Development of UTX-143, a Selective Sodium-hydrogen Exchange Subtype 5 Inhibitor, using Amiloride as a Lead Compound

NHE5, an isoform of the Na/H exchanger (NHE) protein, is an ion-transporting membrane protein that regulates intracellular pH and is highly expressed in colorectal adenocarcinoma. Therefore, we hypothesized that NHE5 inhibitors can be used as anticancer drugs. However, because NHE1 is ubiquitously expressed in all cells, it is extremely important to demonstrate its selective inhibitory activity against NHE5. We used amiloride, an NHE non-selective inhibitor, as a lead compound and created UTX-143, which has NHE5-selective inhibitory activity, using a structure-activity relationship approach. UTX-143 showed selective cytotoxic effects on cancer cells and reduced the migratory and invasive abilities of cancer cells. These results suggest a new concept wherein drugs exhibit cancer-specific cytotoxic effects through selective inhibition of NHE5 and the possibility of UTX-143 as a lead NHE5-selective inhibitor.


Enhancing Effect of Novel Schiff Base Derivatives, UTX-134 and UTX-135, on 5-Aminolevulinic Acid-based Photodynamic Therapy


Structure Activity Relationships of UTX-121 Derivatives for the Development of Novel Matrix Metalloproteinase-2/9 Inhibitors


Development of a novel acetyl glucose-modified gefitinib derivative to enhance the radiosensitizing effect

Various radiosensitizers are being developed to increase the radiation sensitivity of hypoxic cancer cells, which show resistance to radiation. Previously, we demonstrated that an acetyl glucose-modified nitroimidazole derivative showed a high radiosensitizing effect by inhibiting glucose uptake and glycolysis. Based on this finding, we designed and synthesized novel sugar hybrid radiosensitizers, wherein acetyl glucose was introduced into gefitinib. Among them, UTX-114 had higher autophosphorylation and radiosensitizing activity than gefitinib and inhibited glucose uptake. This result supports our hypothesis that an acetyl glucose moiety improves the radiosensitizing effect of the drug, and UTX-114 can be expected to be a leading compound with a radiosensitizing effect.


Photoacoustic in vivo 3D imaging of tumor using a highly tumor-targeting probe under high-threshold conditions.

Three-dimensional (3D) representation of a tumor with respect to its size, shape, location, and boundaries is still a challenge in photoacoustic (PA) imaging using artificial contrast agents as probes. We carried out PA imaging of tumors in mice using 800RS-PMPC, which was obtained by coupling of 800RS, a near-infrared cyanine dye, with PMPC, a highly selective tumor-targeting methacrylate polymer having phosphorylcholine side chains, as a probe. The conjugate 800RS-PMPC forms compact nanoparticles (d

tumor buried 6 mm beneath the skin, the probe 800RS-PMPC selectively accumulates in the tumor and emits PA signals that are strong enough to be unambiguously distinguished from noise signals of endogenous blood/hemoglobin. The PA image thus obtained under high-threshold conditions allows 3D characterization of the tumor in terms of its size, shape, location, and boundaries.


Therapeutic Effect and Mechanism of Action of Low-molecular-weight Whey Protein Capable of Activating Macrophages in Bovine Mastitis

Bovine mastitis is caused by the invasion and propagation of pathogenic microorganisms into the udder and mammary gland tissues of cattle. In this study, the therapeutic effect of a low-molecular-weight whey protein (LMW-WP) on bovine mastitis was evaluated. LMW-WP was orally, intraperitoneally, and vaginally administered to bovine with mastitis. The number of somatic cells in milk was measured 24 h before the administration of LMW-WP. The effect of LMW-WP on cytokine production was measured with a microarray that evaluates the expression of cytokines. In the group that received 1,000 mg intraperitoneally, the somatic cell count was reduced to less than 400,000 at the shipment standard value in three of the four udders, indicating 75% efficacy. The group that received 1,000 mg by vaginal administration showed 67% efficacy. It was confirmed that LMW-WP increased the production of cytokines such as IL-5, IL-6, IL-9, IL-12, MCP-1, and VEGF in mouse macrophage cells, but it did not show any antibacterial activity. LMW-WP may be an effective therapeutic agent for bovine mastitis.


