Water-soluble phosphorescent ruthenium complex with a fluorescent coumarin unit for ratiometric sensing of oxygen levels in living cells.

Dual emission was applied to a molecular probe for the ratiometric sensing of oxygen concentration in a living system. We prepared ruthenium complexes possessing a coumarin unit (Ru-Cou), in which the (3)MLCT phosphorescence of the ruthenium complex was efficiently quenched by molecular oxygen, whereas the coumarin unit emitted constant fluorescence independent of the oxygen concentration. The oxygen status could be determined precisely from the ratio of phosphorescence to fluorescence. We achieved the molecular imaging of cellular oxygen levels using Ru-Cou possessing an alkyl chain, which provided appropriate lipophilicity to increase cellular uptake.

Hypoxic X-irradiation as an external stimulus for conformational change of oligodeoxynucleotides that possess disulfide bond and regulation of DNAzyme function.

We achieved a conformational change of oligodeoxynucleotides and the regulation of DNAzyme function by means of a radiolytic strand exchange reaction of disulfide bond. We designed a system in which the DNAzyme function of RNA cleavage was suppressed by the hybridization of an inhibitor strand that possessed disulfide bond with an active DNAzyme. Hypoxic X-irradiation led to the recovery of RNA cleavage because the strand exchange reaction at the disulfide bond in inhibitor strand resulted in a release of inhibitor strand. This strategy may be applicable to gene regulation by hypoxic X-irradiation.

Enzyme-catalyzed conversion of chemical structures on the surface of gold nanorods.

For developing metal nanoparticles of which surface chemical structures could be altered by enzymes in the cells, functional linkers caged by coumaric acids have been synthesized. Synthesized gold nanorod (GNR) conjugates possessing coumaric acid precursors underwent (porcine liver) esterase-catalyzed hydrolysis on their surface to afford GNRs coated with amino-functionalized polyethylene glycol and fluorescent coumarins as reporter molecules for monitoring the conversion. The chemical structural conversion on the GNR surfaces was successfully observed inside cells by fluorescence microscopy when GNR conjugates were incubated with tumor cells.

Probing DNA mismatched and bulged structures by using 19F NMR spectroscopy and oligodeoxynucleotides with an 19F-labeled nucleobase.

In this study, DNA local structures with bulged bases and mismatched base pairs as well as ordinary full-matched base pairs by using (19)F NMR spectroscopy with (19)F-labeled oligodeoxynucleotides (ODNs) were monitored. The chemical shift change in the (19) F NMR spectra allowed discrimination of the DNA structures. Two types of ODNs possessing the bis(trifluoromethyl)benzene unit (F-unit) at specified uridines were prepared and hybridized with their complementary or noncomplementary strands to form matched, mismatched, or bulged duplexes. By using ODN F1, in which an F-unit was connected directly to a propargyl amine-substituted uridine, three local structures, that is, full-matched, G-U mismatch, and A-bulge could be analyzed, whereas other structures could not be discriminated. A molecular modeling study revealed that the F-unit in ODN F1 interacted little with the nucleobases and sugar backbone of the opposite strand because the linker length between the F-unit and the uridine base was too short. Therefore, the capacity of ODN F1 to discriminate the DNA local structures was limited. Thus, ODN F2 was designed to improve this system; aminobenzoic acid was inserted between the F-unit and uridine base so the F-unit could interact more closely with the opposite strand. Eventually, the G-bulge and T-U mismatch and the three aforementioned local structures could be discriminated by using ODN F2. In addition, the dissociation processes of these duplexes could be monitored concurrently by (19)F NMR spectroscopy.

Ruthenium complexes with hydrophobic ligands that are key factors for the optical imaging of physiological hypoxia.

The phosphorescence emission of ruthenium complexes was applied to the optical imaging of physiological hypoxia. We prepared three complexes with hydrophobic substituents on the phenanthroline ligand and characterized their emission, which was quenched by molecular oxygen. Among the complexes synthesized in this study, a pyrene chromophore-linked ruthenium complex, Ru-Py, exhibited optimal properties for the imaging of hypoxia; the prolonged lifetime of the triplet excited state of the ruthenium chromophore, which was induced by efficient energy distribution and transfer from the pyrene unit, provided the highest sensitivity towards molecular oxygen. The introduction of hydrophobic pyrene increased the lipophilicity of the complex, leading to enhanced cellular uptake. Consequently, the bright phosphorescence of Ru-Py was seen in the cytoplasm of viable hypoxic cells, whereas the signal from aerobic cells was markedly weaker. Thus, we could clearly discriminate between hypoxic and aerobic cells by monitoring the phosphorescence emission. Furthermore, Ru-Py was applied to optical imaging in live mice. An intramuscular injection of Ru-Py successfully visualized ischemia-based hypoxia, which was constructed by leg banding.

