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The O 6 -alkylguanosine adduct O 6 -carboxymethyldeoxyguanosine ( O 6 -CMdG) has been detected at elevated levels in blood and tissue samples from colorectal cancer patients and from healthy volunteers after consuming red meat. The diazo compound l-azaserine leads to the formation of O 6 -CMdG as well as the corresponding methyl adduct O 6 -methyldeoxyguanosine ( O 6 -MedG) in cells and is therefore in wide use as a chemical probe in cellular studies concerning DNA damage and mutation. However, there remain knowledge gaps concerning the chemical basis of DNA adduct formation by l-azaserine. To characterize O 6 -CMdG formation by l-azaserine, we carried out a combination of chemical and enzymatic stability and reactivity studies supported by liquid chromatography tandem mass spectrometry for the simultaneous quantification of O 6 -CMdG and O 6 -MedG. We found that l-azaserine is stable under physiological and alkaline conditions as well as in active biological matrices but undergoes acid-catalyzed hydrolysis. We show, for the first time, that l-azaserine reacts directly with guanosine (dG) and oligonucleotides to form an O 6 -serine-CMdG ( O 6 -Ser-CMdG) adduct. Moreover, by characterizing the reaction of dG with l-azaserine, we demonstrate that O 6 -Ser-CMdG forms as an intermediate that spontaneously decomposes to form O 6 -CMdG. Finally, we quantified levels of O 6 -CMdG and O 6 -MedG in a human cell line exposed to l-azaserine and found maximal adduct levels after 48 h. The findings of this work elucidate the chemical basis of how l-azaserine reacts with deoxyguanosine and support its use as a chemical probe for N-nitroso compound exposure in carcinogenesis research, particularly concerning the identification of pathways and factors that promote adduct formation.

The deuteration of N 2 -ethyl-2'-deoxyguanosine (Et-dG), which is a DNA adduct generated from acetaldehyde, was studied by the addition reaction of acetaldehyde-d 4 to 2'-deoxyguanosine (dG) in deuterium oxide (D 2 O), with the aim to obtain an isotope internal standard for the liquid chromatography/tandem mass spectrometry (LC/MS/MS) quantitation of Et-dG. The replacement of the dG C-8 hydrogen atom by a deuteron atom took place at 50°C in D 2 O and afforded a mixture of Et-dG-d 4 and Et-dG-d 5 . Et-dG-d 4 , which was stable in aqueous solutions, was prepared by incubating the mixture in H 2 O at 60°C for 48 h. The calibration curve was obtained by multiple reaction monitoring (MRM) measurements using a hydrophilic interaction chromatography-electrospray ionization-tandem mass spectrometric (HILIC/ESI-MS/MS) system between the Et-dG concentration, ranging from 1.0 × 10 -10 to 4.0 × 10 -9 M in the sample solutions, and the relative peak areas of Et-dG (m/z: 296.1 → 180.1) to the value of Et-dG-d 4 (m/z: 300.2 → 184.2), with an internal standard showing good linearity (R 2 = 0.995, n = 5).

3'-O-(2-Thio-4,4-pentamethylene-1,3,2-oxathiaphospholane) derivatives of 5'-O-DMT-N6-methyl-deoxyadenosine and 5'-O-DMT-N2,N2-dimethyl-O6-diphenylcarbamoyl-deoxyguanosine (OTP-NY, NY = DMT-m6dA or DMT-m,m2dGDPC) were synthesized, resolved onto pure P-diastereomers, and used in P-stereocontrolled synthesis of dinucleoside 3',5,-phosphorothioates NXPST (NX = m6dA or m,m2dG), in which the absolute configuration of the stereogenic phosphorus atom was established enzymatically. Diastereomerically pure OTP-NY and standard OTP-N (N = DMT-dABz or DMT-dGBz,DPC) were used in the synthesis of chimeric RP-stereodefined phosphorothioate oligomers ((RP-PS)-DN(NX)A) with hampered Watson-Crick base pairings. It was found that the m6dA units slightly reduce the thermodynamic stability of antiparallel duplexes formed with RNA and (2'-OMe)-RNA matrices, whereas m,m2dG units prevent their formation. The m6dA units stabilize (by up to 4.5 °C per modified unit) the parallel duplexes formed by (RP-PS)-DN(NX)A with Hoogsteen-paired (2'-OMe)-RNA templates compared to the analogous reference duplex containing only unmodified nucleobases. In contrast, the m,m2dG units destabilize such duplexes by up to 3 °C per modified unit. Both units prevent the formation of the corresponding parallel triplexes.

We report the synthesis of N 2 -aryl (benzyl, naphthyl, anthracenyl, and pyrenyl)-deoxyguanosine (dG) modified phosphoramidite building blocks and the corresponding damaged DNAs. Primer extension studies using E. coli Pol IV, a translesion polymerase, demonstrate that translesion synthesis (TLS) across these N 2 -dG adducts is error free. However, the efficiency of TLS activity decreases with increase in the steric bulkiness of the adducts. Molecular dynamics simulations of damaged DNA-Pol IV complexes reveal the van der Waals interactions between key amino acid residues (Phe13, Ile31, Gly32, Gly33, Ser42, Pro73, Gly74, Phe76, and Tyr79) of the enzyme and adduct that help to accommodate the bulky damages in a hydrophobic pocket to facilitate TLS. Overall, the results presented here provide insights into the TLS across N 2 -aryl-dG damaged DNAs by Pol IV.

