Polymerization of nucleotide analogues I: Reaction of nucleoside 5′-phosphorimidazolides with 2′-amino-2′-deoxyuridine

Summary 2-Amino-2-deoxyuridine reacts efficiently with nucleoside 5-phosphorimidazolides in aqueous solution. The dinucleoside monophosphate analogues were obtained in yields exceeding 80% under conditions in which little reaction occurs with the natural nucleosides.In a similar way, the 5-phosphorimidazolide of 2-amino-2-deoxyuridine undergoes self-condensation in aqueous solution to give a complex mixture of oligomers.The phosphoramidate bond in the dinucleoside monophosphate analogues is stable for several days at room temperature and pH 7. The mechanisms of their hydrolysis under acidic and alkaline conditions are described.Abbreviations A adenosine - C cytidine - G guanosine - U uridine - T thymidine - U_{N 3} 2-azido-2-deoxyuridine - U_{NH 2} 2-amino-2-deoxyuridine - ImpA adenosine 5-phosphorimidazolide - ImpU uridine 5-phosphorimidazolide - ImpU_{N 3} 2-azido-2-deoxyuridine 5-phosphorimidazolide - ImpU_{NH 2} 2-amino-2-deoxyuridine 5-phosphorimidazolide - pA adenosine 5-phosphate - pU uridine 5-phosphate - pU_{N 3} 2-azido-2-deoxyuridine 5-phosphate - pU_{NH 2} 2-amino-2-deoxyuridine 5-phosphate - UpA uridylyl-[35]-adenosine - UpU uridylyl-[35]-uridine - U_NpA adenylyl-[52]-2-amino-2-deoxy-uridine - U_NpU uridylyl-[52]-2-amino-2-deoxyuridine (pU_N)_n n=2,3,4 [25]-linked oligomers of pU_{NH 2} poly(A) polyadenylic acid - Im imidazole - MeIm l-methylimidazole     

A fluorometric assay of ceramide glycanase with 4-methylumbelliferyl β-d-lactoside derivatives

4-Methylumbelliferyl 6-O-benzyl--d-lactoside (6Bn-MU-Lac) and some related compounds were synthesizedvia different selective reactions including phase-transfer glycosylation. Their suitability as substrates for a fluorometric assay of ceramide glycanase (CGase) was evaluated. Among others, the 6Bn-MU-Lac, which is resistant to exogalactosidase, was found to be a suitable substrate for routine assay of the CGase activity. For American leech CGase, theK _m value is 0.232 mM at pH 5. Abbreviations: CGase, ceramide glycanase; Gal, galactose; Glc, Glucose; Lac, lactose; MU, 4-methylumbelliferone; MU-Lac, 4-methylumbelliferyl -d-lactoside; bBn-Lac, 6-O-benzyl-lactose; 6Bn-MU-Lac, 4-methylumbelliferyl 6-Obenzyl--d-lactoside; 46Bd-MU-Lac, 4-methylumbelliferyl 4,6-O-benzylidene--d-lactoside; MU-Cel, 4-methylumbellifery -d-cellobioside; 46Bd-MU-Cel, 4-methylumbelliferyl 4,6-O-benzylidene--d-cellobioside; TLC, thin layer chromatography;¹H-NMR, proton nuclear magnetic resonance; GSL, glycosphingolipids; CSA, 10-camphorsulfonic acid. See Scheme 1 for chemical structures.     

Carotenoid pigments of Sorangium compositum (Myxobacterales) including two new carotenoid glucoside esters and two new carotenoid rhamnosides

Summary The carotenoid pigments of the myxobacterium Sorangium compositum were analyzed by chromatographical and chemical techniques and by visible, infra red, and mass spectroscopy. Besides -carotene, neurosporene, torulene, lycopene, and 1,2-dihydro-1-hydroxy--carotene, four new carotenoid glycosides were found. These pigments were identified as 1,2-dihydro-1-hydroxy-torulene glucoside ester (I), 1,2-dihydro-3,1-dihydroxy-torulene glucoside ester (III), 1,2-dihydro-1-hydroxy-torulene rhamnoside (II), and 1,2-dihydro-3,1-dihydroxytorulene rhamnoside (IV).Fifth communication on the carotenoids of myxobacteria. Fourth communication see Arch. Mikrobiol. 76, 364–380 (1971).     

