N3 Of Adenine Research Articles

Determinants of Selectivity in Alkylation of Nucleosides and DNA by Secondary Diazonium Ions: Evidence for, and Consequences of, a Preassociation Mechanism

Reactions have been carried out in which 1,3-diisopropyltriazene or N-isopropyl-N-(1-hydroxyethyl)nitrosamine has been decomposed in neutral, buffered aqueous media in the presence of (15N2)2'-deoxyguanosine and (15N6)2'-deoxyadenosine. The products of covalent attachment of the isopropyl cation, derived from the isopropyl diazonium ion, to the heteroatoms of the purines have been separated and quantified by HPLC/electrospray mass spectrometry by employing isotopically distinct synthetic standards. The results indicate that the two different precursors of the isopropyl cation result in the formation of different yields of products in the reactions at all of the heteroatoms of both purines, outside experimental error, except possibly in the case of the N3 position of dAdo. For the different alkylating agents, the ratios of yields at any two sites vary as well. This leads to the conclusion that isopropylation occurs by a preassociation mechanism in which the isopropyl cation intermediate reacts in the solvation shell in which it is generated from its precursors. The reaction of N-isopropyl-N-(1-hydroxyethyl)nitrosamine results in alkylation of 2'-deoxyguanosine in preference to 2-deoxyadenosine, by a factor of 3-4. In this reaction, the yields for reaction at N1, N3, N6, and N7 of adenine vary over less than a factor of 2, whereas the yields for N2, N3, O6, and N7 of guanine vary over less than a factor of 4. The N1 atom of guanine is disfavored over the major product, the O6 adduct, by a factor of <8. The reaction of 1,3-diisopropyltriazene shows a similar preference for alkylation of 2-deoxyguanosine, with a similar range of product distribution in the reactions at adenine heteroatoms and a somewhat larger range of distribution at guanine heteroatoms. In particular, the yield of 1-isopropylguanosine is 50-fold lower than that of O6-isopropylguanosine. The comparable yields of products of reaction at the "hard" and "soft" sites of the purines lead to the conclusion that nucleophilicity is unimportant in site selectivity of alkylation by the isopropyl cation. The noteworthy selectivities, above, are rationalized by: differences in the association constants of the precursors of the cations with the two nucleosides; steric, statistical, and electrostatic effects that favor reaction of the O6 atom of guanine; and larger steric and/or desolvation requirements for association of the 1,3-diisopropyltriazenium cation with the N1 atom of guanine. The reaction of N-isopropyl-N-(1-hydroxyethyl)nitrosamine with double-stranded DNA has been similarly analyzed. The product distribution is remarkably similar in profile to that observed for the nucleosides in solution. In particular, exocyclic amino groups are competitive with the more traditional sites of diazonium ion-mediated alkylation. A comparison to earlier literature data on alkylation by methyl- and ethyl-diazonium ions illustrates some fundamental differences between the reaction of the diazonium ions and the isopropyl cation derived from the isopropyl diazonium ion.

Titration of poly(dA-dT) . poly(dA-dT) in solution at variable NaCl concentration.

CD and uv absorption data showed that high molecular weight poly(dA-dT) . poly(dA-dT), at 298 K, undergoes an acid-induced transition from B-double helix to random coil in NaCl solutions of different concentrations, ranging from 0.005 to 0.600M. Similarly, titration of the polynucleotide with a strong base causes duplex-to-single strands transition. The base- and acid-induced transitions were both reversible by back-titration (with an acid or, respectively, with a base): the apparent pKa were the same in both directions. However, the number of protons per titratable site (adenine N1) required to reach half-denaturation was in great excess over the stoichiometric value; to a much larger extent, the same effect was observed also for the deprotonation of the N3H sites of thymine. Moreover, in the basic denaturation experiments, at low salt concentrations ([NaCl]< or =0.300M) less acid than calculated was needed to back-titrate the base excess to half-denaturation. Both effects could be qualitatively justified on the basis of the counterion condensation theory of polyelectrolytes and considering the energy barrier created by the negatively charged phosphodiester groups to the penetration of the OH- ions inside the double helix and the screening effect of the Na+ ions on such charges, in the deprotonation experiments.

