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DNA constituents are effectively decomposed via dissociative electron attachment (DEA). However, the DEA contribution to radiation damage in living tissues is a subject of ongoing discussion. We address an essential question, how aqueous environment influences the DEA to DNA. In particular, we report experimental fragmentation patterns for DEA to microhydrated 2-deoxycytidine 5-monophosphate (dCMP). Isolated dCMP was previously set as a model to describe mechanisms of DNA-strand breaks induced by secondary electrons and decomposes primarily by dissociation of the C-O phosphoester bond. We show that hydrated molecules decompose via dissociation of the C-N glycosidic bond followed by dissociation of the P-O bond. This significant change of the proposed mechanism can be interpreted by a reactive role of water in the postattachment dynamics. Comparison of the fragmentation with previous macroscopic irradiation studies suggests that the actual contribution of DEA to DNA radiation damage in living tissue is rather small. [ABSTRACT FROM AUTHOR]

The dCTP pyrophosphatase 1 (dCTPase) is a nucleotide pool "housekeeping" enzyme responsible for the catabolism of canonical and noncanonical nucleoside triphosphates (dNTPs) and has been associated with cancer progression and cancer cell stemness. We have identified a series of piperazin-1-ylpyridazines as a new class of potent dCTPase inhibitors. Lead compounds increase dCTPase thermal and protease stability, display outstanding selectivity over related enzymes and synergize with a cytidine analogue against leukemic cells. This new class of dCTPase inhibitors lays the first stone toward the development of drug-like probes for the dCTPase enzyme. [ABSTRACT FROM AUTHOR]

Several modified bases have been observed in the genomic DNA of bacteriophages, prokaryotes, and eukaryotes that play a role in restriction systems and/or epigenetic regulation. In our efforts to understand the consequences of replacing a large fraction of a canonical nucleoside with a modified nucleoside, we previously replaced around 75% of thymidine (T) with 5'- hydroxymethyl-2'-deoxyuridine (5hmU) in the Escherichia coli genome. In this study, we engineered the pyrimidine nucleotide biosynthetic pathway using T4 bacteriophage genes to achieve approximately 63% replacement of 2'- deoxycytidine (dC) with 5-hydroxymethyl-2'-deoxycytidine (5hmC) in the E. coli genome and approximately 71% replacement in plasmids. We further engineered the glucose metabolic pathway to transform the ShmC into glucosyl-5-hydroxymethyl-2'-deoxycytidine (5-gmC) and achieved 20% 5-gmC in the genome and 45% 5-gmC in plasmid DNA. [ABSTRACT FROM AUTHOR]

Computational results have been reported for 2′-deoxycytidine (dC), its gas phase isomers, tautomers, and their conformers, as well as for the crystalline phase. In addition to the neutral gas phase molecules, we have also considered associated radical anions and cations. The structural calculations were performed at the density functional and MP2 levels of theory. Vertical electron ionization energies and excess electron binding energies were determined using electron propagator theory. The α-anomer proved to be more stable by a fraction of kcal/mol than the biologically relevant canonical β-anomer. The conformational space of canonical dC has been systematically probed. dC in the crystalline phase or DNA structures favors canonical anti conformations. These structures were used in past computational studies to model gas phase characteristics of dC. Our findings indicate, however, that the gas phase dC favors syn conformations. It has repercussions for earlier interpretations of gas phase experimental results based on these computational results. The thermodynamic dominance of syn conformations results from the formation of an intramolecular O5′-H13···O2 hydrogen bond. The IR spectra of the most stable syn and anti canonical conformers differ markedly in the region of frequencies corresponding to NH/OH stretching modes. The MP2 value of deprotonation enthalpy of dC of 1411.7 kJ/mol is in very good agreement with the experimental value of 1409 ± 2.5 kJ/mol. The most stable valence anions are characterized by electron vertical detachment energies (VDE) in the 0.8-1.0 eV range, in good agreement with the experimental VDE of 0.87 eV. The barrier for the glycosidic bond cleavage is significant in the neutral canonical dC, 40.0 kcal/mol, and it is reduced to 22 and 16 kcal/mol for the anionic and cationic radicals of dC, respectively. The cleavage reaction is exothermic by 4 kcal/mol for dC- and endothermic by 7 and 9 kcal/mol for dC+ and dC, respectively. We decomposed the crystal cohesive energy into repulsive one-body terms associated with the syn-anti conformational changes, and the attractive intermolecular interaction term. We exposed that the syn-anti conformational changes are very favorable for intermolecular interactions; in particular they make the imino-amino side of the cytosine residue accessible to intermolecular interactions. [ABSTRACT FROM AUTHOR]

