Independent Generation and Time-Resolved Detection of 2′-Deoxyguanosin-N2-yl Radicals

Guanine radicals are important reactive intermediates in DNA damage. Hydroxyl radical (HO^.) has long been believed to react with 2′-deoxyguanosine (dG) generating 2′-deoxyguanosin-N1-yl radical (dG(N1-H)^.) via addition to the nucleobase π-system and subsequent dehydration. This basic tenet was challenged by an alternative mechanism, in which the major reaction of HO^. with dG was proposed to involve hydrogen atom abstraction from the N2-amine. The 2′-deoxyguanosin-N2-yl radical (dG(N2-H)^.) formed was proposed to rapidly tautomerize to dG(N1-H)^.. We report the first independent generation of dG(N2-H)^. in high yield via photolysis of 1 . dG(N2-H)^. is directly observed upon nanosecond laser flash photolysis (LFP) of 1 . The absorption spectrum of dG(N2-H)^. is corroborated by DFT studies, and anti- and syn-dG(N2-H)^. are resolved for the first time. The LFP experiments showed no evidence for tautomerization of dG(N2-H)^. to dG(N1-H)^. within hundreds of microseconds. This observation suggests that the generation of dG(N1-H)^. via dG(N2-H)^. following hydrogen atom abstraction from dG is unlikely to be a major pathway when HO^. reacts with dG.     

MECHANISMS OF QUENCHING OF THE FLUORESCENCE OF A BENZO[a]PYRENE TETRAOL METABOLITE MODEL COMPOUND BY 2'-DEOXYNUCLEOSIDES

Abstract— The hydrophobic interactions of bulky polycyclic aromatic hydrocarbons with nucleic acid bases and the formation of noncovalent complexes with DNA are important in the expressions of the mutagenic and carcinogenic potentials of this class of compounds. The fluorescence of the polycyclic aromatic residues can be employed as a probe of these interactions. In this work, the interactions of the (+)-trans stereoisomer of the tetraol 7,8,9,10-tetrahydroxytetrahydrobenzo[a]pyrene (BPT), a hydrolysis product of a highly mutagenic and carcinogenic diol epoxide derivative of benzo[a]pyrene, were studied with 2′-deoxynucleosides in aqueous solution by fluorescence and UV spectroscopic techniques. Ground-state complexes between BPT and the purine derivatives 2′-deoxyguanosine (dG), 2′-deoxyadenosine (dA), and 2′-deoxyinosine (dI) are formed with association constants in the range of ～40–130 M^?1 Complex formation with the pyrimidine derivatives 2′-deoxythymidine (dT), 2′-deoxycytidine (dC), and 2′-deoxyuridine (dU) is significantly weaker. Whereas dG is a strong quencher of the fluorescence of BPT by both static and dynamic mechanisms (dynamic quenching rate constant k_dyn= [2.5 ± 0.41 × 10⁹M¹ s ¹, which is close to the estimated diffusion-controlled value of ～ 5 × 10⁹M^{? 1} s^?1), both dA and dI are weak quenchers and form fluorescenceemitting complexes with BPT. The pyrimidine derivatives dC, dU, and dT are efficient dynamic fluorescence quenchers (K_dyn～ [1.5–3.0] × 10⁹M^?1 s^?1), with a small static quenching component due to complex formation evident only in the case of dT. None of the four nucleosidcs dG, dA, dC and dT are dynamic quenchers of BPT in the triplet excited state; the observed lower yields of triplets are attributed to the quenching of single excited states of BPT by 2′-deoxynucleosides without passing through the triplet manifold of BPT. Possible fluorescence quenching mechanisms involving photoinduced electron transfer are discussed. The strong quenching of the fluorescence of BPT by dG, dC and dT accounts for the low fluorescence yields of BPT-native DNA and of pyrene-DNA complexes.     

Detection of Trace Amounts of 8-Hydroxy-2′-Deoxyguanosine in Commercial 2′-Deoxyguanosine by Means of HPLC Analysis and Electrochemical Analysis

Abstract

A commercial sample of 2′-deoxyguanosine (dG) was submitted to reversed phase HPLC analysis. The column effluent was analyzed by UV detection set at 293 nm coupled to an electrochemical detector at 600 mV oxidation potential. When both detectors were set at their highest sensitivity, the UV detector did not show the presence of the 8-hydroxy-2′-deoxyguanosine but the electrochemical detector was capable of detecting this oxidatively modified 2′-deoxyguanosine. When planning in vitro experiments with 2′-deoxyguanosine in order to detect the oxidatively modified nucleoside it is necessary to assess the presence of this C8-hydroxylated derivative in the commercial product.     

