Properties of Some Diuridine Phosphate Analogues

Abstract

The hydrolytic stability of oligoribonucleotides containing 2′- amino nucleophile is due to poor leaving characteristic of 5′-nucleoside, replacement of 5′-leaving group by thio or amino results in considerable instability towards hydrolysis.     

The interaction of methylene blue,azure B,and thionine with DNA: Formation of complexes with polynucleotides and mononucleotides as model systems

The interactions of methylene blue, azure B, and thionine with calf thymus DNA, [poly (dG-dC)]₂, [poly(dA-dT)]₂, and the constituent mononucleotides 2′-deoxyguanosine-5′-monophosphate(dGMP), 2′-deoxyadenosine-5′-monophosphate(dAMP), 2′-deoxycytidine-5′-monophosphate(dCMP), and thymidine-5′-monophosphate(dTMP) have been studied by steady-state absorption spectroscopy and with equilibrium dialysis. Scatchard plots for binding of the dyes to the nucleic acid polymers were convex downward at low binding ratios, characteristic of intercalation, and binding constants for this mode were calculated under conditions of varying ionic strength. For each of the dyes, binding constants with [poly(dG-dC)]₂ and [poly(dA-dT)]₂ were of the same order of magnitude, so that previously reported (G-C) preferentially is not very marked. At high binding ratios, the Scatchard plots did not return to the abscissa but curved upward, indicative of a weaker cooperative binding mode, occurring under conditions where the dye is in excess, which is suggested to be external stacking of the dye molecules promoted by the polyanion. The dependence of the absorption spectra on added salt demonstrated a shift in the strong binding mode for the three dyes with [poly(dA-dT)]₂ with increasing ionic strength, while with [poly(dG-dC)]₂ this does not occur. The dyes were found to bind to purine but not pyrimidine mononucleotides with dGMP and dAMP, 1:1 complexes were formed initially and also 1:2 dye/nucleotide complexes with increasing nucleotide concentrations. Under low salt conditions, binding to dAMP was slightly stronger than to dGMP for the three dyes studied, while at high ionic strength, when the binding constants are significantly lower, all binding constants become very similar. Binding to mononucleotides is suggested to be primarily stabilised by π-π stacking interactions between the planar dyes and the nucleobases: for thionine and azure B there also appears to be H-bonds between the exocyclic amines and the sugar–phosphates conferring extra stability. Neither increasing the number of phosphate groups on the nucleotides nor changing from deoxyribose to ribose sugars had any significant effect on the binding constants. © 1995 John Wiley & Sons, Inc.     

Rational Approaches to the Design of Mechanism-Based Inhibitors of S-Adenosylhomocysteine Hydrolase

Abstract

Crucial to the rational design of inhibitors of S-adenosyl-L-homocysteine (AdoHcy) hydrolase was the elucidation of its mechanism of catalysis by Palmer and Abeles (J. Biol. Chem. 254, 1217–1226, 1979). This mechanism involves an NAD⁺-dependent oxidation (oxidative activity) of the 3′-hydroxyl group of AdoHcy followed by elimination of homocysteine (Hcy) to form 4′,5′-didehydro-3′-keto-Ado. Addition of water at the 5′-position (hydrolytic activity) of this tightly bound intermediate followed by an NADH-dependent reduction results in the formation of adenosine (Ado). Many inhibitors of this enzyme have been shown to serve as substrates [e.g., 9-(trans-2-trans-3-dihydroxycyclopent-4-en-1-yl)adenine, DHCeA)] for the oxidative activity of AdoHcy hydrolase, affording the 3′-keto-derivative (e.g., 3′-keto-DHCeA), which is tightly bound to the enzyme, and converting the enzyme from its active form (NAD⁺) to its inactive form (NADH) (Type I mechanism-based inhibitors; Wolfe and Borchardt, J. Med. Chem. 34, 1521–1530, 1991). More recently, substrates [e.g., (E)-5.,6′-didehydro-6′-deoxy-6′-fluorohomoadenosine, EDDFHA] for the hydrolytic activity of AdoHcy hydrolase have been identified by our laboratories. Identification of hydrolytic substrates affords a new strategy for the design of more potent and more specific inhibitors of AdoHcy hydrolase.     

Molecular cloning and expression analysis of an HINT1 homologue from maize (Zea mays L.)

