Enzymatic epimerization of d-erythro-dihydroneopterin triphosphate to l-threo-dihydroneopterin triphosphate

An enzyme has been discovered in Escherichia coli that catalyzes the conversion of the triphosphate ester of 2-amino-4-hydroxy-6-(d-erythro-1′,2′,3′-trihydroxypropyl)-7,8-dihydropteridine, (i.e. d-erythro-dihydroneopterin triphosphate) to an epimer of this compound, l-threo-dihydroneopterin triphophate. The enzyme, which is here named “d-erythro-dihydroneopterin triphosphate 2′-epimerase,” needs a divalent cation (Mg²⁺ or Mn²⁺ is most effective) for maximal activity. Its molecular weight is estimated at 87 000–89 000. Little or no activity can be detected if either the monophosphate or the phosphate-free form of the substrate is incubated with the enzyme. Evidence is presented to establish that all three phosphate residues of the substrate are retained in the product and that the product is of the l-threo configuration.     

Enzymatic Synthesis of the Crithidia Factors in Cell-free Extracts of Serratia indica

The concomitant production of formic acid and pterin compounds from guanosine-5′-triphosphate (GTP) has been found in cell-free extracts of Serratia indica. Among the pterin compounds, l-threo-neopterin–the major Crithidia factor in S. indica–, a cyclic phosphate of neopterin (cNP), d-erythro-neopterin and 6-hydroxymethyl pterin were detected and isolated. Formate-¹⁴C elimination from GTP-8-¹⁴C was quantitatively distributed in the ethyl acetate layer in the ehyl acetate-hydrochloric acid partition system. Carbon 8 of GTP was released as formic acid. Enzymatic production of formate and cNP was linear for 2 hr at 37°C. Formate production was proportional to the enzyme concentration. The optimum pH for formate elimination was observed around pH 8.6. Optimum temperature for the production of formate and cNP was 50°C. The apparent Km value of GTP for formate production was 6.2×10^?bm. Formate eliminating activity was activated by disodium phosphate but was inhibited by Mg²⁺ or AMP. Incorporation of GTP-U-¹⁴C into pterin compounds was also regulated with disodium phosphate. Effective incorporation into cNP and d-erythro-neopterin occurred in the presence of phosphate. When phosphate was omitted from the system, however, effective incorporation into 6-hydroxymethyl pterin was observed. The biosynthetic process of the Crithidia factors, i.e. l-threo-neopterin and cNP, from GTP in S. indica is also discussed.     

Isolation and Characterization of a Cyclic Phosphate of Neopterin and Other 6-Alkylpterin Compounds Enzymatically Synthesized from GTP by Cell-free Extracts of Serratia indica

A cell-free system for the biosynthesis of l-threo-neopterin, a growth factor for protozoan, Crithidia fasciculata from guanosine-5′-triphosphate (GTP) was obtained from extracts of Serratia indica IFO 3759. This preparation catalyzed the production of a specific pteridine from GTP, which was isolated and characterized as a cyclic phosphate of neopterin (cNP). Among the other products, l-threo-neopterin, as the Crithidia factor, 6-hydroxymethylpterin, and erythro-neopterin were tentatively identified. Requirements for the synthesis of these products include GTP, Mg²⁺, and disodium phosphate. Fluorescence formation was inhibited by purine nucleotides.

When a disodium phosphate was included in the reaction system, cNP and erythro-neopterin were effectively synthesized from GTP. On the other hand, when the phosphate was omitted 6-hydroxymethylpterin was formed.

The possible biosynthetic process of l-threo-neopterin was discussed.     

Synthesis of New 5″-Sulfonylamido Derivatives of 3″-Azido-3″-Deoxythymidine (AZT)

Abstract

A series of 5′-N-methanesulfonyl derivatives of 3′-azido-5′-(alkylamino)-3′,5′-dideoxythymidine was synthesised. The first step of the synthesis involved the reaction of 1-(2,5-dideoxy-5-O-tosyl-β-D-threo-pentofuranosyl)thymine 1 with an appropriate amine to give 1-[5-(alkylamino)-2,5-dideoxy-β-D-threo-pentofuranosyl]thymines 2a-e and 1-(2,5-dideoxy-β-threo-pent-4-enofuranosyl)thymine 3 as a by-product. Compounds 2a-e were treated with an excess of methanesulfonyl chloride to yield intermediates 1-[5-(dimethylamino)-3-O-methanesulfonyl-2,3,5-trideoxy-β-D-threo-pentofuranosyl]-thymine 4a and 1-[5-(N-alkyl-N-methanesulfonyl)-3-O-methanesulfonyl-2,3,5-trideoxy-β-D-threo-penfuranosyl]thymines 4b-e. The reaction of 4a-e with lithium azide in dimethyl-formamide afforded the final compounds 1-[3-azido-5-(N-methyl-N-methanesulfonyl)-2,3,5-trideoxy-β-D-erythro-penofuranosyl]thymine 5a and 1-[3-azido-5-(N-alkyl-N-methanesulfonyl)-2,3,5-trideoxy-β-D-erythro-penofuranosyl]thymines 5b-e. The independent synthesis of 4′,5′-unsaturated product 3 was also described.     