Synthesis of Oligonucleotides Containing the N6-(2-Deoxy-α,β-d-erythropentofuranosyl)-2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy⋅dG) Oxidative Damage Product Derived from 2′-Deoxyguanosine

N⁶-(2-Deoxy-α,β-d -erythropentofuranosyl)-2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy⋅dG) is a major DNA lesion produced from 2′-deoxyguanosine under oxidizing conditions. Fapy ⋅ dG is produced from a common intermediate that leads to 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-OxodGuo), and in greater quantities in cells. The impact of Fapy ⋅ dG on DNA structure and function is much less well understood than that of 8-OxodGuo. This is largely due to the significantly greater difficulty in synthesizing oligonucleotides containing Fapy ⋅ dG than 8-OxodGuo. We describe a synthetic approach for preparing oligonucleotides containing Fapy ⋅ dG that will facilitate intensive studies of this lesion in DNA. A variety of oligonucleotides as long as 30 nucleotides are synthesized. We anticipate that the chemistry described herein will provide an impetus for a wide range of studies involving Fapy ⋅ dG.     

Photodynamic properties of toluidine blue characterized by fluorescence and absorption spectroscopy

Red light photolysis studies of the production of singlet oxygen by toluidine blue dye photosensitization were measured by the photo-oxidation rate of tryptophan. Absorption spectra of toluidine blue together with each of the four synthetic nucleosides of DNA have been obtained. After irradiation, only deoxyguanosine showed any noticeable reaction at neutral pH.Abreviations (see Fig. 1): TB, toluidine blue; ¹O₂, singlet oxygen; TRP, tryptophan; dA, 2′-deoxyadenosine; dC, 2′-deoxycytidine; dT, 2′-deoxythymidine; dG, 2′-deoxyguanosine; NaN₃, sodium azide.     

Independent Generation and Time‐Resolved Detection of 2′‐Deoxyguanosin‐N2‐yl Radicals

Guanine radicals are important reactive intermediates in DNA damage. Hydroxyl radical (HO^.) has long been believed to react with 2′‐deoxyguanosine (dG) generating 2′‐deoxyguanosin‐N1‐yl radical (dG(N1‐H)^.) via addition to the nucleobase π‐system and subsequent dehydration. This basic tenet was challenged by an alternative mechanism, in which the major reaction of HO^. with dG was proposed to involve hydrogen atom abstraction from the N2‐amine. The 2′‐deoxyguanosin‐N2‐yl radical (dG(N2‐H)^.) formed was proposed to rapidly tautomerize to dG(N1‐H)^.. We report the first independent generation of dG(N2‐H)^. in high yield via photolysis of 1 . dG(N2‐H)^. is directly observed upon nanosecond laser flash photolysis (LFP) of 1 . The absorption spectrum of dG(N2‐H)^. is corroborated by DFT studies, and anti‐ and syn‐dG(N2‐H)^. are resolved for the first time. The LFP experiments showed no evidence for tautomerization of dG(N2‐H)^. to dG(N1‐H)^. within hundreds of microseconds. This observation suggests that the generation of dG(N1‐H)^. via dG(N2‐H)^. following hydrogen atom abstraction from dG is unlikely to be a major pathway when HO^. reacts with dG.     

