin (Johnson, J. L., and Rajagopalan, K. V. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 6856-6860) as a 6-alkylpterin with a 4-carbon side chain containing an enedithiol on C-1' and C-2', a secondary alcohol on C-3', and a phosphorylated primary alcohol on C-4'. As isolated, the di-(carboxamido-methyl)molybdopterin was found to be a 5,6,7,8-tetrahydropterin.     

7-deazainosine derivatives: synthesis and characterization of 7- and 7,8-substituted pyrrolo [2,3-d]pyrimidine ribonucleosides

The synthesis of model 7 deazapurine derivatives related to tubercidin and toyocamycin has been performed. Tubercidin derivatives were obtained by simple conversion of the amino group of the heterocyclic moiety of the starting 7-deazadenosine compounds, into a hydroxyl group. Preparation of toyocamycin derivatives was accomplished by treatment of the silylated 6-bromo-5-cyanopyrrolo[2,3-d]pyrimidin-4-one with 1-O-acetyl-2,3,5-tri-O-benzoyl-beta-d-ribofuranose. The glycosylation reaction afforded a mixture of 8-bromo 7-cyano 2',3',5' tri-O-benzoyl 7-deazainosine and 6-bromo-5-cyano-3-(2',3',5'-tri-O-benzoyl-beta-d-ribofuranosyl)pyrrolo[2,3-d]-pyrimidin-4-one isomers: The structures were assigned on the basis of NMR spectroscopy studies. Next deprotection treatment gave the novel 7-deazainosine ribonucleosides.     

Microbiological degradation of bile acids. Metabolites formed from 3-(3a alpha-hexahydro-7a beta-methyl-1,5-dioxoindan-4 alpha-yl) propionic acid by Streptomyces rubescens.

1. The metabolism of 3-(3a alpha-hexahydro-7a beta-methyl-1,5-dioxoindan-4 alpha-yl)propionic acid (III), which is a possible precursor of 2,3,4,6,6a beta, 7,8,9,9a alpha,9b beta-decahydro-6a beta-methyl-1H-cyclopenta[f]quinoline-3,7-dione (II) formed from cholic acid (I) by streptomyces rubescens, was investigated by using the same organism. 2. This organism effected amide bond formation, reduction of the carbonyl groups, trans alpha beta-desaturation and R-oriented beta-hydroxylation of the propionic acid side chain and skeleton cleavage, and the following metabolites were isolated as these forms or their derivatives: compound (II), 1,2,3,4 a beta,-5,6,6a beta,7,8,9a alpha,9b beta-dodecahydro-6a beta -methylcyclopental[f][1]benzopyran-3,7-dione (IVa), (1R)-1,2,3,4a beta,5,6,6a beta,7,8,9.9a alpha,9b beta-dodecahydro-1-hydroxy-6a beta-methylcyclopenta[f][1]benzopyran-3,7-dione (IVb), (E)-3-(3aalpha-hexahydro-5 alpha-hydroxy-7a beta-methyl-l-oxo-indan-4 alpha-yl)prop-2-enoic acid (V), (+)-(5R)-5-methyl-4-oxo-octane-1,8-dioic acid (VI), 3-(4-hydroxy-5-methyl-2-oxo-2H-pyran-6-yl)propionic acid (VII) and 3-(3a alpha-hexahydro-1 beta-hydroxy-7a beta-methyl-5-oxoindan-4 alpha-yl)propionic acid (VIII). The metabolites (IVb), (V), (VI) and (VII) were new compounds, and their structures were established by chemical synthesis. 3. The question of whether these metabolites are true degradative intermediates is discussed, and a degradative pathway of compound (III) to the possible precursor of compound (VII), 7-carboxy-4-methyl-3,5-dioxoheptanoyl-CoA (IX), is tentatively proposed. The further degradation of compound (IX) to small fragments is also considered.     

Design and synthesis of a metabolically stable and potent antitussive agent, a novel delta opioid receptor antagonist, TRK-851

We have previously reported on antitussive effect of (5R,9R,13S,14S)-17-cyclopropylmethyl-6,7-didehydro-4,5-epoxy-5',6'-dihydro-3-methoxy-4'H-pyrrolo[3,2,1-ij]quinolino[2',1':6,7]morphinan-14-ol(1b) methanesulfonate (TRK-850), a selective delta opioid receptor antagonist which markedly reduced the number of coughs in a rat cough model. We designed TRK-850 based on naltrindole (NTI), a typical delta opioid receptor antagonist, to improve its permeability through the blood-brain barrier by introducing hydrophobic moieties to NTI. The ED(50) values of NTI and compound 1b by intraperitoneal injections were 104 microg/kg and 2.07 microg/kg, respectively. This increased antitussive potency probably resulted from the improved brain exposure of compound 1b. However, 1b was extremely unstable toward metabolism by cytochrome P450. In this study, we designed and synthesized compound 1b derivatives to improve the metabolic instability, which resulted in affording highly potent and metabolically stable oral antitussive agent (5R,9R,13S,14S)-17-cyclopropylmethyl-6,7-didehydro-4,5-epoxy-8'-fluoro-5',6'-dihydro-4'H-pyrrolo[3,2,1-ij]quinolino[2',1':6,7]morphinan-3,14-diol (1c) methanesulfonate (TRK-851).     