Environmental exposure and cellular metabolism can give rise to DNA alkylation, which can occur on the nitrogen and oxygen atoms of nucleobases, as well as on the phosphate backbone. Although O 6 -alkyl-2'-deoxyguanosine ( O 6 -alkyl-dG) lesions are known to be associated with cancer, not much is known about how the alkyl group structures in these lesions affect their repair and replicative bypass in vivo or how translesion synthesis DNA polymerases influence the latter process. To answer these questions, here we synthesized oligodeoxyribonucleotides harboring seven O 6 -alkyl-dG lesions, with the alkyl group being Me, Et, n Pr, i Pr, n Bu, i Bu, or s Bu, and examined the impact of these lesions on DNA replication in Escherichia coli cells. We found that replication past all the O 6 -alkyl-dG lesions was highly efficient and that SOS-induced DNA polymerases play redundant roles in bypassing these lesions. Moreover, these lesions directed exclusively the G → A mutation, the frequency of which increased with the size of the alkyl group on the DNA. This could be attributed to the varied repair efficiencies of these lesions by O 6 -alkylguanine DNA alkyltransferase (MGMT) in cells, which involve the MGMT Ogt and, to a lesser extent, Ada. In conclusion, our study provides important new knowledge about the repair of the O 6 -alkyl-dG lesions and their recognition by the E. coli DNA replication machinery. Our results suggest that the lesions' carcinogenic potentials may be attributed, at least in part, to their strong mutagenic potential and their efficient bypass by the DNA replication machinery., (© 2018 Wang and Wang.)

Acrolein, an α,β-unsaturated aldehyde, is generated in vivo as the end product of lipid peroxidation and from metabolic oxidation of polyamines, and it is a ubiquitous environmental pollutant. The reaction of acrolein with the N2 of guanine in DNA leads to the formation of γ-hydroxy-1- N 2 -propano-2' deoxyguanosine (γ-HOPdG), which can exist in DNA in a ring-closed or a ring-opened form. Here, we identified the translesion synthesis (TLS) DNA polymerases (Pols) that conduct replication through the permanently ring-opened reduced form of γ-HOPdG ((r) γ-HOPdG) and show that replication through this adduct is mediated via Rev1/Polη-, Polι/Polκ-, and Polθ-dependent pathways, respectively. Based on biochemical and structural studies, we propose a role for Rev1 and Polι in inserting a nucleotide (nt) opposite the adduct and for Pols η and κ in extending synthesis from the inserted nt in the respective TLS pathway. Based on genetic analyses and biochemical studies with Polθ, we infer a role for Polθ at both the nt insertion and extension steps of TLS. Whereas purified Rev1 and Polθ primarily incorporate a C opposite (r) γ-HOPdG, Polι incorporates a C or a T opposite the adduct; nevertheless, TLS mediated by the Polι-dependent pathway as well as by other pathways occurs in a predominantly error-free manner in human cells. We discuss the implications of these observations for the mechanisms that could affect the efficiency and fidelity of TLS Pols., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

A new environmentally responsive fluorescent nucleoside, 3-(naphthalen-1-ylethynyl)-3-deaza-2'-deoxyguanosine ( 3nz G), has been synthesized. The nucleoside, 3nz G, exhibited solvatochromic properties and when introduced into ODN probes it was able to recognize 2'-deoxycytidine in target strands by a distinct change in its emission wavelength through probing microenvironmental changes in the DNA minor groove. Thus, 3nz G has the potential for use as a fluorescent probe molecule for micro-structural studies of nucleic acids including the detection of single-base alterations in target DNA sequences.

Bromonucleosides constitute a significant class of molecules and are well known for their biological activity. 5-Bromouridine, 5-bromo-2'-deoxyuridine, 5-bromouridine-5'-triphosphate, and nucleotides containing 5-bromouridine have been tested and used for numerous biological studies. 8-Bromopurine nucleosides have been used as essential precursors for the synthesis of nucleosides with fluorescent properties. This unit describes protocols for the synthesis of bromonucleosides using sodium monobromoisocyanurate (SMBI) in a straightforward way. Reactions are carried out at room temperature, and aqueous solvent mixtures are used to dissolve the nucleosides. Sodium azide is used as catalyst for the bromination of pyrimidine nucleosides, and no catalyst is necessary for the bromination of purine nucleosides. Unprotected 2'-deoxy pyrimidine and 2'-deoxy purine nucleosides are treated with SMBI to afford C-5 bromo pyrimidine and C-8 bromo purine nucleosides, respectively. This methodology has been found to be efficient for the synthesis of bromonucleosides on gram scale with consistently high yields. © 2017 by John Wiley & Sons, Inc., (Copyright © 2017 John Wiley & Sons, Inc.)

We report the synthesis and self-assembly of two lipophilic 2'-deoxyguanosine (G) derivatives whose fluorescence intensity is modulated by self-assembly into supramolecular G-quadruplexes (SGQs). Whereas both derivatives self-assemble isostructurally, one shows up to 100% emission enhancement while the other shows an initial enhancement, followed by 10% quenching. Thus, the rotational restrictions resulting from self-assembly are enough to induce significant changes in emission, but it is critical to consider the specific interactions between fluorophores since they will determine the ultimate emission signature. These findings could open the door to the development of luminescent supramolecular sensors and probes.

A methodology is reported to conjugate human O 6 -alkylguanine-DNA-alkyltransferase (hAGT) to the 3'-end of DNA in excellent yields with short reaction times by using intrastrand cross-linked (IaCL) DNA probes. This strategy exploited the substrate specificity of hAGT to generate the desired DNA-protein covalent complex. IaCL DNA linking two thymidine residues, or linking a thymidine residue to a 2'-deoxyguanosine residue (either in a 5'→3' or 3'→5' fashion), lacking a phosphodiester linkage at the cross-linked site, were prepared using a phosphoramidite strategy followed by solid-phase synthesis. All duplexes containing the model IaCL displayed a reduction in thermal stability relative to unmodified control duplexes. The O 4 -thymidine-alkylene-O 4 -thymidine and the (5'→3') O 6 -2'-deoxyguanosine-alkylene-O 4 -thymidine IaCL DNA adducts were not repaired by any of the AGTs evaluated (human AGT and Escherichia coli homologues, OGT and Ada-C). The (5'→3') O 4 -thymidine-alkylene-O 6 -2'-deoxyguanosine IaCL DNA containing a butylene or heptylene tethers were efficiently repaired by the human variant, whereas Ada-C was capable of modestly repairing the heptylene IaCL adduct. The IaCL strategy has expanded the toolbox for hAGT conjugation to DNA strands, without requiring the presence of a complementary DNA sequence. Finally, hAGT was functionalized with a fluorescently-labelled DNA sequence to demonstrate the applicability of this conjugation method.