Isolation of D-erythro-neopterin 2′:3′-cyclic phosphate from Photobacterium phosphoreum

A new naturally occurring pteridine has been isolated from Photobacterium phosphoreum. This compound is shown by degradative experiments and by comparison with authentic material to be D-erythro-neopterin 2′:3′-cyclic phosphate. The possible biochemical significance of the compound is discussed.     

Enzymatic epimerization of d-erythro-dihydroneopterin triphosphate to l-threo-dihydroneopterin triphosphate

An enzyme has been discovered in Escherichia coli that catalyzes the conversion of the triphosphate ester of 2-amino-4-hydroxy-6-(d-erythro-1′,2′,3′-trihydroxypropyl)-7,8-dihydropteridine, (i.e. d-erythro-dihydroneopterin triphosphate) to an epimer of this compound, l-threo-dihydroneopterin triphophate. The enzyme, which is here named “d-erythro-dihydroneopterin triphosphate 2′-epimerase,” needs a divalent cation (Mg²⁺ or Mn²⁺ is most effective) for maximal activity. Its molecular weight is estimated at 87 000–89 000. Little or no activity can be detected if either the monophosphate or the phosphate-free form of the substrate is incubated with the enzyme. Evidence is presented to establish that all three phosphate residues of the substrate are retained in the product and that the product is of the l-threo configuration.     

Radioimmunoassay for biopterin in body fluids and tissues

Specific antibodies against l-erythro-biopterin have been prepared in rabbits using the conjugates to bovine serum albumin. The antiserum against l-erythro-biopterin distinguished among l-erythro-tetrahydro- or 7,8-dihydro-biopterin, the other three stereoisomers of biopterin, d-erythro-neopterin, folic acid, and other synthetic pteridines. Using the specific antiserum against l-erythro-biopterin, a radioimmunoassay has been developed to measure the biopterin concentrations in urine, serum, cerebrospinal fluid, and tissues. The conjugate of l-erythro-biopterin with tyramine, 4-hydroxy-2-[2-(4-hydroxyphenyl)ethylamino]-6-(l-erythro-1,2-dihydroxypropyl)pteridine (BP-TYRA), was synthesized and labeled with ¹²⁵I as the labeled ligand for the radioimmunoassay. BP-¹²⁵I-TYRA had similar binding affinity as the natural l-erythro-biopterin and was thus permitted to establish a highly sensitive radioimmunoassay for biopterin. The limit of sensitivity of the radioimmunoassay with BP-¹²⁵I-TYRA as labeled ligand was 0.5 pmol. The total concentration of biopterins, i.e., biopterin, 7,8-dihydro-, quinonoid dihydro and tetrahydrobiopterins, in the biological samples was obtained by iodine oxidation under acidic conditions prior to the radioimmunoassay, whereas iodine oxidation under alkaline conditions gave the concentration only of the former two. Biopterin in urine could be measured directly using 1 μl of urine, but a pretreatment with a small Dowex 50-H⁺ column was required for serum, cerebrospinal fluid, and brain tissues.     

Plastid development in primary leaves of Phaseolus vulgaris

Summary The development of increased activities of ribulosediphosphate carboxylase (EC 4.1.1.39) and of phosphoribulokinase (EC 2.7.1.19) in greening bean leaves was completely inhibited by D-threo chloramphenicol but unaffected by L-threo chloramphenicol. This indicates that these enzymes are synthesized by the ribosomes of the developing plastids. A different mechanism appears to be responsible for the development of activity of NADP-dependent triosephosphate dehydrogenase (EC 1.2.1.13) where the D-threo isomer gave 45% inhibition and the L-threo isomer gave 18% inhibition. Thus both specific (D-threo isomer) and unspecific (both isomers) inhibition occurred. It is suggested that the development of NADP-dependent triosephosphate dehydrogenase activity may result from the allosteric activation, in the plastids, of the NAD-dependent enzyme (Müller et al., 1969) which has been synthesized by cytoplasmic ribosomes. Neither isomer inhibited the development of five other enzymes of the photosynthetic carbon cycle namely ribosephosphate isomerase (EC 5.3.1.6), phosphoglycerate kinase (EC 2.7.2.3), triosephosphate isomerase (EC 5.3.1.1), tructosediphosphate aldolase (EC 4.1.2.13) and transketolase (EC 2.2.1.1), but there was a significant stimulation of the activity of transketolase by D-threo chloramphenicol.     

