Imidazole Nucleosides IV Nucleosides of 5 (4)-Formamido-imidazole-4 (5)carboxamide

Abstract

Nucleosides of 5(4)-aminoimidazole-4(5)-carboxamide were formylated with sodium formate, formic acid and acetic anhydride to the β-D-ribo-, α-D-arabino-, α-L-arabino- and β-D-xylofuranosides of 5-formamidoimidazole-4-carboxamide, and to the β-D-ribo-, β-D-arabi-no-, α-D-arabino- and α-L-arabinopyranosides of 4-formamidoimidazole-5-carboxamide.     

Circular dichroism spectra of DNA quadruplexes [d(G(5)T(5))](4) as formed with G(4) and T(4) tetrads and [d(G(5)T(5)). d(A(5)C(5))]2 as formed with Watson-Crick-like (G-C)(2) and (T-A)(2) tetrads

We utilize electrophoresis and find that a thermally treated equimolar mixture of the oligonucleotide d(G(5)T(5)) and its complementary oligonucleotide d(A(5)C(5)) exhibits either two bands or a single band in one lane, depending on the conditions of the incubation solutions. The thermally treated d(G(5)T(5)) solution loaded in a different lane exhibits a single band of the parallel quadruplex [d(G(5)T(5))](4), which is composed of homocyclic hydrogen-bonded G(4) and T(4) tetrads previously proposed. For the thermally treated equimolar mixture of d(G(5)T(5)) and d(A(5)C(5)), the fast band is assigned to a Watson-Crick d(G(5)T(5)). d(A(5)C(5)) duplex, so that the slow band with the same low mobility as that of [d(G(5)T(5))](4) may be assigned to either [d(G(5)T(5))](4) itself or a [d(G(5)T(5)). d(A(5)C(5))](2) quadruplex. If the latter compound is true, this may be the antiparallel quadruplex composed of the heterocyclic hydrogen-bonded G-C-G-C and T-A-T-A tetrads proposed previously. After removing these three bands for the duplex and two kinds of hypothetical quadruplexes, we electrophoretically elute the corresponding compounds in the same electrophoresis buffer using an electroeluter. The eluted compounds are ascertained to be stable by electrophoresis. The circular dichroism (CD) and UV absorption spectra measured for the three isolated compounds are found to be clearly different. For the electrophoretic elution of the hypothetical [d(G(5)T(5))](4) quadruplex, the result of the molecularity of n = 4 obtained from the CD melting curve analysis provides further support for the formation of the parallel [d(G(5)T(5))](4) quadruplex already proposed. For the thermally treated equimolar mixture of d(G(5)T(5)) and d(C(5)A(5)), the fast band with a molecularity of n = 2 corresponds to the Watson-Crick duplex, d(G(5)T(5)). d(A(5)C(5)). The slow band with a molecularity of n = 4 indicates the antiparallel quadruplex [d(G(5)T(5)). d(A(5)C(5))](2), whose observed CD and UV spectra are different from those of [d(G(5)T(5))](4). By electrophoresis, after reannealing the eluted compound [d(G(5)T(5)). d(A(5)C(5))](2), a distinct photograph showing the band splitting of this quadruplex band into the lower duplex and upper quadruplex bands is not possible; but by a transilluminator, we occasionally observe this band splitting with the naked eye. The linear response polarizability tensor calculations for the thus determined structures of the [d(G(5)T(5))](4) quadruplex, the McGavin-like [d(G(5)T(5)). d(A(5)C(5))](2) quadruplex, and the Watson-Crick d(G(5)T(5)). d(A(5)C(5)) duplex are found to qualitatively predict the observed CD and UV spectra.     

Studies on the Accumulation of 5(4)-Amino-4(5)-imidazolecarboxamide Riboside by Escherichia coli

The acculnulation of 5 (4) -amino-4 (5) -imidazolecarboxamide riboside (AICA-R) in the culture medium of sulfonamide-inhibited Escherichia coli, and E. coli-like bacteria was studied. E. coli strain Band 32 strains of E. coli-like bacteria accumulated more than 50 μmoles of AICA-R in test tube scale experiments, and one of E. coli-like bacteria accumulated 358 μmoles. E. coli B-96 (purine-requiring mutant) had ability to accumulate AICA-R in the glucose-salt medium containing purine bases, especially xanthine. The addition of glycine alone or together with glutamic acid to the glucose-salt medium increased the accumulation of AICA-R by sulfadiazine-inhibited E. coli strain B. The accumulation was considerably increased by the addition of polypeptone or casein hydrolysate.

AICA-R accumulated during sulfadiazine bacteriostasis of E. coli strain B was purified and crystallized according to the procedure of Greenberg and Spilman, and light amber colored crystals were obtained.     