Spectral properties and G-quadruplex DNA binding selectivities of a series of unsymmetrical perylene diimides

A series of new unsymmetrical perylene diimides have been synthesized to investigate their binding selectivities to G-quadruplex DNA structure, a unique four-stranded DNA motif, which is significant to the regulation of telomerase activity. The structures of the perylene diimides have been characterized by IR spectrophotometer, ¹H NMR, ¹³C NMR, MS, TGA and time-resolved instruments. Spectrochemical behaviors have been investigated by visible absorption and fluorescence emission spectra. The spectral characterization of the compounds has been investigated in five common organic solvents of different polarity and in water (in 170 mM phosphate buffer at pH 6). Marked red shifts of absorbance and fluorescence emission bands of the compounds in aqueous solution are compared with the other organic solutions. The fluorescence quantum yields are determined low in more polar solvents and also calculated to be about less than about 0.05 in aqueous solution because of the aggregation effects. Photodegradation rate constants (k_p) of the synthesized compounds have been compared under xenon lamp irradiation in acetonitrile solution.Binding abilities of the synthesized perylene diimides to different form of DNA strands have been investigated by visible absorption and fluorescence spectroscopy in the phosphate buffer solutions. Also, pH-dependent aggregation and G-quadruplex DNA binding selectivity of these ligands have been compared. Among these ligands, N-(2,6-diisopropylphenyl)-N′-(4-pyridyl)-perylene-3,4,9,10-tetracarboxylic diimide (PYPER) has been found to be the most selective interactive ligand for G-quadruplex formed in the G4′-DNA structure. PYPER has shown a significant selectivity to G4′-DNA which is comprised of d(TTAGGG) repeats, known as human telomeres, in the phosphate buffer at pH 6. The absorption maximum of the PYPER/G4′-DNA complex has given bathochromic shift of 7 nm with respect to the absorption maximum of DNA-free solution of PYPER in phosphate buffer at pH 6. Fluorescence quenching experiments between PYPER and G4′-DNA show that PYPER demonstrates about a 9.3-fold selectivity for binding to G4′-DNA versus ds-DNA base pairs with the bimolecular rate constant of 0.95 × 10¹² M⁻¹ s⁻¹.     

Studies on the Base-Pairing Properties of N7-(2-Deoxy-β-D-erythro-pentofuranosyl)guanine (N7Gd)

The base-pairing properties of N⁷-(2-deoxy-β-D -erythro-pentofuranosyl)guanine (N⁷G_d; 1 ) are investigated. The nucleoside 1 was obtained by nucleobase-anion glycosylation. The glycosylation reaction of various 6-alkoxy-purin-2-amines 3a - i with 2-deoxy-3,5-di-O-(4-toluoyl)-α-D -erythro-pentofuranosyl chloride ( 8 ) was studied. The N⁹/N⁷-glycosylation ratio was found to be 1:1 when 6-isopropoxypurin-2-amine ( 3d ) was used, whereas 6-(2-methoxyethoxy)purin-2-arnine ( 3i ) gave mainly the N⁹-nucleoside (2:1). Oligonucleotides containing compound 1 were prepared by solid-phase synthesis and hybridized with complementary strands having the four conventional nucleosides located opposite to N⁷G_d. According to T_m values and enthalpy data of duplex formation, a base pair between N⁷G_d and dG is suggested. From the possible N⁷G_d dG base pair motives, Hoogsteen pairing can be excluded as 7-deaza-2′-deoxyguanosine forms the same stable base pair with N⁷G_d as dG.     

O6‐Alkylguanine DNA Alkyltransferase Repair Activity Towards Intrastrand Cross‐Linked DNA is Influenced by the Internucleotide Linkage

Oligonucleotides containing an alkylene intrastrand cross‐link (IaCL) between the O⁶‐atoms of two consecutive 2′‐deoxyguanosines (dG) were prepared by solid‐phase synthesis. UV thermal denaturation studies of duplexes containing butylene and heptylene IaCL revealed a 20 °C reduction in stability compared to the unmodified duplexes. Circular dichroism profiles of these IaCL DNA duplexes exhibited signatures consistent with B‐form DNA. Human O⁶‐alkylguanine DNA alkyltransferase (hAGT) was capable of repairing both IaCL containing duplexes with slightly greater efficiency towards the heptylene analog. Interestingly, repair efficiencies of hAGT towards these IaCL were lower compared to O⁶‐alkylene linked IaCL lacking the 5′‐3′‐phosphodiester linkage between the connected 2′‐deoxyguanosine residues. These results demonstrate that the proficiency of hAGT activity towards IaCL at the O⁶‐atom of dG is influenced by the backbone phosphodiester linkage between the cross‐linked residues.     

