Tracking and recording of intracellular oxygen concentration changes in cell organelles: preparation and function of azide-modified fluorescent probes.

Tracking hypoxic environments and changes in oxygen levels contribute to the elucidation of pathological mechanisms. In this study, we attempted to design molecular probes that can be activated to show fluorescence under hypoxic conditions and that can move to specific cell organelles. Considering that azide groups were selectively reduced to primary amines by reductases under hypoxic conditions, we prepared Hoechst and fluorophore Cy-5 derivatives with azide groups (Hoechst-N3 and Cy-N3) as hypoxia probes. Hoechst-N3 and Cy-N3 showed weak fluorescence, but once activated in the cytosol of hypoxic cells, they exhibited robust fluorescence and then moved to their target organelles, the cell nucleus and mitochondria. In addition, when these probes were administered to the cells in the proper sequence, each probe was activated in response to the intracellular oxygen concentration at that point and exhibited oxygen concentration-dependent fluorescence at the target organelle. By measuring the fluorescence intensity of the cell nucleus and mitochondria, we successfully traced the history of changes in intracellular oxygen levels. Thus, we achieved tracking and recording of oxygen status in the cells.

A detection system using sensing motif-tethered oligodeoxynucleotides for multiplex biomolecular analysis.

We developed a system to detect multiple target biomolecules through sensing motif-tethered oligodeoxynucleotides. DNA-based molecular probes gave the primary amine motif upon reaction with the target biomolecules, glutathione (GSH) and H2O2. After labelling with biotin, the product DNAs were selectively collected to be quantified by qPCR.

Excited state properties of 5-fluoro-4-thiouridine derivative†.

The excited state properties of thionated 5-fluorouridine (2',3',5'-tri-O-acetyl-5-fluoro-4-thiouridine; ta5F4TUrd), synthesized with Lawesson's reagent, have been intensively investigated with nanosecond transient absorption spectroscopy, time-resolved thermal lensing, near-infrared emission, and quantum chemical calculation. The intrinsic triplet lifetime of ta5F4TUrd was determined to be 4.2 ± 0.7 µs in acetonitrile, and the formation quantum yield of the excited triplet state was as large as 0.79 ± 0.01 . The quenching rate constants of the triplet ta5F4TUrd by the dissolved oxygen molecule and by the self-quenching process were found to be nearly equal to the diffusion-controlled rate of acetonitrile. The quantum yield of the singlet molecular oxygen produced through energy transfer between the triplet ta5F4TUrd and the dissolved oxygen, Φ Δ , was successfully determined to be 0.61 ± 0.02 under the oxygen-saturated condition. From the oxygen concentration dependence of the Φ Δ value, the fraction of triplet ta5F4TUrd quenched by dissolved oxygen which gives rise to the 1 O2 * formation, S Δ , was successfully obtained to be 0.78 ± 0.01 , which was the largest among the thionucleobases and the thionucleosides reported so far. This could be due to the lower energy and/or the ππ* character of the triplet state.

Hoechst-Modification on Oligodeoxynucleotides for Efficient Transport to the Cell Nucleus and Gene Regulation.

Various artificial oligodeoxynucleotides (ODNs) that contribute to gene regulation have been developed and their diversity and multifunctionality have been demonstrated. However, few artificial ODNs are actively transported to the cell nucleus, despite the fact that gene regulation also takes place in both the cell nucleus and the cytoplasm. In this study, to prepare ODNs with the ability to accumulate in the cell nucleus, we introduced Hoechst molecules into ODNs that act as carriers of functional molecules to the cell nucleus (Hoe-ODNs). We synthesized Hoe-ODNs and confirmed that they bound strongly to DNA duplexes. When single-stranded Hoe-ODNs or double-stranded ODNs consisting of Hoe-ODNs and its complementary strand were administered into living cells, both ODNs were efficiently accumulated in the cell nucleus. In addition, antisense ODNs, which were tethered with Hoechst unit, were delivered into the cell nucleus and efficiently suppressed the expression of their target RNA. Thus, we constructed a delivery system that enables the transport of ODNs into cell nucleus.

