Design and Synthesis of a Library of BODIPY-Based Environmental Polarity Sensors Utilizing Photoinduced Electron-Transfer-Controlled Fluorescence ON/OFF Switching

We systematically examined the mechanism of the solvent polarity dependence of the fluorescence ON/OFF threshold of the BODIPY (boron dipyrromethene) fluorophore and the role of photoinduced electron transfer (PeT). In a series of BODIPY derivatives with variously substituted benzene moieties at the 8-position, the oxidation potential of the benzene moiety became more positive and the reduction potential of the BODIPY fluorophore became more negative as the solvent polarity was decreased; consequently, the free energy change of PeT from the benzene moiety becomes larger in a more nonpolar environment. Utilizing this finding, we designed and synthesized a library of probes in which the threshold of fluorescence ON/OFF switching corresponds to different levels of solvent polarity. These environment-sensitive probes were used to examine bovine serum albumin (BSA) and living cells. The polarity at the surface of albumin was concluded to be similar to that of acetone, while the polarity of the internal membranes of HeLa cells was similar to that of dichloromethane

Mechanism-Based Molecular Design of Highly Selective Fluorescence Probes for Nitrative Stress

Nitrative stress is implicated in various pathogenic processes, including neurodegenerative disorders, but there is no practical fluorescence probe which can monitor the generation of nitrative stress with high selectivity. To design a suitable fluorescence probe, we have first focused on the fluorescence quenching mechanism of the nitro group, which has been believed to be a unique quencher of fluorescent dyes. We found that nitro group-based fluorescence quenching could be explained in terms of an electron transfer process, from the excited fluorophore to the electron-deficient aromatic nitro moiety. By utilizing this result, we succeeded in developing novel fluorogenic probes, NiSPYs, which can selectively monitor the generation of nitrative stress based on aromatic nitration. NiSPYs showed strong fluorescence enhancement upon the reaction with nitrating agents, including peroxynitrite, but showed little or no fluorescence augmentation in the presence of other reactive oxygen species. NiSPYs should be potentially useful as tools to study the role of nitrative stress in various biological applications

Development of a Highly Specific Rhodamine-Based Fluorescence Probe for Hypochlorous Acid and Its Application to Real-Time Imaging of Phagocytosis

The tetramethylrhodamine (TMR) fluorophore is a useful platform for fluorescence probes, being applicable, for example, to biological investigations utilizing fluorescence microscopy, owing to its excellent photochemical properties in aqueous media. We have developed new TMR derivatives that show different dependences of their behavior upon the environment. Among them, HMTMR showed unique characteristics, and its putative spirocyclic structure was confirmed by X-ray crystallography. Utilizing this discovery, we have established a strategy to modulate the fluorescence of TMR by regulating the spirocyclization, and we have obtained a new fluorescence probe that can detect hypochlorous acid specifically. This probe, HySOx, can work in 99.9% aqueous solution at pH 7.4 and was confirmed to be able to detect hypochlorous acid being generated inside phagosomes in real time. HySOx is tolerant to autoxidation and photobleaching under bioimaging conditions. Regulation of the spirocyclization of rhodamines, as we describe here, provides a new approach to the rational development of novel fluorescence probes

Development of a Highly Specific Rhodamine-Based Fluorescence Probe for Hypochlorous Acid and Its Application to Real-Time Imaging of Phagocytosis

The tetramethylrhodamine (TMR) fluorophore is a useful platform for fluorescence probes, being applicable, for example, to biological investigations utilizing fluorescence microscopy, owing to its excellent photochemical properties in aqueous media. We have developed new TMR derivatives that show different dependences of their behavior upon the environment. Among them, HMTMR showed unique characteristics, and its putative spirocyclic structure was confirmed by X-ray crystallography. Utilizing this discovery, we have established a strategy to modulate the fluorescence of TMR by regulating the spirocyclization, and we have obtained a new fluorescence probe that can detect hypochlorous acid specifically. This probe, HySOx, can work in 99.9% aqueous solution at pH 7.4 and was confirmed to be able to detect hypochlorous acid being generated inside phagosomes in real time. HySOx is tolerant to autoxidation and photobleaching under bioimaging conditions. Regulation of the spirocyclization of rhodamines, as we describe here, provides a new approach to the rational development of novel fluorescence probes

Development of a Ratiometric Fluorescent Zinc Ion Probe in Near-Infrared Region, Based on Tricarbocyanine Chromophore

Novel ratiometric fluorescent probes for Zn2+ in the near-infrared region, based on a tricarbocyanine chromophore, have been designed, synthesized, and evaluated. Upon addition of Zn2+, a 44 nm red shift of the absorption maximum was observed, which indicates that this probe could work as a ratiometric probe for Zn2+. This change is due to the difference in the electron-donating ability of the amine substituent before and after reaction with Zn2+. This fluorescence modulation of amine-substituted tricarbocyanines should be applicable to dual-wavelength measurement of various biomolecules or enzyme activities