Transient suppression of F-plasmid incompatibility in a strain of Escherichia coli

Summary The incompatibility between F plasmids is transiently suppressed in Escherichia coli strain CSH54. As a result this strain is able to maintain two F factors or an F factor and a mini-F plasmid for considerably longer periods than normal strains. When selective pressure for two markers carried by two separate Fs (or an F and mini-F) is imposed on normal strains, the two plasmids tend to form a cointegrate structure which can be detected genetically by the joint transfer of both the markers upon mating. This does not happen in CSH54; instead, the two plasmids are maintained and transferred independently. Physical evidence for the maintenance of an F and a mini-F plasmid is provided by agarose gel electrophoresis.     

Monomers for Oligonucleotide Synthesis with Linkers Carrying Reactive Residues: II. The Synthesis of Phosphoamidites on the Basis of Uridine and Cytosine and Containing a Linker with Methoxyoxalamide Groups in Position 2′

A convenient preparative synthesis of 2-amino-2-deoxyuridine was developed. Starting from 2-amino-2-deoxyuridine and 2-amino-2-deoxycytidine, monomers for the phosphoamidite oligonucleotide synthesis were obtained that carry a linker with methoxyoxalamide groups in position 2.     

Cleavage of nucleotides by Ce4+ and the lanthanide metal complexes

The cleavage of adenosine-5-monophosphate (5-AMP) and guanosine-5-monophosphate (5-GMP) by Ce⁴⁺ and lanthanide complex of 2-carboxyethylgermanium sesquioxide (Ge-132) in acidic and near neutral conditions was investigated by NMR , HPLC and measuring the liberated inorganic phosphate at 37°C and 50°C. The results showed that 5-GMP and 5-AMP was converted to guanine (G), 5-monophosphate (depurination of 5-GMP), ribose (depurination and dephosphorylation of 5-GMP), phosphate and adenine (A), 5-monophosphate (depurination of 5-AMP), ribose (depurination and dephosphorylation of 5-AMP), phosphate respectively by Ce⁴⁺. In presence of lanthanide complexes, 5-GMP and 5-AMP were converted to guanosine (Guo) and phosphate and adenosine (Ado) and phosphate respectively. The mechanism of cleaving 5-GMP and 5-AMP is hydrolytic scission     

The effect of the methyl substituent on the bioconversion of cyclohexene derivatives

Summary The absence of the methyl substituent at the 2position of the cyclohexene ring of TCHP enhances the conversion rate as well as the yields of the 3-hydroxy product obtained byStreptomyces natalensis and the 3-keto product obtained byMycobacterium smegmatis.Abbreviations TCHP 1-(2-thienyl)-3-(1-cyclohexen-1-yl)-1-propanone - TCHP-OH 1-(2-thienyl)-3-(3-hydroxyl-1-cyclohexen-1-yl)-1-propanone - TCHP-ketone 1-(2-thienyl)-3-(1-cyclohexen-1-yl-3-one)-1-propane - TMCHP 1-(2-thienyl)-3-(2-methyl-1-cyclohexen-1-yl)-propanone     

The formation of peptides from the 2′(3′)-glycyl ester of a nucleotide

Summary We have synthesized 2(3)-O-(glycyl)-adenosine-5-(O-methylphos-phate), an analogue of the 3-terminus of aminoacylated tRNA. A 0.4M solution of this compound maintained at pH 8.2, yields 5.5% of diglycine and 11.5% of diketopiperazine, in addition to the hydrolysis products glycine and adenosine-5-(O-methylphosphate). Under the same conditions, glycine ethyl ester reacts much more slowly, but ultimately gives similar yields of diglycine and diketopiperazine.The aminolysis of 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) by free glycine is relatively inefficient, but serine reacts 20 times more rapidly and yields up to 50% of N-glycylserine. The prebiotic significance of these reactions is discussed.Abbreviations MepA adenosine-5-(O-methylphosphate) - MepA-gly 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) - MepA-bis-gly 2,3-O-(bis-glycyl)-adenosine-5-(O-methylphosphate) - DKP diketopiperazine - gly Et glycine ethyl ester - gly-ser N-glycylserine - O-gly-ser O-glycylserine - O-(gly)-gly-ser O-(glycyl)-glycylserine - Boc-gly N-tert-butyloxycarbonylglycine - MepA-Boc-gly 2(3)-O-(Boc-glycyl)-adenosine-5-(O-methylphosphate) - MepA-bis-Boc-gly 2,3-O-(bis-Boc-glycyl)-adenosine-5(O-methylphosphate) - (gly)₂ diglycine - (gly)₃ triglycine     