IRMPD action spectroscopy studies of protonated 2'-deoxycytidine and cytidine, [dCyd+H]+ and [Cyd+H]+, have established that both N3 and O2 protonated conformers coexist in the gas phase. Threshold collision-induced dissociation (CID) of [dCyd+H]+ and [Cyd+H]+ is investigated here using guided ion beam tandem mass spectrometry techniques to elucidate the mechanisms and energetics for N-glycosidic bond cleavage. N-Glycosidic bond cleavage is observed as the major dissociation pathways resulting in competitive elimination of either protonated or neutral cytosine for both protonated cytosine nucleosides. Electronic structure calculations are performed to map the potential energy surfaces (PESs) for both N-glycosidic bond cleavage pathways observed. The molecular parameters derived from theoretical calculations are employed for thermochemical analysis of the energy-dependent CID data to determine the minimum energies required to cleave the N-glycosidic bond along each pathway. B3LYP and MP2(full) computed activation energies for N-glycosidic bond cleavage associated with elimination of protonated and neutral cytosine, respectively, are compared to measured values to evaluate the efficacy of these theoretical methods in describing the dissociation mechanisms and PESs for N-glycosidic bond cleavage. The 2'-hydroxyl of [Cyd+H]+ is found to enhance the stability of the N-glycosidic bond vs that of [dCyd+H]+. O2 protonation is found to control the threshold energies for N-glycosidic bond cleavage as loss of neutral cytosine from the O2 protonated conformers is found to require ~25 kJ/mol less energy than the N3 protonated analogues, and the activation energies and reaction enthalpies computed using B3LYP exhibit excellent agreement with the measured thresholds for the O2 protonated conformers. [ABSTRACT FROM AUTHOR]

The dCTPase pyrophosphatase 1 (dCTPase) regulates the intracellular nucleotide pool through hydrolytic degradation of canonical and noncanonical nucleotide triphosphates (dNTPs). dCTPase is highly expressed in multiple carcinomas and is associated with cancer cell stemness. Here we report on the development of the first potent and selective dCTPase inhibitors that enhance the cytotoxic effect of cytidine analogues in leukemia cells. Boronate 30 displays a promising in vitro ADME profile, including plasma and mouse microsomal half-lives, aqueous solubility, cell permeability and CYP inhibition, deeming it a suitable compound for in vivo studies. [ABSTRACT FROM AUTHOR]

One common oxidative DNA lesion, 8-oxo-7,8- dihydro-2'-deoxyguanine (8-oxoG), is highly mutagenic in vivo due to its anti-conformation forming a Watson--Crick base pair with correct deoxycytidine 5'-triphosphate (dCTP) and its syn-conformation forming a Hoogsteen base pair with incorrect deoxyadenosine 5'-triphosphate (dATP). Here, we utilized time-resolved X-ray crystallography to follow 8-oxoG bypass by human DNA polymerase β (hPol//). In the 12 solved structures, both Watson--Crick (anti-8-oxoG:antidCTP) and Hoogsteen (syn-8-oxoG:anti-dATP) base pairing were clearly visible and were maintained throughout the chemical reaction. Additionally, a third Mg2+ appeared during the process of phosphodiester bond formation and was located between the reacting α- and β-phosphates of the dNTP, suggesting its role in stabilizing reaction intermediates. After phosphodiester bond formation, hPol/Z reopened its conformation, pyrophosphate was released, and the newly incorporated primer 3'-terminal nucleotide stacked, rather than base paired, with 8-oxoG. These structures provide the first real-time pictures, to our knowledge, of how a polymerase correctly and incorrectly bypasses a DNA lesion. [ABSTRACT FROM AUTHOR]