A Thiol-Reactive Fluorescence Probe Based on Donor-Excited Photoinduced Electron Transfer: Key Role of Ortho Substitution

We designed and synthesized a novel thiol-reactive fluorescence probe based on the BODIPY fluorophore. The fluorescence of this probe is strongly quenched by donor-excited photoinduced electron transfer (d-PeT) from BODIPY to maleimide, but after reaction with thiol, the fluorescence of BODIPY is restored, affording a 350-fold intensity increase

Creation of Superior Carboxyfluorescein Dyes by Blocking Donor-Excited Photoinduced Electron Transfer

Carboxyfluoresceins are widely utilized as fluorescence labeling reagents, but we recently found that their emission intensity is markedly decreased after esterification. On the basis of our hypothesis that the fluorescence decrease is due to a donor-excited photoinduced electron transfer (d-PeT) process, we have developed novel carboxyfluorescein derivatives in which the d-PeT process is hampered, and the emission intensity is not decreased upon esterification. These novel dye derivatives display high quantum yields and are expected to be useful as labeling agents

Design and Synthesis of Fluorescent Probes for Selective Detection of Highly Reactive Oxygen Species in Mitochondria of Living Cells

We designed and synthesized fluorescent probes for the sensitive and selective detection of highly reactive oxygen species (hROS) in mitochondria of living cells and confirmed their usefulness in vitro. MitoAR and MitoHR are highly selective for hROS (•OH, ONOO-, OCl-) over other ROS. They have exellent properties, including tolerance to autoxidation and photobleaching by laser irradiation during fluorescence microscopy and should be useful tools for biological and pathological applications

Selective Zinc Sensor Molecules with Various Affinities for Zn²⁺, Revealing Dynamics and Regional Distribution of Synaptically Released Zn²⁺ in Hippocampal Slices

We have developed a series of fluorescent Zn2+ sensor molecules with distinct affinities for Zn2+, because biological Zn2+ concentrations vary over a wide range from sub-nanomolar to millimolar. The new sensors have Kd values in the range of 10-8−10-4 M, compared with 2.7 nM for ZnAF-2. They do not fluoresce in the presence of other biologically important metal ions such as calcium or magnesium, and they can detect Zn2+ within 100 ms. In cultured cells, the fluorescence intensity of ZnAF-2 was saturated at low Zn2+ concentration, while that of ZnAF-3 (Kd = 0.79 μM) was not saturated even at relatively high Zn2+ concentrations. In hippocampal slices, we measured synaptic release of Zn2+ in response to high-potassium-induced depolarization. ZnAF-2 showed similar levels of fluorescence increase in dentate gyrus (DG), CA3 and CA1, which were indistinguishable. However, ZnAF-3 showed a fluorescence increase only in DG. Thus, by using a combination of sensor molecules, it was demonstrated for the first time that a higher Zn2+ concentration is released in DG than in CA3 or CA1 and that we can easily visualize Zn2+ concentration over a wide range. We believe that the use of various combinations of ZnAF family members will offer unprecedented versatility for fluorescence-microscopic imaging of Zn2+ in biological applications

Development of a Zinc Ion-Selective Luminescent Lanthanide Chemosensor for Biological Applications

Detection of chelatable zinc (Zn2+) in biological studies has attracted much attention recently, because chelatable Zn2+ plays important roles in many biological systems. Lanthanide complexes (Eu3+, Tb3+, etc.) have excellent spectroscopic properties for biological applications, such as long luminescence lifetimes of the order of milliseconds, a large Stoke's shift of >200 nm, and high water solubility. Herein, we present the design and synthesis of a novel lanthanide sensor molecule, [Eu-7], for detecting Zn2+. This europium (Eu3+) complex employs a quinolyl ligand as both a chromophore and an acceptor for Zn2+. Upon addition of Zn2+ to a solution of [Eu-7], the luminescence of Eu3+ is strongly enhanced, with high selectivity for Zn2+ over other biologically relevant metal cations. One of the important advantages of [Eu-7] is that this complex can be excited with longer excitation wavelengths (around 340 nm) as compared with previously reported Zn2+-sensitive luminescent lamthanide sensors, whose excitation wavelength is at too high an energy level for biological applications. The usefulness of [Eu-7] for monitoring Zn2+ changes in living HeLa cells was confirmed. This novel Zn2+-selective luminescent lanthanide chemosensor [Eu-7] should be an excellent lead compound for the development of a range of novel luminescent lanthanide chemosensors for biological applications