The free nucleoside 2'-deoxyguanosine (dGuo) is the most susceptible to oxidation by reactive oxygen species (ROS) compared to the other free nucleosides, and its oxidation product 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) has been used as a biomarker to quantify oxidative stress damage. We investigated different reactions using Fe(2+) or Cu(2+) and H2O2 in order to identify the reaction with the best yield. HPLC coupled with a UV detector and micrOTOF mass spectrometry were used to detect and confirm the identity of 8-oxodGuo. The optimized reaction synthesized 8-oxodGuo with a yield of 72.0%, much higher than that previously described in the literature. Our improved method for 8-oxodGuo synthesis could be extremely useful for assays that require the synthesis of internal standards labeled with stable isotopes.

The synthesis of a highly efficient, water soluble [Pd(Sacc)2 (TPA)2 ] complex for C-C bond formation is described. Additionally, application of the [Pd(Sacc)2 (TPA)2 ] complex for Suzuki-Miyaura arylation of all four nucleosides (5-iodo-2'-deoxyuridine [5-IdU], 5-iodo-2'-deoxycytidine [5-IdC], 8-bromo-2'-deoxyadenosine, and 8-bromo-2'-deoxyguanosine) with various aryl/heteroaryl boronic acids in plain water under milder conditions is demonstrated. © 2016 by John Wiley & Sons, Inc., (Copyright © 2016 John Wiley & Sons, Inc.)

A series of non-hydrolysable 5'-aryl substituted GDP analogs has been synthesized by reacting 5'-azido-5'-deoxyguanosine with different aryl- and benzyloxy-alkynes. Cu(I) nanoparticles in water were found to be the most efficient catalyst, producing the desired 5'-arylguanosines with good yields. The synthesized compounds were screened for in vitro antileishmanial activity against Leishmania donovani axenic amastigotes and intramacrophage amastigotes stages. The 4-(3-nitrobenzyl)-1,2,3-triazole 5'-substituted guanosine analog was found to be the most active in the series with an IC50 of 8.6 μM on axenic amastigotes. Despite a rather low in vitro antileishmanial activity on the intramacrophage amastigotes, the absence of cytotoxicity on RAW 264.7 macrophages justifies further pharmacomodulations making this antileishmanial series promising.

Mitomycin C (MC) and Decarbamoylmitomycin C (DMC) - a derivative of MC lacking the carbamate on C10 - are DNA alkylating agents. Their cytotoxicity is attributed to their ability to generate DNA monoadducts as well as intrastrand and interstrand cross-links (ICLs). The major monoadducts generated by MC and DMC in tumor cells have opposite stereochemistry at carbon one of the guanine-mitosene bond: trans (or alpha) for MC and cis (or beta) for DMC. We hypothesize that local disruptions of DNA structure from trans or cis adducts are responsible for the different biochemical responses produced by MC and DMC. Access to DNA substrates bearing cis and trans MC/DMC lesions is essential to verify this hypothesis. Synthetic oligonucleotides bearing trans lesions can be obtained by bio-mimetic methods. However, this approach does not yield cis adducts. This report presents the first chemical synthesis of a cis mitosene DNA adduct. We also examined the stereopreference exhibited by the two drugs at the mononucleotide level by analyzing the formation of cis and trans adducts in the reaction of deoxyguanosine with MC or DMC using a variety of activation conditions. In addition, we performed Density Functional Theory calculations to evaluate the energies of these reactions. Direct alkylation under autocatalytic or bifunctional conditions yielded preferentially alpha adducts with both MC and DMC. DFT calculations showed that under bifunctional activation, the thermodynamically favored adducts are alpha, trans, for MC and beta, cis, for DMC. This suggests that the duplex DNA structure may stabilize/oriente the activated pro-drugs so that, with DMC, formation of the thermodynamically favored beta products are possible in a cellular environment., (Copyright © 2016 Elsevier Inc. All rights reserved.)

C8-N-arylamine adducts of 2'-deoxyguanosine (2'-dG) play an important role in the induction of the chemical carcinogenesis caused by aromatic amines. C8-N-acetyl-N-arylamine dG adducts that differ in their substitution pattern in the aniline moiety were converted by cycloSal technology into the corresponding C8-N-acetyl-N-arylamine-2'-deoxyguanosine-5'-triphosphates and C8-NH-arylamine-2'-deoxyguanosine-5'-triphosphates. Their conformation preference has been investigated by NOE spectroscopy and DFT calculations. The substrate properties of the C8-dG adducts were studied in primer-extension assays by using Klenow fragment exo(-) of Escherichia coli DNA polymerase I and human DNA polymerase β. It was shown that the incorporation was independent of the substitution pattern in the aryl moiety and the N-acetyl group. Although the triphosphates were poor substrates for the human polymerases, they were incorporated twice before the termination of the elongation process occurred; this might demonstrate the importance of C8-N-arylamine-2'-deoxyguanosine-5'-triphosphates in chemical carcinogenesis., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