A novel celecoxib analog UTX-121 inhibits HT1080 cell invasion by modulating membrane-type 1 matrix metalloproteinase

We designed and synthesized a celecoxib derivative UTX-121 to enhance its anti-tumor activity. Similar to celecoxib, this compound could also inhibit matrix metalloproteinase (MMP)-9 activity. In addition, UTX-121 suppressed membrane-type 1 MMP (MT1-MMP)-mediated pro-MMP-2 activation by disturbing the cell surface expression of MT1-MMP. UTX-121 also impeded the glycosylation of cell surface proteins, resulting in the suppression of cell attachment to fibronectin. This inhibition by UTX-121 caused the reduction of fibronectin-stimulated focal adhesion kinase activation, Akt activation, and cell migration. Consequently, UTX-121 treatment significantly inhibited fibronectin-induced HT1080 cell invasion into the Matrigel. UTX-121 may be a potent lead compound that can be used to develop a novel anti-tumor drug.

Biochemical and Biophysical Research Communications


Enzymatic activation of indolequinone-substituted 5-fluorodeoxyuridine prodrugs in hypoxic cells

Among the various enzymes, reductases that catalyze one-electron reduction are involved in the selective activation of functional compounds or materials in hypoxia, which is one of the well-known pathophysiological characteristics of solid tumors. Enzymatic one-electron reduction has been recognized as a useful reaction that can be applied in the design of tumor hypoxia-targeting drugs. In this report, we characterized the enzymatic reaction of 5-fluorodeoxyuridine (FdUrd) prodrug bearing an indolequinone unit (IQ-FdUrd), which is a substrate of reductases. IQ-FdUrd was activated to release FdUrd under hypoxic conditions after treatment with cytochrome NADPH P450 reductase. We also confirmed that IQ-FdUrd showed selective cytotoxicity in hypoxic tumor cells.

Bioorganic & Medicinal Chemistry Letters


Concentration-dependent Activation of Inflammatory/Anti-inflammatory Functions of Macrophages by Hydrolyzed Whey Protein

Whey protein is a mixture of globulins isolated from whey and mainly composed of β-lactoglobulin, α-lactoalbumin, and lactoferrin. In this study, whey protein was hydrolyzed using various proteases, and the macrophage activation was evaluated. Hydrolyzed whey protein was prepared using various proteases to evaluate phagocytic activity and cytokine productivity. The results of SDS-PAGE and gel permeation chromatography (GPC) analysis indicated that the molecular weight of whey protein was reduced using various proteases. The hydrolyzed whey protein showed a concentration-dependent induction of macrophage phagocytic activity. In addition, the hydrolyzed whey protein significantly enhanced the production of the inflammatory cytokine, TNF-α. Production of the anti-inflammatory cytokine, IL-10, was not observed at concentrations up to 1 μg, but significant production was confirmed at 100 μg. Hydrolyzed whey protein can induce the phagocytic activity of macrophages and activation of the inflammatory/anti-inflammatory functions of the macrophages depends on the concentration of the hydrolyzed whey protein.


Radiosensitizing Effect of 5-Aminolevulinic Acid and Protoporphyrin IX on Carbon-ion Beam Irradiation

Carbon-ion beam is one of the most advanced radiations used for cancer treatment. However, there are tumors that are difficult to suppress with carbon-ion beam alone, thus necessitating development of drugs that can enhance its therapeutic effect. In this regard, the radiosensitizing effect of 5-aminolevulinic acid (ALA) and protoporphyrin IX (PpIX), that is a metabolic intermediate of ALA, on carbon-ion beam was investigated. Radiosensitizing activity, mitochondrial ROS and DNA double-strand break production of ALA and PpIX were evaluated by irradiation with 1.0 or 1.5-Gy carbon-ion beam to mouse mammary EMT6 tumor cells. Combination of carbon-ion beam and ALA or PpIX showed a significant enhancement of its cytotoxic activity through a significant increase in ROS production in mitochondria. Furthermore, the combined activity of carbon-ion beam and ALA resulted in a significant increase in DNA double-strand breakage. ALA selectively accumulates in the mitochondria and PpIX synthesized from ALA reacts with carbon-ion beam to produce ROS that exert antitumor activity.