Medicinal Chemistry and Pharmacology of Cyclic ADP‐Ribose

AbstractFor Abstract see ChemInform Abstract in Full Text.

Adenine-containing DNA-DNA cross-links of antitumor nitrogen mustards.

Nitrogen mustards (NMs) are useful chemotherapeutic agents in the treatment of lymphoma, leukemia, multiple myeloma, and ovarian carcinoma. The antitumor activity of NMs has been attributed to their ability to cross-link the twin strands of DNA. The resulting bifunctional lesions, if not repaired, can inhibit DNA replication and transcription, eventually leading to cell cycle arrest, apoptosis, and the inhibition of tumor growth. The predominant bifunctional DNA lesions of NM have been reported to involve the distal guanine bases in the opposite strands of 5'-GNC sequences. In the present work, the formation of guanine-adenine and adenine-adenine adducts of N,N-bis(2-chloroethyl)methylamine (mechlorethamine) in double-stranded DNA is demonstrated. Guanine-adenine cross-links of mechlorethamine were identified as N-(2-[N3-adenyl]ethyl)-N-(2-[N7-guanyl]ethyl)methylamine (N3A-N7G-EMA), N-(2-[N1-adenyl]ethyl)-N-(2-[N7-guanyl]ethyl)methylamine, and N-(2-[N(6)-adenyl]ethyl)-N-(2-[N7-guanyl]ethyl)methylamine. All three adducts were produced interstrand, while N3A-N7G-EMA was the dominant intrastrand G-A cross-link. The prevalent adenine-adenine mechlorethamine lesions have the structure of N,N-bis(2-[N3-adenyl]ethyl)methylamine (bis-N3A-EMA). DNA-derived lesions have the same HPLC retention times, UV spectra, and MS/MS fragmentation patterns as the authentic standards prepared independently. bis-N3A-EMA lesions were produced in a concentration-dependent manner in calf thymus DNA treated with increasing amounts of mechlorethamine. Furthermore, HPLC-ESI-MS/MS analysis was used to demonstrate the formation of analogous N3-N3 adenine lesions in DNA treated with aromatic nitrogen mustards, N,N-bis(2-chloroethyl)-p-aminophenylbutyric acid and L-phenylalanine mustard. The presence of cross-linked adenine-adenine lesions may explain the enhanced cytotoxicity and mutagenicity of NMs in cells deficient in N3-alkyladenine glycosylase.

Medicinal chemistry and pharmacology of cyclic ADP-ribose.

Cyclic ADP-ribose (cADPR) is a signaling molecule that has been shown to regulate calcium mobilization from intracellular stores in a wide variety of biological systems. Synthesis of structural analogs of cADPR has provided insights into structure-activity relationships as well as produced pharmacological research tools with useful properties such as, hydrolysis-resistance and cell permeability. The first generation of cADPR analogs was synthesized by a chemo-enzymatic approach that took advantage of the broad substrate specificity of Aplysia ADP-ribosyl cyclase. Analogs synthesized by this approach provided useful structure-activity information, including the importance of the 8-position of the adenine in determining agonistic or antagonistic activity and of the 3'-hydroxyl group of the southern ribose for activity. Hydrolysis resistant analogs were generated by replacing the southern ribose with a carbocyclic structure or by replacing the adenine ring with 7-deaza- or 3-deaza-adenine. Approaches to synthesize cADPR analogs by total chemical approaches have been recently reported. These approaches allow the synthesis of analogs with stable linkages between N1 of adenine and the northern ribose (or surrogate) that are not possible with the enzymatic strategy. This review will focus on the synthesis and properties of analogs that have been shown to have utility in dissecting the role of cADPR in calcium signaling.