Synthesis of oligonucleotides containing 7-(2-deoxy-β-d-erythro-pentofuranosyl)guanine and 8-amino-2′-deoxyguanosine

The synthesis of oligonucleotides containing 7-(2-deoxy-β-D-erythro-pentofuranosyl)guanine and 8-amino-2′-deoxyguanosine was accomplished. The viable intermediate N²-isobutyryl-7-(2-deoxy-β-D-erythro-pentofuranosyl)guanine ( 6 ) was prepared via a four step deoxygenation procedure from 7-β-D-ribofuranosylguanine ( 1 ). The 5′-hydroxyl group of 6 was protected as 4,4′-dimethoxytrityl ether and then converted to the target phosphoramidite ( 8 ) via conventional phosphitylation procedure. The amino groups of 8-amino-2′-deoxyguanosine ( 9 ) were protected in the form of N-(dimethylainino)methylene functions to give the protected nucleoside 10 , which was subsequently converted to the target phosphoramidite 12 via dimethoxytritylation followed by phosphitylation. The phosphoramidites 8 and 12 were incorporated into a 26-mer and a 31-mer G-rich oligonucleotide using solid-support, phosphoramidite methodology. Analysis of antiparallel triplex formation by the oligonucleotides containing 7-(2-deoxy-β-D-erythro-pentofura-nosyl)guanine in place of 2′-deoxyguanosine showed no enhancement in triple helix formation.     

Synthesis,spectral-luminescent properties of B(III) and Zn(II) complexes with alkyl- and aryl-substituted dipyrrins and azadipyrrins

Effect of complexing atom, molecular structure of dipyrrolylmethene and its aza analog on spectral-luminescent properties of heteroleptic boron(III) and homoleptic zinc(II) complexes with 3,3′,5,5′-tetramethyl-2,2′-dipyrrolylmethene, 3,3′,5,5′-tetraphenyl-2,2′-dipyrrolylmethene, and 3,3′,5,5′-tetraphenyl-ms-aza-2,2′-dipyrrolylmethene in organic solvent solutions was studied. The complexes were found to exhibit strong chromophore (λ = 350–690 nm, ? ～ 10⁵ L/mol cm) and fluorescent properties. Quantum yield (γ^fl) for fluoroborate complexes reaches 100% and is weakly dependent on medium nature. The value of γ^fl for phenyl- and alkyl-substituted zinc(II) dipyrrolylmethenates in nonpolar solvents is not higher 0.3 and 0.03, respectively; complete fluorescence quenching is observed in electron-donating solvents. Aza-substitution at the meso spacer causes considerable shift of electronic absorption and fluorescence spectra to the red region but completely quenches fluorescence of zinc(II) chelates and decreases γ^fl of boron(III) complex to 0.04.     

Nucleoside–metallacarborane conjugates for multipotential electrochemical coding of DNA

Metallacarboranes as electrochemical labels are proposed. The electrochemical properties of nucleoside conjugates, derivatives of thymidine (T), 2′-deoxycytidine (dC), 2′-deoxyadenosine (dA) and 2′-deoxyguanosine (dG), containing metallacarborane complex of cobalt or iron are described. A multielectrochemical detection using specific metallacarborane tags is shown. The proposed labelling of nucleosides lays the foundations for electrochemical coding of DNA with metallacarborane complexes and simultaneous detection of several DNA targets.     

Enrichment of trace anions from water with 2,2'-diaminodiethylamine cellulose filters

Cellulose filters with immobilized 2,2'-diaminodiethylamine (DEN) functional groups are studied for trace anion preconcentration from aqueous solution, with subsequent x-ray fluorescence measurements. For most oxoanions with a central metal atom, nearly quantitative collection can be achieved by 10-cm² DEN filters under the following optimized conditions: pH 3–6, filtration rate up to 0.5 ml cm^-2 min^-1, and sample volume up to 100 ml cm^-2. The collection yield is independent of the trace oxoanion concentration up to at least 1.5 μmol cm^-2. Although the DEN filter exhibits some selectivity towards oxoanions with a central metal atom, ionic strength affects the results; the collection efficiency is strongly depressed with salt (e.g. NaCl) concentrations above 0.01 M. The applicability of the DEN filter in anion collection is therefore limited to dilute solutions.     