Development of a novel Schiff base derivative for enhancing the anticancer potential of 5-aminolevulinic acid-based photodynamic therapy.

5-Aminolevulinic acid (ALA), a precursor of protoporphyrin IX (PpIX), is now widely used for photodynamic diagnosis (ALA-PDD) and photodynamic therapy (ALA-PDT) of various cancers. Recently, we found that treatment of cancer cells with the Schiff base derivative TX-816 along with ALA could significantly increase the efficacy of ALA-PDT. This enhancing effect of TX-816 on ALA-PDT is attributed to 3,5-dichlorosalicylaldehyde (DCSA), a molecule produced by the degradation of TX-816. Similar to TX-816, DCSA significantly enhances the effect of ALA-PDT. Furthermore, DCSA could restore the sensitivity of cancer cells that acquired resistance to ALA-PDT. These results indicate that DCSA, as well as TX-816, is a potent lead compound for the development of an ALA-PDT sensitizer. TX-816 might be a useful compound for designing prodrug-type ALA-PDT enhancers.


Development of a novel Schiff base derivative for enhancing the anticancer potential of 5-aminolevulinic acid-based photodynamic therapy


Polymeric (1) H MRI Probes for Visualizing Tumor In Vivo.

Magnetic resonance imaging (MRI) has become a prominent non- or low-invasive imaging technique, providing high-resolution, three-dimensional images as well as physiological information about tissues. Low-molecular-weight Gd-MRI contrast agents (CAs), such as Gd-DTPA (DTPA: diethylenetriaminepentaacetic acid), are commonly used in the clinical diagnosis, while macromolecular Gd-MRI CAs have several advantages over low-molecular-weight Gd-MRI CAs, which help minimize the dose of CAs and the risk of side effects. Accordingly, we developed chiral dendrimer Gd-MRI CAs, which showed high r1 values. The association constant values (Ka ) of S-isomeric dendrimer CAs to bovine serum albumin (BSA) were higher than those of R-isomeric dendrimer CAs. Besides, based on a totally new concept, we developed (13) C/(15) N-enriched multiple-resonance NMR/MRI probes, which realized highly selective observation of the probes and analysis of metabolic reactions of interest. This account summarizes our recent study on developing both chiral dendrimer Gd-MRI CAs, and self-traceable (13) C/(15) N-enriched phosphorylcholine polymer probes for early detection of tumors.


Design and Synthesis of Novel Anti-metastatic Hypoxic Cytotoxin TX-2137 Targeting AKT Kinase.

The hypoxic microenvironment plays a crucial role in the malignant progression of tumor cells. Moreover, AKT, a serine/threonine kinase, is activated by various extracellular growth factors and is important for cell growth, survival, and motility of leukocytes, fibroblasts, endothelial cells, and tumor cells. Therefore, we aimed to design an anti-metastatic hypoxic cytotoxin which has inhibitory effects on AKT.TX-2137 was designed and synthesized based on the structural similarity of a preexisting AKT1/2 kinase inhibitor and a hypoxic cytotoxin tirapazamine. TX-2137 effectively reduced the expression of phosphorylated AKT and matrix metalloproteinase 9 (MMP9) and showed strong inhibition of the proliferation of B16-F10, HT-1080, and MKN-45 cells. In addition, TX-2137 exhibited hypoxia-selective cytotoxicity towards A549 cells and inhibited liver metastasis of B16-F10 cells in a xenograft chick embryo model in the same way as doxorubicin.TX-2137 may be a potent lead compound in the development of a novel anti-metastatic AKT kinase inhibitor.


