Preparation and enzymatic hydrolysis of dinucleoside monophosphates and DNA modified with aromatic residues

The following procedures have been used to prepare fifteen modified dinucleoside monophosphates: (a) bisulfite-catalyzed transamination with aniline to give an N4-phenylcytidine (CPh), (b) bisulfite-catalyzed transamination with beta-naphthylamine to give an N4-beta-naphthylcytidine (CbetaN), (c) alkylation with 7-bromomethylbenz[a] anthracene to afford a 7(benz[a]anthryl-7-methyl)guanosine (GMBA), and (d) reaction with N-acetoxy-2-acetylaminofluorene to give an 8-(N-2-fluorenylacetamido)guanosine (GAAF). The compounds prepared were A-CPh, CPh-A, CPh-G, U-CPh, CPh-U, A-CbetaN, CbetaN-A, G-CbetaN, CbetaN-G, U-CbetaN, CbetaN-U, GMBA-U, U-GMBA, GAAF-U, and U-GAAF. All of the modified compounds were hydrolyzed to the expected monomers with venom and spleen exonucleases. Hydrolysis by micrococcal nuclease was inhibited in the following cases: A-CPh, A-CbetaN, U-GMBA, and U-GAAF. The first three reactions above were applied to denatured calf thymus DNA to prepare modified DNA samples containing from 0.3 to 2.0% bound aromatic residues. The modified nucleic acids were completely hydrolyzed to nucleosides by the combination of venom exonuclease, deoxyribonuclease I and alkaline phosphatase. The same results were obtained with a combination of spleen exonuclease, deoxyribonuclease II, and alkaline phosphatase. Hydrolysis of the modified nucleic acids by micrococcal nuclease and alkaline phosphatase afforded primarily nucleosides, with some dinucleoside monophosphates. The amount of the latter did not exceed that found in the hydrolysis of control DNA, however. Other workers have observed inhibition of enzymatic hydrolysis of nucleic acids modified by aromatic carcinogens. We postulated that their results may have been caused by cross-links, which were avoided in our studies.     

The preparation and properties of 4-thiouridine containing 2''-5'' and 3''-5'' dinucleoside monophosphates.

2'-5' and 3'-5' dinucleoside monophosphates containing 4-thiouridine were prepared by the thiolation of the cytosine containing compounds and purified by chromatography on a DEAE-Sephadex column. The chromatographic and optical properties of the isomers are compared.     

Circular dichroism study of 2'-5' dinucleoside monophosphates modified with N-2-acetylaminofluorene.

The conformational properties of four 2′ – 5′ dinucleoside monophosphates modified with $\text{N}$-2-acetylaminofluorene have been studied by circular dichroism spectroscopy. Covalent binding of this chemical carcinogen at the C₈ position of guanosine in the 2′ – 5′ dinucleoside monophosphates induces striking changes in their circular dichroic spectra depending on their base sequence and composition. The changes in CD spectra, redshift of the extrema and change of their polarity, not observed in the spectra of corresponding 3′ – 5′ derivatives modified with $\text{N}$-2-acetylaminofluorene are correlated with the difference in the configuration of 2′ – 5′ and 3′ – 5′ dinucleoside monophosphates and discussed in respect to the intramolecular stacking interactions.     

Osmium (VI) complexes of the 3', 5'-dinucleoside monophosphates, ApU and UpA.

The dinucleoside monophosphates, ApU and UpA, react with potassium osmate (VI) and 2,2'-bipyridyl to form the corresponding oxo-osmium (VI) bipyridyl sugar ester in which the osmate group is bonded to the terminal 2',3'-glycol. Osmium (VIII) tetroxide and 2,2'-bipyridyl react with the dinucleosides to form the corresponding oxo-osmium (VI) bipyridyl heterocyclic esters which result from addition of the tetroxide to the 5,6-double bond of the uracil residue. Although capable of transesterification reactions, these heterocyclic esters are exceptionally stable toward exchange reactions in solution. No apparent exchange was observed after 1 month. This reaction thus seems promising for single-site osmium labeling in polynucleotides.     

Reactivity and selectivity in light-induced free radical reactions of 2-propanol with purine and pyrimidine mononucleotides and dinucleoside monophosphates.