8,5' cyclopurine deoxynucleosides (cPu) are locally distorting DNA base lesions corrected by nucleotide excision repair (NER) and proposed to play a role in neurodegeneration prevalent in genetically defined Xeroderma pigmentosum (XP) patients. In the current study, purified recombinant helicases from different classifications based on sequence homology were examined for their ability to unwind partial duplex DNA substrates harboring a single site-specific cPu adduct. Superfamily (SF) 2 RecQ helicases (RECQ1, BLM, WRN, RecQ) were inhibited by cPu in the helicase translocating strand, whereas helicases from SF1 (UvrD) and SF4 (DnaB) tolerated cPu in either strand. SF2 Fe-S helicases (FANCJ, DDX11 (ChlR1), DinG, XPD) displayed marked differences in their ability to unwind the cPu DNA substrates. Archaeal Thermoplasma acidophilum XPD (taXPD), homologue to the human XPD helicase involved in NER DNA damage verification, was impeded by cPu in the non-translocating strand, while FANCJ was uniquely inhibited by the cPu in the translocating strand. Sequestration experiments demonstrated that FANCJ became trapped by the translocating strand cPu whereas RECQ1 was not, suggesting the two SF2 helicases interact with the cPu lesion by distinct mechanisms despite strand-specific inhibition for both. Using a protein trap to simulate single-turnover conditions, the rate of FANCJ or RECQ1 helicase activity was reduced 10-fold and 4.5-fold, respectively, by cPu in the translocating strand. In contrast, single-turnover rates of DNA unwinding by DDX11 and UvrD helicases were only modestly affected by the cPu lesion in the translocating strand. The marked difference in effect of the translocating strand cPu on rate of DNA unwinding between DDX11 and FANCJ helicase suggests the two Fe-S cluster helicases unwind damaged DNA by distinct mechanisms. The apparent complexity of helicase encounters with an unusual form of oxidative damage is likely to have important consequences in the cellular response to DNA damage and DNA repair.

Malondialdehyde (MDA), an endogenous genotoxic product formed upon lipid peroxidation and prostaglandin biosynthesis, can react with DNA to form stable adducts. These adducts may contribute to the development of such inflammation-mediated diseases as cancer and cardiovascular and neurodegenerative diseases. The predominant MDA-derived DNA adduct formed under physiological conditions is 3-(2-deoxy-β-d-erythro-pentafuranosyl)pyrimido[1,2-α]purin-10(3H)-one deoxyguanosine (M1dG). In this study, we developed a novel liquid chromatography (LC)-nanoelectrospray ionization (NSI)-high-resolution tandem mass spectrometry (HRMS/MS) method for the analysis of M1dG in human leukocyte DNA. After enzymatic hydrolysis of DNA, M1dG and the added internal standard [(13)C3]M1dG were reduced to their 5,6-dihydro derivatives by addition of sodium borohydride to the hydrolysate and purified by solid-phase extraction and column chromatography. The 5,6-dihydro derivatives in the purified samples were analyzed by LC-NSI-HRMS/MS using higher-energy collisional dissociation (HCD) fragmentation, isolation widths of 1 Da for both the analyte and internal standard, and a resolution of 50 000. The detection limit of the developed method is 5 amol on-column, and the limit of quantitation is 0.125 fmol/mg DNA starting with 200 μg of DNA. Method accuracy and precision were characterized. The developed method was further applied to the analysis of leukocyte DNA from 50 human subjects. M1dG was detected in all samples and ranged from 0.132 to 275 fmol/mg DNA, or 0.004 to 9.15 adducts per 10(8) bases. This unique and highly sensitive HRMS/MS-based method can be used in future studies investigating the pathophysiological role of M1dG in human diseases.

DNA duplexes containing 8-cyclopropyl-2'-deoxyguanosine ((8CP) G) were synthesized to investigate the effect of the C8-modified deoxyguanosine as a kinetic trap for transient hole occupancy on guanines during DNA-mediated hole transport (HT). Thermal denaturation and CD spectra show that DNA duplexes containing (8CP) G are able to form stable B-form duplexes. Photoirradiation of terminal tethered anthraquinone can induce oxidative decomposition of (8CP) G through DNA HT along adenine tracts with lengths of up to 4.8 nm. Shallow and periodic distance dependence was observed in a long adenine tract with intervening guanines. The efficient charge transport indicates that (8CP) G can electronically couple well with a DNA bridge and form HT-active conformational domains to facilitate transient hole delocalization over an adenine tract., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

N7-methyl-9-deaza-dG was synthesized and incorporated into oligonucleotides. Thermal melting studies showed that replacement of dG by N7-methyl-9-deaza-dG only slightly decreased DNA duplex stability. Replication of DNA templates containing N7-methyl-9-deaza-dG and the related 7-methyl-7-deaza-dG and 7-deaza-dG by the Klenow fragment of Escherichia coli DNA polymerase I was examined. The dNTP misinsertion frequencies on all three templates were comparably low, although the 7-methyl group significantly slowed down the turnover rates of the polymerase when dCTP was incorporated. The stabilities of N7-methyl-9-deaza-dG and 7-methyl-7-deaza-dG against the actions of formamidopyrimidine DNA glycosylase (Fpg) and human alkyladenine DNA glycosylase (hAAG) were also examined. N7-methyl-9-deaza-dG was stable in the presence of both enzymes. In contrast, 7-methyl-7-deaza-dG was cleaved by Fpg, and possibly by hAAG but at an extremely slow rate. This study suggests that N7-alkyl-9-deaza-dG is a better analogue than 7-alkyl-7-deaza-dG for cellular studies., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

During the synthesis of a series of 2'-deoxy-9-deaza nucleosides using Heck methodology, the necessity for a pyrrole protecting group was discovered. The results of this brief study revealed that the benzyloxymethyl (BOM) group proved optimal, and Heck coupling using Jeffery conditions increased the coupling yield significantly. The results are reported herein.

