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.

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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.

Hypoxia is an important feature of many diseases such as malignant solid tumors, inflammatory diseases and cardiac ischemia. We herein focused on the development of a novel hypoxia-sensitive fluorescent probe, IQ-R, consisting of an indolequinone unit and a rhodol fluorophore. IQ-R has good solubility in water and longer wavelength for absorption and emission, which are favorable for cellular bioimaging. While the fluorescence of rhodol in the IQ-R conjugate was quenched by the function of intramolecular indolequinone unit, it was restored under hypoxic conditions through the enzymatic one-electron reduction of IQ-R by NADPH:cytochrome P450 reductase to release the nonconjugated free rhodol. When administered to A549 cells, IQ-R was activated and reduced by endogenous reductase preferentially under hypoxic conditions, thereby visualizing hypoxic cancer cells by robust fluorescence.

A characteristic feature of the reactivity of indolequinone derivatives, substituents of which can be removed by one-electron reduction under hypoxic conditions, was applied to the development of a new class of fluorescent probes for disease-relevant hypoxia. A reducing indolequinone parent molecule conjugated with fluorescent coumarin chromophores could suppress efficiently the fluorescence emission of the coumarin moieties by an intramolecular electron-transfer quenching mechanism and a conventional internal-filter effect. Under hypoxic conditions, however, the conjugate, denoted IQ-Cou, underwent a one-electron reduction triggered by X irradiation or the action of a reduction enzyme to release a fluorescent coumarin chromophore, whereupon an intense fluorescence emission with a maximum intensity at 420 nm was observed. The one-electron reduction of IQ-Cou was suppressed by molecular oxygen under aerobic conditions. IQ-Cou also showed intense fluorescence in a hypoxia-selective manner upon incubation with a cell lysate of the human fibrosarcoma cell line HT-1080. The IQ-Cou conjugate has several unique properties that are favorable for a fluorescent probe of hypoxia-specific imaging.