Anthraquinone-sensitized photooxidation of 5-methylcytosine in DNA leading to piperidine-induced efficient strand cleavage

One-electron photooxidations of 5-methyl-2'-deoxycytidine (d(m)C) and 5-trideuteriomethyl-2'-deoxycytidine ([D(3)]d(m)C) by sensitization with anthraquinone (AQ) derivatives were investigated. Photoirradiation of an aerated aqueous solution containing d(m)C and anthraquinone 2-sulfonate (AQS) afforded 5-formyl-2'-deoxycytidine (d(f)C) and 5-hydroxymethyl-2'-deoxycytidine (d(hm)C) in good yield through an initial one-electron oxidation process. The deuterium isotope effect on the AQS-sensitized photooxidation of d(m)C suggests that the rate-determining step in the photosensitized oxidation of d(m)C involves internal transfer of the C5-hydrogen atom of a d(m)C-tetroxide intermediate to produce d(f)C and d(hm)C. In the case of a 5-methylcytosine ((m)C)-containing duplex DNA with an AQ chromophore that is incorporated into the backbone of the DNA strand so as to be immobilized at a specific position, (m)C underwent efficient direct one-electron oxidation by the photoexcited AQ, which resulted in an exclusive DNA strand cleavage at the target (m)C site upon hot piperidine treatment. In accordance with the suppression of the strand cleavage at 5-trideuterio-methylcytosine observed in a similar AQ photosensitization, it is suggested that deprotonation at the C5-methyl group of an intermediate (m)C radical cation may occur as a key elementary reaction in the photooxidative strand cleavage at the (m)C site. Incorporation of an AQ sensitizer into the interior of a strand of the duplex enhanced the one-electron photooxidation of (m)C, presumably because of an increased intersystem crossing efficiency that may lead to efficient piperidine-induced strand cleavage at an (m)C site in a DNA duplex.     

Synthesis and characterization of DNA duplexes containing an N(4)C-ethyl-N(4)C interstrand cross-link

Short DNA duplexes containing an N(4)C-ethyl-N(4)C interstrand cross-link, C-C, were synthesized on controlled pore glass supports. Duplexes having two, three, or four A/T base pairs on either side of the C-C cross-link and terminating with a C(4) overhang at their 5'-ends were prepared. The cross-link was introduced using a convertible nucleoside approach. Thus, an oligonucleotide terminating at its 5'-end with O(4)-triazoyl-2'-deoxyuridine was first prepared on the support. The triazole group of support-bound oligomer was displaced by the aminoethyl group of 5'-dimethoxytrityl-3'-O-tert-butyldimethylsilyl-N(4)-(2-aminoethyl)deoxycytidine to give the cross-link. The dimethoxytrityl group was removed, and the upper and lower strands of the duplex were extended from two 5'-hydroxyl groups of the cross-link using protected nucleoside 3'-phosphoramidites. The tert-butyldimethylsilyl group of the resulting partial duplex was then removed, and the chain was extended in the 3'-direction from the resulting 3'-hydroxyl of the cross-link using protected nucleoside 5'-phosphoramidites. The cross-linked duplexes were purified by HPLC and characterized by enzymatic digestion and MALDI-TOF mass spectrometry. Duplexes with three or four A/T base pairs on either side of the C-C cross-link gave sigmoidal shaped A(260) profiles when heated, a behavior consistent with cooperative denaturation of the A/T base pairs. Each cross-linked duplex could be ligated to an acceptor duplex using T4 DNA ligase, a result that suggests that the C-C cross-link does not interfere with the ligation reaction, even when it is located only two base pairs from the site of ligation. The ability to synthesize duplexes with a defined interstrand cross-link and to incorporate these duplexes into longer pieces of DNA should enable preparation of substrates that can be used for a variety of biophysical and biochemical experiments, including studies of DNA repair.     

Synthesis and characterization of oligodeoxynucleotides containing the major DNA adducts formed by 1,6- and 1,8-dinitropyrene

[reaction: see text] An efficient method for the synthesis of oligonucleotides containing a site-specific DNA adduct formed by the carcinogens 1,6- and 1, 8-dinitropyrene has been developed. Palladium-catalyzed amination provided a straightforward route for the synthesis of aminonitropyrenes which, following separation, were reduced to the nitrosonitropyrenes. The N-hydroxyaminonitropyrene, generated in situ from each nitrosonitropyrene, was used successfully to introduce the dinitropyrene-derived DNA adduct at a defined site in an oligonucleotide.     