Histidine triad nucleotide binding protein (HINT1) belongs to a histidine triad (HIT) superfamily, which contains a highly conserved His-X-His-X-His-XX motif (X is a hydrophobic amino acid) and plays an important role in many biological processes. In this study, we have isolated the full-length cDNA of an HINT1 homologue from maize (Zea mays L.), designated as Zm-HINT1. The full-length cDNA of Zm-HINT1 consists of 729 bp with an ORF encoding a 138-amino acid protein. The deduced amino acid sequence of Zm-HINT1 shows high sequence homology to the mammalian HINT1 and contains conserved domains including the HIT motif, helical regions and β-strands, which are the characteristics of HINT1 proteins. The phylogenetic analysis has revealed that Zm-HINT1 is branched along with Caenorhabditis elegans HINT1. RT-PCR analysis has revealed that Zm-HINT1 is ubiquitously expressed in maize tissues but not in the pericarp, thus suggesting that Zm-HINT1 may not be related to the production of fibrin. Furthermore, expression levels of Zm-HINT1 have increased rapidly following treatment with salicylic acid. Taken together, these results indicate that Zm-HINT1 is a mammalian HINT1 homologue and may be involved in the immune response of maize.     

Labelling of DNA with a Non-Radioactive Analogue of dGTP

Abstract

8-Bromo-2′-deoxyguanosine-5′ phosphate reacts with 2-mercaptoethylamine. Oxidation of the reaction mixture generates a disulphide with free aliphatic amino group. Biotinylation yields an analogue of dGMP, Bio-15-dGMP. The triphosphate. Bio-15-dGTP. may be incorportated into DNA by DNA polymerase I of Escherischia coli and detected by reagents conjugated conjugated to avidin.     

Interaction of Purine Nucleotides with Cobalt-Hexammine,Cobalt-Pentammine and Cobalt- Tetrammine Cations. Evidence for the Rigidity of Adenosine and Flexibility of Guanosine and Deoxyguanosine Sugar Conformations

Abstract

The interaction of adenosine-5′-monophosphate (5′-AMP), guanosine-5′-monophosphate (5′-GMP) and 2′-deoxyguanosine-5′-monophosphate (5′-dGMP) with the [Co(NH₃)₆]³⁺, [CO(NH₃)₅C1]²⁺ and [CO(NH₃)₄C1₂]⁺ cations has been investigated in aqueous solution with metal/nucleotide ratios (r) of 1/2, 1 and 2 at neutral pH. The solid complexes have been isolated and characterized by FT-IR and ¹H-NMR spectroscopy.

The complexes are polymeric in nature both in the crystalline solid and aqueous solution. The binding of the cobalt-hexammine cation is indirectly (via NH₃) through the N-7 and the PO₃ ^2- groups of the AMP and via O-6, N-7 and the PO₃ ^2- of the GMP and dGMP anions (outer-sphere). The cobalt-pentammine and cobalt-tetrammine bindings are through the phosphate groups (inner-sphere) and the N-7 site (outer-sphere) of these nucleotide anions. The ribose moiety shows C2′-endo/anti conformation, in the free AMP and GMP anions as well as in the cobalt-ammine - AMP complexes, whereas a mixture of the C2′-endo/anti and C3′-endo/anti sugar puckers were observed for the Co(NH₃)₆-GMP, Co(NH₃)₅-GMP and a C3′-endo/anti conformer for the Co(NH₃)₄-GMP complexes. The deoxyribose showed an O4′-endo/anti conformation for the free dGMP anion and a C3′-endo/anti for the Co(NH₃)₆-dGMP, Co(NH₃)₅-dGMP and Co(NH₃)₄-dGMP complexes.     

Binding of Bis-linked Netropsin Derivatives in the Parallel-Stranded Hairpin Form to DNA

Abstract

Cis-diammine Pt(II)- bridged bis-netropsin and oligomethylene-bridged bis-netropsin in which two monomers are linked in a tail-to-tail manner bind to the DNA oligomer with the sequence 5′-CCTATATCC-3′ in a parallel-stranded hairpin form with a stoichiometry 1:1. The difference circular dichroism (CD) spectra characteristic of binding of these ligands in the hairpin form are similar. They differ from CD patterns obtained for binding to the same duplex of another bis-netropsin in which two netropsin moieties were linked in a head-to-tail manner. This reflects the fact that tail-to-tail and head-to-tail bis-netropsins use parallel and antiparallel side-by-side motifs, respectively, for binding to DNA in the hairpin forms. The binding affinity of cis -diammine Pt(II)- bridged bis-netropsin in the hairpin form to DNA oligomers with nucleotide sequences 5′-CCTATATCC-3′ (I), 5′-CCTTAATCC-3′ (II), 5′-CCTTATTCC-3′ (III), 5′-CCTTTTTCC-3′ (IV) and 5′-CCAATTTCC-3′ (V) decreases in the order I = II > III > IV> V. The binding of oligomethylene-bridged bis-netropsin in the hairpin form follows a similar hierarchy. An opposite order of sequence preferences is observed for partially bonded monodentate binding mode of the synthetic ligand.     