Specific phosphorylase from Euglena gracilis splits diadenosine 5',5''-P(1),P(4)-tetraphosphate (Ap(4)A) and diadenosine 5',5''-P(1),P(3)-triphosphate (Ap(3)A)

1. Phosphorolytic cleavage of Ap(4),A was demonstrated in cell-free extracts from two protozoan organisms, Euglena gracilis and Acanthamoeba castellanii. 2. A specific dinucleoside oligophosphate (DNOP) alpha, beta-phosphorylase which degrades substrates with formation of corresponding nucleoside 5'-diphosphate (NDP) as one of the reaction products was purified 625-fold from Euglena gracilis cells. 3. In addition to Ap(4)A, the phosphorylase degrades AP(3)A, Ap(5)A, Gp(4)G and one of phosphonate analogs, ApppCH(2)pA. The K(m) values for Ap(4), A and Ap(3) A are 27 and 25 micron, and relative velocities 100 and 14, respectively. The K(m) for phosphate is 0.5 mM. 4. Some anions (arsenate, chromate, molybdate and vanadate) can substitute for phosphate in the catalyzed reactions and in their presence the DNOPs yield corresponding nucleoside 5'-monophosphate as one of the reactions' product. The enzyme supports also an anion-dependent dephosphorylation of NDPs. 5. Molecular weight of the native Euglena phosphorylase is 30,000. Optimum pH for its activity is at 8.0 Divalent metal cations are essential for the phosphorolysis of DNOPs but are not for the NDP dephosphorylation mentioned.     

(2-Aryl-5-methylimidazol-4-ylcarbonyl)guanidines and (2-aryl-5-methyloxazol-4-ylcarbonyl)guanidines as NHE-1 inhibitors

A series of (2-aryl-5-methylimidazol-4-ylcarbonyl)guanidines and (2-aryl-5-methyloxazol-4-ylcarbonyl)guanidines were synthesized and evaluated as NHE-1 inhibitors. The structure–activity relationships well matched those of furan derivatives, which were previously investigated. The (2,5-disubstituted)phenyl compounds showed better activities than the other analogues in both imidazole and oxazole compounds. Especially, 2-(2,5-dichlorophenyl)imidazole 52, and 2-(2-methoxy-5-chlorophenyl)imidazole 54 compounds exhibited potent cardioprotective efficacy both in vitro and in vivo as well as high NHE-1 inhibitory activities.     

Studies on the Accumulation of 5(4)-Amino-4(5)-imidazolecarboxamide by Escherichia coli in a Shaking Culture

In the previous paper the author reported that 5(4)-amino-4(5) -imidazolecarboxamide (AICA) was accumulated in peptone medium by Escherichia coli strain B grown as a shaking culture in the absence of sulfonamide inhibitor and glucose. It appeared from the further investigations that l-tryptophan would not replace peptone for the accumulation. However, it was found that indole produced from l-tryptophan by E. coli gave pink color by the Bratton nad Marshall method. In order to eliminate the effect of indole, the procedure by petroleum-ether treatment was applied and it was ascertained that E. coli had an ability to accumulate AICA and AICA-riboside in the peptone or L-tryptophan medium without glucose and sulfonamide inhibitor. But the concentrations of AICA and AICA-riboside measured by the above procedure were smaller than those determined by the Bratton and Marshall method which measured indole produced by the bacteria at the same time.     

Right- and left-handed (Z) helical conformations of the hairpin d(C-G)5T4(C-G)5 monomer and dimer

The partial self-complementary 24-mer oligodeoxynucleotide d(C-G)5T4(C-G)5 forms a hairpin which can be enzymatically dimerized to a dumbbell structure. The blunt-ended nature of the hairpin is indicated by its ability to inhibit the T4 DNA ligase catalyzed joining of phi X174 HaeIII fragments. The hairpin monomer and dimer (dumbbell) undergo a reversible B to Z transition as shown by ultraviolet, circular dichroism, and 31P NMR spectroscopy. The Z form of the hairpin monomer and dimer is supported by monovalent ions (Na+), divalent ions (Mg2+ but not Mn2+), and dehydrating (ethanol) conditions. The conformational transition of d(C-G)5T4(C-G)5 monomer requires higher ionic or dehydrating conditions than those necessary for the corresponding linear oligomer d(C-G)5. The contribution of the loop (-T4-) of the hairpin to the apparent free energy change for the B to Z conformational transition at the midpoint was calculated to be 3.8 kJ mol-1.     