5-Aminolevulinic Acid Enhances Ultrasound-mediated Antitumor Activity via Mitochondrial Oxidative Damage in Breast Cancer

5-Aminolevulinic acid (5-ALA), a precursor of protoporphyrin IX (PpIX), is now used for photodynamic therapy (PDT) of pre-cancers of the skin and photodynamic diagnosis (PDD) of brain tumors. Sonodynamic therapy (SDT) of cancers with ultrasound has been studied using 5-ALA as a sonosensitizer. In this article, we evaluated the sonosensitizing activity and mode of action of 5-ALA/PpIX by using mouse mammary tumor EMT6 cells.5-ALA-SDT showed significant antitumor effects toward EMT6 cells in vitro and in vivo. The fluorescence of MitoSOX Red, an indicator specific for mitochondrial superoxide, was significantly increased by 5-ALA-SDT. Moreover, the fluorescence derived from JC-1, an indicator of mitochondrial membrane potential, was also significantly increased by 5-ALA-SDT. These findings suggest that mitochondria are one of the target organelles of 5-ALA-SDT. PpIX enhanced reactive oxygen species (ROS) production from tert-butyl hydroperoxide (tBHP), suggesting that PpIX might stabilize or promote ROS generation from tBHP.5-ALA-SDT showed an antitumor effect in mouse mammary tumor EMT6 cells through oxidation of the mitochondrial membrane via ROS production.


RADIOSENSITIZING EFFECT OF PROTOPORPHYRIN IX WITH CARBON ION BEAM AGAINST MOUSE MAMMARY BREAST TUMOR CELL.

5-Aminolevulinic acid (ALA), a precursor ofprotoporphyrin IX (PpIX), has been utilized as a potent sensitizerfor photodynamic therapy (PDT) of cancer through productionof reactive oxygen species (ROS). ALA also has been proposed asa possible radiosensitizer with low linear energy transfer (LET)radiation because antitumor activity of low-LET radiation dependson ROS mainly. In this paper, we investigated the radiosensitizingactivity of PpIX in carbon ion irradiation as a high LET radiation.PpIX was detected in an EMT6 mouse mammary breast tumor cellseven at 24 h post treatment. The cytotoxicity of PpIX against EMT6cells was IC50 = 48.0 m. The combined treatment of 1.0 m of PpIXwith 2 Gy of carbon ion irradiation (290 MeV/nucleon, 85.1 keV/m)showed a more potent antitumor activity than carbon ion irradiationalone. We demonstrated that PpIX showed a radiosensitizing effectwith carbon ion beam against mouse mammary breast tumor cells.

Journal of Advanced Manufacturing Technology


13C/15N-Enriched L-Dopa as a Triple-Resonance NMR Probe to Monitor Neurotransmitter Dopamine in the Brain and Liver Extracts of Mice


Pharmacokinetics of Chiral Dendrimer-Triamine-Coordinated Gd-MRI Contrast Agents Evaluated by in Vivo MRI and Estimated by in Vitro QCM


Degalactosylated/Desialylated Bovine Colostrum Induces Macrophage Phagocytic Activity Independently of Inflammatory Cytokine Production.

Similar to the use of GcMAF, degalactosylated/desialylated bovine colostrum can be used as a potential macrophage activator for various immunotherapies.


Magnetic Resonance Imaging of Tumor with a Self-Traceable Polymer Conjugated with an Antibody Fragment

A (13)C-enriched phosphorylcholine polymer ((13)C-PMPC) as a self-traceable MR (magnetic resonance) tag was conjugated with a fragment (scFv) of Herceptin, a clinical antibody against antigen Her2. When injected in model mice bearing Her2(+) (gastric) and Her2(-) (pancreatic) tumors, the antibody-tag conjugate (13)C-PMPC-scFv selectively accumulated in the Her2(+) tumor with a rapid build-up/decay (accumulation/clearance) profile and, with the use of the (1)H-(13)C double-resonance (heteronuclear correlation) technique, the Her2(+) gastric tumor was clearly MR imaged.

Bioorganic & Medicinal Chemistry Letters


Magnetic resonance imaging of tumor with a self-traceable phosphorylcholine polymer.