Mitochondrial thymidine kinase 2 (TK2) is a nuclear gene-encoded protein, synthesized in the cytosol and subsequently translocated into the mitochondrial matrix, where it catalyzes the phosphorylation of thymidine (dT) and deoxycytidine (dC). The kinetics of dT phosphorylation exhibits negative cooperativity, but dC phosphorylation follows hyperbolic Michaelis— Menten kinetics. The two substrates compete with each other in that dT is a competitive inhibitor of dC phosphorylation, while dC acts as a noncompetitive inhibitor of dT phosphorylation. In addition, TK2 is feedback inhibited by dTTP and dCTP. TK2 also phosphorylates a number of pyrimidine nucleoside analogues used in antiviral and anticancer therapy and thus plays an important role in mitochondrial toxicities caused by nucleoside analogues. Deficiency in TK2 activity due to genetic alterations causes devastating mitochondrial diseases, which are characterized by mitochondrial DNA (mtDNA) depletion or multiple deletions in the affected tissues. Severe TK2 deficiency is associated with early-onset fatal mitochondrial DNA depletion syndrome, while less severe deficiencies result in lateonset phenotypes. In this review, studies of the enzyme kinetic behavior of TK2 enzyme variants are used to explain the mechanism of mtDNA depletion caused by TK2 mutations, thymidine overload due to thymidine phosphorylase deficiency, and mitochondrial toxicity caused by antiviral thymidine analogues. [ABSTRACT FROM AUTHOR]

Recently 5-hydroxymethyl-2′-deoxycytidine(5hmdC), 5-formyl-2′-deoxycytidine(5fdC), and 5-carboxyl-2′-deoxycytidine (5cadC) were discoveredin mammalian DNA as oxidation products of 5-methyl-2′-deoxycytidine(5mdC) induced by the ten-eleven translocation family of enzymes.These oxidized derivatives of 5mdC may not only act as intermediatesof active cytosine demethylation in mammals but also serve as epigeneticmarks on their own. It remains unclear how 5hmdC, 5fdC, and 5cadCaffect DNA replication in mammalian cells. Here, we examined the effectsof the three modified nucleosides on the efficiency and accuracy ofDNA replication in HEK293T human kidney epithelial cells. Our resultsdemonstrated that a single, site-specifically incorporated 5fdC or5cadC conferred modest drops, by approximately 30%, in replicationbypass efficiency without inducing detectable mutations in human cells,whereas replicative bypass of 5hmdC is both accurate and efficient.The lack of pronounced perturbation of these oxidized 5mdC derivativeson DNA replication is consistent with their roles in epigenetic regulationof gene expression. [ABSTRACT FROM AUTHOR]

Newly proposed approach involvingcomputational analysis of multistepchemical reactions has been successfully applied to study the interactionbetween 2′-deoxycytidine and cis-2-butene-1,4-dial,a metabolite of furan. The new method comprises a combination of fewsteps. They include the prediction of the reaction mechanism, calculationof Gibbs free energies for the reaction pathway, and conversion ofbarrier energies to rate constants. On the basis of the results ofprevious steps, corresponding kinetic equations are constructed andsolved. Such a procedure allows one to indicate the definite concentrationof reaction species (reactants, intermediates, and products) at anymoment in time. Obtained results show that 2′-deoxycytidinereacts with cis-2-butene-1,4-dial to form primaryproducts, which are represented by four polycyclic diastereomers.These primary products further transform to more stable secondaryproduct by dehydration, which is catalyzed by acid. The obtained datademonstrate that cis-2-butene-1,4-dial plays a keyrole in furan-induced carcinogenesis. [ABSTRACT FROM AUTHOR]