Chemical ligation of oligodeoxynucleotides by X-irradiation and its application to regulation of G-quadruplex formation.

We demonstrated radiolytic ligation of oligodeoxynucleotides (ODNs) possessing disulfide bond and its application to regulation of DNA quadruplex formation. G-rich hexamer ODNs had poor ability to form quadruplex, while X-irradiation of the ODNs induced interstrand exchange reaction at disulfide bond to form ligated 12 mer ODNs, leading to the ready formation of quadruplex due to the entropic effect. Since complexation of the ligated ODNs with hemin in the presence of K(+) showed strong soret band absorption and also catalyzed the H(2)O(2)-mediated oxidation of luminol, it appears that the quadruplex formed from ligated ODNs showed a function similar to native DNA quadruplex.

Stepwise regulation of hole transport in DNA by control of triplex formation.

A functionality for regulating hole transport efficiency is a prerequisite for the utilization of DNA duplexes as nanodevices. Herein, we report the regulation of hole transport in anthraquinone-tethered DNA with dual triplex forming sites. Long-range photooxidation experiments showed that hole transport was effectively suppressed by the formation of triplex at low temperature, while it was recovered by dissociation to the duplex at higher temperature. Variation of temperature induced the formation and dissociation of the third strand at each triplex region individually, leading to the stepwise regulation of hole transport in DNA.

Radiolytic reduction characteristics of drug-encapsulating DNA aggregates possessing disulfide bond.

Radiation-responsive aggregates consisting of artificial DNA were demonstrated. We prepared DNA amphiphiles (DAMs) consisting of oligodeoxynucleotides (ODNs) as the hydrophilic part and an alkyl chain as the hydrophobic part, which were linked by a radiation-sensitive disulfide bond. DAMs assembled to form nanosized aggregates in aqueous solution that encapsulated hydrophobic dyes and drugs. X-irradiation, which triggered selective reduction at the disulfide bond in the DAMs, induced an exchange reaction, resulting in dissociation of the aggregates and release of encapsulated dyes and drugs. Among the DAMs synthesized in this study, DAM 1 possessing 5 mer ODNs showed favorable properties as a nanocarrier; for example, the large amount of drug was encapsulated in the aggregates consisting of DAM 1, and the aggregates showed the high sensitivity to radiolytic reduction. Biological experiments using living A549 cells revealed that the aggregates smoothly penetrated into the cells without the need for transfection reagents, while the drugs inside the aggregates were inert because of the encapsulation. X-irradiation enhanced the cytotoxicity because of dissociation of the aggregates and concomitant release of drugs.

Aggregate formation and radiolytic degradation of amphiphilic DNA block copolymer possessing disulfide bond.

We have designed a novel aggregate of DNA block copolymer (DBC) that is sensitive to hypoxic X-irradiation. The DBC consists of tetrahydropyrane-protected 2-hydroxyethyl methacrylate as a hydrophobic unit and oligodeoxynucleotides as a hydrophilic unit, which are linked to a radiation-sensitive disulfide bond. The DBC self-assembled efficiently to form aggregates that encapsulated small molecules such as nile red and pyrene. Hypoxic X-irradiation could then induce reductive degradation of the DBC aggregates via an exchange reaction of the disulfide bond to release guest molecules.

Reductive activation of 5-fluorodeoxyuridine prodrug possessing azide methyl group by hypoxic X-irradiation.

We prepared a 5-fluorodeoxyuridine (5-FdUrd) derivative possessing azide methyl group (N(3)-FdUrd) as a novel radiation-activated prodrug. The parent antitumor agent, 5-FdUrd, was released efficiently from N(3)-FdUrd by hypoxic X-irradiation. On the other hand, the activation of N(3)-FdUrd was suppressed upon X-irradiation under aerobic conditions. A biological assay using A549 cells revealed that the cytotoxicity of N(3)-FdUrd was significantly enhanced by hypoxic X-irradiation.

Synthesis and photooxidation of oligodeoxynucleotides containing 5-dimethylaminocytosine as an efficient hole-trapping site in the positive-charge transfer through DNA duplexes.