Mutagenic N-Acyloxy-N-alkoxyamides: Probes for Drug - DNA Interactions

N-acyloxy-N-alkoxyamides are direct-acting mutagens in Salmonella typhimurium TA100 and react with DNA at N7 of guanine and N3 of adenine. From extensive mutagenicity data a quantitative structure–activity relationship (QSAR) has been derived that predicts activity to be dependent upon hydrophobicity (represented by log P) and stability to chemical reactions (through the pKA value of the leaving carboxylic acid group). Sterically bulky substituents (represented by Taft ES parameters) reduce activity. Deviations from this QSAR highlight structural features that can enhance or impede association of small molecules with DNA. Naphthyl, pyrenyl, and fluorenyl substituents raise activity significantly, possibly through an intercalative effect. The influence of a single sterically bulky tert-butyl group remote from the reactive nitrogen is adequately modelled by the QSAR. However, substrates with two or more such groups show radically reduced activity, most likely through a groove exclusion process. Branching close to the reactive centre strongly reduces activity in line with a steric inhibition of reaction with DNA.

Inhibition of Werner Syndrome Helicase Activity by Benzo[c]phenanthrene Diol Epoxide dA Adducts in DNA Is Both Strand-and Stereoisomer-dependent

Helicases are among the first enzymes to encounter DNA damage during DNA processing within the cell and thus are likely to be targets for the adverse effects of DNA lesions induced by environmental chemicals. Here we examined the effect of cis- and trans-opened 3,4-diol 1,2-epoxide (DE) DNA adducts of benzo[c]phenanthrene (BcPh) at N6 of adenine on helicase activity. These adducts are derived from the highly tumorigenic (-)-(1R,2S,3S,4R)-DE as well as its less carcinogenic (+)-(1S,2R,3R,4S)-DE enantiomer in both of which the benzylic 4-hydroxyl group and epoxide oxygen are trans. The hydrocarbon portions of these adducts intercalate into DNA on the 3' or the 5' side of the adducted deoxyadenosine for the 1S- and 1R-adducts, respectively. These adducts inhibited the human Werner (WRN) syndrome helicase activity in a strand-specific and stereospecific manner. In the strand along which WRN translocates, cis-opened adducts were significantly more effective inhibitors than trans-opened isomers, indicating that WRN unwinding is sensitive to adduct stereochemistry. WRN helicase activity was also inhibited but to a lesser extent by cis-opened BcPh DE adducts in the displaced strand independent of their direction of intercalation, whereas inhibition by the trans-opened stereoisomers in the displaced strand depended on their orientation, such that only adducts oriented toward the advancing helicase inhibited WRN activity. A BcPh DE adduct positioned in the helicase-translocating strand did not sequester WRN, nor affect the rate of ATP hydrolysis relative to an unadducted control. Although the Bloom (BLM) syndrome helicase was also inhibited by a cis-opened adduct in a strand-specific manner, this helicase was not as severely affected as WRN. Because BcPh DEs form substantial amounts of deoxyadenosine adducts at dA, their adverse effects on helicases could contribute to genetic damage and cell transformation induced by these DEs. Thus, the unwinding activity of RecQ helicases is sensitive to the strand, orientation, and stereochemistry of intercalated polycyclic aromatic hydrocarbon adducts.

Characterization and Elucidation of Coordination Requirements of Adenine Nucleotides Complexes with Fe(II) Ions

In spite of the significant role of iron ions-nucleotide complexes in living cells, these complexes have been studied only to a limited extent. Therefore, we fully characterized the ATP:Fe(II) complex including stoichiometry, geometry, stability constants, and dependence of Fe(II)-coordination on pH. A 1:1 stoichiometry was established for the ATP:Fe(II) complex based on volumetric titrations, UV and SEM/EDX measurements. The coordination sites of ferrous ions in the complex with ATP, established by 1H-, 31P-, and 15N-NMR, involve the adenine N7 as well as Pα, Pβ, and Pγ. Coordination sites remain the same within the pH range of 3.1–8.3. By applying fluorescence monitored Fe(II)-titration, we established a log K value of 5.13 for the Fe(ATP)2− complex, and 2.31 for the Fe(HATP)− complex. Ferrous complexes of ADP3− and AMP2− were less stable (log K 4.43 and 1.68, respectively). The proposed major structure for the Fe(ATP)2− complex is the ‘open’ structure. In the minor ‘closed’ structure N7 nitrogen is probably coordinated with Fe(II) through a bridging water molecule. The electronic and stereochemical requirements for Fe(II)-coordination with ATP4− were probed using a series of modified-phosphate or modified-adenine ATP analogues. We concluded that: Fe(II) coordinates solely with the phosphate-oxygen atom, and not with sulfur, amine, or borane in the cases of phosphate-modified analogues of ATP; a high electron density on N7 and an anti conformation of the adenine-nucleotide are required for enhanced stability of ATP analogues:Fe(II) complexes as compared to ATP complexes (up to more than 100-fold); there are no stereochemical preferences for Fe(II)-coordination with either Rp or Sp isomers of ATP-α-S or ATP-α-BH3 analogues.