Regioselective preparation of 2′, 3′-di-O-acylribonucleosides carrying lipophilic acyl groups through a lipase-catalysed alcoholysis

Candida antarctica B lipase-catalysed alcoholysis of 2, 3, 5-tri-O-hexanoyluridine (1a), 2, 3, 5-tri-O-dodecanoyluridine (1b), 2, 3, 5-tri-O-hexanoylinosine (1c) and 2, 3, 5-tri-O-dodecanoylinosine (1d) proceeded regioselectively to produce the corresponding 2, 3-di-O-acylribonucleosides 2a–d, providing a simple and efficient access to these new lipophilic compounds. Contrasting to the alcoholysis, enzymatic hydrolysis of 1a–d using different enzymes and experimental conditions did not proceed regioselectively.     

Determination of the backbone torsion angle ɛ in nucleic acids

Summary The multiplet structure of cross peaks in double-quantum-filtered COSY NMR spectra is analysed for those resonances that include passive heteronuclear couplings. Interestingly, the cross peak involving the sugar-ring protons H2 and H3 in nucleic acids display an E.COSY-type appearance exclusively when the backbone torsion angle (C4-C3-O3-P) adopts a gauche(-) conformation. This observation allows an unambiguous analysis of the conformation around , without the knowledge of ³J_cp coupling constants.     

Localization of 5′-nucleotidase in bovine brain myelin fraction and myelin subfractions

Purified myelin from fresh calf brain white matter was subfractionated in a discontinuous sucrose gradient; significant recovery of protein and 2,3-cyclic nucleotide 3-phosphohydrolase (CNP) and 5-nucleotidase (5N) activities occurred in all three obtained subfractions, the highest recovery being in the light subfraction; highest 5N and CNP specific activities were in medium myelin. Purified myelin was also subfractionated in a continuous sucrose gradient, with a similar localization of protein; CNP activity and 5N activity maxima suggest that myelin may be a predominant locus of 5N in bovine brain white matter. Freezing of brain white matter caused an increase in protein and in CNP and 5N total activity recoveries in denser myelin subfractions. Cytochemistry showed the reaction product of 5N in the whole myelin fraction to be associated with the innermost, outermost and medial compact myelin layers. Effects of non-ionic detergent (Lubrol WX) on 5N activity were studied, and the results also suggest the intrinsic nature of 5N as an ectoenzyme in myelin membranes. Lubrol WX was viewed as an advisable detergent for the stimulation of myelin 5N activity, but not for the solubilization of this enzyme.     

Phagostimulation of the mediterranean fruit fly, Ceratitis capitata by ribonucleotides and related compounds

Females of the medfly, Ceratitis capitata, prefer sucrose solutions containing ribonucleotides to sucrose solutions without them. The order of preference for the nucleotides was: 5GMP>GTP>5CMP>5IMP >dGMP>5UMP>5AMP>5XMP=ATP=2 & 3GMP=RP>3AMP.2AMP, guanosine, inosine, adenine and 5TMP produced no significant stimulation. Females sterilized by irradiation showed reduced attraction to 5GMP as compared to non-irradiated females.Optimal molecular configuration for phagostimulation includes: phosphorylation at the 5 position of the ribose, free hydroxyl groups at 2 and 3 on the ribose, and an NH₂ group at the 2 position of the aromatic ring of purine.It is proposed that the 5GMP in yeast hydrolyzate can be used as a measure of the suitability of the hydrolyzate as a bait.