Fluorescence of meso-tetrakis(4-(carboxy)phenyl)porphine covalently bound to oligonucleotides d(CG)5 and d(TA)5

The amino-reactive derivative of tetraphenylporphine meso-tetrakis[4-(carboxy)phenyl]porphine (TCPP) was synthesized, which is characterized by a high molar absorption coefficient (epsilon 416 = 36,500 M-1.cm-1). TCPP was covalently attached to oligonucleotides d(CG)5 [d(CG)5-TCPP] and d(TA)5 [d(TA)5-TCPP]. The spectral characteristics of these complexes were studied in 0.01 M phosphate buffer, pH 7 at 23 degrees C. UV-visible absorption spectra of these complexes have a clearly pronounced Soret band at (414 +/- 1) nm for d(CG)5-TCPP and at (412 +/- 1) nm for d(TA)5-TCPP. The fluorescence spectra of these complexes have maxima at (648 +/- 2) nm for d(CG)5-TCPP and at (658 +/- 2) nm for d(TA)5-TCPP. In this study we also determined fluorescence quantum yields q and fluorescence lifetimes tau [q = 0.099 +/- 0.011, tau = (9.0 +/- 0.3) ns for d(CG)5-TCPP and q = 0.080 +/- 0.011, tau = (8.7 +/- 0.3) ns for d(TA)5-TCPP]. A temperature rise from 5 to 50 degrees C produced only slight (within 23%) emission changes in both samples studied. Taking into account: a) high fluorescence yields (q), b) weak dependence of q on temperature, c) weak q dependence of q on the oligonucleotide type, we conclude that TCPP may be used as a sensitive fluorescence label in DNA studies.     

Expression of ribosomal protein L4 (rpL4) during neurogenesis and 5-azacytidine (5AzC)-induced apoptotic process in the rat

5-Azacytidine (5AzC) induces neuronal apoptosis in rat and mouse fetuses. 5AzC also induces apoptosis in undifferentiated PC12 cells, and ribosomal protein L4 (rpL4) mRNA expression increases prior to apoptosis. To clarify the roles of rpL4 during neurogenesis, we first examined the distribution of rpL4 mRNA in the developing rat brain by in situ hybridization and RT-PCR, and compared the results to the distribution of TUNEL- or PCNA-positive cells. rpL4 mRNA expression was strong in the ventricular zone (VZ), subventricular zone (SVZ), cortical plate (CP), cerebral cortex, granule cell layer (GCL), pyramidal cell layer (Py) and external granular layer (EGL) during embryonic and early postnatal days, and it was remarkably weakened thereafter. A lot of PCNA-positive cells were observed in VZ, SVZ, and EGL during embryonic and early postnatal days, and such distribution of PCNA-positive cells was almost identical to rpL4 mRNA distribution. Only few TUNEL-positive cells were observed in VZ, SVZ, cerebral cortex, EGL, and hippocampus during embryonic and early postnatal days, and the regions with TUNEL-positive cells were not identical to rpL4 mRNA distribution. Next, the changes of rpL4 mRNA expression in the brain of 5AzC-treated rat fetuses were examined by in situ hybridization and RT-PCR. Apoptotic cells appeared at 9 to 24 hours after treatment (HAT). However, the rpL4 mRNA expression was unchanged during the apoptotic process. From the results, it is suggested that rpL4 would have certain roles in cell proliferation and differentiation during neurogenesis, but have no roles in 5AzC-induced apoptosis in the fetal brain.     

Studies on the Accumulation of 5(4)-Amino-4(5)-imidazole- carboxamide by Escherichia coli in a Shaking Culture

Various attempts were made to accumulate 5 (4) -amino-4 (5) -imidazolecarboxamide (abreviated as AICA; in this paper, separate analyses for the riboside forms were not attempted, and hence AICA and AICA-riboside will be presented as total AICA) in a shaking culture medium by Escherichia coli strain B. The accumulation of non-acetylatable, diazotizable amines was accomplished by the addition of 0.01% of sulfadiazine, 0.2% of glucose, and 2% of peptone in the medium for sixteen hours at 30°C. E coli strain B was able to accumulate the amines in the pepton medium, even when glucose and the sulfonamide inhibtor were omitted. Although paper chromatographic and spectrophotometric analyses proved the accumulation of AICA and AICA-riboside by E. coli train B in the medium, another substance colored by the Bratton and Marshall method was also accumulated.     

Synthesis and antibacterial activities of 5-hydroxy-4-amino-2(5H)-furanones

Starting from the mucohalogen acids 1a and b 5-hydroxy-2(5H)-furanones 2a-h have been prepared and tested. These novel 4-amino-5-hydroxy 2(5H)-furananones have shown a broad antibiotic activity against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 in the micromolar range. A one step synthesis from mucohalogen acids towards the antibacterials 2a-h was developed, in which the target was obtained from 1a and b under reflux in toluene in presence of a catalytic amount of sulfuric acid. The derivatives 2b and c displayed a MIC and MBC of 4/8mug/ml, against Staphylococcus aureus with a selectivity towards the resistant strains.     

Cloning and characterization of the rat cytochrome P450 4F5 (CYP4F5) gene

The analysis of a non-redundant set of human proteins, for which both the crystallographic structures and the corresponding gene sequences are available, show that bases at third codon position are non-uniformly distributed along the coding sequences. Significant compositional differences are found by comparing the gene regions corresponding to the different secondary structures of the proteins. Inter-and intra-structure differences were most pronounced in the GC-richest genes. These results are not compatible with any proposed hypotheses based on a neutral process of formation/maintenance of the high GC₃ levels of the genes localized in the GC-richest isochores of the human genome.