Structural differentiation of diastereomeric benzo[ghi]fluoranthene adducts of deoxyadenosine by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and postsource decay.

The product ion formation characteristics of four diastereomeric deoxyadenosine adducts formed by the reaction of the syn and anti diastereomers of trans-3,4-dihydroxy-5,5a-epoxy-3,4,5,5a-tetrahydrobenzo[ghi]fluoranthene are studied by matrix-assisted laser desorption ionization and postsource decay (PSD) to determine fragmentation pathways that may permit differentiation of their structures. The two adducts derived from each diol-epoxide with DNA differ in structure based on the cis/trans arrangement of the 3'-hydroxyl group on the benzo[ghi]fluoranthene (B[ghi]F) and the adenine base bound to the B[ghi]F 5a carbon. The two adduct diastereomers with the cis adenine-3'-hydroxyl configuration produce product ions at m/z 394 and m/z 510 formed by the loss of water that are not observed in the PSD spectra of the two trans isomers. The data suggest a mechanism of water loss that is initiated by a hydrogen-bonding interaction between the charge-bearing proton on the N1 atom and the 3'-hydroxyl oxygen on the polycyclic aromatic hydrocarbon (PAH). Fragmentation is initiated by the transfer of the adenine N1 proton from the nitrogen to the PAH 3'-hydroxyl oxygen and inductive cleavage of the C3-O(3) bond to form a benzylic carbocation on B[ghi]F. The proposed mechanism is supported by semiempirical molecular modeling calculations.

Selectivity of purine alkylation by a quinone methide. Kinetic or thermodynamic control?

The alkylation reaction of 9-methyladenine and 9-methylguanine (as prototype substrates of deoxy-adenosine and -guanosine), by the parent o-quinone methide (o-QM), has been investigated in the gas phase and in aqueous solution, using density functional theory at the B3LYP/6-311+G(d,p) level. The effect of the medium on the reactivity, and on the stability of the resulting adducts, has been investigated by using the C-PCM solvation model to assess which adduct arises from the kinetically favorable path, or from an equilibrating process. The calculations indicate that the most nucleophilic site of the methyl-substituted nucleobases in the gas phase is the guanine oxygen atom (O(6)) (DeltaG()(gas) = 5.6 kcal mol(-)(1)), followed by the adenine N1 (DeltaG)(gas) = 10.3 kcal mol(-)(1)), while other centers exhibit a substantially lower nucleophilicity. The bulk effect of water as a solvent is the dramatic reduction of the nucleophilicity of both 9-methyladenine N1 (DeltaG)(solv) = 14.5 kcal mol(-)(1)) and 9-methylguanine O(6) (DeltaG)(solv) = 17.0 kcal mol(-)(1)). As a result there is a reversal of the nucleophilicity order of the purine bases. While O(6) and N7 nucleophilic centers of 9-methylguanine compete almost on the same footing, the reactivity gap between N1 and N7 of 9-methyladenine in solution is highly reduced. Regarding product stability, calculations predict that only two of the adducts of o-QM with 9-methyladenine, those at NH(2) and N1 positions, are lower in energy than reactants, both in the gas phase and in water. However, the adduct at N1 can easily dissociate in water. The adducts arising from the covalent modification of 9-methylguanine are largely more stable than reactants in the gas phase, but their stability is markedly reduced in water. In particular, the oxygen alkylation adduct becomes slightly unstable in water (DeltaG(solv) = +1.4 kcal mol(-)(1)), and the N7 alkylation product remains only moderately more stable than free reactants (DeltaG(solv) = -2.8 kcal mol(-)(1)). Our data show that site alkylations at the adenine N1 and the guanine O(6) and N7 in water are the result of kinetically controlled processes and that the selective modification of the exo-amino groups of guanine N2 and adenine N6 are generated by thermodynamic equilibrations. The ability of o-QM to form several metastable adducts with purine nucleobases (at guanine N7 and O(2), and adenine N1) in water suggests that the above adducts may act as o-QM carriers.