Thecoupling of gemcitabine with functionalized carboxylic acids (C9–C13)or reactions of 4-N-tosylgemcitabine with the correspondingalkyl amines afforded 4-N-alkanoyl and 4-N-alkyl gemcitabine derivatives. The analogues with a terminalhydroxyl group on the alkyl chain were efficiently fluorinated underconditions that are compatible with protocols for 18F labeling.The 4-N-alkanoylgemcitabines showed potent cytostaticactivities in the low nanomolar range against a panel of tumor celllines, whereas cytotoxicity of the 4-N-alkylgemcitabineswere in the low micromolar range. The cytotoxicity for the 4-N-alkanoylgemcitabine analogues was reduced approximatelyby 2 orders of magnitude in the 2′-deoxycytidine kinase (dCK)-deficientCEM/dCK–cell line, whereas cytotoxicity of the4-N-alkylgemcitabines was only 2–5 times lower.None of the compounds acted as efficient substrates for cytosolicdCK; therefore, the 4-N-alkanoyl analogues need tobe converted first to gemcitabine to display a significant cytostaticpotential, whereas 4-N-alkyl derivatives attain modestactivity without measurable conversion to gemcitabine. [ABSTRACT FROM AUTHOR]

1-Chloro-3-buten-2-one(CBO) is a potential metabolite of 1,3-butadiene (BD), a carcinogenicair pollutant. CBO is a bifunctional alkylating agent that readilyreacts with glutathione (GSH) to form mono-GSH and di-GSH adducts.Recently, CBO and its precursor 1-chloro-2-hydroxy-3-butene (CHB)were found to be cytotoxic and genotoxic in human liver cells in culturewith CBO being approximately 100-fold more potent than CHB. In thepresent study, CBO was shown to react readily with 2′-deoxycytidine(dC) under in vitro physiological conditions (pH 7.4, 37 °C)to form four dC adducts with the CBO moieties forming fused ringswith the N3 and N4atomsof dC. The four products were structurally characterized as 2-hydroxy-2-hydroxymethyl-7-(2-deoxy-β-d-erythro-pentofuranosyl)-1,2,3,4-tetrahydro-6-oxo-6H,7H-pyrimido[1,6-a]pyrimidin-5-ium(dC-1and dC-2, a pair of diastereomers),4-chloromethyl-4-hydroxy-7-(2-deoxy-β-d-erythro-pentofuranosyl)-1,2,3,4-tetrahydro-6-oxo-6H,7H-pyrimido[1,6-a]pyrimidin-5-ium(dC-3), and 2-chloromethyl-2-hydroxy-7-(2-deoxy-β-d-erythro-pentofuranosyl)-1,2,3,4-tetrahydro-6-oxo-6H,7H-pyrimido[1,6-a]pyrimidin-5-ium(dC-4). Interestingly, dC-1and dC-2were stable under our experimental conditions (pH 7.4, 37 °C,and 6 h) and existed in equilibrium as indicated by HPLC analysis,whereas dC-3and dC-4were labile with thehalf-lives being 3.0 ± 0.36 and 1.7 ± 0.06 h, respectively.Decomposition of dC-4produced both dC-1and dC-2, whereas acid hydrolysis of dC-1/dC-2and dC-4in 1 M HCl at 100 °Cfor 30 min yielded the deribosylated adducts dC-1H/dC-2Hand dC-4H, respectively. Because fused-ring dC adductsof other chemicals are mutagenic, the characterized CBO-dC adductscould be mutagenic and play a role in the cytotoxicity and genotoxicityof CBO and its precursors, CHB and BD. The CBO-dC adducts may alsobe used as standards to characterize CBO–DNA adducts and todevelop potential biomarkers for CBO formation in vivo. [ABSTRACT FROM AUTHOR]