Polymers are concentration-amplified with respect to the monomeric units. We show here that a phosphorylcholine polymer enriched with (13)C/(15)N at the methyl groups is self-traceable by multiple-resonance (heteronuclear-correlation) NMR in tumor-bearing mice inoculated with the mouse rectal cancer cell line (colon 26). Preliminary measurements indicated that the present polymeric nanoprobe was satisfactorily distinguished from lipids and detectable with far sub-micromolar spectroscopic and far sub-millimolar imaging sensitivities. Detailed ex vivo and in vivo studies for the tumor-bearing mice administered the probe with a mean molecular weight of 63 000 and a mean size of 13 nm, revealed the following: (1) this probe accumulates in the tumor highly selectively (besides renal excretion) and efficiently (up to 30% of the injected dose), (2) the tumor can thus be clearly in vivo imaged, the lowest clearly imageable dose of the probe being 100 mg/kg or 2.0 mg/20-g mouse, and (3) the competition between renal excretion and tumor accumulation is size-controlled; that is, the larger (higher molecular-weight) and smaller (lower molecular-weight) portions of the probe undergo tumor accumulation and renal excretion, respectively. The observed size dependence suggests that the efficient tumor-targeting of the present probe is stimulated primarily by the so-called enhanced permeability and retention (EPR) effect, that is, size-allowed invasion of the probe into the tumor tissue via defective vascular wall. Self-traceable polymers thus open an important area of magnetic resonance imaging (MRI) of tumors and may provide a highly potential tool to visualize various delivery/localization processes using synthetic polymers.

Journal of the American Chemical Society


Simple, Selective, and Practical Synthesis of 2-Substituted 4(3H)-Quinazolinones by Yb(OTf3)-Catalyzed Condensation of 2-Aminobenzamide with Carboxyamides


Novel Biocompatible Cobalt Oxide Nanoparticles for Use in Dual Photoacoustic and Magnetic Resonance Imaging

JSM Biotechnology & Biomedical Engineering


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.


Transporting excess electrons along potential energy gradients provided by 2'-deoxyuridine derivatives in DNA.


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.


Substrate/Product-targeted NMR monitoring of pyrimidine catabolism and its inhibition by a clinical drug.

We report the application of one-dimensional triple-resonance NMR to metabolic analysis and thereon-based evaluation of drug activity. Doubly (13)C/(15)N-labeled uracil ([(15)N1,(13)C6]-uracil) was prepared. Its catabolic (degradative) conversion to [(13)C3,(15)N4]--alanine and inhibition thereof by gimeracil, a clinical co-drug used with the antitumor agent 5-fluorouracil, in mouse liver lysates were monitored specifically using one-dimensional triple-resonance ((1)H-{(13)C-(15)N}) NMR, but not double-resonance ((1)H-{(13)C}) NMR, in a ratiometric manner. The administration of labeled uracil to a mouse resulted in its non-selective distribution in various organs, with efficient catabolism to labeled -alanine exclusively in the liver. The co-administration of gimeracil inhibited the catabolic conversion of uracil in the liver. In marked contrast to in vitro results, however, gimeracil had practically no effect on the level of uracil in the liver. The potentiality of triple-resonance NMR in the analysis of in vivo pharmaceutical activity of drugs targeting particular metabolic reactions is discussed.


Preparation of Functionalized Oligodeoxynucleotides and Photochemical One-electron Oxidation of 5-Methylcytosine in DNA

光機能性修飾核酸の合成とDNA中メチル化シトシンに選択的な光-電子酸化反応

Journal of Synthetic Organic Chemistry, Japan


In Situ Analysis of [8-13C-7-15N]-Double-Labelled Theophylline by a Triple Resonance NMR Technique


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.


Photoinduced Electron Injection into DNA by N-Cyclopropyl-1-aminonaphthalene

Journal of Photochemistry and Photobiology A: Chemistry


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.

Journal of the American Chemical Society


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.


DNA hairpins containing a diaminostilbene derivative as a photoinduced electron donor for probing the effects of single-base mismatches on excess electron transfer in DNA.

To investigate the effects of local structural disorder induced by a single-base mismatch on excess electron transfer (EET) in DNA, a novel hairpin DNA containing diaminostilbene (DAS) as a photoinducible electron donor has been developed. It was clearly demonstrated that EET efficiency depends on the electron injection modes from the electron donors and redox properties of the mismatched bases.


Photoinduced electron transfer reaction in diaminostilbene-tethered DNA duplexes.

