N6-substituted adenine derivatives and RNA primitive catalysts

In our search for primitive RNA catalysts, we noticed that N6-ribosyl-adenine, a compound easily synthesized under presumed prebiotic conditions, has a free imidazole group. We showed that it is, as a catalyst, a potential analogue of histidine. Furthermore, among the chemical groups involved in protein catalysis, the imidazole ring of histidine has no equivalent in the RNA world. We have synthesized aliphatic amino groups containing polymers with adenine rings linked to macromolecules by their 6-amino group. These polymers exhibit pronounced catalytic activities in the hydrolysis of p-nitrophenylacetate. We discuss here the fact that in primitive catalysis the imidazole group could have been replaced by N6-substituted adenine derivatives.     

Studies on Griseolic Acid Derivatives X. Synthesis and Phosphodiesterase Inhibitory Activity of 2-Substituted Derivatives of Griseolic Acid

Abstract

Griseolic acid derivatives which were modified at the 2-and/or 6-positions were first synthesized from griseolic acid by a ring opening—reclosure reaction of the adenine ring. Among these derivatives, the 2-amino-6-deamino-6-hydroxyl (guanine) derivative showed 3.3 and 45 times stronger inhibitory activity against cAMP and cGMP PDE, respectively, than those of griseolic acid. Structure-activity relationships among these derivatives are also discussed.     

Reduction of the toxicity and mutagenicity of aziridine in mammalian cells harboring the Escherichia coli fpg gene.

Aziridine (ethyleneimine) reacts with DNA in vitro, mainly at the N7 position of guanine and N3 of adenine, then imidazole ring opening of the modified guanine results in formation of formamidopyrimidine (FaPy) residues. The Escherichia coli fpg gene encodes a DNA glycosylase that removes FaPy residues from DNA. To determine whether aziridine produces FaPy lesions in mammalian cells we have expressed the E.coli fpg gene in CHO cells. The transfected cells, expressing high levels of the bacterial protein, are more resistant to the toxic and mutagenic effects of aziridine than the control population. Less DNA damage was measured by quantitative PCR analysis in transfected than in control cells treated with equimolar concentrations of aziridine. The results suggest that aziridine produces in vivo FaPy residues that could account for the deleterious effects of this compound.     

Synthesis and 1H NMR spectroscopic characterization of trans-[Pt(NH3)2[d(ApGpGpCpCpT)-N7-A(1),N7-G(3)]]

The reaction of trans-[Pt(NH3)2Cl2] with the sodium salt of [d(ApGpGpCpCpT)]2 in aqueous solution at 37 degrees C was monitored by reversed-phase high-performance liquid chromatography and UV spectroscopy. Two intermediates, most likely monofunctional adducts, were observed, which subsequently formed one predominant single-stranded product, as well as several polymeric species proposed to be interstrand cross-linked products. The single-stranded adduct was structurally characterized by 1H NMR spectroscopy. From the pH dependence of the chemical shifts, two-dimensional homonuclear chemical shift correlation (COSY) spectroscopy, and one- and two-dimensional nuclear Overhauser effect (NOESY) experiments, the platinum(II) moiety was found to be coordinated to the N7 positions of adenine(1) and guanine(3), with the intervening guanine(2) base destacked from its neighboring residues. This intrastrand 1,3 adduct induces changes in the backbone torsion angles and causes the deoxyribose ring of adenine(1) to switch from a C2'-endo to a predominantly C3'-endo conformation. The other deoxyribose rings retain B DNA type conformations. The structure of trans-[Pt(NH3)2[d(ApGpGpCpCpT)-N7-A(1),N7-G(3)]] differs from those previously reported for cis-DDP 1,2- and 1,3-intrastrand oligonucleotide adducts but is consistent with the structures of trans-DDP 1,3-intrastrand adducts of two previously reported trinucleotides.     