We have designed and synthesized DNA duplexes containing 5-dimethylaminocytosine ((DMA)C) to investigate the effects of C(5)-substituted cytosine bases on the transfer and trapping of positive charge (holes) in DNA duplexes. Fluorescence quenching experiments revealed that a (DMA)C base is more readily one-electron oxidized into a radical cation intermediate as compared with other natural nucleobases. Upon photoirradiation of the duplexes containing (DMA)C, the photosensitizer-injected hole migrated through the DNA bases and was trapped efficiently at the (DMA)C sites, where an enhanced oxidative strand cleavage occurred by hot piperidine treatment. The (DMA)C radical cation formed by hole transfer may undergo specific hydration and subsequent addition of molecular oxygen, thereby leading to its decomposition followed by a predominant strand cleavage at the (DMA)C site. This remarkable property suggests that the modified cytosine (DMA)C can function as an efficient hole-trapping site in the positive-charge transfer in DNA duplexes.

Synthesis and one-electron reduction characteristics of radiation-activated prodrugs possessing two 5-fluorodeoxyuridine units.

Two molecules of an antitumor agent, 5-fluorodeoxyuridine (5-FdUrd), were connected by a 2-oxoalkyl linker (Oxo-linker) at the N(3) position to obtain radiation-activated prodrugs, FdUrd(2) A and FdUrd(2) B. The prodrugs in this study released 5-FdUrd via one-electron reduction initiated by hypoxic X-irradiation. The release of 5-FdUrd from FdUrd(2) A and FdUrd(2) B proceeded more efficiently than that of previous prodrug, Oxo-FdUrd, which possessed one molecule of 5-FdUrd. FdUrd(2) A exhibited increased cytotoxicity against A549 cells when the FdUrd(2) A solution had been irradiated with a large dose of X-rays before administration to the cells. However, we observed no effect on cytotoxicity when the cells were X-irradiated under hypoxic conditions in the presence of FdUrd(2) A because the amount of 5-FdUrd released in the cells seemed to be too low to induce cytotoxic activity.

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.

(13)C-labeled indolequinone-DTPA-Gd conjugate for NMR probing cytochrome:P450 reductase-mediated one-electron reduction.

We designed and synthesized a new class of (13)C-labeled NMR probe, (13)C-IQ-Gd, to monitor one-electron reductions by cytochrome:P450 (CYP450) reductase under hypoxic conditions. (13)C-IQ-Gd consisted of a Gd(3+)-diethylene triamine pentaacetic acid (DTPA) complex unit and an indolequinone ((13)C-IQ) unit bearing a (13)C-labeled methoxy group. The (13)C NMR signal of (13)C-IQ-Gd was suppressed because of the intramolecular paramagnetic effect of Gd(3+), whereas enzymatic reduction mediated by CYP450 reductase under hypoxic conditions yielded an intensed (13)C NMR signal due to enzymatic activation of the IQ unit followed by release of the DTPA-Gd unit from (13)C-IQ-Gd. This (13)C NMR spectral change allowed the monitoring of CYP450 reducatase-mediated one-electron reduction.

αvß3-Integrin-targeting lanthanide complex: synthesis and evaluation as a tumor-homing luminescent probe.

The application of lanthanide complexes in the time-resolved fluorescence imaging of living cells has emerged in the last few decades, providing high-contrast images of cells through detection of the delayed emission. In the present study, we synthesized novel trivalent lanthanide complexes containing the cyclic peptide c(RGDfK) to visualize the α(v)ß(3)-integrin-expressing tumor cells. Conjugation of c(RGDfK) with the macrocyclic bipyridine ligand had little effect on the fluorescence properties of the complex, indicating that the coordinated lanthanide ion was well isolated from the peptide. Bright luminescence images of α(v)ß(3)-integrin-expressing U87-MG cells were successfully obtained by employing the probes.

Radiolytic cyclization of stem-and-loop structured oligodeoxynucleotide with neighboring arrangement of α,ω-bis-disulfides.

Upon X-ray irradiation of hypoxic aqueous solution, modified oligodeoxynucleotides (ODNs) bearing a pair of disulfides at both ends of the strand that forms a stem-and-loop structure with a neighboring arrangement of α,ω-bis-disulfides underwent efficient cyclization via an intramolecular exchange reaction at the disulfide moieties with a multiple turnover process. Mechanistic studies revealed that hydrogen atoms generated in the radiolysis of water are key active species initiating a chain reaction to produce cyclic ODN disulfides, in which addition of hydrogen atom results in dissociation of the original disulfide bond to generate a thiyl radical intermediate as the chain carrier for the succeeding disulfide exchange into cyclization. The properties were also assessed for the resultant cyclic ODN disulfide that has several favorable features for use in the transcriptional decoy strategy. The cyclic ODN disulfides produced by the present radiolytic method showed high thermal stability, resistance to nuclease, and high binding activity to a representative transcriptional factor of nuclear factor κB.