Photoalkylation reactions with 2-propanol, initiated with di-tert-butyl peroxide, of a variety of purine and pyrimidine mononucleotides and dinucleoside monophosphates lead to the substitution of an alpha-hydroxyisopropyl group for the H-8 atom of adenosine and the addition of the alcohol across the 5,6-double bond of the pyrimidines. Adenosine moieties blocked at their 3'-hydroxyl group are alkylated faster than those blocked at their 5'-hydroxyl. The reactivity of the uridine moieties of 3'-UMP, 5'-UMP, and uridylyl-(3',5')-uridine is not affected by the location of the phosphate group. However, the uridine moiety of uridylyl-(3',5')-adenosine is modified faster than that of adenylyl-(3',5')-uridine. It is suggested that steric hindrance imposed by the phosphate group determines the reactivity of adenosine moieties, while base stacking involving adenosine determines the reactivity of uridine moieties. These two effects play a major role in controlling the nature and degree of the selectivity of these photoalkylation reactions for either adenosine or uridine. Cytidine has been found to be inert in these reactions.     

Syntheses, structures and vibrational spectroscopy of some unusual silver(I) (pseudo-) halide/unidentate nitrogen base polymers

The meagre (structurally defined) array of 1:2 silver(I) (pseudo-)halide:unidentate nitrogen base adducts is augmented by the single-crystal X-ray structural characterization of the 1:2 silver(I) thiocyanate:piperidine (‘pip’) adduct. It is of the one-dimensional ‘castellated polymer’ type previously recorded for the chloride: ?Ag(pip)₂(μ-SCN)Ag(pip)₂? a single bridging atom (S) linking successive silver atoms. By contrast, in its copper(I) counterpart, also a one-dimensional polymer, the thiocyanate bridges as end-bound SN-ambidentate: ?CuSCNCuSCN? A study of the 1:1 silver(I) bromide:quinoline (‘quin’) adduct is recorded, as the 0.25 quin solvate, isomorphous with its previous reported ‘saddle polymer’ chloride counterpart.Recrystallization of 1:1 silver(I) iodide:tris(2,4,6-trimethoxyphenyl)phosphine (‘tmpp’) mixtures from py and quinoline (‘quin’)/acetonitrile solutions has yielded crystalline materials which have also been characterized by X-ray studies. In both cases the products are salts, the cation in each being the linearly coordinated silver(I) species [Ag(tmpp)₂]⁺, while the anions are, respectively, the discrete [Ag₅I₇(py)₂]²⁻ species, based on the already known but unsolvated [Cu₅I₇]²⁻ discrete, and the polymeric, arrays, and polymeric . The detailed stereochemistry of the [Ag(tmpp)₂]⁺ cation is a remarkably constant feature of all structures, as is its tendency to close-pack in sheets normal to their P-Ag-P axes.The far-IR spectra of the above species and of several related complexes have been recorded and assigned. The vibrational modes of the single stranded polymeric AgX chains in [XAg(pip)₂]_(∞|∞) (X = Cl, SCN) are discussed, and the assignments ν(AgX) = 155, 190 cm⁻¹ (X = Cl) and 208 cm⁻¹ (X = SCN) are made. The ν(AgX) and ν(AgN) modes in the cubane tetramers [XAg(pip)]₄ (X = Br, I) are assigned and discussed in relation to the assignments for the polymeric AgX:pip (1:2) complexes, and those for the polymeric [XAg(quin)]_(∞|∞) (X = Cl, Br) compounds. The far-IR spectra of [Ag(tmpp)₂]₂[Ag₅I₇(py)₂] and its corresponding 2-methylpyridine complex show a single strong band at about 420 cm⁻¹ which is assigned to the coordinated tmpp ligand in [Ag(tmpp)₂]⁺, and a partially resolved triplet at about 90, 110 and 140 cm⁻¹ which is assigned to the ν(AgI) modes of the [Ag₅I₇L₂]²⁻ anion. An analysis of this pattern is given using a model which has been used previously to account for unexpectedly simple ν(CuI) spectra for oligomeric iodocuprate(I) species.     

Recombination in Escherichia coli. VI. Characterization of a recombination-deficient mutation with unusual properties

A detailed study of the rec-34 mutation shows that this mutation is located near pheA on the E. coli chromosome, like the recA and recH genes. The rec-34 recipients, which are radiation-sensitive and “reckless”, yield about 0.1–1% of the number of recombinants obtained with a Rec⁺ recipient in conjunction experiments. The recombinants obtained in conjunction experiments, in which rec-4⁺ allele transfer is avoided, are still recombination deficient.rec-34 definitely is not a mutation in the recH gene. Although definite proof is lacking we suggest that rec-34 is an unusual recA mutation.     

Conformation of mononucleotides and dinucleoside monophosphates. P[H] and H[H] nuclear Overhauser effects.