How flexible are DNA constituents? The quantum-mechanical study

Relaxed force constants (RFCs) and vibrational root-mean-square deviations have been evaluated by the original calculation method for conformational parameters of the DNA structural units and their constituents: nucleic acid bases (uracile, thymine, cytosine, adenine and guanine) and their 'building blocks' (benzene, pyrimidine, imidazole and purine molecules), as well as the DNA backbone structural units: tetrahydrofuran, 1,2-dideoxyribose, methanol and orthophosphoric acid. It has been found that the RFCs for nomenclature torsions beta, gamma, epsilon; and sugar pseudorotation angle P in 1,2-dideoxyribose are sensible to the molecule conformation and their values are in the range of 1-25 kcal/(mole·rad2) obeying the inequality K(γ)> K(ε) > K(ρ) > K(β). The RFCs values for endocyclic torsions of nucleic acid bases six-member rings lie within 15-45?kcal/(mole·rad2) in pyrimidines and within 20-60?kcal/(mole·rad2) in purines. It is shown that the quantum zero-point motion effectively neglects the amino group non-planarity in cytosine, adenine and partially in guanine.     

Rates of heat-induced DNA purine alterations in synthetic polydeoxyribonucleotides

Damage to DNA by heat can occur at physiological conditions. The effects of the varying conformational states adopted by double-stranded DNA on the incidences and distributions of thermally induced hydrolytic purine alterations are unknown. The possible role of conformational changes on damage by heat to purines in DNA polymers was therefore investigated. Model compounds used were the synthetic alternating copolymer poly(dG-dC):poly(dG-dC) and the homopolymer poly(dG):poly(dC). Base damages were assayed by high performance liquid chromatography using polymers radioactively labeled in guanine. Conformational states were assayed by circular dichroic spectral changes. Incubation and heating of the polymers in 1 mM Mn2+ caused the spectral shift reported for the left-handed Z-DNA conformation in the alternating copolymer and the change reported for the triple helix in the homopolymer. After incubation at 85 degrees C., incidences of base damages were compared between the polymers. No deamination of guanine to xanthine was observed under any conditions. The presence of manganese reduced depurination in both polymers. Rates of guanine imidazole ring openings to yield 2,6-diamino-4-hydroxy-5-formamidopyrimidine were increased in the presence of the cation and constituted the chief form of purine damage in the homopolymer. Therefore, the distribution of heat-induced DNA alterations within the genome may be determined by DNA conformational states. This observed opening of purine imidazole rings in the presence of manganese ions may have mutagenic consequences and may be involved in carcinogenesis by metals.     

Stacking conformation of 9-[omega-(thymin-1-yl)alkyl]adenine in aqueous solution

An intramolecular stacking between adenine and thymine rings of 9-[omega-(thymin-1-yl)alkyl]adenine was investigated by means of NMR and UV spectroscopy. The stacking conformation was presumed on the basis of the temperature dependence on the chemical shifts of the adenine and thymine ring protons in the buffer solution at pD 7.0.     

Enzymatic excision from gamma-irradiated polydeoxyribonucleotides of adenine residues whose imidazole rings have been ruptured

The main forms of base damage in polydeoxyadenylic acid gamma-irradiated under hypoxic conditions are due to saturation and fragmentation of the adenine imidazole ring. An irradiated polymer was annealed with an equimolar amount of poly (dT) to generate a double-stranded polydeoxyribonucleotide containing scattered damaged base residues. On incubation of the latter with partially purified cell extracts of E.coli, imidazole ring-opened adenine, i.e. 4,6-diamino-5-formamidopyrimidine, was released in free form by a DNA glycosylase activity. The enzyme has been purified 4,500-fold, has Mr = 29,000, and appears to be identical with the previously described DNA repair enzyme formamidopyrimidine-DNA glycosylase.     

Structures of the adducts formed between [Pt(dien)Cl]Cl and DNA in vitro.

The products of the reaction between [Pt(dien)Cl]Cl and salmon sperm DNA have been purified and their structures determined. [Pt(dien)Cl]Cl binds at the N7 position of guanine for levels of fixation below 0.1 platinum per DNA base. Above this level of binding, [Pt(dien)Cl]Cl also reacts at the N7 position of adenine. 1,7-[Pt(dien)]2Ade was observed when more than 0.3 platinum per base were bound to the DNA. Platination at the N7 position of guanosine, unlike alkylation, stabilized the glycosyl linkage and did not lead to fission of the imidazole ring at high pH.     