Unsymmetrical DNA Cross-Linking Agents: Combination of the CBI and PBD Pharmacophores

A set of 10 compounds, each combining the seco-1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one (seco-CBI) and pyrrolo[2,1-c][1,4]benzodiazepine (PBD) pharmacophores, was designed and prepared. These compounds were anticipated to cross-link between N3 of adenine and N2 of guanine in the minor groove of DNA. The compounds, which differ in the chain length separating the two alkylation subunits, and the configuration of the CBI portion, showed great variation in cellular toxicity (over 4 orders of magnitude in a cell line panel) with the most potent example exhibiting IC50s in the pM range. Cytotoxicity correlated with the ability of the compounds to cross-link naked DNA. Cross-linking was also observed in living cells, at much lower concentrations than for a related symmetrical PBD dimer. A thermal cleavage assay was used to assess sequence selectivity, demonstrating that the CBI portion controlled the alkylation sites, while the PBD substituent increased the overall efficiency of alkylation. Several compounds were tested for in vivo activity using a tumor growth delay assay against WiDr human colon carcinoma xenografts, with one compound (the most cytotoxic and most efficient cross-linker) showing a statistically significant increase in survival time following a single iv dose.

Functional Analysis of BamHI DNA Cytosine-N4 Methyltransferase

We show that the kinetic mechanism of the DNA (cytosine-N4-)-methyltransferase M.BamHI, which modifies the underlined cytosine (GGATCC), differs from cytosine C5 methyltransferases, and is similar to that observed with adenine N6 methyltransferases. This suggests that the obligate order of ternary complex assembly and disassembly depends on the type of methylation reaction. In contrast, the single-turnover rate of catalysis for M.BamHI (0.10s−1) is closer to the DNA (cytosine-C5-)-methyltransferases (0.14s−1) than the DNA (adenine-N6-)-methyltransferases (>200s−1). The nucleotide flipping transition dominates the single-turnover constant for adenine N6 methyltransferases, and, since the disruption of the guanine-cytosine base-pair is essential for both types of cytosine DNA methyltransferases, this transition may be a common, rate-limiting step for methylation for these two enzyme subclasses. The similar overall rate of catalysis by M.BamHI and other DNA methyltransferases is consistent with a common rate-limiting catalytic step of product dissociation. Our analyses of M.BamHI provide functional insights into the relationship between the three different classes of DNA methyltransferases that complement both prior structural and evolutionary insights.

Internal degrees of freedom, structural motifs, and conformational energetics of the 5'-deoxyadenosyl radical: implications for function in adenosylcobalamin-dependent enzymes. A computational study.

The potential energy surface of the free 5'-deoxyadenosyl radical in the gas phase is explored using density functional and second-order Møller-Plesset perturbation theories with 6-31G(d) and 6-31++G(d,p) basis sets and interpreted in terms of attractive and repulsive interactions. The 5',8-cyclization is found to be exothermic by approximately 20 kcal/mol but kinetically unfavorable; the lowest cyclization transition state (TS) lies about 7 kcal/mol higher than the highest TS for conversion between most of the open isomers. In open isomers, the two energetically most important attractive interactions are the hydrogen bonds (a) between the 2'-OH group and the N3 adenine center and (b) between the 2'-OH and 3'-OH groups. The relative ribose-adenine rotation about the C1'-N9 glycosyl bond in a certain range changes the energy by as much as 10-15 kcal/mol, the origin being (i) the repulsive 2'-H.H-C8 and O1'.N3 and (ii) the attractive 2'-OH.N3 ribose-adenine interactions. The hypothetical synergy between the glycosyl rotation and the Co-C bond scission may contribute to the experimentally established labilization of the Co-C bond in enzyme-bound adenosylcobalamin. The computational results are not inconsistent with the rotation about the C1'-N9 glycosyl bond being the principal coordinate for long-range radical migration in coenzyme B(12)-dependent enzymes. The effect of the protein environment on the model system results reported here remains an open question.