A novel rhodamine-based turn-on probe for fluorescent detection of Au3+ and colorimetric detection of Cu2+

In this work, we design and synthesize the novel probe RC through introduction the 1-aza-4,13-dithia-15-crown-5 ring into the structure of rhodamine 6G hydrazide, where the N atom of crown ring is responsible for quenching of rhodamine fluorescence. The compound obtained behaves as multifunctional cation sensor providing selective fluorescent response to Au³⁺ and selective colorimetric response to Cu²⁺ ions in aqueous acetonitrile (1/1, v/v) at pH 7.0. The use of 10^?5?M RC solution allowed reliable determination of target cations in the presence of a wide range of environmentally relevant ions with detection limits of 2?×?10^?6?M and 5?×?10^?7?M for gold and copper, respectively.     

Optimization of fluorescence property of the 8-oxodGclamp derivative for better selectivity for 8-oxo-2′-deoxyguanosine

2′-Deoxyguanosine (dG) suffers from oxidation by reactive oxygen species (ROS) to form 8-oxo-2′-deoxyguanosine (8-oxo-dG), which is regarded as a marker of oxidative stress in the cells. In our continuous study for the recognition molecule of 8-oxo-dG, 8-oxoGclamp and its derivatives have been identified as the selective fluorescent probe. However, it is an obstacle for further application that dG also forms a complex with 8-oxoGclamp, resulting in fluorescence quenching in less polar solvents. Quenching of the fluorescence of 8-oxoGclamp is thought to involve photo-induced electron transfer in the complex. It was hypothesized that the energy level of the excited state of 8-oxoGclamp and the HOMO energy of dG are the preliminary determinant of the quenching efficiency. Thus, fluorescence properties of the substituted derivatives at the 7-position of the 1,3-diazaphenoxazine part of 8-oxoGclamp were investigated. Among the new derivatives, fluorescence of the 7-phenyl substituted 8-oxoGclamp was not quenched by dG even in the stable complex, exhibiting the highest selectivity for 8-oxo-dG.     

On <Emphasis Type="Italic">Trans</Emphasis>-Resveratrol in Aqueous Solutions

A thermodynamic study on aqueous solutions of trans-Resveratrol (3,5,4′-trihydroxy-trans-stilbene) at 25.00 ± 0.02 °C, in 0.5 mol·dm^?3 NaCl, has been conducted. The protolysis equilibria and the complex formation between trans-Resveratrol and a metal(II), namely Mn²⁺ and Cu²⁺, have been investigated. The experimental method consists of potentiometric, spectrophotometric (absorption and emission) acid–base titrations. The pH range investigated is 2.5 ≤ pH ≤ 13 for the binary system, while for the ternary system it is 2.5 ≤ pH ≤ 6. The results of the graphical and numerical methods adopted indicate, for all the systems investigated, the formation of a predominant Me(II)–trans-Resveratrol mononuclear complex. UV–Vis absorption spectra and desorption/ionization time of flight mass spectra show the occurrence of hydrolytic species exhibiting a higher molecular weight than the Resveratrol molecule, becoming more evident as the pH and the time increased. Moreover, high performance liquid chromatography analysis and infrared spectroscopy of aqueous cis/trans-Resveratrol solutions upon excitation at 300 nm have highlighted a highly fluorescent compound, with absorption maximum at 250 nm, and a blue shift in the fluorescence emission that have not previously been reported.     

Sulfonation of 3,3′-Diformyl-BINOL for Enantioselective Fluorescent Recognition of Amino Acids in Water

Reaction of (R)-3,3′-diformyl-1,1′-bi-2-naphthol with concentrated sulfuric acid gives the corresponding 6,6′-disulfonated compound (R)- 2 selectively. This provides a simple and efficient method to convert a water-insoluble compound to a water-soluble fluorescent probe. It is found that (R)- 2 in combination with Zn²⁺ shows a highly enantioselective fluorescent response toward various amino acids in the aqueous HEPES buffer solution at pH 7.4. For example, an enantioselective fluorescence enhancement ratio [ef=ΔI_D/ΔI_L] up to 35.8 is observed for the recognition of asparagine. NMR and mass spectroscopic investigations are conducted to explore the reaction of (R)- 2 with asparagine.     