A kinetic study is reported for the acid-catalyzed hydrolysis of oxygen (O)-linked biaryl ether 8-2'-deoxyguanosine (dG) adducts produced by phenolic toxins following metabolism into phenoxyl radical intermediates. Strikingly, the reaction rate of hydrolysis at pH 1 decreases as electron-withdrawing chlorine (Cl) substituents are added to the phenoxyl ring. The Hammett plot for hydrolysis at pH 1 shows a linear negative slope with ρX = -0.65, implying that increased Cl-substitution diminishes the rate of hydrolysis by lowering N(7) basicity. Spectrophotometric titration provided an N(7)H(+) pKa value of 1.1 for the unsubstituted adduct 8-phenoxy-dG (Ph-O-dG). Model pyridine compounds suggest N(7)H(+) pKa values of 0.92 and 0.37 for 4-Cl-Ph-O-dG and 2,6-dichloro-Ph-O-dG (DCP-O-dG), respectively. Density functional theory (DFT) calculations also highlight the ability of the 8-phenoxy substituent to lower N(7) basicity and predict a preference for N(3)-protonation for highly chlorinated O-linked 8-dG adducts in water. The calculations also provide a rationale for the hydrolytic reactivity of O-linked 8-dG adducts in the gas-phase, as determined using electrospray mass spectrometry (ESI-MS). The inclusion of our data now establishes that the order of hydrolytic reactivity at neutral pH for bulky 8-dG adducts is N-linked > C-linked > O-linked, which correlates with their relative ease of N(7)-protonation.

To facilitate NMR studies and low-level detection in biological samples by mass spectrometry, [1,3, NH2-(15)N3] (5'S)-8,5'-cyclo-2'-deoxyguanosine was synthesized from imidazole-4,5-dicarboxylic acid in 21 steps. The three (15)N isotopes were introduced during the chemo-enzymatic preparation of [1,3, NH2-(15)N3]-2'-deoxyguanosine using an established procedure. The (15)N-labeled 2'-deoxyguanosine was converted to a 5'-phenylthio derivative, which allowed the 8-5' covalent bond formation via photochemical homolytic cleavage of the C-SPh bond. SeO2 oxidation of C-5' followed by sodium borohydride reduction and deprotection gave the desired product in good yield. The isotopic purity of the [1,3, NH2-(15)N3] (5'S)-8,5'-cyclo-2'-deoxyguanosine was in excess of 99.94 atom% based on liquid chromatography-mass spectrometry measurements., (Copyright © 2013 John Wiley & Sons, Ltd.)

Photoirradiation of a hexadecameric supramolecular G-quadruplex leads to a diastereoselective [2 + 2] cyclodimerization of half of its constituent subunits, which in turn shifts the equilibrium toward the formation of a precise heteromeric octamer.

We recently reported the use of PSCl3 for the thiophosphorylation of alkylamines where the resulting N-thiophosphoramidate ions could be readily S-alkylated (Chem. Commun., 2011, 47, 6156-6158.). Herein we report the development of this methodology using amino acid, amino sugar, aminonucleoside and aniline substrates. The hydrolysis properties of N-thiophosphoramidate ions and their reactivities towards alkylating agents are also explored. In addition, we demonstrate the application of our approach to the preparation of a small library of compounds, including quinoline-based N,S-dialkylthiophosphoramidates which were tested for antileishmanial activity.

Rel proteins in bacteria synthesize the signal molecules (p)ppGpp that trigger the Stringent Response, responsible for bacterial survival. Inhibiting the activity of such enzymes prevents the Stringent Response, resulting in the inactivation of long-term bacterial survival strategies, leading to bacterial cell death. Herein, we describe a series of deoxyguanosine-based analogs of the Relacin molecule that inhibit in vitro the synthetic activity of Rel proteins from Gram positive and Gram negative bacteria, providing a deeper insight on the SAR for a better understanding of their potential interactions and inhibitory activity. Among the inhibitors evaluated, compound 2d was found to be more effective and potent than our previously reported Relacin., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Reactions of reducing sugars with free amino groups of proteins can form advanced glycation end products (AGEs). While the formation of nucleoside AGEs has been studied in detail, no extensive work has been carried out to assess DNA Amadori and DNA advanced glycation end products. In this study, we report biophysical/chemical characterization of glucose-induced changes in DNA, as well as DNA Amadori and DNA advanced glycation end products. Glucose treated DNA exhibited hyperchromicity, decrease in melting temperature, and enhanced emission intensity in a time dependent manner. Formation of DNA Amadori product and DNA advanced glycation end products, mainly CEdG (N²-carboxyethyl-2'-deoxyguanosine), were the major outcome of the study., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The nano-sized particles present in coal fly ash (CFA) were characterized through the X-ray diffraction (XRD), transmission and scanning electron microscopy (TEM, SEM), atomic force microscopy (AFM) and Fourier transform infrared spectroscopy (FTIR) analyses. The XRD data revealed the average crystallite size of the CFA nanoparticles (CFA-NPs) as 14 nm. TEM and SEM imaging demonstrated predominantly spherical and some polymorphic structures in the size range of 11 to 25 nm. The amount of heavy metal associated with CFA particles (μg/g) were determined as Fe (34160.0±1.38), Ni (150.8±0.78), Cu (99.3±0.56) and Cr (64.0±0.86). However, the bioavailability of heavy metals in terms of percent release was in the order as Cr>Ni>Cu>Fe in CFA-dimethyl sulfoxide (DMSO) extract. The comet and cytokinesis blocked micronucleus (CBMN) assays revealed substantial genomic DNA damage in peripheral blood mononuclear (PBMN) cells treated with CFA-NPs in Aq and DMSO extracts. About 1.8 and 3.6 strand breaks per unit of DNA were estimated through alkaline unwinding assay at 1:100 DNA nucleotide/CFA ppm ratios with the Aq and DMSO extracts, respectively. The DNA and mitochondrial damage was invariably greater with CFA-DMSO extract vis-à-vis -Aq extract. Generation of superoxide anions (O(2)•(-)) and intracellular reactive oxygen species (ROS) through metal redox-cycling, alteration in mitochondrial potential and 8-oxodG production elucidated CFA-NPs induced oxidative stress as a plausible mechanism for CFA-induced genotoxicity., (Copyright © 2012 Elsevier B.V. All rights reserved.)