DNA duplexes containing diaminostilbene (DAS) as a photoinduced electron donor were synthesized to investigate mechanisms of electron injection into DNA and the succeeding electron transfer in the duplexes. DAS-Capped hairpin DNA showed a high structural stability thereby attains large interaction between DAS and the terminal base pair. DAS-Tethered DNA by a single linker at the end of the duplex was also synthesized and the yields of photoinduced electron transfer through mismatched base pairs were quantified. Both duplexes showed similar electron transfer efficiencies depending on the base pairs, which suggests DAS stacks well on the "pi-way" of the duplex DNA.


Photoelectrochemical identification of 5-methylcytosine modification in DNA: combination of photosensitization and enzymatic cleavage.

We present a novel photoelectrochemical approach for discriminating between cytosine and 5-methylcytosine in DNA when used in combination with enzymatic digestion. A photosensitizer-linked DNA duplex bearing 5-methylcytosine or cytosine at a given restriction site of the strand was immobilized on a gold electrode and digested with enzyme, and the photocurrent response was measured. We observed high photocurrent density that was selective for the methylated duplex.


Efficient photooxidative strand cleavage at 5-methylcytosine in DNA by sensitization with 9,10-anthraquinone-tethered oligonucleotides.

Photoirradiation and subsequent hot piperidine treatment of the duplex consisting of 5-methylcytosine ((m)C)-containing DNA and 9,10-anthraquinone (AQ)-tethered complementary oligodeoxynucleotide led to selective oxidative strand cleavage at the target (m)C site, whereas no strand cleavage was observed for the duplex containing normal cytosine instead of (m)C. Incorporation of an AQ sensitizer into the interior of a strand induced an enhanced one-electron photooxidation, presumably because of a much larger intersystem crossing efficiency, leading to an efficient strand cleavage at the target (m)C site in DNA. Optimization of the photosensitizer could afford more strand cleavage at (m)C in DNA, thereby allowing for the more sensitive detection of the target (m)C on a sequencing gel.


pH effect on the one-electron photooxidation of 5-methylcytosine with naphthoquinone sensitizer.

The pH effect on the one-electron photooxidation of 5-methyl-2'-deoxycytidine (d(m)C) by sensitization with 1,4-naphthoquinone (NQ) was investigated. Upon photoirradiation of d(m)C in the presence of NQ under slightly acidic conditions such as pH 5.0, 5-formyl-2'- deoxycytidine (d(f)C) was formed efficiently, whereas similar NQ-photosensitized oxidation of d(m)C proceeded to lesser extent under neutral or basic conditions. Under slightly acidic conditions, dmC radical cation favors to undergo irreversible deprotonation at the C(5)-methyl group to form a methyl-centered radical, leading to a higher yield of the alkali-labile oxidation products including d(f)C. In contrast, the d(m)C radical cation competitively undergoes deprotonation at the exocyclic N(4)-amino group under neutral or basic conditions, resulting in a decreased yield of NQ-photosensitized oxidation products.


Radiolytic ligation of oligodeoxynucleotides possessing disulfide bond.

Hypoxic X-radiolysis of diluted aqueous solutions was performed to generate hydrated electrons that induced one-electron reduction of oligodeoxynucleotides (ODNs) possessing a disulfide bond. Upon hypoxic irradiation, reductive ligation reaction between two types of ODNs occurred in the presence of a template ODN strand, resulting in the formation of a prescribed ODN in substantial amounts.


The pH effect on the naphthoquinone-photosensitized oxidation of 5-methylcytosine.

The pH effect on the one-electron photooxidation of 5-methyl-2'-deoxycytidine (d(m)C) by sensitization with 2-methyl-1,4-naphthoquinone (NQ) was investigated. Photoirradiation of an aqueous solution containing d(m)C and NQ under slightly acidic conditions of pH 5.0 efficiently produced 5-formyl-2'-deoxycytidine, whereas similar NQ-photosensitized oxidation of d(m)C proceeded to a lesser extent under more acidic or basic conditions. Fluorescence-quenching experiments revealed that the less-efficient photooxidation at pH values below 4.5 is attributed to the decreased rate of one-electron oxidation of d(m)C owing to protonation at the N(3)-position. The NQ-photosensitized oxidation of an N(4)-dimethyl-substituted d(m)C derivative under various pH conditions also suggests that the pH change in the range of 5.0 to 8.0 may be responsible for a reversible deprotonation-protonation equilibrium at the N(4)-exocyclic amino group of the d(m)C radical cation. In accord with the photochemical reactivity of monomeric d(m)C, the 5-methylcytosine residue in oligodeoxynucleotides was oxidized efficiently by photoexcited NQ-tethered oligodeoxynucleotides under slightly acidic conditions to form an alkali-labile 5-formylcytosine residue.