Alkyne-tethered oligodeoxynucleotides that allow simultaneous detection of multiple DNA/RNA targets using Raman spectroscopy.

Detection of multiple DNA/RNA targets is essential for understanding cellular function. Herein, we propose a general method for the simultaneous detection of plural nucleic acids based on surface-enhanced Raman scattering (SERS) using gold nanoparticles bearing functional oligodeoxynucleotides (ODNs) on their surface. Modified ODNs bearing an acetylene tag hybridized with their complementary ODNs on the surface of the gold nanoparticles, inducing a strong SERS signal of the acetylene tag. The addition of the target nucleic acid to the system resulted in a spontaneous displacement of the strand on the particle and dissociation of the alkyne-tagged ODN from the particle, resulting in a dramatic decrease in signal intensity. By using an alkyne tag for each of the multiple target nucleic acids, each target could be detected simultaneously. In addition, we successfully detected cellular microRNA. Different targets showed changes with different wavenumbers in the Raman spectra, allowing for the detection of multiple nucleic acids.

Synthesis of Red-Light-Responsive Pheophorbide-a Tryptamine Conjugated Photosensitizers for Photodynamic Therapy.

Six methyl pheophorbide-a derivatives were prepared by linking a tryptamine side chain at the C-131 , C-152 and C-173 positions of pheophorbide-a. Prepared conjugates were characterized and evaluated for their photocytotoxicity against A549 cells. The conjugate 6 a with strong absorption at 413 nm (Soret band), 663-671 nm (Q bands) and comparable fluorescence quantum yield (0.26) was found to exhibit significant cytotoxicity (659 nM). Molecular integration of pheophorbide-a and tryptamines showed synergistic effects as the most potent conjugate 6 a was identified with enhanced photocytotoxicity when compared to methyl pheophorbide-a. The conjugate 6 a was smoothly taken up by A549 cells and exhibited intracellular localization predominantly to lysosome in the cytoplasm. Upon photoirradiation 6 a generated singlet oxygen to show potent cytotoxicity toward A549 cells.

Synthesis of azafluoranthenes by iridium-catalyzed [2 + 2 + 2] cycloaddition and evaluation of their fluorescence properties.

We report a method for the synthesis of azafluoranthenes under neutral reaction conditions in a highly atom-economical manner by the iridium-catalyzed [2 + 2 + 2] cycloaddition of 1,8-dialkynylnaphthalenes with nitriles. A variety of nitriles react with methyl- or phenyl-substituted 1,8-dialkynylnaphthalenes to give a wide range of azafluoranthenes. Azafluoranthenes bearing an amino group show intense fluorescence at around 500 nm. Comparison of the fluorescence properties of amine-substituted azafluoranthenes with related compounds revealed the importance of the amine moiety for obtaining a high fluorescence quantum yield. The choice of the solvent affected the emission maxima and the fluorescence quantum yield. Azafluoranthenes bearing pyrrolidine exhibited blue-shifted emission bands in a non-polar solvent and gave a fluorescence quantum yield of 0.76 in toluene. A Lippert-Mataga plot and computational studies provide insight into the origin of the fluorescence of azafluoranthenes. Furthermore, cellular experiments using human breast adenocarcinoma cells SK-BR-3 demonstrated the feasibility of using azafluoranthenes as fluorescent probes.

Raman Signal Enhancement by DABCYL-Substitution on DNA Aptamer for Identification of Cellular ATP.