NMR characterization of the DNA binding properties of a novel Hoechst 33258 analogue peptide building block.

A novel aryl-bis-benzimidazole amino acid analogue of the DNA-binding compound Hoechst 33258 has recently been designed for incorporation in peptide combinatorial libraries by replacing the N-methylpiperazine group with a carboxyl group and the hydroxy group with an amino-methyl group. The DNA-binding properties of the aryl-bis-benzimidazole monomer with the C-terminus derivatized with 3-(dimethylamino)-propylamine has been investigated in this paper by (1)H NMR studies of two different complexes with two different DNA sequences: A(5) d(5'-GCCA(5)CG-3'):d(5'-CGT(5)GGC-3') and A(3)T(3) d(5'-CGA(3)T(3)CG-3')(2). Chemical shift footprinting shows that the ligand binds at the center of the A(3)T(3) sequence but at the 3'-end of A(5). A large number of NOEs show a well-defined complex with the ligand situated at the center of the palindromic A(3)T(3) but with the asymmetric A(5) the ligand binds with an orientational preference with the bis-benzimidazole moiety displaced toward the 3'-end from the center of the duplex. Two families of models of the complexes with A(5) and A(3)T(3) were derived with restrained molecular dynamics based on a large set of 70 and 61, respectively, intermolecular ligand NOEs. Both models give a picture of a tightly fitting ligand with close van der Waals contacts with the walls of the minor groove and with the two benzimidazole and the amide hydrogens involved in bifurcated cross-strand hydrogen bonds to adenine N3 and thymine O2. The minor groove width of the models correlate well with the binding site of the ligand, and the orientational preference is argued to be a consequence of the minor groove width and hydrogen bonding.

Different Substrate Recognition Motifs of Human and Trypanosome Nucleobase Transporters: SELECTIVE UPTAKE OF PURINE ANTIMETABOLITES

The therapeutic index of antimetabolites such as purine analogues is in large part determined by the extent to which it is selectively accumulated by the target cell. In the current study we have compared the transport of purine nucleobase analogues by the H2 transporter of bloodstream form Trypanosoma brucei brucei and the equilibrative nucleobase transporter of human erythrocytes. The H2 transporter forms hydrogen bonds with hypoxanthine at positions N3, N7, N(1)H, and N(9)H of the purine ring, with apparent Delta G(0) of 7.7-12.6 kJ/mol. The transporter also appears to H-bond with the amine group of adenine. The human transporter forms hydrogen bonds that form to (6)NH(2) and N1 of adenine. An H-bond is also formed with N3 and the 6-keto and amine groups of guanine but not with the protonated N1, thus explaining the low affinity for hypoxanthine. N7 and N9 do not directly interact with the human transporter in the form of H-bonds, and it is proposed that pi-pi stacking interactions contribute significantly to permeant binding. The potential for selective uptake of antimetabolites by the parasite transporter was demonstrated.

A theoretical examination of the factors controlling the catalytic efficiency of the DNA-(adenine- N 6 )-methyltransferase from Thermus aquaticus