Reactions of the 3-oxime 2-phenylhydrazone and mixed bishydrazones of dehydro-L-ascorbic acid: Conversion into substituted triazoles and pyrazolinediones

L-threo-2,3-Hexodiulosono-1,4-lactone 2-phenylhydrazone(1) reacted with hydroxylamine to give the 3-oxime 2-phenylhydrazone(2). On boiling with acetic anhydride,2 was dehydrated to 4-[L-threo-2,3-diacetoxy-(1-hydroxypropyl)]-2-phenyl-1,2,3-triazole-5-car?ylic acid lactone(3), which was converted into 2-phenyl-4-(L-threo-1,2,3-trihydroxypropyl)-1,2,3-triazole-5-car?amide(4) with liquid ammonia. The structure of compound4 was confirmed by acetylation to 2-phenyl-4-(L-threo-1,2,3-triacetoxypropyl)-1,2,3-triazole-5-car?amide(5), and by periodate oxidation followed by reduction, to give 4-(hydroxymethyl)-2-phenyl-1,2,3-triazole-5-car?amide(6). Treatment of compound1 with aryl- or aroyl-hydrazines afforded mixed bishydrazones(7–14), which were acetylated to15–21, and treated with hydrazine to give pyrazolinediones22 and23     

Bromine-Induced Selective N-Monodemethylation of N6, N6-Dimethyl-2′,3′-O-isopropylideneadenosine

Abstract

Bromination of the title compound 1 with bromine in phosphate buffer has led to 8-bromo-N⁶, N⁶-dimethyl-2′,3′-0-isopropylidene-adenosine (2) and 2′,3′-0-isopropylidene-N⁶-methyladenosine (3). Under similar conditions, compound 2 gave 8-bromo-2′,3′-0-isopropylidene-N⁶-methyladenosine (4). The transformations 1 → 3 and 2 → 4 represent biomimetic models of in vivo N⁶-demethylation of antibiotic puromycin.     

Isolation and identification of products in the reaction of 2-acetamido-3,4,6-tri-O-acetyl-1,5-anhydro-2-deoxy-d-arabino-hex-1-enitol with theophylline

Fusion of 2-acetamido-3,4,6-tri-O-acetyl-1,5-anhydro-2-deoxy-d-arabino-hex-1-enitol with theophylline, in the presence of boron trifluoride etherate as the catalyst, caused condensation to occur. This reaction afforded a variety of products of nucleosidic character, which were successively separated by repeated chromatography on silica gel. The structures of the products were determined, on the basis of X-ray crystallographic analysis (for three compounds) and by means of n.m.r.-spectral data and mass spectrometry, as the following: 7-(2-acetamido-4,6-di-O-acetyl-2,3-dideoxy-β-d-erythro-hex-2-enopyranosyl)theophylline, the corresponding α- and β-d-threo derivatives, and 7-(2-acetamido-6-O-acetyl-2,3-dideoxy-α-d-threo-hex-2-enopyranosyl)theophylline and its β anomer.In addition to these 2′,3′-unsaturated nucleosides having the base linked at C-1′, three products of a new type, having the base attached at C-4′, were also isolated: 7-(methyl 2-acetamido-6-O-acetyl-2,3,4-trideoxy-β-d-erythro-hex-2-enopyranosid-4-yl)theophylline, and the corresponding α-d-threo and α-d-erythro isomers.The correlation of the data obtained by X-ray structure analysis and proton nuclear magnetic spectroscopy, together with their application for the determination of configuration and conformation of these compounds, are discussed. It appears that the ¹H-n.m.r. data alone do not suffice for unambiguous and correct structure determination for these classes of compounds.     

Phenolic glucoside gallates from quercus mongolica and q. acutissima

Six phenolic glucoside gallates: D-threo-guaiacylglycerol 8-O-, L-threo-guaiacylglycerol 8-O-, 3-methoxy-4-hydroxyphenol 1-O-, gentisic acid 5-O-, 3,5- dimethoxy-4-hydroxyphenol 1-O- and cis-coniferyl alcohol 4-O-β-D-(6′-O-galloyl)glucopyranosides were isolated from Quercus mongolica and Q. acutissima.     