Three-dimensional structure of a mutant ribonuclease T1 (Y45W) complexed with non-cognizable ribonucleotide, 2'AMP, and its comparison with a specific complex with 2'GMP.

The crystal structure of a mutant ribonuclease T1 (Y45W) complexed with a non-cognizable ribonucleotide, 2'AMP, has been determined and refined to an R-factor of 0.159 using X-ray diffraction data at 1.7 A resolution. A specific complex of the enzyme with 2'GMP was also determined and refined to an R-factor of 0.173 at 1.9 A resolution. The adenine base of 2'AMP was found at a base-binding site that is far apart from the guanine recognition site, where the guanine base of 2'GMP binds. The binding of the adenine base is mediated by a single hydrogen bond and stacking interaction of the base with the imidazole ring of His92. The mode of stacking of the adenine base with His92 is similar to the stacking of the guanine base observed in complexes of ribonuclease T1 with guanylyl-2',5'-guanosine, reported by Koepke et al., and two guanosine bases, reported by Lenz et al., and in the complex of barnase with d(GpC), reported by Baudet & Janin. These observations suggest that the site is non-specific for base binding. The phosphate group of 2'AMP is tightly locked at the catalytic site with seven hydrogen bonds to the enzyme in a similar manner to that of 2'GMP. In addition, two hydrogen bonds are formed between the sugar moiety of 2'AMP and the enzyme. The 2'AMP molecule adopts the anti conformation of the glycosidic bond and C-3'-exo sugar pucker, whereas 2'GMP is in the syn conformation with C-3'-endo-C'-2'-exo pucker. The mutation enhances the binding of 2'GMP with conformational changes of the sugar ring and displacement of the phosphate group towards the interior of the catalytic site from the corresponding position in the wild-type enzyme complex. Comparison of two crystal structures obtained provides a solution to the problem that non-cognizable nucleotides exhibit unexpectedly strong binding to the enzyme, compared with high specificity in nucleolytic activity. The results indicate that the discrimination of the guanine base from the other nucleotide bases at the guanine recognition site is more effective than that estimated from nucleotide-binding experiments so far.     

Studies on the interaction between Cd(2+) ions and nucleobases and nucleotides

Cadmium is a potent carcinogen in rodents and has recently been accepted by the International Agency for Research on Cancer as a category 1 (human) carcinogen, but the molecular mechanism of its action remains largely unclear. It has however been suggested that cadmium-induced carcinogenesis may involve either direct or indirect interaction of Cd²⁺ with DNA. In this study it is found that when Cd²⁺ is allowed to interact with adenine and guanine, there is a marked change in the high performance liquid chromatography (HPLC) retention time for adenine but not for guanine. Since Cd²⁺ is believed to bind covalently to adenine and guanine, the changes in retention time but absence of any cadmium in the peak fraction point to the following: (i) lability of cadmium-nucleobase adducts, and (ii) introduction of some kind of chemical modification in adenine but not in guanine as a result of covalent binding. This result is different from that for Ni²⁺ in which case a change in retention time was observed for guanine but not for adenine.     

1H nuclear magnetic resonance studies of histidine-containing di- and tripeptides. Estimation of the effects of charged groups on the pKa value of the imidazole ring.

The nmr titration curves of chemical shifts versus pH were observed for the protons of various histidine-containing di- and tripeptides. With these results, the macroscopic pK_a values and the chemical shifts intrinsic to each ionic species were determined by a computer curve-fitting based on a simple acid dissociation sequence. The pK_a value of the imidazole ring in N-acetyl-L -histidine methylamide was assumed to represent the intrinsic (or unperturbed) pK_a of the imidazole rings of histidine having peptide linkages at both the CO and NH sides. The pK_a values of the imidazole rings observed for most di- and tripeptides were reasonably reproduced by simple calculations using the intrinsic value and the perturbations due to the CO₂^? and NH₃⁺ groups located at various positions. Some other factors affecting the pK_a value of the imidazole ring are also discussed.     

