Bismuth-rhodamine (BiR) was developed as a new photosensitizer scaffold, and its photophysical properties were evaluated. BiR showed significant red-shifted absorption and emission compared with other xanthene-based photosensitizers, together with an efficient quantum yield for the generation of 1O2. BiR showed efficient cell-permeability as well as photo-triggered generation of 1O2 in cells.

•IRP2 expression was high in iron-rich regions of ovarian endometrial cysts.•Hypoxia prevented the degradation of IRP2 despite an abundance of iron.•Increase of IRP2 expression provides aberrant uptake of iron.Ovarian endometrial cysts cause some kinds of ovarian cancer, and iron is considered as one factor of carcinogenesis. In contrast, hypoxia is associated with progression, angiogenesis, metastasis, and resistance to therapy in cancer. We investigated hypoxia-induced perturbation of iron homeostasis in terms of labile iron, iron deposition, and iron regulatory protein (IRP) in ovarian endometrial cysts. Iron deposition, expression of IRPs, and a protein marker of hypoxia in human ovarian endometrial cysts were analyzed histologically. The concentration of free iron and the pO2 level of the cyst fluid of human ovarian cysts (n = 9) were measured. The expression of IRP2 under hypoxia was investigated in vitro by using Ishikawa cells as a model of endometrial cells. Iron deposition and the expression of IRP2 and Carbonic anhydrase 9 (CA9) were strong in endometrial stromal cells in the human ovarian endometrial cysts. The average concentration of free iron in the cyst fluid was 8.1 ± 2.9 mg/L, and the pO2 was 22.4 ± 5.2 mmHg. A cell-based study using Ishikawa cells revealed that IRP2 expression was decreased by an overload of Fe(II) under normoxia but remained unchanged under hypoxia even in the presence of excess Fe(II). An increase in the expression of IRP2 caused upregulation of intracellular iron as a result of the response to iron deficiency, whereas the protein was degraded under iron-rich conditions. We found that iron-rich regions existed in ovarian endometrial cysts concomitantly with the high level of IRP2 expression, which should generally be decomposed upon an overload of iron. We revealed that an insufficient level of oxygen in the cysts is the main factor for the unusual stabilization of IRP2 against iron-mediated degradation, which provides aberrant uptake of iron in ovarian endometrial stromal cells and can potentially lead to carcinogenesis.

Iron (Fe) species play a number of biologically and pathologically important roles. In particular, iron is a key element in oxygen sensing in living tissue where its metabolism is intimately linked with oxygen metabolism. Regulation of redox balance of labile iron species to prevent the generation of iron-catalyzed reactive oxygen species (ROS) is critical to survival. However, studies on the redox homeostasis of iron species are challenging because of a lack of a redox-state-specific detection method for iron, in particular, labile Fe2+. In this study, a universal fluorogenic switching system is established, which is responsive to Fe2+ ion based on a unique N-oxide chemistry in which dialkylarylamine N-oxide is selectively deoxygenized by Fe2+ to generate various fluorescent probes of Fe2+–CoNox-1 (blue), FluNox-1 (green), and SiRhoNox-1 (red). All the probes exhibited fluorescence enhancement against Fe2+ with high selectivity both in cuvette and in living cells. Among the probes, SiRhoNox-1 showed an excellent fluorescence response with respect to both reaction rate and off/on signal contrast. Imaging studies were performed showing the intracellular redox equilibrium shift towards labile iron in response to reduced oxygen tension in living cells and 3D tumor spheroids using SiRhoNox-1, and it was found that the hypoxia induction of labile Fe2+ is independent of iron uptake, hypoxia-induced signaling, and hypoxia-activated enzymes. The present studies demonstrate the feasibility of developing sensitive and specific fluorescent probes for Fe2+ with refined photophysical characteristics that enable their broad application in the study of iron in various physiological and pathological conditions.