Some Properties of GTP Cyclohydrolase from Serratia indica and the Pterin Product by Its Enzymatic Reaction

GTP cyclohydrolase which catalyzes the formation of formic acid and a pterin compound from guanosine-5′-triphosphate (GTP) has been partially purified from extracts of Serratia indica IFO 3759. ¹⁴C-Formic acid eliminated from (8-¹⁴C)GTP is oxidized with mercury acetate to ¹⁴CO₂, which is trapped by β-phenylethylamine. The molecular weight of the enzyme is approximately 170,000 and the enzyme is relatively heat-stable. The enzyme activity is strongly inhibited by GDP and ATP, but not by other nucleotides. Inhibition by GDP is competitive with GTP. Metals, such as Fe²⁺, Co²⁺, Ni²⁺, Zn²⁺, Cd²⁺, Al³⁺, Hg²⁺ and p-chloromercuribenzoate strongly inhibit the enzyme activity. The activity is also inhibited by . The pterin product has been characterized as a derivative of neopterin triphosphate by enzymatic degradations, ultraviolet spectra, fluorescence and excitation spectra, thin-layer chromatography and thin-layer electrophoresis. The product is estimated to differ from d-erythro-neopterin triphosphate prepared from the enzyme system of Escherichia coli B, since (1) only one mole of phosphate can be liberated by alkaline phosphatase and two moles of phosphates by phosphodiesterase and alkaline phosphatase from the product, and (2) the retention time of the product on high-performance liquid chromatography is different from that of d-erythro-neopterin triphosphate.     

Absolute stereochemistry of methanopterin and 5,6,7,8-tetrahydromethanopterin

The configuration at the C-9 of methanopterin (MPT) has been determined by comparing the circular dichroism (CD) spectra of MPT and its hydrolytic fragment, 1-[4-[[1-(2-amino-7-methyl-4-hydroxy-6-pteridinyl)-ethyl]amino]phenyl]-1-deoxy-D -ribitol (HP-1), with the CD spectra of a series of model compounds of known stereochemistry. These compounds included (S)-6-[1-(4-carboxymethylanilino)ethyl]pterin, (S-6(1-hydroxyethyl)-7-methylpterin, (S-6-(1-hydroxyethyl)pterin, (R)-6-(1-phenoxyethyl)pterin, D (+)-neopterin, and L -biopterin. From this comparison it was concluded that MPT has the R configuration at C-9 and is thus configurationally related to D (+)-neopterin, which has the S configuration at C-1. From previous work establishing the relative stereochemistry at C-6, C-7, and C-9 of N⁵-N¹⁰-methenyl-5,6,7,8-tetrahydromethanopterin (N⁵-N¹⁰-methenyl-H₄MPT) as R, S, and R, respectively, it is clear that the remaining asymmetric carbons at C-6 and C-7 of H₄MPT have the S and S configuration, respectively. Comparison of these latter two positions to the equivalent carbons in 5,6,7,8-tetrahydrofolate (H₄folate) show that the steps involved in the biological reduction of MPT to H₄MPT occur with the same stereochemical outcome as those involved in the biological reduction of folate to H₄folate. © 1996 Wiley-Liss, Inc.     

Synthesis and Antiviral Evaluation of the β-L-Enantiomers of Some Thymine 3′-Deoxypentofuranonucleoside Derivatives

Abstract

3′-Deoxy-β-L-erythro- (3), 3′-deoxy-β-L-thero- (6), 2′-fluoro- (7) and 2′-azido-2′,3′-dideoxy-β-L-erythro- (10) pentofuranonucleoside derivatives of thymine have been synthesized and their antiviral properties examined. All these derivatives were stereospecifically prepared by glycosylation of thymine with a suitable peracylated 3-deoxy-L-erythro-pentofuranose sugar (1), followed by appropriate chemical modifications. The prepared compounds were tested for their activity against HIV, but they did not show an antiviral effect.     

Synthesis of a Novel C-Nucleoside, 2-Amino-7-(2-deoxy-β- D-erythro-pentofuranosyl)-3H,5H-pyrrolo-[3,2-d]pyrimidin-4-one (2′-Deoxy-9-deazaguanosine)

Abstract

A synthesis of the C-nucleoside, 2-amino-7-(2-deoxy-β-D-erythro-pentofuranosyl)-3H,5H-pyrrolo[3,2-d]pyrimidin-4-one (9-deaza-2′-deoxyguanosine) was achieved starting from 2-amino-6-metnyl-3H-pyrimidin-4-one (5) and methyl 2-deoxy-3,5-di-O-(p-nitrobenzoyl)- D-erythro-pento-furanoside (11). The anomeric configuration of the C-nucleoside was established by ¹H NMR, NOEDS and ROESY. This C-nucleoside did not inhibit the growth of T-cell lymphoma cells.     