Ab initio and density functional calculations have been carried out to more fully understand the factors controlling the catalytic activity of the Thermus aquaticus DNA methyltransferase (MTaqI) in the N-methylation at the N(6) of an adenine nucleobase. The noncatalyzed reaction was modeled as a methyl transfer from trimethylsulfonium to the N(6) of adenine. Activation barriers of 32.0 kcal/mol and 24.0 kcal/mol were predicted for the noncatalyzed reaction in the gas phase by MP2/6-31+G(d,p)//HF/6-31+G(d,p) and B3LYP/6-31+G(d,p) calculations, respectively. Calculations performed to evaluate the effect of substrate positioning in the active site of MTaqI on the reaction determine the barrier to be 23.4 kcal/mol and 17.3 kcal/mol for the MP2/6-31+G(d,p)//HF/6-31+G(d,p) and B3LYP/6-31+G(d,p) gas phase calculations, respectively. The effect of hydrogen bonding between the N(6) of adenine and the terminal oxygen of Asn-105 on the activation barrier was also studied. A formamide molecule was modeled into the system to mimic the function of active site residue Asn-105. The activation barrier for this reaction was found to be 21.8 kcal/mol and 15.8 kcal/mol as determined from the MP2/6-31+G(d,p)//HF/6-31+G(d,p) and B3LYP/6-31+G(d,p) calculations, respectively. This result predicts a contribution of less than 2 kcal/mol to the lowering of the activation barrier from amide hydrogen bonding between formamide and N(6) of adenine. Comparison of the reaction coordinates suggest that it is not the hydrogen bonding of the Asn-105 that lends to the catalytic prowess of the enzyme since the organization of the substrates in the active site of the enzyme has a far greater effect on reducing the activation barrier. The results also suggest a stepwise mechanism for the removal of the hydrogen from the N(6) of adenine as opposed to a concerted reaction in which a proton is abstracted simultaneously with the transfer of the methyl group. The hydrogen on the N(6) of the intermediate methyl adenine product is far more acidic than in the reactant complex and may be subsequently abstracted by basic groups in the active site that are too weak to abstract the proton before the full sp(3) hybridization of the attacking nitrogen.

Novel hydrolysis-resistant analogues of cyclic ADP-ribose: modification of the "northern" ribose and calcium release activity.

Three novel analogues modified in the "northern" ribose (ribose linked to N1 of adenine) of the Ca(2+) mobilizing second messenger cyclic adenosine diphosphoribose, termed 2"-NH(2)-cyclic adenosine diphosphoribose, cyclic adenosine diphospho-carbocyclic-ribose, and 8-NH(2)-cyclic adenosine diphospho-carbocyclic-ribose, were synthesized (chemoenzymatically and by total synthesis) and spectroscopically characterized, and the pK(a) values for the 6-amino/imino transition were determined in two cases. The biological activity of these analogues was determined in permeabilized human Jurkat T-lymphocytes. 2"-NH(2)-cyclic adenosine diphosphoribose mediated Ca(2+) release was slightly more potent than that of the endogenous cyclic adenosine diphosphoribose in terms of the concentration-reponse relationship. Both compounds released Ca(2+) from the same intracellular Ca(2+) pool. In addition, the control compound 2"-NH(2)-adenosine diphosphoribose was almost without effect. In contrast, only at much higher concentrations (> or =50 microM) did the "northern" carbocyclic analogue, cyclic adenosine diphospho-carbocyclic-ribose, significantly release Ca(2+) from permeabilized T cells, whereas the previously reported "southern" carbocyclic analogue, cyclic aristeromycin diphosphoribose, was slightly more active than the endogenous cyclic adenosine diphosphoribose. Likewise, 8-NH(2)-cyclic adenosine diphospho-carbocyclic-ribose, expected to antagonize Ca(2+) release as demonstrated previously for 8-NH(2)-cyclic adenosine diphosphoribose, did not inhibit cyclic adenosine diphosphoribose mediated Ca(2+) release. This indicates that the 2"-NH(2)-group substitutes well for the 2"-OH-group it replaces; it may be oriented toward the outside of the putative cyclic adenosine diphosphoribose receptor binding domain and/or it can potentially also engage in H bonding interactions with residues of that domain. In sharp contrast to this, replacement of the endocyclic furanose oxygen atom by CH(2) in a carbocyclic system obviously interferes with a crucial element of interaction between cyclic adenosine diphosphoribose and its receptor in T-lymphocytes.