Raman probes have attracted widespread attention for the visualization and identification of biomolecules, because they can be applied to identify detailed chemical structures, detect multiple molecules simultaneously, and visualize cellular functional molecules. However, the biological application of Raman probes is still limited because of their weak signal intensity. Herein, we present a molecular system that shows an enhanced Raman signal using a nonfluorescent dye. We introduced a DABCYL molecule bearing an acetylene unit into thymidine at the 5-position. The resulting modified nucleobase, dDAU, showed a robust signal around 2200 cm-1, which was attributed to the acetylene unit, due to resonance Raman induced by the DABCYL group. We further prepared a DNA aptamer modified with dDAU, and characterized the change of the Raman spectra. Combination with gold nanoparticles, which enhanced the Raman signal by surface-enhanced Raman scattering (SERS), allowed sensitive detection of cellular adenosine derivatives including ATP. Thus, the present system is a promising tool for the detection of biological materials by Raman spectroscopy.

Preparation of a multifunctional photoactivated prodrug on a streptavidin scaffold bearing a DNA aptamer.

Prodrugs that present strong cytotoxicity toward specific cells have been utilized for cell-type selection and purification. In this study, we designed and prepared a multifunctional, photoactivated prodrug based on a streptavidin scaffold. Biotin-labeled DNA aptamer that recognizes the membrane antigen EpCAM, and biotin-labeled photoactivated prodrug bearing the antitumor camptothecin, were prepared. Both molecules were linked to the streptavidin scaffold by simple mixing. The resulting prodrug bound to the EpCAM-overexpressing SK-BR-3 target cells and showed cytotoxic effects upon photoirradiation, corresponding to cytotoxic drug release.

pH-responsive aggregates consisted of oligodeoxynucleotides bearing nitrophenol group that deliver drugs into acidic cells.

A decrease in pH is observed in most solid tumors, thus, the development of drug delivery systems that respond to slightly acidic extracellular pH environment is important in providing tumor-targeted therapies. DNA aggregates can act as useful drug delivery agents, and therefore, we designed an artificial oligodeoxynucleotides (ODNs) that formed an aggregate only under acidic conditions in this study. In other words, we expected that if we could make DNA aggregates that form only in an acidic environment and that encapsulate drugs, it would be possible to transport drugs to tumor tissues selectively. Nitrophenol derivatives, which underwent protonation and deprotonation in response to pH changes, was introduced into ODNs. The ODNs formed aggregates under weakly acidic conditions because of expression of amphiphilicity, which was induced by protonation of nitrophenol unit, and were smoothly taken up into cells. We also found that the aggregates transported anticancer drug, 5FU, into acidified cells to show cytotoxic effects.

Changes of C≡C Triple Bond Vibration that Disclosed Non-Canonical Cytosine Protonation in i-Motif-Forming Oligodeoxynucleotides.

Non-canonical protonation at cytosine (C) in DNA is related to a formation of second order DNA structures such as i-motif, which has a role in gene regulation. Although the detailed structural information is indispensable for comprehension of their functions in cells, the protonation status of C in complicated environments is still elusive. To provide a reporter system of non-canonical protonation, we focused on the molecular vibration that could be monitored using the Raman spectroscopy. We prepared a cytosine derivative (PC) with an acetylene unit as a Raman tag, and found that the Raman signal of acetylene in PC in oligodeoxynucleotides (ODNs) changed due to protonation at the cytosine ring which shortened an acetylene bond. The signal change in i-motif-forming ODNs was also observed in crowded environments with polyethylene glycol, evidencing protonation in i-motif DNA in complicated environments. This system would be one of tracking tools for protonation in DNA structures.

Phosphonated mesoporous silica nanoparticles bearing ruthenium complexes used as molecular probes for tracking oxygen levels in cells and tissues.