We describe the synthesis of a fluorescent deoxyguanosine derivative that co-assembles (in water) with an unlabeled analogue into a heteromeric supramolecular G-quadruplex, which forms a host-guest complex with doxorubicin as evidenced by FRET experiments.

A simple method for the etherification at the O(6)-position of silyl-protected inosine, guanosine, and 2'-deoxyguanosine is described. Typically, a THF solution of the silylated nucleoside is treated with 1H-benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP) and Cs(2)CO(3) under a nitrogen atmosphere. Conversion to the O(6)-(benzotriazol-1-yl) ethers occurs within about 10 min for inosine, and within about 60 min for guanosine and 2'-deoxyguanosine. Then, for reaction with alcohols, the reaction mixture is evaporated and the O(6)-(benzotriazol-1-yl) ether is treated with Cs(2)CO(3) and an appropriate alcohol, at room temperature. On the other hand, for reaction with phenols, Cs(2)CO(3) and the appropriate phenol are added to the reaction mixture without evaporation, and the reaction is carried out at 70°C. Subsequently, workup, isolation, and purification lead to the requisite O(6)-alkyl or O(6)-aryl ethers in good to excellent yields.

A synthetic strategy that utilizes O6-protected 8-bromoguanosine gives broad access to C8-guanine derivatives with phenyl, pyridine, thiophene, and furan substituents. The resulting 8-substituted 2'-deoxyguanosines are push-pull fluorophores that can exhibit environmentally sensitive quantum yields (Φ=0.001-0.72) due to excited-state proton-transfer reactions with bulk solvent. Changes in nucleoside fluorescence were used to characterize metal-binding affinity and specificity of 8-substituted 2'-deoxyguanosines. One derivative, 8-(2-pyridyl)-2'-deoxyguanosine (2PyG), exhibits selective binding of Cu(II), Ni(II), Cd(II), and Zn(II) through a bidentate effect provided by the N7 position of guanine and the 2-pyridyl nitrogen atom. Upon incorporation into DNA, 2-pyridine-modified guanine residues selectively bind to Cu(II) and Ni(II) with equilibrium dissociation constants (K(d)) that range from 25 to 850 nM; the affinities depend on the folded state of the oligonucleotide (duplex>G-quadruplex) as well as the identity of the metal ion (Cu>Ni≫Cd). These binding affinities are approximately 10 to 1 000 times higher than for unmodified metal binding sites in DNA, thereby providing site-specific control of metal localization in alternatively folded nucleic acids. Temperature-dependent circular-dichroism studies reveal metal-dependent stabilization of duplexes, but destabilization of G-quadruplex structures upon adding Cu(II) to 2PyG-modified oligonucleotides. These results demonstrate how the addition of a single pyridine group to the C8 position of guanine provides a powerful new tool for studying the effects of N7 metalation on the structure, stability, and electronic properties of nucleic acids., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The 2'-hydroxyl groups within RNA contribute in essential ways to RNA structure and function. Previously, we designed an atomic mutation cycle (AMC) that uses ribonucleoside analogues bearing different C-2'-substituents, including -OCH(3), -NH(2), -NHMe, and -NMe(2), to identify hydroxyl groups within RNA that donate functionally significant hydrogen bonds. To enable AMC analysis of the nucleophilic guanosine cofactor in the Tetrahymena ribozyme reaction and at other guanosines whose 2'-hydroxyl groups impart critical functional contributions, we describe here the syntheses of 2'-methylamino-2'-deoxyguanosine (G(NHMe)) and 2'-N,N-dimethylamino-2'-deoxyguanosine (G(NMe(2))) and their corresponding phosphoramidites. The key step in obtaining the nucleosides involved S(N)2 displacement of 2'-β-triflate from an appropriate guanosine derivative by methylamine or dimethylamine. We readily obtained the G(NMe(2)) phosphoramidite and incorporated it into RNA. However, the G(NHMe) phosphoramidite posed a significantly greater challenge due to lack of a suitable -2'-NHMe protecting group. After testing several strategies, we established that allyloxycarbonyl (Alloc) provided suitable protection for 2'-N-methylamino group during the phosphoramidite synthesis and the subsequent RNA synthesis. This work enables AMC analysis of guanosine's 2'-hydroxyl group within RNA.

DNA oligonucleotide-conjugated pyrrole polyamide-2'-deoxyguanosine hybrids were synthesized and examined as novel gene expression control compounds. The T(m) values and circular dichroism spectral analyses showed that the oligonucleotide-conjugated hybrids possess high DNA recognition and a very high binding affinity for DNA that includes the pyrrole polyamide binding sequence.

The radical-based alkylation of 8-bromoguanosine (1a) and 8-bromo-2'-deoxyguanosine (1b) at the C8 position has been investigated in aqueous solutions. Alkyl radicals were generated by scavenging of the primary species of γ-radiolysis by the alcohol substrate. These reactions result in the efficient formation of intermolecular C-C bonds in aqueous media, by using the reactivity of α-hydroxyalkyl radicals derived from alcohols with 1a and 1b. A mechanism for the formation of C8 guanine alkylated adducts has been proposed, based on the quantification of radiation chemical yields for the disappearance of starting material and the formation of all products. Two α-hydroxyalkyl radicals are needed to form an alkylated guanine, the first one adding to C8 followed by ejection of Br(-) with formation of guanyl adduct and the second one acting as reducing agent of the guanyl adduct.