Duplex stability of DNA.DNA and DNA.RNA duplexes containing 3'-S-phosphorothiolate linkages

3'-S-Phosphorothiolate (3'-SP) linkages have been incorporated into the DNA strand of both a DNA.RNA duplex and a DNA.DNA duplex. Thermal melting (T(m)) studies established that this modification significantly stabilises the DNA.RNA duplex with an average increase in T(m) of about 1.4 degrees C per modification. For two or three modifications, the increase in T(m) was larger for an alternating, as compared to the contiguous, arrangement. For more than three modifications their arrangement had no effect on T(m). In contrast to the DNA.RNA duplex, the 3'-S-phosphorothiolate linkage destabilised the DNA.DNA duplex, irrespective of the arrangement of the 3'-SP linkages. The effect of ionic strength on duplex stability was similar for both the phosphorothiolate-substituted and the unmodified RNA.DNA duplexes. The results are discussed in terms of the influence that the sulfur atom has on the conformation of the furanose ring and comparisons are also drawn between the current study and those previously conducted with other modifications that have a similar conformational effect.     

Synthesis and triplex forming properties of pyrrolidino pseudoisocytidine containing oligodeoxynucleotides

Pyrrolidino pseudo-C-nucleosides are isosteres of natural deoxynucleosides which are protonated at the pyrrolidino ring nitrogen under physiological conditions. As constituents of a triplex forming oligodeoxynucleotide (TFO), the positive charge is expected to stabilise DNA triple helices via electrostatic interactions with the phosphodiester backbone of the target DNA. We describe the synthesis of the pyrrolidino isocytidine pseudonucleoside and the corresponding phosphoramidite building block and its incorporation into TFOs. Such TFOs show substantially increased DNA affinity compared to unmodified oligodeoxynucleotides. The increase in affinity is shown to be due to the positive charge at the pyrrolidino subunit.     

Synthesis of DNA oligos containing 2'-deoxy-2'-fluoro-D-arabinofuranosyl-5-carboxylcytosine as hTDG inhibitor

As an important step of the active demethylation of 5-methylcytosine (5mC), human thymine DNA glycosylase (hTDG) efficiently excises 5-carboxylcytosine (5caC) from double-stranded DNA (dsDNA). Here, we present synthesis of DNA oligos containing a 2'-deoxy-2'-fluoro-D-arabinofuranosyl-5-carboxylcytidine (F-5caC) modification that act as hTDG inhibitors. The glycosylase activity assay showed that F-5caC oligos were resistant to excision by the hTDG catalytic domain (hTDG(cat), residues 111-308) and they could inhibit the excision of DNA oligos containing 5caC. The electrophoretic mobility shift assay confirmed that DNA oligos containing F-5caC could bind well with unmodified hTDG(cat) to form a stable complex, which makes it possible to obtain the crystal structure of the complex to reveal details on how hTDG(cat) recognizes the DNA substrate.     

Synthesis and DNA triplex formation of an oligonucleotide containing an urocanamide

An urocanamide nucleoside designed and previously tested as its protected ribose derivative in aprotic solvents for binding a cytosine-guanine (CG) Watson-Crick base pair was successfully incorporated into a triplex forming oligonucleotide. Binding affinity and specificity of this nonnatural nucleoside were studied in a triple helix with duplex targets containing all four possible Watson-Crick base pairs opposite the nucleoside analog in the third strand. UV melting experiments indicate the formation of a well-defined triplex with specific binding of the urocanamide analog to a CG inversion of the homopurine-homopyrimidine target. However, binding affinities in the triplex are weak and much lower when compared to the canonical base triads.     

Synthesis and pairing properties of oligodeoxynucleotides containing bicyclo-RNA and bicyclo-ANA modifications

The synthesis of the ribo(bc-rT)- and arabino(bc-araT)-version of bicyclothymidine (bc-dT) has been achieved. A conformational analysis by X-ray and/or (1)H NMR spectroscopy on the corresponding 3',5'-benzyl-protected nucleosides featured a rigid C(2')-endo conformation for the furanose ring, irrespective of the configuration of the OH group at C(2'). The conformation of the carbocyclic ring in these nucleosides was found to be less defined and thus more flexible. Both nucleosides were converted into the corresponding phosphoramidites and incorporated into oligodeoxynucleotides by standard DNA chemistry. T(m)-data of duplexes with cDNA and RNA revealed that a bc-rT unit strongly destabilized duplexes with cDNA and RNA by 6-8 °C/mod, while bc-araT was almost T(m) neutral. A rationale based on a previous structure of a bc-DNA mini duplex suggests that the strong destabilization caused by a bc-rT unit arises from unfavorable steric interactions of the equatorial 2'-OH group with the sugar residue of the 3'-neighboring nucleotide unit.     