Strategically Functionalized Adenosines: Agonists for Adenosine Receptors

Abstract

The syntheses of three classes of adenosine analogues involving cyclosubstitution at the 6-position and functionalization at the 2-position are reported. The target molecules synthesized are stable with respect to hydrolytic deamination by mammalian adenosine deaminase, and, because of major structural changes at the 2- and 6-positions, these compounds are expected to be poor phosphorylation substrates for the kinases. Adenosine receptor binding data reveal that several of the compounds synthesized show excellent A₁ receptor affinity and A₂/A₁ selectivity.     

Synthesis and Anti‐HIV Activity of 4′‐Cyano‐2′,3′‐didehydro‐3′‐deoxythymidine

A new anti‐HIV agent 4′‐cyano‐2′,3′‐didehydro‐3′‐deoxythymidine (9) was synthesized by allylic substitution of the 3′,4′‐unsaturated nucleoside 14, having a leaving group at the 2′‐position, with cyanotrimethylsilane in the presence of SnCl₄. Evaluation of the anti‐HIV activity of 9 showed that this compound is much less potent than the recently reported 2′,3′‐didehydro‐3′‐deoxy‐4′‐(ethynyl)thymidine (1).     

Synthesis of 2′,3′-Dideoxypurinenucleosides via the Palladium Catalyzed Reduction of 9-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-d-xylofuranosyl)purine Derivatives

Abstract

Practical method to produce 2′,3′-dideoxypurinenucleosides from 9-(2,5-di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)purines (1) was developed. High ratio of 2′,3′-dideoxynucleoside to 3′-deoxyribonucleoside was obtained by selecting the reaction conditions (solvent, pH and/or base), or changing 2′-acyloxy leaving group. The reaction mechanism was studied by deuteration experiments of 1a and 1-(3,5-di-O-acety1-2-bromo-2-deoxy-β-D-ribofuranosyl)thymine (12).

     

Arabidopsis SCP1-like small phosphatases differentially dephosphorylate RNA polymerase II C-terminal domain

RNA polymerase II carboxyl-terminal domain (pol II CTD) phosphatases that can dephosphorylate both Ser2-PO₄ and Ser5-PO₄ of CTD have been identified in animals and yeasts, however, only Ser5-PO₄-specific CTD phosphatases have been identified in plants. Among predicted Arabidopsis SCP1-like small phosphatases (SSP), SSP4, SSP4b, and SSP5 form a unique group with long N-terminal extensions. While SSPs’ expression showed similar tissue-specificities, SSP4 and SSP4b were localized exclusively in the nuclei, whereas SSP5 accumulated in both nuclei and cytoplasm. Detailed characterization of SSP activities using various peptides and full-length Arabidopsis pol II CTD substrates established that SSP4 and SSP4b could dephosphorylate both Ser2-PO₄ and Ser5-PO₄ of CTD, whereas SSP5 dephosphorylated only Ser5-PO₄. These results indicate that Arabidopsis SSP gene family encodes active CTD phosphatases like animal SCP1 family proteins, with distinct substrate specificities.     

Phosphorylation of dGMP analogs by vaccinia virus TMP kinase and human GMP kinase

Vaccinia virus thymidylate kinase, although similar in sequence to human TMP kinase, has broader substrate specificity and phosphorylates (E)-5-(2-bromovinyl)-dUMP and dGMP. Modified guanines such as glyoxal-dG, 8-oxo-dG, O⁶-methyl-dG, N²-ethyl-dG and N⁷-methyl-dG were found present in cancer cell DNA. Alkylated and oxidized dGMP analogs were examined as potential substrates for vaccinia TMP kinase and also for human TMP and GMP kinases. Molecular models obtained from structure-based docking rationalized the enzymatic data. All tested nucleotides are found surprisingly substrates of vaccinia TMP kinase and also of human GMP kinase. Interestingly, O⁶-methyl-dGMP is the only analog specific for the vaccinia enzyme. Thus, O⁶-Me-dGMP could be useful for designing new compounds of medical interest either in antipoxvirus therapy or in experimental combined gene/chemotherapy of cancer. These results also provide new insights regarding dGMP analog reaction with human GMP kinase and their slow recycling by salvage pathway nucleotide kinases.     

Methodology for the Synthesis of Dinucleoside Monophosphates Containing A 2′-Deoxy-3-Isoadenosine Unit: 3-iso-dApT and Tp(3-iso-dA)

Abstract

2′ -Deoxy-3-isoadenylyl(3′-5′)thymidine and thymidylyl-(3′-5′)-2′-deoxy-3-isoadenosine have been synthesized by mild protection/deprotection methodology that circumvents facile N3-Cl′ hydrolytic cleavage of the 2′-deoxy-3-isoadenosine moiety.