Combinedinhibition of ribonucleotide reductase and deoxycytidinekinase (dCK) in multiple cancer cell lines depletes deoxycytidinetriphosphate pools leading to DNA replication stress, cell cycle arrest,and apoptosis. Evidence implicating dCK in cancer cell proliferationand survival stimulated our interest in developing small moleculedCK inhibitors. Following a high throughput screen of a diverse chemicallibrary, a structure–activity relationship study was carriedout. Positron Emission Tomography (PET) using 18F-L-1-(2′-deoxy-2′-FluoroArabinofuranosyl) Cytosine(18F-L-FAC), a dCK-specific substrate, was used to rapidlyrank lead compounds based on their ability to inhibit dCK activity in vivo.Evaluation of a subset of the most potent compoundsin cell culture (IC50= ∼1–12 nM) using the 18F-L-FAC PET pharmacodynamic assay identified compounds demonstratingsuperior in vivo efficacy. [ABSTRACT FROM AUTHOR]

Fluorogenic hydroxylamine reagents were used for detecting 5-fC through a labeling pathway. Chemical synthesis, HPLC, denaturing PAGE, and DNA MS were applied to testify that the probe reacted with 5-fC with oligodeoxynucleotide selectivity to achieve 5-fC detection conveniently and quantificationally with the method of fluorescence. The feasibility of fluorescently detecting 5-fC in a genome was also investigated. [ABSTRACT FROM AUTHOR]

Cation radicals of DNA nucleosides, 2'-deoxyadenosine, 2'-deoxyguanosine, 2'-deoxycytidine, and 2'-deoxythymidine, can exist in standard canonical forms or as noncanonical isomers in which the charge is introduced by protonation of the nucleobase, whereas the radical predominantly resides in the deoxyribose moiety. Density functional theory as well as correlated ab initio calculations with coupled clusters (CCSD(T)) that were extrapolated to the complete basis set limit showed that noncanonical nucleoside ion isomers were thermodynamically more stable than their canonical forms in both the gas phase and as water-solvated ions. This indicated the possibility of exothermic conversion of canonical to noncanonical forms. The noncanonical isomers were calculated to have very low adiabatic ion-electron recombination energies (RE ad ) for the lowest-energy isomers 2'-deoxy-(N-3 H )adenos-1'-yl (4.74 eV), 2'-deoxy-(N-7 H )guanos-1'-yl (4.66 eV), 2'-deoxy-(N-3 H )cytid-1'-yl (5.12 eV), and 2'-deoxy-5-methylene-(O-2 H )uridine (5.24 eV). These were substantially lower than the RE ad value calculated for the canonical 2'-deoxyadenosine, 2'-deoxy guanosine, 2'-deoxy cytidine, and 2'-deoxy thymidine cation radicals, which were 7.82, 7.46, 8.14, and 8.20 eV, respectively, for the lowest-energy ion conformers of each type. Charge and spin distributions in noncovalent cation-radical dA⊂dT and dG⊂dC nucleoside pairs and dAT, dCT, dTC, and dGC dinucleotides were analyzed to elucidate the electronic structure of the cation radicals. Born-Oppenheimer molecular dynamics trajectory calculations of the dinucleotides and nucleoside pairs indicated rapid exothermic proton transfer from noncanonical T + · to A in both dAT + · and dA⊂dT + · , leading to charge and radical separation. Noncanonical T + · in dCT + containing canonical neutral and noncanonical ionized deoxycytidine.· and dTC + · initiated rapid proton transfer to cytosine, whereas the canonical dCT + · dinucleotide ion retained the cation radical structure without isomerization. No spontaneous proton transfer was found in dGC + · and dG⊂dC + · containing canonical neutral and noncanonical ionized deoxycytidine.