Synthesis and characterization of oligodeoxynucleotides containing formamidopyrimidine lesions and nonhydrolyzable analogues

Oligodeoxynucleotides containing formamidopyrimidine lesions and C-nucleoside analogues at defined sites were prepared by solid-phase synthesis and in some cases enzymatic ligation. Formamidopyrimidine lesions were introduced as dinucleotides to prevent rearrangement to their pyranose isomers. Oligodeoxynucleotides containing single diastereomers of C-nucleoside analogues of Fapy.dA were introduced by using the respective phosphoramidites. The formamidopyrimidine lesions reduce the T(M) of dodecamers relative to their unmodified nucleotide counterparts when opposite the nucleotide proper base-pairing partner. However, duplexes containing Fapy.dG-dA mispairs melt significantly higher than those comprised of dG-dA. All duplexes containing Fapy.dA-dX or its C-nucleoside analogue melt lower than the respective complexes containing dA-dX. Studies of the alkaline lability of oligodeoxynucleotides containing formamidopyrimidine lesions indicate that Fapy.dA is readily identified as an alkali-labile lesion with use of piperidine (1.0 M, 90 degrees C, 20 min), but Fapy.dG is less easily identified in this manner.     

Syntheses of 5-formyl- and 5-carboxyl-dC containing DNA oligos as potential oxidation products of 5-hydroxymethylcytosine in DNA

To investigate the potential oxidation products of 5-hydroxymethylcytosine (5-hmC)-containing DNA, we present here efficient syntheses of 5-formyl- and 5-methoxycarbonyl-2'-deoxycytidine phosphoramidites. The 5-formyl group in III was easy to introduce and was compatible with phosphoramidite and DNA syntheses. An additional treatment of ODN1 with NaBH(4) produced the corresponding ODN2 quantitatively. Phosphoramidite V was also incorporated into DNA, and the methyl ester could be hydrolyzed under mild basic conditions to afford ODN3.     

Highly selective binding of naphthyridine with a trifluoromethyl group to cytosine opposite an abasic site in DNA duplexes

We report on highly selective binding of a naphthyridine derivative with a trifluoromethyl group to cytosine opposite an abasic site in DNA duplexes; the binding-induced fluorescence quenching is applicable to the analysis of a C-related single-base mutation in DNAs amplified by PCR.     

Head-to-tail oxime cyclization of oligodeoxynucleotides for the efficient synthesis of circular DNA analogues

A convenient strategy for the synthesis of the analogue of cyclic oligodeoxyribonucleotides is presented. The cyclization of the oligonucleotide was accomplished through intramolecular oxime bond formation between a 5'-oxyamine moiety and a 3'-aldehydic group.     

Charge transfer excited-state dynamics in DNA duplexes substituted with an ethynylpyrenyldeoxyuridine electron source and a fluorodeoxyuridine electron trap

Studies of six 5-(pyren-1-yl-ethynyl)-2'-deoxyuridine (U(PY))-substituted DNA duplexes in this work test and support the conclusions reported by Gaballah et al. (J. Phys. Chem. B 2005, 109, 5927-5934) based on investigations of 5-(2-pyren-1-yl-ethylenyl)-2'-deoxyuridine (U(PE))-substituted DNA hairpins. As expected because of the rigid ethynyl linker in U(PY) (compared to the flexible ethylenyl linker in U(PE)), U(PY)-substituted duplexes do not show enhanced charge transfer (CT) emission quantum yields for duplexes with 5-fluorodeoxyuridine (U(F)) electron traps near U(PY) compared to duplexes without traps. Furthermore, the average CT lifetime and emission quantum yield of U(PY)-substituted duplexes is independent of the U(F) trap location. These new results strongly suggest that the excess electron in the PY*+/dU*- CT state of U(PY) is restrained from hopping to nearby U(F) traps due to attraction to PY*+.     