We synthesized various substituted 8-styryl-2'-deoxyguanosine and 8-styryl-2'-deoxyadenosine derivatives. Among them only acetyl substituted 8-styryl-2'-deoxyguanosine analog 5b showed a remarkable solvatochromicity (Δλ(max)(em)=91 nm),that is, strong fluorescence at 477 nm in 1,4-dioxane, but in methanol the fluorescence was red shifted to 558 nm with very low intensity. Such environmentally sensitive solvatochromic fluorescent guanosine analogs may be useful as a sensor for investigating interactions of DNA with DNA binding proteins., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

N(2)-alkyl analogues of 8-oxo-7,8-dihydro-2'-deoxyguanosine (OG) were synthesized (alkyl = propyl, benzyl) via reductive amination of the protected OG nucleoside and incorporated into various positions of an RNA strand. Thermal stability studies of duplexes containing A or C opposite a single modified base revealed only moderate destabilization. Both OG as well as its N(2)-alkyl analogues can pair opposite A or C with nearly equal stability, potentially offering a new means of modulating RNA-protein interactions in the minor vs major grooves.

O(6)-2'-Deoxyguanosine-alkyl-O(6)-2'-deoxyguanosine interstrand DNA cross-links (ICLs) with a four and seven methylene linkage in a 5'-GNC- motif have been synthesized and their repair by human O6-alkylguanine-DNA alkyltransferase (hAGT) investigated. Duplexes containing 11 base-pairs with the ICLs in the center were assembled by automated DNA solid-phase synthesis using a cross-linked 2'-deoxyguanosine dimer phosphoramidite, prepared via a seven step synthesis which employed the Mitsunobu reaction to introduce the alkyl lesion at the O(6) atom of guanine. Introduction of the four and seven carbon ICLs resulted in no change in duplex stability based on UV thermal denaturation experiments compared to a non-cross-linked control. Circular dichroism spectra of these ICL duplexes exhibited features of a B-form duplex, similar to the control, suggesting that these lesions induce little overall change in structure. The efficiency of repair by hAGT was examined and it was shown that hAGT repairs both ICL containing duplexes, with the heptyl ICL repaired more efficiently relative to the butyl cross-link. These results were reproducible with various hAGT mutants including one that contains a novel V148L mutation. The ICL duplexes displayed similar binding affinities to a C145S hAGT mutant compared to the unmodified duplex with the seven carbon containing ICLs displaying slightly higher binding. Experiments with CHO cells to investigate the sensitivity of these cells to busulfan and hepsulfam demonstrate that hAGT reduces the cytotoxicity of hepsulfam suggesting that the O(6)-2'-deoxyguanosine-alkyl-O(6)-2'-deoxyguanosine interstrand DNA cross-link may account for at least part of the cytotoxicity of this agent.

Selenium modification of nucleic acids is of great importance in X-ray crystal structure determination and functional study of nucleic acids. Herein, we describe a convenient synthesis of a new building block, the 2'-SeMe-modified guanosine (G(Se)) phosphoramidite, and report the first incorporation of the 2'-Se-G moiety into DNA. The X-ray crystal structure of the 2'-Se-modified octamer DNA (5'-GTG(Se)TACAC-3') was determined at a resolution of 1.20 A. We also found that the 2'-Se modification points to the minor groove and that the modified and native structures are virtually identical. Furthermore, we observed that the 2'-Se-G modification can significantly facilitate the crystal growth with respect to the corresponding native DNA.

We describe the synthesis of protected phosphoramidites of deoxyriboguanosine and guanosine derivatives containing a thiopropyl tether at the guanine N2 (7a,b) for site-specific crosslinking from the minor groove of either DNA or RNA to a thiol of a protein or another nucleic acid. The thiol is initially protected as a tert-butyl disulfide that is stable during oligonucleotide synthesis. While the completed oligonucleotide is still attached to the support, or after purification, the tert-butyl thiol can readily be removed or replaced by thioethylamine or 5-thio-2-nitrobenzoic acid, which have more favorable crosslinking rates.

The synthesis of 3'-amino-3'-deoxyguanosine and 3'-amino-3'-deoxyxyloguanosine monophosphate HepDirect prodrugs from guanosine is reported. Initial incorporation of N,N-dibenzylformamidino protection of the C2-amino of guanosine masked the reactivity of that group and simplified purification of subsequent analogues. The first key intermediate, 9-(2,5-bis-O-tert-butyldimethylsilyl-beta-D-ribofuranosyl)-2-N-(N,N-dibenzylformamidino)guanine (3a), was prepared in 60% yield after recycling of the undesired 3',5'-bis-O-protected byproduct (4a) by simple equilibration in methanol to a mixture of the two bis-O-protected compounds. Thus, protected, the 3'-position was manipulated to form the 3'-deoxyribo- or 3'-deoxyxylo-3'-azido derivatives (9 or 16, respectively). Further selective manipulations provided the cis-5'-monophosphate (3-chlorophenyl)-1,3-propanyl diester prodrugs (HepDirect prodrugs), 15 and 21. These HepDirect prodrugs were demonstrated to activate to their respective NTPs in rat hepatocytes.

6-S-[2-[(2-ethylhexyl)oxycarbonyl]ethyl)}-3',5'-O-bis(tert-butyldimethylsilyl)-2'-deoxy-6-thiogua nosine (2) was synthesized in high yield from the corresponding 6-O-mesitylenesulfonyl derivative by the reaction with 2-ethylhexyl 3-mercapto-propionate. The phosphoramidite precursor derived from 2 was successfully applied to an automated DNA synthesizer to produce 2'-deoxy-6-thioguanosine containing ODN. The results showed that 2-ethylhexyl 3-mercaptopropionate is useful as an odor less reagent and also as an S-protecting group of 2'-deoxy-6-thioguanosine.