Molecular oxygen plays an important role in living organisms. Its concentration and fluctuation in cells or tissues are related to many diseases. Therefore, there is a need for molecular systems that can be used to detect and quantify oxygen levels in vitro and in vivo. In this study, we synthesized phosphonated mesoporous silica nanoparticles bearing ruthenium complexes in their pores (pM-Rus) and evaluated their photophysical and biological properties. The pM-Rus were highly soluble in water and showed robust phosphorescence under hypoxic conditions, while the addition of oxygen suppressed this emission. Cellular experiments revealed that pM-Rus with a size of 100 nm showed efficient cellular uptake to emit phosphorescence in hypoxic cells. In addition, pM-Rus have negligible toxicity to cells due to the blockage of direct contact between ruthenium complexes and intracellular biomolecules and the deactivation of singlet oxygen (1O2) generated by photoexcitation of ruthenium complexes before leaking out of the pores. Animal experiments confirmed that pM-Rus showed robust emission at hypoxic regions in mice. Thus, pM-Rus are promising oxygen probes for living systems.

Excited States of Thio-2'-deoxyuridine Bearing an Extended π-Conjugated System: 3',5'-Di-O-acetyl-5-phenylethynyl-4-thio-2'-deoxyuridine.

A new thio-2'-deoxyuridine with an extended π-conjugated group was successfully synthesized: 3',5'-di-O-acetyl-5-phenylethynyl-4-thio-2'-deoxyuridine. The thio-2'-deoxyuridine derivative has a large red-shifted absorption band in the UVA region and also shows fluorescence, a rare photo-property among thionucleobases/thionucleosides. The triplet-triplet absorption spectrum and the rate constants (the intrinsic decay rate constant of the triplet state, the self-quenching rate constant, and the quenching rate constant of the triplet state by an oxygen molecule) of the thio-2'-deoxyuridine were obtained by transient absorption spectroscopy. The quantum yield of intersystem crossing and the quantum yield of singlet molecular oxygen formation (ϕΔ) under an oxygen atmosphere were also determined. The Ï•Δ value of the new thio-2'-deoxyuridine was found to be substantially higher than all reported values of other thio-2'-deoxyribonucleosides in low oxygen concentrations similar to cancer cell environments. The fluorescence quantum yield depended on the excitation wavelength, revealing certain photochemical reactions in the higher excited singlet states. However, when excited into the higher excited state with non-resonant two-photon absorption, the Ï•Δ of the thio-2'-deoxyuridine derivative was found to remain sufficiently large. These findings should be very useful for the development of thio-2'-deoxyribonucleoside-based pharmaceuticals as DNA-specific photosensitizers for photochemotherapy.

Modulation of cell membrane functionalization with aggregates of oligodeoxynucleotides containing alkyl chain-modified uridines.

In this study, we prepared oligodeoxynucleotides (ODNs) containing the uridine base modified by an alkyl chain at the 5-position (AU) and characterized their aggregate formation, localization, and functions in cells. These experiments revealed that aggregates of these ODNs were readily transported into cells, but their localization was dependent upon the number of hydrophobic units. ODNs with one modified AU were transported in the cytosol, while ODNs with multiple AU modifications resulted in their accumulation at the cell membrane. We also examined the ability of the AU-modified ODNs to capture small molecules at the cell membrane and their cellular uptake. We positioned a thioflavin-T (ThT)-binding aptamer on the cell membrane by means of hybridization with ODNs with three AUs at the strand end. Treatment with ThT resulted in its efficient uptake into cells, due to the capture of the ThT by the aptamers on the cell membrane. Thus, we demonstrated the functionalization of cell membranes with modified ODNs and the efficient delivery of small molecules into the cells.

Monitoring intracellular metal ion complexation with an acetylene-tagged ligand by Raman spectroscopy.