Photoelectrochemical Approach to Detection of Methylation Status of Cytosine Base in DNA


Photocurrent response after enzymatic treatment of DNA duplexes immobilized on gold electrodes: electrochemical discrimination of 5-methylcytosine modification in DNA.

We demonstrate a photoelectrochemical approach to the detection of the methylation status of cytosine bases in DNA. We prepared anthraquinone (AQ) photosensitizer-tethered oligodeoxynucleotide (ODN) duplexes bearing 5-methylcytosine (mC) or the corresponding cytosine (C) at a restriction site of the ODN strand immobilized on gold electrodes, and measured their photocurrent responses arising from hole transport after enzymatic digestion. Treatment with HapII or HhaI of the duplexes bearing normal C led to strand cleavage, and the photosensitizer unit was eliminated from the ODN strand immobilized on the gold electrode, exclusively reducing the photocurrent density. With a similar treatment, the duplexes bearing mC showed higher photocurrent responses arising from hole transport through the duplex. This significant difference in the photocurrent response between mC and normal C residues in DNA on the gold electrodes is potentially applicable to the detection of mC modification in DNA.


Fluorometric identification of 5-methylcytosine modification in DNA: combination of photosensitized oxidation and invasive cleavage.

An efficient fluorometric detection system of DNA methylation has been developed by a combination of a photooxidative DNA cleavage reaction with 2-methyl-1,4-naphthoquinone (NQ) chromophore and an invasive cleavage reaction with human Flap endonuclease-1. Enzymatic treatment of a mixture of photochemically fragmented target oligodeoxynucleotides (ODNs) at 5-methylcytosine mC) and hairpin-like probe oligomer possessing a fluorophore (F) and a quencher (D) resulted in a dramatic enhancement of fluorescence. In contrast, fluorescence emission for the ODN containing cytosine but not mC at the target sequence was extremely weak. In addition, by monitoring the fluorescence change, this system allows for the detection of mC in DNA at subfemtomole amounts. This system would provide a highly sensitive protocol for determining the methylation status in DNA by fluorescence emission.


One-electron photooxidation and site-selective strand cleavage at 5-methylcytosine in DNA by sensitization with 2-methyl-1,4-naphthoquinone-tethered oligonucleotides.

Photosensitized one-electron oxidation was applied to discriminate a specific base site of 5-methylcytosine (mC) generated in DNA possessing a partial sequence of naturally occurring p53 gene, using a sensitizing 2-methyl-1,4-naphthoquinone (NQ) chromophore tethered to an interior of oligodeoxynucleotide (ODN) strands. Photoirradiation and subsequent hot piperidine treatment of the duplex consisting of mC-containing DNA and NQ-tethered complementary ODN led to oxidative strand cleavage selectively at the mC site, when the NQ chromophore was arranged so as to be in close contact with the target mC. The target mC is most likely to be one-electron oxidized into the radical cation intermediate by the sensitization of NQ. The resulting mC radical cation may undergo rapid deprotonation and subsequent addition of molecular oxygen, thereby leading to its degradation followed by strand cleavage at the target mC site. In contrast to mC-containing ODN, ODN analogs with replacement of normal cytosine, thymine, adenine, or guanine at the mC site underwent less amount of such an oxidative strand cleavage at the target base site, presumably due to occurrence of charge transfer and charge recombination processes between the base radical cation and the NQ radical anion. Furthermore, well designed incorporation of the NQ chromophore into an interior of ODN could suppress a competitive strand cleavage at consecutive guanines, which occurred as a result of positive charge transfer. Thus, photosensitization by an NQ-tethered ODN led to one-electron oxidative strand cleavage exclusively at the target mC site, providing a convenient method of discriminating mC in naturally occurring DNA such as human p53 gene as a positive band on a sequencing gel.