Synthesis and properties of oligodeoxynucleotides incorporating a conformationally rigid uridine unit having a cyclic structure at the 5'-terminal site

A 2'-O-methyluridylic acid derivative 3 having a cyclic structure linked between the 5-position of the uracil residue and the 5'-phosphate group was synthesized. The NMR analysis suggests that this cyclouridylic acid derivative has exclusively the C3'-endo conformation that is in favor of duplex formation with RNA. Two oligonucleotides ?pc3Um(pT)(9) and pc3Um(pU)(9) incorporating this cyclouridylic acid unit at the 5'-terminal site were synthesized by using the fully protected cyclouridylic acid 3'-phosphoramidite derivative 11 in the solid-phase synthesis. To examine the actual effect of this cyclic structure on the thermal stability of duplexes between the modified oligonucleotides and their complementary oligonucleotides, two oligonucleotides ?pUm(pT)(9) and pUm(pU)(9) having an acyclic structure were also synthesized. As the complementary oligonucleotides, dA(pdA)(9) and A(pA)(9) were used for T(m) experiments with these 5'-terminal modified oligonucleotides. The T(m) values of all the possible duplexes were measured. These results clearly show that the duplex of pc3Um(pT)(9)-A(pA)(9) has a higher T(m) value by 5.5 degrees C than that of A(pA)(9)-T(pT)(9). This is rather significant compared with all other cases. Moreover, the T(m) value of pc3Um(pT)(9)-A(pA)(9) is 4.5 degrees C higher than that of pUm(pT)(9)-A(pA)(9). This result suggests that the cyclic structure can considerably contribute to stabilization of the duplex only in the case of the modified oligomer (DNA) and decaadenylate (RNA).     

Synthesis and photophysical processes of an anthracene derivative containing hole transfer groups

A novel luminescent compound 9,10-di-(N-carbazovinylene)anthracene (DCVA) was synthesized by Heck reaction of 9,10-dibromoanthracene and N-vinylcarbazole. The structure was characterized by MS, 1H NMR and Elemental analysis. The photoluminescent properties of DCVA have been carefully investigated by UV-vis absorption and fluorescence emission spectra. The results showed that the luminescent quantum yield of DCVA was 0.73 in THF and it emitted blue-light with the band gap of 3.60 eV estimated from the onset absorption. In addition, the light-emission of DCVA can be quenched by electron acceptor (dimethyl terephthalate), however, the fluorescent intensities of DCVA were slowly increased with the addition of electron donor (N,N-dimethylaniline). Furthermore, the molecular interactions of DCVA with fullerene (C60) and carbon nanotubes (CNTs) were also investigated, which indicated the organic luminescent compound can be used as new fluorescent probe.     

Synthesis and characterization of fluorenone-, anthraquinone-, and phenothiazine-labeled oligodeoxynucleotides: 5'-probes for DNA redox chemistry

A facile and automated procedure for the synthesis of oligodeoxynucleotides possessing derivatives of 9-fluoreneone, 9,10-anthraquinone, and phenothiazine is described. The phosphoramidite approach is used to attach these redox and spectroscopic probes to the 5'-terminus of oligodeoxynucleotides in high yield (>95%). Thermal denaturation studies of labeled oligodeoxynucleotides show a slight enhancement in duplex stability relative to the unlabeled control, and circular dichroism spectra confirm B-form helical DNA structure in solution.     

Structural, dynamical, and electronic transport properties of modified DNA duplexes containing size-expanded nucleobases

Among the distinct strategies proposed to expand the genetic alphabet, size-expanded nucleobases are promising for the development of modified DNA duplexes with improved biotechnological properties. In particular, duplexes built up by replacing canonical bases with the corresponding benzo-fused counterparts could be valuable as molecular nanowires. In this context, this study reports the results of classical molecular dynamics simulations carried out to examine the structural and dynamical features of size-expanded DNAs, including both hybrid duplexes containing mixed pairs of natural and benzo-fused bases (xDNA) and pure size-expanded (xxDNA) duplexes. Furthermore, the electronic structure of both natural and size-expanded duplexes is examined by means of density functional computations. The results confirm that the structural and flexibility properties of the canonical DNA are globally little affected by the presence of benzo-fused bases. The most relevant differences are found in the enhanced size of the grooves, and the reduction in the twist. However, the analysis also reveals subtle structural effects related to the nature and sequence of benzo-fused bases in the duplex. On the other hand, electronic structure calculations performed for xxDNAs confirm the reduction in the HOMO-LUMO gap predicted from the analysis of the natural bases and their size-expanded counterparts, which suggests that pure size-expanded DNAs can be good conductors. A more complex situation is found for xDNAs, where fluctuations in the electrostatic interaction between base pairs exerts a decisive influence on the modulation of the energy gap.     

CopperII phthalocyanine as an efficient and reusable catalyst for the N-arylation of nitrogen containing heterocycles

Copper phthalocyanine (Cu^IIPc) was found to be an efficient catalyst for the catalyzed N-arylation of NH heterocycles with aryl iodides and bromides under mild reaction conditions. A variety of hindered and functionalized NH heterocycles and aryl halides were successfully used as the substrates for the given catalytic reaction and were transformed in good to excellent yields.