Iron is an essential metal nutrient that plays physiologically and pathologically important roles in biological systems. However, studies on the trafficking, storage, and functions of iron itself in living samples have remained challenging due to the lack of efficient methods for monitoring labile intracellular iron. Herein, we report a new class of Fe2+-selective fluorescent probes based on the spirocyclization of hydroxymethylrhodamine and hydroxymethylrhodol scaffolds controlled by using our recently established N-oxide chemistry as a Fe2+-selective switch of fluorescence response. By suppressing the background signal, the spirocyclization strategy improved the turn-on rate dramatically, and reducing the size of the substituents of the N-oxide group enhanced the reaction rate against Fe2+, compared with the first generation N-oxide based Fe2+ probe, RhoNox-1. These new probes showed significant enhancements in the fluorescence signal against not only the exogenously loaded Fe2+ but also the endogenous Fe2+ levels. Furthermore, we succeeded in monitoring the accumulation of labile iron in the lysosome induced by transferrin-mediated endocytosis with a turn-on fluorescence response.

Although labile iron plays critical roles in diverse biological processes in living cells, the physiological and pathophysiological functions of iron have not been sufficiently explored, partially due to a lack of methods for visualizing intracellular labile iron. In this edge article, we present a novel turn-on fluorescent probe (RhoNox-1) for the selective detection of Fe2+ based on N-oxide chemistry. Spectroscopic studies combined with DFT calculations and electrochemical studies revealed that fluorescence quenching of RhoNox-1 occurred in physiological conditions, which was attributed to non-radiative deactivation of the excited state of tertiary amine N-oxide substituted xanthene involving a twisted internal charge transfer (TICT) process and partially due to photo-induced electron transfer (PET) from the N-oxide group. RhoNox-1 showed significant enhancement of the fluorescence signal in Fe2+-loaded cells via selective Fe2+-mediated deoxygenation of the N-oxide group and also successfully detected basal and endogenous labile Fe2+ in living cells.

To elucidate the mechanisms of direct transmembrane penetration of pepducins, which are artificial lipopeptide G protein-coupled receptor (GPCR) modulators, we developed two types of FRET-based probes, Pep13-FL-SS-Dab (13) targeting the inner leaflet of the lipid bilayer and Pep13-Dab-SS-FL (14) targeting the cytosol, respectively. They are composed of a pepducin moiety and a fluorescent switch component consisting of 5(6)-carboxyfluorescein (FAM) as a fluorophore and dabcyl as a quencher connected through disulfide bond linkage. When they are internalized into the cytosol, intracellular glutathione can cleave the disulfide bond to release the quencher, which results in a turn-on fluorescence signal. Using these probes, we performed live cell imaging of transbilayer movements of pepducins on MCF-7 cells for the first time. The results suggested that the lipid moiety of the probes facilitated pepducin flipping across and tethering to the membrane. The present study raises the possibility of applying the probe architecture for direct intracellular drug delivery.

We developed a novel near-infrared (NIR) fluorescent probe, GPU-167, for in vivo imaging of tumor hypoxia. GPU-167 comprises a tricarbocyanine dye as an NIR fluorophore and two 2-nitroimidazole moieties as exogenous hypoxia markers that undergo bioreductive activation and then selective entrapment in hypoxic cells. After treatment with GPU-167, tumor cells contained significantly higher levels of fluorescence in hypoxia than in normoxia. In vivo fluorescence imaging specifically detected GPU-167 in tumors 24 h after administration. Ex vivo analysis revealed that fluorescence showed a strong correlation with hypoxia inducible factor (HIF)-1 active hypoxic regions. These data suggest that GPU-167 is a promising in vivo optical imaging probe for tumor hypoxia.