Monitoring of duplex and triplex formation by 19F NMR using oligodeoxynucleotides possessing 5-fluorodeoxyuridine unit as 19F signal transmitter.

We prepared oligodeoxynucleotides (ODNs) possessing a 5-fluorodeoxyuridine (5-FU) unit as a 19F-signal transmitter, and characterized their structures including single strand, duplex, and triplex using 19F NMR. The change in chemical shift induced by incorporation of 5-FU into the ODNs and the formation of higher order structures allowed monitoring of structural changes. Data from UV melting experiments and CD spectra were consistent with the spectral changes in the NMR studies. These 19F-labeled ODNs may be promising molecular probes for the identification of DNA structures in complicated biological conditions.

Monitoring of biological one-electron reduction by (19)F NMR using hypoxia selective activation of an (19)F-labeled indolequinone derivative.

Biological reduction of fluorine-labeled indolequinone derivative (IQ-F) was characterized by (19)F NMR for quantitative molecular understanding. The chemical shift change in (19)F NMR allowed monitoring of the enzymatic reduction of IQ-F. Upon hypoxic treatment of IQ-F with NADPH:cytochrome P450 reductase, IQ-F was activated via catalytic one-electron reduction to release nonafluoro-tert-butyl alcohol (F-OH), while the formation of F-OH was significantly suppressed under aerobic conditions. Similar hypoxia-selective reduction of IQ-F occurred within A549 cells, which expresses NADPH:cytochrome P450 reductase. The kinetic analysis was also performed to propose a reaction mechanism. The molecular oxygen slightly prevents the binding of IQ-F to reductase, while the rate of net reaction was decreased due to oxidation of a semiquinone anion radical intermediate generated by one-electron reduction of IQ-F. The disappearance of IQ-F and appearance of F-OH were imaged by (19)F fast spin echo, thus visualizing the hypoxia-selective reduction of IQ-F by means of MR imaging.

Properties and reactivity of the adenosine radical generated by radiation-induced oxidation in aqueous solution.

The formation of oxidation products of DNA bases induced by hole injection into DNA duplexes is closely related to the characteristics of the radical species generated, especially those from purine bases possessing lower oxidation potentials than pyrimidines. To investigate the reactivities of adenosine base radicals generated from the radical cations of adenosine (Ado), we have conducted extensive pulse radiolysis and steady-state X-radiolysis investigations of Ado under the conditions generating oxidizing radical species such as the sulfate radical anion (SO(4)(-*)). Kinetic analysis of the transient absorption spectra demonstrated that the redox-neutral radical of adenosine [Ado(-H)(*)] decays via a homogeneous bimolecular reaction, recombines with an alkyl alcohol radical, or abstracts hydrogen from the phosphate ion, with rate constants of 2k = 4.2 x 10(8) M(-1) s(-1) and k = 6.4-8.5 x 10(8) and 10(3)-10(4) M(-1) s(-1), respectively. High-dose pulsed electron beam irradiation, which generates high concentrations of Ado(-H)(*) during the radiolysis, yielded 8-oxo-7,8-dihydroadenosine (8-oxo-Ado) with a G value of 0.018 x 10(-7) mol J(-1). It is thus proposed that the disproportionation of Ado(-H)(*) might lead to the formation of 8-oxo-Ado as a minor process, which is in addition to the widely accepted mechanism in which the addition of hydroxyl radical to Ado initiates its formation.

Pathways of excess electron transfer in phenothiazine-tethered DNA containing single-base mismatches.

The effects of local structural disorder on excess electron transfer (EET) in DNA were investigated by evaluating photoinduced electron transfer in phenothiazine (PTZ)-modified oligodeoxynucleotides bearing single-base mismatches. Unexpectedly, more efficient electron transfer was observed for the mismatched duplexes than for the complementary DNA, suggesting that distraction of hydrogen bond interaction at the mismatch site enables electron injection or hopping beyond the mismatch sites. It was also anticipated that water accessibility of the mismatched nucleobases could affect EET because protonation of the electron-captured pyrimidine intermediates became competitive to EET, especially at the mismatch sites.