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Résumé La femelle de la mouche méditerranéenne des fruits, Ceratitis capitata, préfère les solutions de sucrose contenant des ribonucléotides aux simples solutions de sucrose. Lórdre de préférence pour les nucléotides est le suivant: 5GMP>GTP>5CMP>5IMP >dGMP>5UMP>5AMP>5XMP=ATP =2 & 3GMP=RP>3AMP.Le 2AMP, la guanosine, l'inosine, l'adénine et le 5TMP provoquent une stimulation significative. Les femelles montrent aprés stérilisation par irradiation une attirance réduite pour le 5GMP par comparaison avec les femelles non-irradiées.La configuration moléculaire optimale pour la phagostimulation comprend: la phosphorylation en position 5 du ribose; des groupes hydroxyles libres en 2 et 3 sur le ribose; et un groupe NH₂ en position 2 sur le noyau aromatique.Nous proposons que le 5GMP dans l'hydrolysat de levure puisse être utilisé pour mesurer la capacité de l'hydrolysat comme appât.

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Abbreviations 5AMP Adenosine 5-monophosphate - 3AMP Adenosine 3-monophosphate - 2AMP Adenosine 2-monophosphate - dAMP 2-deoxyadenosine 5-monophosphate - ADP Adenosine 5-diphosphate - ATP Adenosine 5-triphosphate - 5GMP Guanosine 5-monophosphate - 2GMP Guanosine 2-monophosphate - 3GMP Guanosine 3-monophosphate - dGMP 2-deoxyguanosine 5-monophosphate - GDP Guanosine 5-diphosphate - GTP Guanosine 5-triphosphate - 5IMP Inosine 5-monophosphate - IDP Inosine 5-diphosphate - ITP Inosine 5-triphosphate - 5XMP Xanthosine 5-monophosphate - 5CMP Cytidine 5-monophosphate - dCMP 2 deoxycytidine 5-monophosphate - CTP Cytidine 5-triphosphate - 5UMP Uridine 5-monophosphate - 5TMP Thymidine 5-monophosphate - RP Ribose 5 monophosphate     

Adenosine-5′-phosphosulfate (APS) as sulfate donor for assimilatory sulfate reduction in Rhodospirillum rubrum

Crude extracts of Rhodospirillum rubrum catalyzed the formation of acid-volatile radioactivity from (³⁵S) sulfate, (³⁵S) adenosine-5-phosphosulfate, and (³⁵S) 3-phosphoadenosine-5-phosphosulfate. An enzyme fraction similar to APS-sulfotransferases from plant sources was purified 228-fold from Rhodospirillum rubrum. It is suggested here that this enzyme is specific for adenosine-5-phosphosulfate, because the purified enzyme fraction metabolized adenosine-5-phosphosulfate, however, only at a rate of 1/10 of that with adenosine-5-phosphosulfate. Further, the reaction with 3-phosphoadenosine-5-phosphosulfate was inhibited with 3-phosphoadenosine-5-phosphate whereas this nucleotide had no effect on the reaction with adenosine-5-phosphosulfate. For this activity with adenosine-5-phosphosulfate the name APS-sulfotransferase is suggested. This APS-sulfotransferase needs thiols for activity; good rates were obtained with either dithioerythritol or reduced glutathione; other thiols like cysteine, 2-3-dimercaptopropanol or mercaptoethanol are less effective. The electron donor methylviologen did not catalyze this reaction. The pH-optimum was about 9.0; the apparent K _m for adenosine-5-phosphosulfate was determined to be 0.05 mM with this so far purified enzyme fraction. Enzyme activity was increased with K₂SO₄ and Na₂SO₄ and was inhibited by 5-AMP. These properties are similar to assimilatory APS-sulfotransferases from spinach and Chlorella.Abbreviations APS adenosine-5-phosphosulfate - PAPS 3-phosphoadenosine-5-phosphosulfate - 5-AMP adenosine-5-monophosphate - 3-AMP adenosine-3-monophosphate - 3-5-ADP 3-phosphoadenosine-5-phosphate (PAP) - DTE dithiorythritol - GSH reduced glutathione - BAL 2-3-dimercaptopropanol     