Synthesis and Characterization of a Water‐Soluble Carboxylated Polyfluorene and Its Fluorescence Quenching by Cationic Quenchers and Proteins

We have developed a new intermediate monomer, 2,7‐[bis(4,4,5,5‐tetramethyl‐1,3,2‐dioxaborolan‐2‐yl)‐9,9‐bis(3‐(tert‐butyl propanoate))]fluorene, that allows the easy synthesis of water‐soluble carboxylated polyfluorenes. As an example, poly[9,9′‐bis(3′′‐propanoate)fluoren‐2,7‐yl] sodium salt was synthesized by the Suzuki coupling reaction, and the properties of the polymer were studied in aqueous solutions of different pH. Fluorescence quenching of the polymer by different cationic quenchers (MV²⁺, MV⁴⁺, and NO₂MV²⁺; MV=methyl viologen) was studied, and the quenching constants were found to be dependent on the charge and electron affinity of the quencher molecule and the pH of the medium. The largest quenching constant was observed to be 1.39×10⁸ M ^?1 for NO₂MV²⁺ at pH 7. The change in polymer fluorescence upon interaction with different proteins was also studied. Strong fluorescence quenching of the polymer was observed in the presence of cytochrome c, whereas weak quenching was observed in the presence of myoglobin and bovine serum albumin. Lysozyme quenched the polymer emission at low protein concentrations, and the quenching became saturated at high protein concentrations. Under similar experimental conditions, the polymer showed improved quenching efficiencies toward cationic quenchers and a more selective response to proteins relative to other carboxylated conjugated polymers.     

Fluorescent derivatives of nucleosides

Fluorescence spectra of formycin anhydronucleosides 5,6,8 and of N-dimethylaminomethylene ribonucleosides 1b, 2, 3a-3c and 4 in aqueous solution at 5-7 × 10^?5 M are reported. Compounds 5 and 6 exhibit a very strong fluorescence emission, ca. 4 and 2 times more intense than that of formycin ( 1a ) accompanied by a bathochromic shift of the emission maximum. Anhydronucleoside 8 also has an increased fluorescence intensity over the parent nucleoside 7 . The level of fluorescence emission is lower in 7 and 8 than in 1a , 5 or 6 . Introduction of N-dimethylaminomethylene group into 1a (compound 1b ) caused a decrease in the fluorescence intensity relative to 1a but a bathochromic shift of the emission maximum. In other instances (compounds 2, 3a-3c, 4 ) the introduction of N-dimethylaminomethylene group led also to fluorescent derivatives. This effect is most pronounced with 2 , whereas the fluorescence intensity of the rest of the group ( 3a-3c and 4 ) is much lower. Compounds 3c and 4 exhibit, however, a profound bathochromic shift in the fluorescence emission maximum relative to 2, 3a or 3b . A possible relationship of the fluorescence emission to the base conformation in formycin and potential use of N-dimethylaminomethylene nucleoside and nucleotide derivatives as fluorescent probes are discussed. J. Heterocyclic Chem., 14 , 135 (1977).     

High-throughput and cost-effective global DNA methylation assay by liquid chromatography-mass spectrometry

An affordable and fast liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the accurate and precise determination of global DNA methylation levels in peripheral blood. Global DNA methylation extent was expressed as the ratio of methylated 2′-deoxycytidine (5MedC) to 2′-deoxyguanosine (dG), which were obtained after DNA extraction and hydrolysis and determined by positive electrospray LC–ESI-MS/MS. The cost-effective internal standards ¹⁵N₃-dC and ¹⁵N₅-dG were incorporated for the accurate quantification of 5MedC and dG, respectively. The desired nucleoside analytes were separated and eluted by LC within 2.5 min on a reverse phase column with a limit of detection of 1.4 femtomole on column for 5MedC. Sample preparation in 96-well format has significantly increased the assay throughput and filtration was found to be a necessary step to assure precision. Precision was performed with repeated analysis of four DNA QC sample over 12 days, with mean intra- and inter-day CVs of 6% and 11%, respectively. Accuracy was evaluated by comparison with a previously reported method showing a mean CV of 4% for 5 subjects analyzed. Furthermore, application of the assay using a benchtop orbitrap LCMS in exact mass full scan mode showed comparable sensitivity to tandem LCMS using multiple reaction monitoring.