Journal of the American Chemical Society


Site-selective strand cleavage at methylated cytosine: regional effect of naphthoquinone chromophore on the one-electron photooxidation of 5-methylcytosine and positive charge transfer in DNA.

Photoirradiation and subsequent hot piperidine treatment of the duplex consisting of 5-methylcytosine ((m)C)-containing DNA and 2-methyl-1,4-naphthoquinone (NQ)-tethered complementary ODN led to oxidative strand cleavage selectively at the (m)C site, when the NQ was arranged so as to be in close contact with the target (m)C. Well designed incorporation of NQ into an interior of ODN could suppress a competitive strand cleavage at consecutive guanines, which occurred as a result of positive charge transfer. In contrast to the ODNs bearing NQ in an interior of the strand, photoirradiation of the duplex with an NQ tethered to a flexible methylene linker at the strand end resulted in not only strong strand cleavage at (m)C but also small amount of strand cleavage at the G doublet. Thus, optimization of the regional position of photosensitizing NQ could provide exclusive strand cleavage at (m)C without unfavorable cleavage at G.


Sensitive discrimination between cytosine and 5-methylcytosine in DNA by a modified invader method.

We designed a detection system of 5-methylcytosine ((m)C) in DNA by a combination of photooxidative DNA cleavage reaction of 2-methyl-1,4-naphthoquinone chromophore with invader technology. Enzymatic treatment of a mixture of photochemically fragmented target oligodeoxynucleotides (ODNs) at (m)C and probe oligomer possessing a fluorophore and a quencher resulted in a dramatic enhancement of fluorescence. In contrast, fluorescence emission for the ODN containing C but not (m)C at the target sequence was extremely weak. We could detect (m)C in sub-femtomol DNA by monitoring the fluorescence change.


Cleavage at 5-methylcytosine in DNA by photosensitized oxidation with 2-methyl-1,4-naphthoquinone tethered oligodeoxynucleotides.

Photosensitized one-electron oxidation of 5-methylcytosine in DNA by 2-methyl-1,4-naphthoquinone, attached to 5'-end of an oligodeoxynucleotide strand, produced 5-formylcytosine and led to selective DNA strand cleavage at the original 5-methylcytosine configuration. This specified photoreaction is useful for positive display of 5-methylcytosine in DNA on a sequencing gel.

Bioorganic & Medicinal Chemistry Letters


Detection of 5-methylcytosine in DNA by photo-functionalized oligodeoxynucleotides possessing 2-methyl-1,4-naphthoquinone chromophore.

We developed a method for detection of 5-methylcytosine in DNA by photosensitized oxidation, using photo-functionalized oligodeoxynucleotides possessing 2-methyl-1,4-naphthoquinone chromophores (NQ) at the interior of the strand. The photooxidation and subsequent hot piperidine treatment of duplex consisting of the NQ-tethered oligodeoxynucleotide and its complementary strand bearing 5-methylcytosine residue led to selective DNA strand cleavage at the 5-methylcytosine residue, whereas no strand cleavage was detected for the control duplex with normal cytosine residue but not 5-methylcytosine.


Remarkable substituent effects on the oxidizing ability of triarylbismuth dichlorides in alcohol oxidation.

Substituent effects on the oxidizing ability of triarylbismuth dichlorides were examined by intermolecular and intramolecular competition experiments on geraniol oxidation in the presence of DBU. It was found that the oxidizing ability of the dichlorides increases with increasing electron-withdrawing ability of the para substituents, and by introduction of a methyl group at the ortho position of the aryl ligands attached to the bismuth. The intermolecular and intramolecular H/D kinetic isotope effects observed for the competitive oxidation of p-bromobenzyl alcohols indicate that the rate-determining step involves C-H bond cleavage. Several primary and secondary alcohols were oxidized efficiently under mild conditions by the combined use of newly developed organobismuth(V) oxidants and DBU.