Influence of cyclic nucleotides on amphibian ectoderm

Summary Isolated amphibian (Triturus alpestris) gastrula ectoderm was treated with cyclic nucleotides for 24 h and cultured up to 12 days. Explants treated with$cyclic N⁶-Monobutyryl-adenosine-35-monophosphate, cyclic Dibutyryladenosine-35-monophosphate and cyclic Dibutyrylguanosine-35-monophosphate in a concentration of 10^–3 and 10^–5 M did not differentiate into mesoderm- or endoderm-derived tissues. The number of explants with small neural and neuroid structures did not exceed the percentage found in the control series. Inductions could also not be obtained when ectoderm was dissociated prior to the treatment with cyclic nucleotides, or when theophylline (which inhibits phosphodiesterase) was added to the culture medium. The results are discussed with regard to the possible mode of action of the vegetalizing factor.     

Expression of a soybean β-conclycinin gene under the control of the Cauliflower Mosaic Virus 35S and 19S promoters in transformed petunia tissues

A gene encoding the -subunit of -conglycinin was ligated to the 19S and 35S promoters of Cauliflower Mosaic Virus and introduced into petunia plants on a disarmed Ti-plasmid using Agrobacterium tumefaciens. Transformed cells were regenerated into whole plants and ummunoreactive polypeptides and hybridizable, polyadenylated mRNA were detected in transformed tissues. Expression from the 35S promoter was 10 to 50 times greater than expression from the 19S promoter. The level of immunodetectable polypeptides was greater in seeds than in leaves or callus tissue. In addition, the pattern of -polypeptide breakdown products was distinctive in seeds and leaves. We conclude that in seeds the higher levels of the -polypeptide reflect enhanced stability of this protein.     

A novel PH-CT-COSY methodology for measuring JPH coupling constants in unlabeled nucleic acids. Application to HIV-2 TAR RNA

A quantitative analysis of J_PH scalar couplings in nucleic acids is difficult due to small couplings to phosphorus, the extreme overlap of the sugar protons and the fast relaxation of the spins involved in the magnetization transfer. Here we present a new methodology that relies on heteronuclear Constant Time Correlation Spectroscopy (CT-COSY). The three vicinal ³J_PH3, ³J_PH5 and ³J_PH5 scalar couplings can be obtained by monitoring the intensity decay of the P_i-H3_{i – 1} peak as a function of the constant time T in a 2D correlation map. The advantage of the new method resides in the possibility of measuring the two ³J_PH5 and ³J_PH5 scalar couplings even in the presence of overlapped H5/H5 resonances, since the quantitative information is extracted from the intensity decay of the P-H3 peak. Moreover, the relaxation of the H3 proton is considerably slower than that of the H5/H5 geminal protons and the commonly populated conformations of the phosphate backbone are associated with large ³J_PH3 couplings and relatively small ³J_{PH5 / H5}. These two facts lead to optimal signal-to-noise ratio for the P-H3 correlation compared to the P-H5/H5 correlation.The heteronuclear CT-COSY experiment is suitable for oligonucleotides in the 10–15 kDa molecular mass range and has been applied to the 30mer HIV-2 TAR RNA. The methodology presented here can be used to measure P-H dipolar couplings (D_PH) as well. We will present qualitative results for the measurement of P-H_base and P-H2 dipolar couplings in the HIV-2 TAR RNA and will discuss the reasons that so far precluded the quantification of the D_PHs for the 30mer RNA.     

Facile synthesis of 2′-substituted lactoses

Isopropylidenation of lactose with 2,2-dimethoxypropane in the presence ofp-toluenesulfonic acid gave two products, which were identified by¹H- and¹³C-NMR as 2,35,63,4-tri-O-isopropylidenelactose dimethyl acetal (1) and its 6-O-(2-methoxy)-isopropyl derivative (2). These products were used for the synthesis of 2-O-methyllactose (7), 2,6-di-O-methyllactose (9) and 2-O-benzyllactose (13).     