Chiral lumazines: Preparation,properties, enantiomeric separation

Optically active lumazines (biolumazine, dictyolumazine, monalumazine, and neolumazine) are prepared from the corresponding pterins by enzymatic reaction, using pterin deaminase excreted by Dictyostelium discoideum. The fluorescence properties, circular dichroism spectra, and chromatographic behavior of these lumazines are studied. D - and L -enantiomers of biolumazine, dictyolumazine, and monalumazine are separated using a chiral flavoprotein column. This column also separates the enantiomeric pterins of the threo form: monapterin and dictyopterin. However, the column does not separate the enantiomeric pterins of the erythro form: neopterin and biopterin. By coupling a reverse-phase column to the flavoprotein column, the separation of pterins and lumazines in function of their hydrophobicity, as well as the separation of the diastereomers, is achieved. This coupled achiral/chiral high-performance liquid chromatography method enables determination of the stereoconfiguration of natural lumazines by comparison with optically pure compounds. A lumazine derivative, present in the extracellular medium of Dictyostelium discoideum, is identified as D -dictyolumazine, i.e., 6-(D -threo-1,2-dihydroxypropyl)-lumazine. © 1994 Wiley-Liss, Inc.     

Synthèse de nucléosides pyrimidiquesàpartir de 1,2-anhydro-4-désoxy-3-O-méthyl-pentopyranoses

Monotosylation of 4-deoxy-3-O-methyl-dl-threo- and -erythro-pentopyranose led, in 62–68% yield, to the 2-O-tosyl derivatives which, on treatment at room temperature with sodium hydride in anhydrous ether, gave quantitatively 1,2-anhydro-4-deoxy-3-O-methyl-dl-threo- and -erythro-pentopyranose, respectively. These epoxides reacted with 2,4-dimethoxypyrimidine in the presence of pyridinium hydrochloride to give, in 68–78% yield, 1-(4-deoxy-3-O-methyl-β-dl-erythro- and -α,β-dl-threo-pentopyranosyl)-4-methoxy-2-pyrimidinone, respectively. Isomers having a trans-1′,2′ configuration were preponderantly formed by an Sn2 reaction.     

The preparation of high specific activity [3H]chloramphenicol base and chloramphenicol labeled in the propanediol side chain

Methods are described for the synthesis of [³H]chloramphenicol and derivatives labeled on carbon 1 of the propanediol side chain, with a specific activity of about 2 mCi/μmol. The labeling step involves the reduction of the I-oxo derivative of N-acetyl chloramphenicol base by KB³H₄ to produce a mixture of the d (?) threo- and d (?) erythro-diastereoisomers, since carbon 1 is an asymmetric carbon atom. The two isomers were separated by thin-layer chromatography after acetylation of the two free hydroxyls. After hydrolysis of the three acetyl groups, the biologically active d (?) threo-[1 ? ³H]chloramphenicol base was converted to chloramphenicol. Modification of the above procedures allows the rapid and simple preparation of the mixed d (?) threo- and d (?) erythro-isomers of [1 ? ³H]chloramphenicol. This mixture can be used where the presence of the inactive d (?) erythro-isomer of chloramphenicol is not important. The modified procedure also allows the preparation of the mixed isomers of [1 ? ³H]chloramphenicol base and of chloramphenicol analogs. Attempts to prepare a 3-aldehyde derivative of chloramphenicol were not successful. If this could be done, reduction of this derivative with KB³H₄ would produce the correct isomer of chloramphenicol since carbon atom 3 on the propanediol side chain is not an asymmetric carbon atom.     

Stereoselective Synthesis of erythro-Serricornin, (4R, 6R, 7S)-, and (4S, 6R, 7S)-4,6-Dimethyl-7-hydroxynonan-3-one,Stereoisomers of the Sex Pheromone of Cigarette Beetle

A stereoselective synthesis of erythro-serricornin [(4RS,6R,7S)-4,6-dimethyl-7-hydroxynonan-3-one] was completed starting from l-(+)-tartaric acid. The relative configuration of C(6)-methyl and C(7)-hydroxyl groups in naturally occurring serricornin was threo.