Boron-containing agents play a key role in successful boron neutron capture therapy (BNCT). Icosahedral boron cluster-Arg-Gly-Asp (RGD) peptide conjugates were designed, synthesized, and evaluated for the biodistribution to develop tumor-selective boron carriers. Integrin αvβ3 is an attractive target for anti-tumor drug delivery because of its specific expression in proliferating endothelial and tumor cells of various origins. We, therefore, selected a c(RGDfK) moiety recognizing αvβ3 as an active tumor-targeting device to conjugate with icosahedral boron-10 clusters, disodium mercaptododecaborate (BSH) or o-carborane as a thermal neutron-sensitizing unit. Preparation of o-carborane derivatives involved microwave irradiation, and resulted in high yields in a short time. An in vitro cell adhesion assay on αvβ3-positive U87MG and SCCVII cells demonstrated the high binding affinity of conjugates to integrin αvβ3 (IC50 = 0.19–2.66 μM). Biodistribution experiments using SCCVII-bearing mice indicated that GPU-201 showed comparable tumor uptake and a significantly longer retention in tumors compared with BSH. These results suggest that GPU-201 is a promising candidate for use in BNCT.

The tumor microenvironment is characterized by hypoxia, low-nutrient levels, and acidosis. A natural product chemistry-based approach was used to discover small molecules that modulate adaptive responses to a hypoxic microenvironment through the hypoxia-inducible factor (HIF)-1 signaling pathways. Five compounds, such as baccharin (3), beturetol (4), kaempferide (5), isosakuranetin (6), and drupanin (9), that modulate HIF-1-dependent luciferase activity were identified from Brazilian green propolis using reporter assay. Compounds 3, 9 and 5 reduced HIF-1-dependent luciferase activity. The cinnamic acid derivatives 3 and 9 significantly inhibited expression of the HIF-1α protein and HIF-1 downstream target genes such as glucose transporter 1, hexokinase 2, and vascular endothelial growth factor A. They also exhibited significant anti-angiogenic effects in the chick chorioallantoic membrane (CAM) assay at doses of 300 ng/CAM. On the other hand, flavonoids 4 and 6 induced HIF-1-dependent luciferase activity and expression of HIF-1 target genes under hypoxia. The contents (g/100 g extract) of the HIF-1-modulating compounds in whole propolis ethanol extracts were also determined based on liquid chromatography–electrospray ionization mass spectrometry as 1.6 (3), 14.2 (4), 4.0 (5), 0.7 (6), and 0.7 (9), respectively. These small molecules screened from Brazilian green propolis may be useful as lead compounds for the development of novel therapies against ischemic cardiovascular disease and cancer based on their ability to induce or inhibit HIF-1 activity, respectively.

A strategy involving palladium-catalysed aromatic C–H functionalisation/intramolecular alkenylation provides a convenient and direct synthesis of 3-alkylideneoxindoles. In the presence of 5 mol% of PdCl2MeCN2 and AgOCOCF3, a wide variety of N-cinnamoylanilines gave 3-alkylideneoxindoles in moderate to good yield.

An efficient method for the synthesis of functionalized benzoxazoles is described that involves a copper(II)-catalyzed regioselective C−H functionalization/C−O bond formation protocol. The use of dichlorobenzene as a solvent at 160 °C allows the use of air as the terminal oxidant in the catalytic synthesis of benzoxazoles in a process that has high functional group tolerance. The presence of a directing group at the meta position markedly improves the reaction efficacy and a variety of 7-substituted benzoxazoles are selectively produced under mild reaction conditions. The mechanism of the reaction is also discussed in this report.

Although labile iron plays critical roles in diverse biological processes in living cells, the physiological and pathophysiological functions of iron have not been sufficiently explored, partially due to a lack of methods for visualizing intracellular labile iron. In this edge article, we present a novel turn-on fluorescent probe (RhoNox-1) for the selective detection of Fe2+ based on N-oxide chemistry. Spectroscopic studies combined with DFT calculations and electrochemical studies revealed that fluorescence quenching of RhoNox-1 occurred in physiological conditions, which was attributed to non-radiative deactivation of the excited state of tertiary amine N-oxide substituted xanthene involving a twisted internal charge transfer (TICT) process and partially due to photo-induced electron transfer (PET) from the N-oxide group. RhoNox-1 showed significant enhancement of the fluorescence signal in Fe2+-loaded cells via selective Fe2+-mediated deoxygenation of the N-oxide group and also successfully detected basal and endogenous labile Fe2+ in living cells.