     

Preparation,Hydrolysis and Intramolecular Transesterification of 3′-Deoxy-3′-thioinosine 3′-S-Dimethylphosphorothiolate

Abstract

The hydrolytic reactions of the dimethyl ester of 3′-deoxy-3′-thioinosine 3′-S-phosphorothiolate have been followed over a wide aciditty range by HPLC. At pH > 3, only hydroxide ion catalyzed isomerization to the 2′-dimethylphosphate takes place, whereas under more acidic conditions hydrolysis to the 2′-monomethylphosphate and 3′-S-monomethylphosphorothiolate competes. The latter is the only product accumulating in very acidic solutions (1 M hydrochloric acid). Mechanisms of the reactions are discussed.     

Synthesis and Enantioselectivity of Cyclopropavir Phosphates for Cellular GMP Kinase

Enantiomeric cyclopropavir phosphates (+)-9 and (?)-9 were synthesized and investigated as substrates for GMP kinase. N²-Isobutyryl-di-O-acetylcyclopropavir (11) was converted to (+)-monoacetate 12 using hydrolysis catalyzed by porcine liver esterase. Phosphorylation via phosphite 13 gave after deacylation, phosphate (+)-9. Acid-catalyzed tetrahydropyranylation of (+)-monoacetate 12 gave, after deacylation, tetrahydropyranyl derivative 14. Phosphorylation via phosphite 15 furnished, after deprotection, enantiomeric phosphate (-)-9. Racemic diphosphate 16 was also synthesized. The phosphate (+)-9 is a relatively good substrate for GMP kinase with a K_M value of 57 μM that is similar to that of the natural substrates GMP (61 μM) and dGMP (82 μM). In contrast, the enantiomer (?)-9 is not a good substrate (K_M 1200 μM) indicating a significant enantioselectivity for the GMP kinase catalyzed reaction of monophosphate to diphosphate.     

Phosphate-Modified Nucleotides: Conversions in Human Serum

Abstract

Mechanisms and rates of hydrolytic dephosphorylation of 5′-hydrogenphosphonates, 5′-phosphorofluoridates, and 5′-phosphates of natural and 3′-substituted thymidines in human serum were studied. The stability of 5′-phosphonates of 2′-deoxy- and 2′,3′-dideoxyadenosines in calf and human sera was found.     

Allosteric effects mediate CHK2 phosphorylation of the p53 transactivation domain

The tumour suppressor p53 is a tetrameric protein that is phosphorylated in its BOX-I transactivation domain by checkpoint kinase 2 (CHK2) in response to DNA damage. CHK2 cannot phosphorylate small peptide fragments of p53 containing the BOX-I motif, indicating that undefined determinants in the p53 tetramer mediate CHK2 recognition. Two peptides derived from the DNA-binding domain of p53 bind to CHK2 and stimulate phosphorylation of full-length p53 at Thr 18 and Ser 20, thus identifying CHK2-docking sites. CHK2 can be fully activated in trans by the two p53 DNA-binding-domain peptides, and can phosphorylate BOX-I transactivation-domain fragments of p53 at Thr 18 and Ser 20. Although CHK2 has a basal Ser 20 kinase activity that is predominantly activated towards Thr 18, CHK1 has constitutive Thr 18 kinase activity that is predominantly activated in trans towards Ser 20. Cell division cycle 25C (CDC25C) phosphorylation by CHK2 is unaffected by the p53 DNA-binding-domain peptides. The CHK2-docking site in the BOX-V motif is the smallest of the two CHK2 binding sites, and mutating certain amino acids in the BOX-V peptide prevents CHK2 activation. A database search identified a p53 BOX-I-homology motif in p21^WAF1 and although CHK2 is inactive towards this protein, the p53 DNA-binding-domain peptides induce phosphorylation of p21^WAF1 at Ser 146. This provides evidence that CHK2 can be activated allosterically towards some substrates by a novel docking interaction, and identify a potential regulatory switch that may channel CHK2 into distinct signalling pathways in vivo.     

HPLC as a Tool to Study the Kinetics of Parallel and Consecutive Reactions: Competitive Interconversion,Dephosphorylation, Deamination and Depyrimidination of Cytidine 2′- and 3′-Monophosphates in Aqueous Acid

Abstract

The applicability of HPLC as a method to study the kinetics of complicated reaction systems of nucleosides and nucleotides has been demonstrated by using the hydrolytic reactions of cytidine 2′- and 3′-monophosphates as an example.