DNA structural perturbation induced by the CPI-derived DNA interstrand cross-linker: molecular mechanisms for the sequence specific recognition

The highly potent cytotoxic DNA-DNA cross-linker consists of two cyclopropa[c]pyrrolo[3, 4-3]indol-4(5H)-ones indoles [(+)-CPI-I] joined by a bisamido pyrrole (abbreviated to "Pyrrole"). The Pyrrole is a synthetic analog of Bizelesin, which is currently in phase II clinical trials due to its excellent in vivo antitumor activity. The Pyrrole has 10 times more potent cytotoxicity than Bizelesin and mostly form DNA-DNA interstrand cross-links through the N3 of adenines spaced 7 bp apart. The Pyrrole requires a centrally positioned GC base pair for high cross-linking reactivity (i.e., 5'-T(A/T)2G(A/T)2A*-3'), while Bizelesin prefers purely AT-rich sequences (e.g., 5'-T(A/T)4 or 5A*-3', where T represents the cross-strand adenine alkylation and A* represents an adenine alkylation) (Park et al., 1996). In this study, the high-field 1H-NMR and rMD studies are conducted on the 11-mer DNA duplex adduct of the Pyrrole where the 5'-TTAGTTA*-3' sequence is cross-linked by the drug. A severe structural perturbation is observed in the intervening sequences of cross-linking site, while a normal B-DNA structure is maintained in the region next to the drug-modified adenines. Based upon these observations, we propose that the interplay between the bisamido pyrrole unit of the drug and central G/C base pair (hydrogen-bonding interactions) is involved in the process of cross-linking reaction, and sequence specificity is the outcome of those interactions. This study suggests a mechanism for the sequence specific cross-linking reaction of the Pyrrole, and provides a further insight to develop new DNA sequence selective and distortive cross-linking agents.

Silver(I) Complexes with DNA and RNA Studied by Fourier Transform Infrared Spectroscopy and Capillary Electrophoresis

Ag(I) is a strong nucleic acids binder and forms several complexes with DNA such as types I, II, and III. However, the details of the binding mode of silver(I) in the Ag-polynucleotides remains unknown. Therefore, it was of interest to examine the binding of Ag(I) with calf-thymus DNA and bakers yeast RNA in aqueous solutions at pH 7.1–6.6 with constant concentration of DNA or RNA and various concentrations of Ag(I). Fourier transform infrared spectroscopy and capillary electrophoresis were used to analyze the Ag(I) binding mode, the binding constant, and the polynucleotides’ structural changes in the Ag-DNA and Ag-RNA complexes. The spectroscopic results showed that in the type I complex formed with DNA, Ag(I) binds to guanine N7 at low cation concentration ( r = 1/80) and adenine N7 site at higher concentrations ( r = 1/20 to 1/10), but not to the backbone phosphate group. At r = 1/2, type II complexes formed with DNA in which Ag(I) binds to the G-C and A-T base pairs. On the other hand, Ag(I) binds to the guanine N7 atom but not to the adenine and the backbone phosphate group in the Ag-RNA complexes. Although a minor alteration of the sugar-phosphate geometry was observed, DNA remained in the B-family structure, whereas RNA retained its A conformation. Scatchard analysis following capillary electrophoresis showed two binding sites for the Ag-DNA complexes with K 1 = 8.3 × 10 4 M −1 for the guanine and K 2 = 1.5 × 10 4 M −1 for the adenine bases. On the other hand, Ag-RNA adducts showed one binding site with K = 1.5 × 10 5 M −1 for the guanine bases.

DNA minor groove alkylating agents.

Recent work on a number of different classes of anticancer agents that alkylate DNA in the minor groove is reviewed. There has been much work with nitrogen mustards, where attachment of the mustard unit to carrier molecules can change the normal patterns of both regio- and sequence-selectivity, from reaction primarily at most guanine N7 sites in the major groove to a few adenine N3 sites at the 3'-end of poly(A/T) sequences in the minor groove. Carrier molecules discussed for mustards are intercalators, polypyrroles, polyimidazoles, bis(benzimidazoles), polybenzamides and anilinoquinolinium salts. In contrast, similar targeting of pyrrolizidine alkylators by a variety of carriers has little effect of their patterns of alkylation (at the 2-amino group of guanine). Recent work on the pyrrolobenzodiazepine and cyclopropaindolone classes of natural product minor groove binders is also reviewed.