Iron (Fe) species play a number of biologically and pathologically important roles. In particular, iron is a key element in oxygen sensing in living tissue where its metabolism is intimately linked with oxygen metabolism. Regulation of redox balance of labile iron species to prevent the generation of iron-catalyzed reactive oxygen species (ROS) is critical to survival. However, studies on the redox homeostasis of iron species are challenging because of a lack of a redox-state-specific detection method for iron, in particular, labile Fe2+. In this study, a universal fluorogenic switching system is established, which is responsive to Fe2+ ion based on a unique N-oxide chemistry in which dialkylarylamine N-oxide is selectively deoxygenized by Fe2+ to generate various fluorescent probes of Fe2+–CoNox-1 (blue), FluNox-1 (green), and SiRhoNox-1 (red). All the probes exhibited fluorescence enhancement against Fe2+ with high selectivity both in cuvette and in living cells. Among the probes, SiRhoNox-1 showed an excellent fluorescence response with respect to both reaction rate and off/on signal contrast. Imaging studies were performed showing the intracellular redox equilibrium shift towards labile iron in response to reduced oxygen tension in living cells and 3D tumor spheroids using SiRhoNox-1, and it was found that the hypoxia induction of labile Fe2+ is independent of iron uptake, hypoxia-induced signaling, and hypoxia-activated enzymes. The present studies demonstrate the feasibility of developing sensitive and specific fluorescent probes for Fe2+ with refined photophysical characteristics that enable their broad application in the study of iron in various physiological and pathological conditions.

A strategy involving palladium-catalysed aromatic C–H functionalisation/intramolecular alkenylation provides a convenient and direct synthesis of 3-alkylideneoxindoles. In the presence of 5 mol% of PdCl2MeCN2 and AgOCOCF3, a wide variety of N-cinnamoylanilines gave 3-alkylideneoxindoles in moderate to good yield.

To elucidate the mechanisms of direct transmembrane penetration of pepducins, which are artificial lipopeptide G protein-coupled receptor (GPCR) modulators, we developed two types of FRET-based probes, Pep13-FL-SS-Dab (13) targeting the inner leaflet of the lipid bilayer and Pep13-Dab-SS-FL (14) targeting the cytosol, respectively. They are composed of a pepducin moiety and a fluorescent switch component consisting of 5(6)-carboxyfluorescein (FAM) as a fluorophore and dabcyl as a quencher connected through disulfide bond linkage. When they are internalized into the cytosol, intracellular glutathione can cleave the disulfide bond to release the quencher, which results in a turn-on fluorescence signal. Using these probes, we performed live cell imaging of transbilayer movements of pepducins on MCF-7 cells for the first time. The results suggested that the lipid moiety of the probes facilitated pepducin flipping across and tethering to the membrane. The present study raises the possibility of applying the probe architecture for direct intracellular drug delivery.

Iron is an essential metal nutrient that plays physiologically and pathologically important roles in biological systems. However, studies on the trafficking, storage, and functions of iron itself in living samples have remained challenging due to the lack of efficient methods for monitoring labile intracellular iron. Herein, we report a new class of Fe2+-selective fluorescent probes based on the spirocyclization of hydroxymethylrhodamine and hydroxymethylrhodol scaffolds controlled by using our recently established N-oxide chemistry as a Fe2+-selective switch of fluorescence response. By suppressing the background signal, the spirocyclization strategy improved the turn-on rate dramatically, and reducing the size of the substituents of the N-oxide group enhanced the reaction rate against Fe2+, compared with the first generation N-oxide based Fe2+ probe, RhoNox-1. These new probes showed significant enhancements in the fluorescence signal against not only the exogenously loaded Fe2+ but also the endogenous Fe2+ levels. Furthermore, we succeeded in monitoring the accumulation of labile iron in the lysosome induced by transferrin-mediated endocytosis with a turn-on fluorescence response.