ATP-analogues as substrates for the leucyl-tRNA synthetase from Escherichia coli MRE 600.

No analogous nucleoside triphosphate was found which acts as well as ATP in binding to and supporting catalysis of leucyl-tRNA synthetase from Escherichia coli MRE 600. However, there are numerous nucleotides which are able to replace ATP, but with lower efficiency. The 6-amino group of the adenine ring and the 2'-hydroxyl group of the ribose ring are essential for binding and catalytic activity. Alterations in the triphosphate moiety of the molecule can cause drastic changes in Km and/or Vmax, whereas alterations of the imidazole ring and substitutions at the 8-position of the adenine ring cause only minor losses of catalytic activity.     

Recognition and interaction of small rings with the ricin A-chain binding site

Ricin A-chain is an N-glucosidase that attacks ribosomal RNA at a highly conserved adenine residue. Our recent crystallographic studies show that not only adenine and formycin, but also pterin-based rings can bind in the active site of ricin. For a better understanding of the means by which ricin recognizes adenine rings, the geometries and interaction energies were calculated for a number of complexes between ricin and tautomeric modifications of formycin, adenine, pterin, and guanine. These were studied by molecular mechanics, semi-empirical quantum mechanics, and ab initio quantum mechanical methods. The calculations indicate that the formycin ring binds better than adenine and pterin better than formycin, a result that is consistent with the crystallographic data. A tautomer of pterin that is not in the low energy form in either the gas phase or in aqueous solution has the best interaction with the enzyme. The net interaction energy, defined as the interaction energy calculated in vacuo between the receptor and an inhibitor minus the solvation energy of the inhibitor, provides a good prediction of the ability of the inhibitor to bind to the receptor. The results from experimental and molecular modeling work suggest that the ricin binding site is not flexible and may only recognize a limited range of adenine-like rings. Proteins 31:33–41, 1998. Published 1998 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Separate regulation of purA and purB loci of Escherichia coli K-12.

We isolated a strain of Escherichia coli K-12 in which the lac structural genes are fused to the purB control region and used this strain to study the regulation of the purA and purB loci. The purA locus was derepressed in response to either limiting adenine or guanine growth conditions in the presence of excess guanine or adenine, respectively. The presence of hypoxanthine in the culture medium did not have any effect on the expression of the purA locus. The purB locus responded to limiting adenine growth conditions in the presence of either excess hypoxanthine or guanine alone but not when both hypoxanthine and guanine were present.     

Solution structure and dynamic character of the histidine-containing phosphotransfer domain of anaerobic sensor kinase ArcB from Escherichia coli

An Escherichia coli sensor kinase, ArcB, transfers a phosphoryl group to a partner response regulator in response to anaerobic conditions. Multidimensional NMR techniques were applied to determine the solution structure of the histidine-containing phosphotransfer signaling domain of ArcB (HPt(ArcB)), which has a phosphorylation site, His717. The backbone dynamics were also investigated by analyses of the (15)N relaxation data and amide hydrogen exchange rates. Furthermore, the protonation states of the histidine imidazole rings were characterized by means of (1)H and (15)N chemical shifts at various pHs. The determined solution structure of HPt(ArcB) contains five helices and forms a four-helix bundle motif like other HPt domains. The obtained order parameters, S (2), [(1)H]-(15)N heteronuclear NOE values, and chemical exchange parameters, R(ex), showed that the alpha-helical regions of HPt(ArcB) are rigid on both picosecond to nanosecond and microsecond to millisecond time scales. On the other hand, helix D, which contains His717, exhibited low protection factors of less than 4000, indicating the presence of fluctuations on a slower time scale in helix D. These results suggest that HPt(ArcB) may undergo a small conformational change in helix D upon phosphorylation. It was also shown that the imidazole ring of His717 has a pK(a) value of 6.76, which is similar to that of a solvent-exposed histidine imidazole ring, and that a pair of deprotonated neutral tautomers are rapidly exchanged with each other. This is consistent with the solution structure of HPt(ArcB), in which the imidazole ring of His717 is exposed to the solvent.     