Complex OsH6(PiPr3)2 (1) deprotonates cyto-sine to give molecular hydrogen and the d4-trihydride derivative OsH3(cytosinate)(PiPr3)2 (2), which in solution exists as a mixture of isomers containing k2-N1,O (2a) and K2-N3,O (2b) amino-oxo and k2-N3,N4 (2c) imino-oxo tautomers. The major isomer 2b associates with the minor one 2c through N--H…N and N--H…O hydrogen bonds to form [2b-2c]2 dimers, which crystallize from saturated pentane solutions of 2. Complex 1 is also able to perform the double deprotonation of cytosine (cytosinate') to afford the dinudear derivative (P'Pr3)2H3Os(cytosinate')OsH3(P iPr3)2 (3), where the anion is coordinated k2-N1,O and k2-N3,N4 to two different OsH3(PiPr3)2 metal fragments. The deprotonation of deoxycytidine and cytidine leads to OsH3(deoxycyndinate)-(PiPr3)2 (4) and OsH3(cytidinate)(PiPr3)2 (5), respectively, containing the anion k2-N3,N4 coordinated. Dimer [2b • 2c]2 and dinuclear complex 3 have been characterized by X-ray diffraction analysis. [ABSTRACT FROM AUTHOR]

B3LYP/6-311+G(2d,p)//B3LYP/6-31+G(d) density functional theory calculations were employed to explore the kinetics and thermodynamics of gas-phase N-glycosidic bond cleavage induced by nucleophilic attack of C1' with a hydroxide ion in 5-substituted 2'-deoxycytidines. The results showed that, among the 5-substituted 2'-deoxycytidine derivatives examined [XdC, where X = H (dC), CH3 (medC), CH2OH (hmdC), CHO (fmdC), COOH (cadC), F (FdC), or Br (BrdC)], fmdC and cadC exhibited the lowest energy barrier and largest exothermicity for N-glycosidic bond cleavage. These results paralleled previously reported nucleobase excision activities of human thymine DNA glycosylase (hTDG) toward duplex DNA substrates harboring a thymine and 5-substituted cytosine derivatives when paired with a guanine. Our study suggests that the inherent chemistry associated with the nucleophilic cleavage of N-glycosidic bond constitutes a major factor contributing to the selectivity of hTDG toward 5-substituted dC derivatives. These findings provided novel insights into the role of TDG in active cytosine demethylation. [ABSTRACT FROM AUTHOR]

Recent experimental and theoretical investigations on resonant electron scattering off DNA and DNA fragments using low-energy electrons (LEEs), to propose the mechanism for single strand breaks (SSBs) and double strand breaks (DSBs), have received considerable attention. It is our purpose here to understand theoretically the comprehensive route to SSB in a selected DNA fragment, namely, 2'-deoxycytidine-3'-monophosphate (3'-dCMPH), induced by LEE (0-3 eV) scattering using the local complex potential based time-dependent wave packet (LCP-TDWP) approach. To the best of our knowledge, there is no time-dependent quantum mechanical study that has been reported in the literature for this DNA fragment to date. Initial results obtained from our calculation in the gas phase provide a good agreement with experimental observation and show the plausibility of SSB at 0.75 eV, which is very close to the highest SSB yield reported from the experimental measurement (0.8 eV) on plasmid DNA in the condensed phase. [ABSTRACT FROM AUTHOR]

Porous liquids are porous materials that have exhibited unique properties in various fields. Herein, we developed a method to synthesize the type I porous liquids via liquefaction of cyclodextrins by chemical modification. The cyclodextrin porous liquids were characterized by Fourier-transform infrared (FTIR) spectroscopy, NMR, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS), circular dichroism (CD), and UV-vis spectroscopy. The measured ionic conductivity of the γ - cyclodextrin porous liquid was 500 times as great as that of its reactants, which was found to be the first instance with such great conductivity for a type I porous liquid. What is more, the γ-cyclodextrin porous liquid had been demonstrated experimentally to have outstanding chiral recognition ability toward pyrimidine nucleosides in water, which was further confirmed by computational simulations. Additionally, enantiomeric excess of the extracted nucleoside was achieved up to 84.81% by convenient extraction from the mixture of racemic nucleosides and γ-cyclodextrin porous liquid. The great features of the novel cyclodextrin porous liquids could bring opportunities in many fields, including the preparation of chiral separation materials, development of new drug screening mechanisms, and construction of chiral response materials.