Oligonucleotides containing a site-specific N(6)-(2-deoxy-d-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine (MeFapy-dGuo) lesion were synthesized, and their in vitro replication by Escherichia coli DNA polymerase I Klenow fragment (exo(-)) and Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) resulted in the misincorporation of Ade, Gua, and Thy opposite the MeFapy-dGuo lesion in addition to the correct insertion of Cyt. However, sequencing of the full-length extension products revealed that the initial insertion of Cyt opposite the lesion was extended most efficiently. Two sequences were examined, and the misincorporation was sequence-dependent. Improvements in the method for the mass spectrometric sequencing of the extension products were developed; a 5'-biotinylated primer strand was used that contained a dUrd near the template-primer junction. The extended primer was immobilized with streptavidin-coated beads, allowing it to be washed free of polymerase, the template strand, and other reagents. The extended primer was cleaved from the solid support with uridine DNA deglycosylase and piperidine treatment, and the extension products were sequenced by LC-ESI-MS-MS. The purification steps afforded by the biotinylated primer resulted in improved sensitivity for the MS analysis. Translesion synthesis of a template with a local 5'-T-(MeFapy-dGuo)-G-3' sequence resulted in only error-free bypass and extension, whereas a template with a local 5'-T-(MeFapy-dGuo)-T-3' sequence also resulted in an interesting deletion product and the misincorporation of Ade opposite the MeFapy-dGuo lesion.

The non-enzymatic repair of DNA oxidative damage can occur in a purely chemical system, but data show that it might also occur in cells. Human hepatoma cells (SMMC-7721) and human hepatocyte cells (LO2) were treated with 200microM H(2)O(2) for 30min to induce oxidative DNA damage quantified by amount of 8-OHdG and degree of DNA strand breaks, without inducing enzymatic repair. The dynamics of enzymatic repair activity quantified by unscheduled DNA synthesis, within 30min after removal of H(2)O(2) enzymatic repair mechanism has not been initiated. However, pre-incubation with low micromolar level polyphenols, quercetin or rutin can significantly attenuate DNA damage in both cell lines, indicating that the polyphenols did not work through an enzymatic mechanism. Unscheduled DNA synthesis after removal of H(2)O(2) was also markedly decreased by quercetin and rutin. Combined with our previous studies of fast reaction chemistry, the inhibitory effect of polyphenols have to be assigned to non-enzymatic repair mechanism rather than to enzymatic repair mechanism or antioxidant mechanism.

Chiral Z- and E-stereoisomers of (1,2-dihydroxyethyl)methylenecyclopropane analogues of 2'-deoxyadenosine and 2'-deoxyguanosine were synthesized, and their antiviral activity was investigated. (S)-Methylenecyclopropylcarbinol (16) was converted in seven steps to reagents 26 and 27, which were used for alkylation-elimination of adenine and 2-amino-6-chloropurine to get ultimately analogues 12a, 12b, 13a, 13b, 14a, 14b, 15a, and 15b. The enantiomeric series ent-12a, ent-12b, ent-13a, ent-13b, ent-14a, ent-14b, ent-15a, and ent-15b was obtained by similar procedures starting from (R)-methylenecyclopropylcarbinol (ent-16). The Z-isomer ent-12b was an inhibitor of two strains of human cytomegalovirus (HCMV) with EC(50) of 6.8 and 7.5 microM and of murine cytomegalovirus (MCMV) with EC(50) of 11.3 microM. It was less active against HCMV with mutated gene UL97. It inhibited Epstein-Barr virus (EBV) with EC(50) of 8 microM. The E-isomers ent-15a, ent-13a, and 15b were less effective. All adenine analogues with the exception of the Z-isomers ent-12a and ent-14a were moderate substrates for adenosine deaminase.

We describe two approaches for the synthesis of 2'-C-beta-methyl-2'-deoxyguanosine (3) via 2'-radical deoxygenation. One approach starts from 1,3,5-tri-O-benzoyl-alpha-d-ribofuranose (6) and gives 3 in 11 steps with 9.7% overall yield. The second approach starts from guanosine and gives 3 in 8 steps with 23% overall yield.

Two different approaches to synthesize oligonucleotides containing the 2 '-deoxyguanosine adducts formed by nitropyrenes are described. A direct reaction of an unmodified oligonucleotide with an activated nitropyrene derivative is a convenient biomimetic approach for generating the major adducts in DNA. A total synthetic approach, by contrast, involves several synthetic steps, including Buchwald-Hartwig Pd-catalyzed coupling, but can be used for incorporating both the major and minor adducts in DNA in high yield.

A phosphoramidite reagent of N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-1,4-dihydro-4-oxo-5-N-methylformamidopyrimidine (MeFapy-dGuo) lesions was synthesized in four steps from 2'-deoxyguanosine. Fapy nucleosides can rearrange to the pyranose form when the 5'-hydroxyl group is unprotected. The phosphoramidite was incorporated into oligonucleotides using solid-phase synthesis by adjusting the deprotection time for removal of the 5'-dimethoxytrityl group of the MeFapy-dGuo nucleotide, thereby minimizing its rearrangement to the ribopyranose. The furanose and pyranose forms were differentiated by a series of two-dimensional NMR experiments.

Fluorinated DNA containing stable fluorine substituents in the "purine" base were synthesized for the first time. For this, the phosphoramidites of 7-fluoro-7-deaza-2'-deoxyadenosine and 7-fluoro-7-deaza-2'-deoxyguanosine were prepared and oligonucleotides were synthesized. The 7-fluoro substitution leads to increased duplex stability and more selective base pairing compared to the non-functionalized 7-deazapurine oligonucleotides. (19)F NMR spectra of fluorinated nucleosides, single stranded oligonucleotides and DNA duplex show only a single signal for one fluorine modification. The NMR sensitive (19)F spin or the positron emitting (18)F isotope make these compounds applicable for DNA detection or imaging in vitro and in vivo.

Photochemical activation of a deoxyribozyme with peroxidase activity was achieved by the synthesis and incorporation of a caged deoxyguanosine.

5'-Amino-5'-deoxyguanosine-5'-N-phosphoramidate (GNHP) was synthesised in four steps from guanosine. Its identity was confirmed using spectroscopic and kinetic studies. A chromatographic method for the purification of this unstable compound was developed. GNHP was found to initiate T7 RNAP promoted transcriptions to afford 5'-O3P-NH-RNA, which hydrolyses readily to yield 5'-H2N-RNA that can be conjugated to activated esters.