Synthesis of 1,3-Dibromopyrene as Precursor of 1-, 3-, 6-, and 8-Substituted Long-Axially Symmetric Pyrene Derivatives.

Pyrene derivatives bearing substituents at positions 1, 3, 6, and 8 find numerous applications, as exemplified by their use in lasers, sensors, and bioimaging probes. However, these derivatives typically have point-symmetric or short-axially symmetric structures, whereas long-axially symmetric derivatives remain underexplored because of the difficulty in obtaining their precursor, 1,3-dibromopyrene. To address this problem, we herein synthesized 1,3-dibromopyrene from 1-methoxypyrene in an overall yield (71 % over four steps) considerably exceeding those of existing methods. 1,3-Dibromopyrene was converted into 13OPA, a long-axially symmetric pyrene dye with electron-donor (alkoxy) groups at positions 1 and 3 and electron-acceptor (formyl) groups at positions 6 and 8. 13OPA exhibited photophysical properties distinct from those of its point-symmetric and short-axially symmetric isomers, featuring a broad and strongly redshifted absorption, strong fluorescence with reduced sensitivity to protic solvents, and small dipole moment change upon photoexcitation. The derivatization of 13OPA into a Schiff base and its functionalization via Lewis acid-base pairing were also demonstrated. Thus, our work expands the design scope of pyrene-based molecules, particularly those used as emitters.

Predictors of therapeutic efficacy of 5-aminolevulinic acid-based photodynamic therapy in human prostate cancer.

BACKGROUND: Photodynamic therapy (PDT) is a minimally invasive cancer therapy. However, its therapeutic efficacy for prostate cancer is not yet fully understood. In this study, the predictors of therapeutic efficacy of 5-aminolevulinic acid-based PDT (ALA-PDT) on prostate cancer cells are investigated. MATERIALS AND METHODS: The human prostate cancer cell lines, PC-3, 22Rv1, DU145, and LNCap were used to investigate the effects of ALA-PDT on protoporphyrin IX (PpIX) intracellular accumulation, which was measured by flow cytometry. The cytotoxicity of ALA-PDT was evaluated by MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay. The levels of porphyrin metabolism-related enzyme and transporter mRNA were comprehensively evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). Protein expression was evaluated by Western blot. A xenograft model was created using PC-3 and 22Rv1, and then, pathological analysis was performed to determine the therapeutic effect of ALA-PDT RESULTS: PC-3 and LNCap cells showed high accumulation of PpIX and high sensitivity to ALA-PDT, while 22Rv1 and DU145 showed low accumulation of PpIX and low sensitivity to ALA-PDT. ALA-PDT-induced cytotoxicity correlated negatively with PpIX accumulation. The in vitro assays identified the ATP-binding cassette transporter subfamily G2 (ABCG2) transporter dimer as a predictor of treatment response. In vivo immunohistochemical staining of ABCG2 transporter showed low expression in PC-3 cells and high expression in 22Rv1 cells, and ALA-PDT-induced tumor tissue degeneration was greater in PC-3 cells than in 22Rv1 cells. CONCLUSION: The ABCG2 transporter is a useful predictor of the therapeutic effect of ALA-PDT on human prostate cancer cells.

Sunitinib with photoirradiation-mediated reactive oxygen species generation induces apoptosis of renal cell carcinoma cells.

BACKGROUND: Photodynamic therapy is a clinically approved, minimally invasive,therapeutic procedure used for the treatment of several cancers. In recent years, sunitinib, one of the tyrosine kinase inhibitors, has also attracted attention as a novel photosensitizer. However, there is currently no data available on the combined cytotoxic effects of sunitinib and photoirradiation on renal cell carcinoma including how the treatment induced cellular toxicity. METHODS: In the present study, we used sunitinib as a photosensitizer and evaluated the effects of sunitinib and photodynamic therapy treatment on renal cancer cell lines, including the induction of cell death. RESULTS: Our study showed that treatment with sunitinib and photoirradiation at 8 mW/cm2 for 30 min resulted in the production intracellular reactive oxygen species (ROS), which is indicated by the increase in mRNA expression levels of PAI-1, NF-κß, and Caspase-3. An increase in rate of apoptotic reaction and increase in the expression level of apoptotic marker were also observed when cells undergo treatment with sunitinib and photoirradiation. CONCLUSIONS: Our findings suggest that combining photodynamic therapy with sunitinib represents a minimally invasive therapeutic procedure with cancer selectivity for renal cell carcinoma.