Synthesis and antibacterial activity of egonol derivatives

Synthesis of egonol derivatives, 5-(3'-chloropropyl)-7-methoxy-2-(3',4'-methylenedioxyphenyl)benzofuran 1, 5-(3'-bromopropyl)-7-methoxy-2-(3',4'-methylenedioxyphenyl)benzofuran 2, 3-[2-(1,3-benzodioxol-5-yl)-7-methoxy-1-benzofuran-5-yl]propanal 3, 5-(3'-iodopropyl)-7-methoxy-2-(3',4'-methylenedioxyphenyl)benzofuran 4, 5-[3-(3'-bromopropyloxy) propyl]-7-methoxy-2-(3',4'-methylenedioxyphenyl)benzofuran 5, 3-[2-(1,3-benzodioxol-5-yl)-7-methoxy-1-benzofuran-5-yl]propylmethanoate 6, 3-[2-(1,3-benzodioxol-5-yl)-7-methoxy-1-benzofuran-5-yl]propyloleate 7, 5-[3'-hydroxypropyl]-6-bromo-7-methoxy-2-(3',4'-methylenedioxyphenyl)benzofuran 8, 4-[2-(1,3-benzodioxol-5-yl)-7-methoxy-1-benzofuran-5-yl]butanenitrile 9, 3-[2-(1,3-benzodioxol-5-yl)-7-methoxy-1-benzofuran-5-yl]propylbenzoate 10, 5-[3'-hydroxypropyl]-7-methoxy-3-nitro-2-(3',4'-methylenedioxyphenyl)benzofuran 11 and their antibacterial activity against Staphylococcus aureus, Bacillus subtilis, Candida albicans and Escherichia coli are reported. The starting material egonol 5-[3'-(hydroxy)propyl]-7-methoxy-2-(3', 4'methylenedioxyphenyl)benzofuran was isolated from seeds of Styrax officinalis L. The structural elucidication of these compounds (1-11) was established using 1D ((1)H, (13)C), 2D NMR (HMBC, HMQC, COSY) and LCMS spectroscopic data. While egonol and some synthesised new compounds show similar antibacterial activity and MIC values against S. aureus, B. subtilis, C. albicans and E. coli, other new derivatives show different activity against S. aureus, B. subtilis, C. albicans and E. coli.     

Benzofurans and another constituent from seeds of Styrax officinalis

The benzofuran constituents of the seeds of Styrax officinalis were investigated. From the hexane extract, two new constituents named 5-(3"benzoyloxypropyl)-7-methoxy-2-(3',4'-methylenedioxyphenyl)-benzofuran (5) and 4-[3"-(1c-methylbutanoyloxy)propyl]-2-methoxy-(3',4'-methylenedioxyphenyl)-1a, 5b-dihydrobenzo-[3,4]-cyclobutaoxirene (6) were isolated together with four known compounds, 5-[3"-(1c-methylbutanoyloxy)propyl]-7-methoxy-2-(3',4'-dimethoxyphenyl)-benzofuran (4), 5-[3"-(1c-methylbutanoyloxy)propyl]-7- methoxy-2-(3',4'-methylenedioxyphenyl)-benzofuran (3), 5-(3"-acetoxypropyl)-7-methoxy2-(3',4'-methylenedioxphenyl)-benzofuran (2) and 5-(3"-hydroxypropyl)-7-methoxy-2-(3',4'-met hylenedioxyphenyl)-benzofuran (1). Although the compounds 1, 2, and 3 have been isolated previously from the seeds of Styrax obassia, this is the first record of their isolation from seeds of Styrax officinalis. The structures of the isolated compounds were established by 1D- and 2D-NMR (HMBC, HMQC, COSY), FABMS and high-resolution ESI FTMS.     

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.     

Synthesis and structure-activity relationships of thioflavone derivatives as specific inhibitors of the ERK-MAP kinase signaling pathway

Condensation of nitrobenzaldehydes 3 and alpha-[o-(p-methoxybenzylthio)benzoyl] sulfoxide 4 gave alpha-sulfinyl enones 5. Treatment of 5 with formic acid caused cyclization followed by debenzylation to afford 3-(methylsulfinyl)thioflavanones 6. Double-bond formation with elimination of methanesulfenic acid was performed by refluxing 6 in benzene, and, finally, the nitro group of 2-phenyl-4H-1-benzothiopyran-4-one (thioflavones) 7 was reduced with tin in tetrafluoroboric acid. Various 2'-aminothioflavones 8 thus prepared were evaluated for their inhibitory effects on the ERK-MAP kinase pathway. In a cell-based assay, 2-(2'-amino-3'-methoxyphenyl)-4H-1-benzothiopyran-4-one (8b) showed a more potent inhibitory effect than the corresponding oxygen compound (PD98059, 1) on the Raf-induced activation of the ERK-MAP kinase pathway as well as cell proliferation. Furthermore, compound 8b selectively and potently inhibited the proliferation of tumor cells in which the ERK-MAP kinase pathway is constitutively activated.     