Heterodimeric molecules including nucleic acid bases and 9-aminoacridine. Spectroscopic studies, conformations, and interactions with DNA

Heterodimeric molecules have been examined in which the 9-amino-6-chloro-2-methoxyacridine ring is linked to the nucleic acid bases adenine, thymine, and guanine by polymethylenic chains (CH2)n of varying length (n = 3, 5, 6). A detailed analysis has been performed, including hypochromism measurement in the UV, chemical shift variations in Fourier transform proton magnetic resonance, and fluorescence emission. All these techniques show that all molecules exist mainly under folded conformations in water in the temperature range 0-90 degrees C, with the acridine and the base rings being stacked one on top of the other. The thermodynamic parameters for the folded in equilibrium unfolded conformational equilibrium were estimated. The geometry of the intramolecular complexes could be determined. (1) All these data give information on the strength and nature of the base-acridine interactions as a function of different parameters such as solvent, temperature, etc. (2) The molecules under study constitute "spectroscopic models" for the drug-base complexes as they occur in DNA. In particular, we show the dramatic influence of the relative orientations of the two stacked chromophores in the complex upon the magnitude of % H, the percent hypochromism. The quenching and enhancement of acridine fluorescence emission induced respectively by guanine and adenine is evidenced and quantitatively estimated. (3) These base-acridine heterodimers bind to DNA to an extent that is inversely proportional to their degree of intramolecular stacking.     

Alkaline opening of imidazole ring of 7-methylguanosine. 2. Further studies on reaction mechanisms and products

High performance liquid chromatography (HPLC) was used to follow the kinetics of the alkaline induced opening of the imidazole ring of 7-methylguanosine (7-meGuo). The kinetics show an initial rapid formation of a major transient intermediate and some minor products that were chromato-graphically separable into seven peaks. This phase of the reaction is followed by the formation of a dominant pyrimidine derivative whose liquid chromatography retention time in a 6% methanol, 0.01 M NH₄H₂PO₄ (pH 5.1) solvent is 6 min; during the rest of the reaction time this dominant species was progressively converted to a co-dominant species that has a 4.5-min column retention. Mass spectroscopy confirmed the existence of two species of ring opened 7-methylguanine (7-meGua), one formylated and another deformylated. Schiff's reaction demonstrated that the species in the second HPLC peak is the formylated one. The ring opened 7-methylguanine (rom⁷Gua) released by formamidopyrimidine (FAPy)-DNA glycosylase was shown to coelute with the formylated species. These results demonstrate that the enzyme excises formylated rom⁷Gua from DNA Analysis of rom⁷Guo by NMR showed that there are two signals assignable to methyl protons and two to formyl protons. These chemical shifts were interpreted as being due to the opening of the imidazole ring at two sites and to the formation of formylated and deformylated rom⁷Gua.     

Properties of 7-substituted deoxyguanosines formed by cis-diamminedichloroplatinum(II)

cis-Diamminedichloroplatinum(II) (cis-Pt) was reacted with deoxyguanosine and guanosine at pH 6 and the reaction products were purified by HPLC. The products were characterized by UV and 1H-NMR spectra and by incubating cis-Pt with 7-methylguanosine, N2-methylguanosine, [8-3H]guanosine and [U-14C]guanosine. The main product was shown to be a cross-link, in which cis-Pt was linked to the N-7 atoms of two guanines. In the other product cis-Pt was bound monofunctionally to the N-7 atom of guanine. The cis-Pt adducts had many unique properties compared to other N-7 alkylated guanosines. When cis-Pt was incubated with [8-3H]guanosine, the products still had their 8-3H-radioactivity. At pH 10 at room temperature the imidazole ring of the monoadduct was not opened in 20 days, while the half-life of imidazole ring-opening for 7-methyldeoxyguanosine was 21.5 min. The half-lives of acid-catalyzed depurination in 0.1 M HCl at 70 degrees C for deoxyguanosine, 7-methyldeoxyguanosine and the monoadduct were 30 s, 48 s and 35 min, respectively.