Polarity Mapping of Cells and Embryos by Improved Fluorescent Solvatochromic Pyrene Probe.

Solvatochromic dyes enable sensing and imaging of biomolecular organization in living systems by monitoring local polarity (lipophilicity), but most such dyes suffer from limited brightness, photostability, lack of a convenient spectral range, and limited sensitivity to polarity. Moreover, the presence of an electron acceptor group, typically a carbonyl, in its push-pull structure raises concerns about its potential chemical reactivity within the biological environment. In order to achieve robust bioimaging, we synthesized a push-pull pyrene probe bearing a ketone acceptor group (PK) and compared it with a recently developed aldehyde analogue (PA). We found that in live cells the aldehyde analogue PA transforms slowly (in ∼100 min) into blue-emissive species, assigned to in situ formation of an imine analogue, whereas the PK probe is stable in the presence of primary amines and inside cells. Like the parent PA, the new probe shows strong solvatochromism and an emission color response to lipid order in membranes (ordered vs disordered liquid phases), while its blue-shifted absorption is more optimal for excitation with 400 nm light sources. In live cells, the PK probe enables high-contrast polarity mapping of organelles using two-color ratiometric detection, suggesting that polarity increases in the following order: lipid droplets < plasma membranes < endoplasmic reticulum. In the zebrafish embryo, polarity imaging with the PK probe reveals a new dimension in visualizing the organization of tissues-lipophilicity distribution, where biomembranes, lipid droplets, cells, yolk, extracellular space, and newly formed organs are revealed by specific emission wavelengths of the probe. The newly developed probe and the proposed approach of polarity mapping open new opportunities for bioimaging at the cellular and animal level.

Bright and two-photon active red fluorescent dyes that selectively move back and forth between the mitochondria and nucleus upon changing the mitochondrial membrane potential.

Since mitochondrial dysfunction was discovered to be the underlying cause of several severe diseases, fluorescent probes with excellent optical properties for visualising and monitoring the mitochondrial membrane potential (MMP) (a parameter of mitochondrial vitality) have been in high demand. Herein, we present novel pyrene-based dyes exhibiting remarkably large two-photon absorption around 900 nm and bright red emission around 620 nm (two-photon brightness (Φσ2) = 425-525 GM), with selective localisation to the mitochondria or nucleus in response to changes in the MMP, providing several advantages over traditional MMP-monitoring probes such as Rhodamine 123 (Φσ2 = 64 GM). The intracellular behavior of the new dyes was investigated in detail. The driving forces for the dyes to dissociate from the mitochondria and migrate toward the nucleus upon decreasing the MMP were two key molecular characteristics: the dyes' permeability to mitochondrial membranes and their affinity to nuclear DNA. The results provide significant insights into improving the molecular design of the dyes.

Development of a high-throughput fluoroimmunoassay for Syk kinase and Syk kinase inhibitors.

Syk is a tyrosine kinase which is indispensable in immunoglobulin Fc receptor- and B cell receptor-mediated signal transduction in various immune cells. This pathway is important in the pathophysiology of allergy. In this study we established a quantitative nonradioactive kinase assay to identify inhibitors of Syk. We used recombinant GST-tagged Syk purified from baculovirus-infected insect cells. As a substrate, biotinylated peptide corresponding to the activation loop domain of Syk, whose tyrosine residues are autophosphorylated upon activation, was employed to screen both ATP- and substrate-competitive inhibitors. After the kinase reaction in solution phase, substrate was trapped on a streptavidin-coated plate, followed by detection of the phosphorylated tyrosine with europium-labeled anti-phosphotyrosine antibody. The kinase reaction in solution phase greatly enhanced phosphorylation of substrate compared to that of plate-coated substrate. High signal-to-background ratio and low data scattering were obtained in the optimized high-throughput screening (HTS) format. Further, several kinase inhibitors showed concentration-dependent inhibition of recombinant Syk kinase activity with almost the same efficacy for immunoprecipitated Syk from a human cell line. These data suggest that this assay is useful to screen Syk kinase inhibitors in HTS.