3'-(4-Aminophenyl phosphate) derivatives of deoxycytidine (dCyd), deoxyadenosine (dAdo), and deoxyguanosine ( dGuo ) were synthesized. The inhibitory effects of these compounds on mammalian and bacterial deoxynucleoside kinases and several other deoxynucleoside-metabolizing enzymes were examined. The same derivatives were coupled to carboxyl-terminal Sepharose CL-6B (3-8 mumol of ligand/mL of gel), and each of the resulting affinity adsorbents was tested with various partially purified enzymes. Reasonable correlation between the inhibitory effect of a soluble deoxynucleoside 3'-phosphate diester and affinity of the corresponding Sepharose adsorbent for the enzyme was observed. Among the three dCyd kinases examined, only the bovine mitochondrial enzyme was adsorbed onto the dCyd-Sepharose column and eluted biospecifically by 1 mM dCyd (1400-fold purification). Its Ki toward the dCyd derivative was relatively low (1.1 mM), whereas no measurable inhibition was seen with mammalian cytosol or bacterial enzymes that did not stick to the column. The Ki of the dAdo derivative toward three dAdo kinases was more than 5 mM in each case, and none of these were retained by dAdo-Sepharose. Among the other dAdo-metabolizing enzymes examined, nucleoside phosphotransferase from barley (Ki = 1.2 mM) was adsorbed to dAdo-Sepharose at pH 5.0 and was biospecifically eluted with dAdo or AMP after suppressing ionic binding by adjusting the pH to 6.0 (480-fold purification to homogeneity). Mammalian mitochondrial dGuo kinase (beef liver) showed the lowest Ki (0.16 mM) among the enzymes tested and was biospecifically purified with dGuo -Sepharose (2800-fold purification).(ABSTRACT TRUNCATED AT 250 WORDS)

Imino proton and 31P NMR studies were conducted on the binding of actinomycin D (ActD) to self-complementary oligodeoxyribonucleotides with adjacent 5'-GC-3' sites. ActD showed very high specificity for binding to GC sites regardless of oligomer length and surrounding sequence. For a first class of duplexes with a central GCGC sequence, a mixture of 1:1 complexes was observed due to the two different orientations of the ActD phenoxazone ring system. Analysis of 1H chemical shifts suggested that the favored 1:1 complex had the benzenoid side of the phenoxazone ring over the G base in the central base pair of the GCGC sequence. This is the first case in which an unsymmetrical intercalator has been shown to bind to DNA in both possible orientations. A unique 2:1 complex, with significantly different 1H and 31P chemical shifts relative to those of the 1:1 complexes, was formed with these same oligomers, again with the benzenoid side of the ActD molecule over the G base of the central GC base pair. There is considerable anticooperativity to binding of the second ActD in a GCGC sequence. In titrations of oligomers with the GCGC sequence, only the two 1:1 complexes are found up to ratios of one ActD per oligomer. Increasing the ActD concentration, however, resulted in stoichiometric formation of the unique 2:1 adduct. Spectrophotometric binding studies indicated that the apparent binding equilibrium constant for a GC site adjacent to a bound site is reduced by approximately a factor of 20 relative to the ActD binding constant to an isolated GC site.