The phosphorus-proton nuclear Overhauser effect (NOE) was used to investigate the quantitative distribution of rotamers about the C3'--O3' bond (phi') of 3'-AMP and 2',3'-cyclic-CMP and the C4'--C5', C5'--O5' bonds (psi, phi) of 5'-AMP. Phosphorus-proton and proton-proton NOE's were used to provide a qualitative insight into the backbone conformation and the glycosyl angle torsions of adenosylyl-(3' leads to 5')-adenosine (ApA). The major psi rotamer in 5'-AMP is the 60 degree (gg) form, while the major phi rotamer is the 180 degrees (g'g') form. The constrained model, 2',3'-cyclic-CMP, manifests the C3'endo furanose pucker predominantly. The results from these two models are consistent with nuclear magnetic resonance (NMR) J coupling analyses. The phi; distribution of 3'-AMP is dominated (77%) by the 180 degrees g- rotamer. The 3'-AMP results are consistent with phosphorus-hydrogen coupling constant analyses, but do not accord with phosphorus-carbon coupling constant results. The phosphorus-proton NOE reveals that the phosphorus of ApA occupies a region of conformation space not seen in 5'-AMP. The proton-proton NOE on APA shows a significant portion of syn rotamer in both X distributions and detects a cross-purine ring interaction consistent with base stacking known to exist in this system.     

Syntheses of oligonucleotide derivatives with P(V) porphyrin and their properties.

Two types of oligonucleotide derivatives which are substituted by P(V) porphyrin at the phosphorus atom of an internucleotidic linkage and at the 5'-terminal internucleotidic linkage via a spacer were synthesized (Fig. 1), and hybridization capabilities of them with complementary oligonucleotides were evaluated. A novel method for a sensing of oligonucleotide by the fluorescence quenching via photo-induced electron transfer between the P(V) porphyrin labeled oligonucleotide and pyrene-labeled one on the oligonucleotide template is reported.     

Synthesis and properties of photolabile (caged) phosphotriester derivatives of dinucleoside phosphates

Dinucleoside phosphates that harbor phosphate groups transiently blocked (caged) byo-nitrobenzyl oro-nitroveratryl residues were synthesized. It was shown that the conditions of the UV-induced deprotection largely depend on the nature of the protective group. The phosphotriesters obtained were resistant toward snake venom phosphodiesterase and nucleases of the cellular extract. The synthesis of the dinucleoside phosphates containing a photolabile group preceeded the incorporation of the modified blocks into extended oligonucleotides by the phosphoramidite method.     

Interaction of metal ions with humic-like models. Part 6. Molecular structure,spectral and thermal properties of diaquabis(2,6-dimethoxybenzoato)dioxouranium(VI) monohydrate

The crystal and molecular structure of [UO₂(DMB)₂(H₂O)₂]·H₂O (DMB = 2,6-dimethoxybenzoate), complex I, has been detcrmined by X-ray diffraction and refined to a final R value of 0.0411. The compound belongs to the space group P2₁/a with cell constants a = 12.649(4), b = 14.418(5), c = 13.460(4) Å and Z = 4. As in the analogous complex [UO₂(DHB)₂(H₂O)₂]·8H₂O (DHB = 2,6- dihydroxybenzoate), compound II, the uranyl ion is bound to two bidentate carboxylate groups and two water molecules, but the point-symmetry is lower because the carboxylates, and the water molecules, are in vicinal positions. The lack of hydrogen- bonds between carboxylate groups and ortho-methoxy substituents and, possibly, steric factors account for the rotation of the phenyl rings with respect to the equatorial plane of the metal, the dihedral angle between the ‘best planes’ being about 77°. Detectable changes in the bond distances and angles within the carboxylate groups are produced by the non-planarity of the ligand. Spectroscopic and thermal properties of complexes I and II are also compared.     

Syntheses, characterisation, infrared and Mo NMR spectroscopy of some coordinated oxo—molybdenum(VI) complexes

Adducts with MoO₄²⁻ tetrahedra coordinated to Cr(III) or Co(III) complexes have been synthesized and studied by IR and high resolution ⁹⁵Mo NMR spectroscopy. The ⁹⁵Mo chemical shifts of the adducts with cobalt(III) lie in the range −33.2 to + 49.4 ppm. This may be compared with an overall known chemical shift range in excess of 7000 ppm and implies a similarity in the molybdenum environment in all cases. For adducts with chelated cobalt(III) complexes several rather broad ⁹⁵Mo singnals are obtained with linewidths up to 260 Hz.