Chemo-enzymatic synthesis of C-9 acetylated sialosides

A chemo-enzymatic synthesis of [(5-acetamido-9-O-acetyl-3,5-dideoxy-D-glycero-alpha-D-galacto-2-nonulopyranosylonic acid)-(2-->3)-O-(beta-D-galactopyranosyl)-(1-->3)-O-(2-acetamido-2-deoxy-alpha-D-galactopyranosyl)]-l-serine acetate (1) has been accomplished by a regioselective chemical acetylation of Neu5Ac (2) to give 9-O-acetylated sialic acid 3, which was enzymatically converted into CMP-Neu5,9Ac(2) (4) employing a recombinant CMP-sialic acid synthetase from Neisseria meningitis [EC 2.7.7.43]. The resulting compound was then employed for the enzymatic glycosylation of the C-3' hydroxyl of chemically prepared glycosylated amino acid 10 using recombinant rat alpha-(2-->3)-O-sialyltransferase expressed in Spodooptera frugiperda [EC 2.4.99.4] to give, after deprotection of the N(alpha)-benzyloxycarbonyl (CBz)-protecting group of serine, target compound 1. The N(alpha)-CBz-protected intermediate 11 can be employed for the synthesis of glycolipopeptides for immunization purposes.     

Microbiological degradation of bile acids. The preparation of some hypothetical metabolites involved in cholic acid degradation.

1. To identify the intermediates involved in the degradation of cholic acid, the further degradation of (4R)-4-[4alpha-(2-carboxyethyl)-3aalpha-hexahydro-7abeta-methyl-5-oxoindan-1beta-yl]valeric acid (IVa) by Arthrobacter simplex was attempted. The organism could not utilize this acid but some hypothetical intermediate metabolities of compound (IVa) were prepared for later use as reference compounds. 2. The nor homologue (IIIa) and the dinor homologue (IIIb) of compound (IVa) were prepared by exposure of 3-oxo-24-nor-5beta-cholan-23-oic acid (I) and (20S)-3beta-hydroxy-5-pregnene-20-carboxylic acid (II) to A. simplex respectively. These compounds correspond to the respective metabolites produced by the shortening of the valeric acid side chain of compound (IVa) in a manner analogous to the conventional fatty acid alpha- and beta-oxidation mechanisms. Their structures were confirmed by partial synthesis. 3. The following authentic samples of reduction products of the oxodicarboxylic acids (IIIa), (IIIb) and (IVa) were also synthesized as hypothetical metabolities: (4R)-4-[3aalpha-hexahydro-5alpha-hydroxy-4alpha-(3-hydroxypropyl)-7abeta-methylindan-1beta-yl]valeric acid (Vb) and its nor homologue (VIIa) and dinor homologue (IXa);(4R)-4-[3Aaalpha-hexahydro-5alpha-hydroxy-4alpha-(3-hydroxypropyl)-7abeta-methylindan-1beta-yl]-pentan-1-ol (Vc); and their respective 5beta epimers (Ve), (VIIc), (IXc) and (Vf). 4. In connexion with the non-utilization of compound (IVa) by A. simplex, the possibility that not all the metabolites formed from cholic acid by a certain micro-organism can be utilized by the same organism is considered.     

Crystal and molecular structure of methyl 4-O-methyl-beta-D-glucopyranosyl-(1-->4)-beta-D-glucopyranoside

The cellulose model compound methyl 4-O-methyl-beta-D-glucopyranosyl-(1-->4)-beta-D-glucopyranoside (6) was synthesised in high overall yield from methyl beta-D-cellobioside. The compound was crystallised from methanol to give colourless prisms, and the crystal structure was determined. The monoclinic space group is P2(1) with Z=2 and unit cell parameters a=6.6060 (13), b=14.074 (3), c=9.3180 (19) A, beta=108.95(3) degrees. The structure was solved by direct methods and refined to R=0.0286 for 2528 reflections. Both glucopyranoses occur in the 4C(1) chair conformation with endocyclic bond angles in the range of standard values. The relative orientation of both units described by the interglycosidic torsional angles [phi (O-5' [bond] C-1' [bond] O-4 [bond] C-4) -89.1 degrees, Phi (C-1' [bond] O-4 [bond] C-4 [bond] C-5) -152.0 degrees] is responsible for the very flat shape of the molecule and is similar to those found in other cellodextrins. Different rotamers at the exocyclic hydroxymethyl group for both units are present. The hydroxymethyl group of the terminal glucose moiety displays a gauche-trans orientation, whereas the side chain of the reducing unit occurs in a gauche-gauche conformation. The solid state (13)C NMR spectrum of compound 6 exhibits all 14 carbon resonances. By using different cross polarisation times, the resonances of the two methyl groups and C-6 carbons can easily be distinguished. Distinct differences of the C-1 and C-4 chemical shifts in the solid and liquid states are found.