The isolation and structural characterization are described of the major monoaddition products formed in the photoreaction of two naturally occurring psoralens, 8-methoxypsoralen and 4,5',8-trimethylpsoralen, with high molecular weight, double-stranded DNA. Hydrolysis of the psoralen-modified DNA and subsequent chromatography resulted in the isolation of four modified nucleosides from each psoralen. Structural characterization was accomplished by mass spectrometry and 1H NMR analysis. The major products, accounting for 44-52% of the covalently bound psoralen, are two diastereomeric thymidine adducts formed by cycloaddition between the 5,6 double bond of the pyrimidine and the 4',5' (furan) double bond of the psoralen. A minor product, less than 2% of the covalently bound psoralen, is a furan-side adduct to deoxyuridine, derived from an initially formed deoxycytidine adduct by hydrolytic deamination. A fourth product is a thymidine adduct where cycloaddition has taken place between the 5,6 double bond of the pyrimidine and the 3,4 (pyrone) double bond of the psoralen. This pyrone-side adduct accounts for 19% of the covalently bound 8-methoxypsoralen but for less than 3% of the covalently bound 4,5'8-trimethylpsoralen. All of the isolated adducts have cis-syn stereochemistry. The stereochemistry and product distribution of the adducts are determined in part by the constraints imposed by the DNA helix on the geometry of the noncovalent intercalation complex formed by psoralen and DNA prior to irradiation.

Forty-three pyrimidine derivatives, mainly containing the 4-aminopyrimidine system, have been prepared and evaluated as inhibitors of deoxycytidine kinase. The most effective inhibitors were 2-alkylthio-4-aminopyrimidines and 1-alky-1-cytosines. The best inhibitors in both groups were those with large alkyl substituents, which indicate that hydrophobic bonding is occurring, possibly in the same area adjacent to the active site.

Solid-state 13C NMR spectroscopy of a series of crystalline nucleosides and nucleotides allows direct measurement of the effect of the deoxyribose ring conformation on the carbon chemical shift. It is found that 3'-endo conformers have 3' and 5' chemical shifts significantly (5-10 ppm) upfield of comparable 3'-exo and 2'-endo conformers. The latter two conformers may be distinguished by smaller but still significant differences in the carbon chemical shifts at the C-2' and C-4' positions. High-resolution solid-state NMR of three modifications of fibrous calf thymus DNA shows that these trends are maintained in high-molecular-weight DNA and confirms that the major ring pucker in A-DNA is 3'-endo, while both B-DNA and C-DNA are largely 2'-endo. The data show that 13C NMR spectroscopy is a straightforward and useful probe of DNA ring pucker in both solution and the solid state.

Deoxyadenosine-deoxycytidine pairing at symmetrically related dA X dC mismatch sites in the d(C1-G2-C3-G4-A5-A6-T6-T5-C4-A3-C2-G1) self-complementary duplex (henceforth called 12-mer AC) has been investigated by proton and phosphorus NMR studies in aqueous solution. We demonstrate that base pairing is maintained on either side of the mismatch site in the 12-mer AC duplex at low temperature. The proton chemical shifts and NOE measurements rule out models in which the H-2 proton of adenosine at the mismatch site is stacked over adjacent dG X dC base pairs. A comparison of the hydrogen-exchange kinetics in the d(C-G-C-G-A-A-T-T-C-G-C-G) duplex (henceforth called 12-mer) with standard dG X dC base pairs at position 3 from either end with the 12-mer AC duplex, which contains dA X dC mismatches at these positions, demonstrates kinetic destabilization at dG X dC base pair 4 adjacent to the mismatch site and at dA X dT base pairs 5 and 6 far from this site. This contrasts with previous hydrogen-exchange studies on the 12-mer GT and 12-mer GA duplexes where the kinetic destabilization was localized to base pair 4, which is adjacent to the mispairing site. The melting temperature of the 12-mer AC duplex in 0.1 M phosphate is approximately 30 degrees C lower than the corresponding value for the 12-mer duplex.(ABSTRACT TRUNCATED AT 250 WORDS)