We propose to monitor molecular vibrations to identify metal ion-ligand complexation by means of Raman spectroscopy, which has been applied to track vibrational modes of molecules and to obtain a structural fingerprint. We prepared ligand molecules for Zn2+ ion complexation with a dipycolylaminoethyl aniline (DPEA) skeleton and phenylacetylene unit as the Raman tag which showed a typical band around 2200 cm-1. Among the labeled ligands synthesized in this study, A-DPEA showed a strong band attributed to the acetylene unit at 2212 cm-1, while the addition of Zn2+ ion resulted in a band shift to 2220 cm-1 due to complex formation. The addition of other metal ions and titration experiments showed that A-DPEA bound with Zn2+ selectively with a dissociation constant (K d) that was estimated to be 0.22 µM. We also conducted cellular experiments and found that complexation between A-DPEA and Zn2+ also occurred in cells, with a shift in the Raman signal of the ligand from 2212 to 2215 cm-1. Thus, complex formation of the metal ion was identified by monitoring the Raman band shift.

Hybridizing Oligonucleotides with Hydrophobic Peptide Nucleic Acids Assists Their Cellular Uptake through Aggregate Formation.

We applied hybridization between hydrophobic peptide nucleic acids (PNAs) and oligodeoxynucleotides (ODNs) to achieve their cellular uptake without any need for transfection reagents. We employed a pyrenyl unit as a hydrophobic functional group and introduced it at the terminus of the PNA strand. The pyrene-tethered PNA (PyPNA) strongly bound with its complementary ODNs to generate amphiphiles; the resulting hybrids formed aggregates that showed efficient cellular uptake and high biological stability. Aggregates containing a functional DNA aptamer that bound to the PyPNA penetrated the cell membrane smoothly, with the aptamer exerting its original function in living cells. Thus, PyPNA efficiently assisted the additive-free cellular uptake of ODNs.

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.

Aggregate Formation of BODIPY-Tethered Oligonucleotides That Led to Efficient Intracellular Penetration and Gene Regulation.

Exogenous nucleic acids showed low efficiency regarding cellular uptake and low stability in biological conditions; therefore, a number of techniques have been developed to improve their basic properties. One of the best solutions is the application of nanosized particles consisting of oligonucleotides that penetrate the cell membrane without any additives and exhibit high stability in cells. In this report, we employed a simple approach to address the basic properties of nanoparticles of oligonucleotides in biological systems. We prepared BODIPY-labeled oligonucleotides that carried an exclusive modification at the strand end. BODIPY shows high hydrophobicity and fluorescent emission; therefore, the oligonucleotides formed nanosized aggregates in aqueous solution and their behaviors in cells or tissues were easily tracked. Detailed experiments revealed that aggregate formation was indispensable for the high cellular uptake of the oligonucleotides via scavenger-receptor-mediated endocytosis. In addition, the aggregates provided an efficient gene regulation in living cells and tumor tissues transplanted into mice.

Biological reduction of nitroimidazole-functionalized gold nanorods for photoacoustic imaging of tumor hypoxia.

Tumor-selective accumulation of gold nanorods (GNR) has been demonstrated for visualization of tumor hypoxia by photoacoustic imaging. We prepared GNRs with hypoxia-targeting nitroimidazole units (G-NI) on their surface. Biological experiments revealed that G-NI produced a strong photoacoustic signal in hypoxic tumor cells and tissues.

Confinement of Singlet Oxygen Generated from Ruthenium Complex-Based Oxygen Sensor in the Pores of Mesoporous Silica Nanoparticles.

We synthesized mesoporous silica nanoparticles bearing ruthenium complexes in their pores (MSN-Ru) and characterized their photochemical properties. The ruthenium complexes that were immobilized in the pores showed oxygen-dependent phosphorescence, similar to the complexes that were not tethered to nanoparticles. Cellular imaging and in vivo experiments revealed that hypoxic cells and tissues could be visualized by monitoring the phosphorescence of MSN-Ru. Our most important finding was that the toxic effect of singlet oxygen (1O2), which was generated by excitation of the complexes, was effectively suppressed by the deactivation before leaking out from the pores. In addition, we observed a negligible toxic effect of the ruthenium complexes themselves due to the blockage of their direct interaction with intracellular biomolecules. Thus, MSN-Ru is a promising molecular probe of oxygen levels in living cells and tissues.