A unique surface membrane anchored purine-salvage enzyme is conserved among a group of primitive eukaryotic human pathogens

Previously, we isolated and characterized the gene encoding the 3-Nucleotidase/Nuclease (Ld3NT/NU) from the human pathogen, Leishmania donovani. This unique cell surface enzyme has been shown to be involved in the salvage of host-derived purines, which are essential for the survival of this important protozoan parasite. In this report, we assessed whether the 3-Nucleotidase/Nuclease was conserved amongst other pathogenic Leishmania and related trypanosomatid parasites. Results of pulsed field gel electrophoresis and Southern blotting showed that a Ld3NT/NU gene homolog was present in each of the visceral and cutaneous Leishmania species tested (i.e. isolates of L. donovani, L. infantum, L. tropica, L. major and L. mexicana, respectively). Further, results of colorimetric assays using 3-adenosine monophosphate as substrate demonstrated that each of these organisms also expressed significant levels of 3-nucleotidase enzyme activity. In addition, we showed that a Ld3NT/NU gene homolog was expressed in each of these Leishmania species as a > 40 kDa 3-nucleotidase enzyme activity. A Ld3NT/NU gene homolog was also identified in two Crithidia species (C. fasciculata and C. luciliae) and Leptomonas seymouri but was only marginally detectable in Trypanosoma brucei, Trypanosoma cruzi and Phytomonas serpens. Cumulatively, results of this study showed that an Ld3NT/NU homolog was conserved amongst pathogenic Leishmania sp. which suggests that this enzyme must play an critical role in purine salvage for all members of this group of human pathogens.     

AMP synthesis in aqueous solution of adenosine and phosphorus pentoxide

Possible formation of a P₄O₁₀ molecule in magma, the stability of the molecule in hydrous volcanic gas at high temperatures and a possible prebiotic phosphate cycle were discussed in relation to chemical evolution. To demonstrate the utility of phosphorus pentoxide as a phosphorylating agent, aqueous solutions of adenosine (0.02M) and phosphorus pentoxide (0.2M) were incubated at 37°C for 5 months. The pH of the solutions was adjusted every day or every few days to each fixed value (9.0, 10.5, 11.5, 12.5) with 10 N NaOH. The HPLC analysis showed the formation of 2-AMP, 3-AMP, 5-AMP, cyclic (2–3)-AMP and cyclic (3–5)-AMP. The main components of the products were 2- and 3-AMP, though cyclic (2–3)-AMP was the main component in the early period of the incubation at pH 9.0. The yields (conversion rate of adenosine to AMPs) were increased almost linearly with the incubation time for 5 months in the case of pH 9.0. The final yields were about 3% (pH 9.0), 6% (pH 9.0, 1 M NaCl), 5% (pH 9.0, 0.01 M CaCl₂, 0.01 M MgCl₂), 7% (pH 9.0, 0.5 M NaCl, 0.01 M CaCl₂, 0.01 M MgCl₂), 9% (pH 9.0, 1 M NaCl, 0.01 M CaCl₂, 0.01 M MgCl₂), 32% (pH 10.5), 43% (pH 11.5), 35% (pH 12.5).     

Oligomerization reactions of deoxyribonucleotides on montmorillonite clay: The effect of mononucleotide structure,phosphate activation and montmorillonite composition on phosphodiester bond formation

2-d-5-GMP and 2-d-5-AMP bind 2 times more strongly to montmorillonite 22A than do 2-d-5-CMP and 5-TMP. The dinucleotide d(pG)₂ forms in 9.2% yield and the cyclic dinucleotide c(dpG)₂ in 5.4% yield in the reaction of 2-d-5-GMP with EDAC in the presence of montmorillonite 22A. The yield of d(pC)₂ (2.0%) is significantly lower but comparable to that obtained from 5-TMP. The yield of dimers which contain the phosphodiester bond decreases as the reaction medium is changed from 0.2 M NaCl to a mixture of 0.2 M NaCl and 0.075 M MgCl₂. A low yield of d(pA)₂ was observed in the condensation reaction of 5-ImdpA on montmorillonite 22A. The cyclic nucleotide (3,5-cdAMP) was obtained in 14% yield from 3-ImdpA. The yield of d(pA)₂ obtained when EDAC is used as the condensing agent increases with increasing iron content of the Na⁺-montmorillonite used as catalyst. Evidence is presented which shows that the acidity of the Na⁺-montmorillonite is a necessary but not sufficient factor for the montmorillonite catalysis of phosphodiester bond formation.