Toshitada Yoshihara

Phosphorescent light-emitting iridium complexes serve as a hypoxia-sensing probe for tumor imaging in living animals.

Iridium complex is a promising organic light-emitting diode material for next generation video displays that emits phosphorescence quenched by oxygen. We used this oxygen-quenching feature for imaging tumor hypoxia. Red light-emitting Ir(btp)(2)(acac) (BTP) presented hypoxia-dependent light emission in culture cell lines, whose intensity was in parallel with hypoxia-inducible factor-1alpha images. BTP was further applied to imaging five nude mouse transplanted with tumors. All tumors presented a bright BTP-emitting image even 5 minutes after injection. The minimal image recognition size was approximately 2 mm in diameter. By morphologic examination and phosphorescence lifetime measurement, BTP appeared to localize to the tumor cells. Because BTP is easily modifiable, we synthesized BTP analogues with a longer excitation/emission wavelength. One of them, BTPHSA, depicted clear imaging from tumors transplanted 6 to 7 mm deep from the skin surface. We suggest that iridium complex materials have a vast potential for imaging hypoxic lesions such as tumor tissues.

A spontaneously blinking fluorophore based on intramolecular spirocyclization for live-cell super-resolution imaging

Single-molecule localization microscopy is used to construct super-resolution images, but generally requires prior intense laser irradiation and in some cases additives, such as thiols, to induce on-off switching of fluorophores. These requirements limit the potential applications of this methodology. Here, we report a first-in-class spontaneously blinking fluorophore based on an intramolecular spirocyclization reaction. Optimization of the intramolecular nucleophile and rhodamine-based fluorophore (electrophile) provide a suitable lifetime for the fluorescent open form, and equilibrium between the open form and the non-fluorescent closed form. We show that this spontaneously blinking fluorophore is suitable for single-molecule localization microscopy imaging deep inside cells and for tracking the motion of structures in living cells. We further demonstrate the advantages of this fluorophore over existing methodologies by applying it to nuclear pore structures located far above the coverslip with a spinning-disk confocal microscope and for repetitive time-lapse super-resolution imaging of microtubules in live cells for up to 1 h.

Temperature-dependent fluorescence lifetime of a fluorescent polymeric thermometer, poly(N-isopropylacrylamide), labeled by polarity and hydrogen bonding sensitive 4-sulfamoyl-7-aminobenzofurazan.

Fluorescent molecular thermometers showing temperature-dependent fluorescence lifetimes enable thermal mapping of small spaces such as a microchannel and a living cell. We report the temperature-dependent fluorescence lifetimes of poly(NIPAM-co-DBD-AA), which is a random copolymer of N-isopropylacrylamide (NIPAM) and an environment-sensitive fluorescent monomer (DBD-AA) containing a 4-sulfamoyl-7-aminobenzofurazan structure. The average fluorescence lifetime of poly(NIPAM-co-DBD-AA) in aqueous solution increased from 4.22 to 14.1 ns with increasing temperature from 30 to 35 degrees C. This drastic change in fluorescence lifetime (27% increase per 1 degrees C) is the sharpest ever reported. Concentration independency, one of the advantages of fluorescence lifetime measurements, was seen in average fluorescence lifetime (13.7 +/- 0.18 ns) of poly(NIPAM-co-DBD-AA) at 33 degrees C over a wide concentration range (0.005-1 w/v%). With increasing temperature, polyNIPAM units in poly(NIPAM-co-DBD-AA) change their structure from an extended form to a globular form, providing apolar and aprotic environments to the fluorescent DBD-AA units. Consequently, the environment-sensitive DBD-AA units translate the local environmental changes into the extension of the fluorescence lifetime. This role of the DBD-AA units was revealed by a study of solvent effects on fluorescence lifetime of a model environment-sensitive fluorophore.

Bicarbazoles: Systematic Structure–Property Investigations on a Series of Conjugated Carbazole Dimers

A large series of conjugated carbazole dimers, namely bicarbazoles 1-12, were synthesized by Suzuki-Miyaura, Sonogashira, Hay, and McMurry coupling reactions. In 1-12, the two carbazole moieties are linked at the 1-, 2-, or 3-position directly or via an acetylenic or olefinic spacer. The structure-property relationships, particularly the effects of the conjugation connectivity and the π-conjugated spacers on the electronic, photophysical, and electrochemical properties of 1-12, were studied by extensive UV-vis and fluorescence spectroscopic measurements, cyclic voltammetry (CV), and theoretical calculations as well as X-ray crystallographic analyses. The connection at the 1-position of carbazole ensures high extent of π-conjugation, while that at the 3-position enhances the electron-donating ability. Both acetylenic and olefinic spacers allow the extension of π-conjugation, and the latter also causes the increase of the donor ability. Moreover, the structural variations were found to affect the fluorescence quantum yields significantly, which are up to 0.84.

Environment‐Sensitive Fluorophores with Benzothiadiazole and Benzoselenadiazole Structures as Candidate Components of a Fluorescent Polymeric Thermometer

An environment-sensitive fluorophore can change its maximum emission wavelength (λ(em)), fluorescence quantum yield (Φ(f)), and fluorescence lifetime in response to the surrounding environment. We have developed two new intramolecular charge-transfer-type environment-sensitive fluorophores, DBThD-IA and DBSeD-IA, in which the oxygen atom of a well-established 2,1,3-benzoxadiazole environment-sensitive fluorophore, DBD-IA, has been replaced by a sulfur and selenium atom, respectively. DBThD-IA is highly fluorescent in n-hexane (Φ(f) =0.81, λ(em) =537 nm) with excitation at 449 nm, but is almost nonfluorescent in water (Φ(f) =0.037, λ(em) =616 nm), similarly to DBD-IA (Φ(f) =0.91, λ(em) =520 nm in n-hexane; Φ(f) =0.027, λ(em) =616 nm in water). A similar variation in fluorescence properties was also observed for DBSeD-IA (Φ(f) =0.24, λ(em) =591 nm in n-hexane; Φ(f) =0.0046, λ(em) =672 nm in water). An intensive study of the solvent effects on the fluorescence properties of these fluorophores revealed that both the polarity of the environment and hydrogen bonding with solvent molecules accelerate the nonradiative relaxation of the excited fluorophores. Time-resolved optoacoustic and phosphorescence measurements clarified that both intersystem crossing and internal conversion are involved in the nonradiative relaxation processes of DBThD-IA and DBSeD-IA. In addition, DBThD-IA exhibits a 10-fold higher photostability in aqueous solution than the original fluorophore DBD-IA, which allowed us to create a new robust molecular nanogel thermometer for intracellular thermometry.

Intracellular and in vivo oxygen sensing using phosphorescent Ir(III) complexes with a modified acetylacetonato ligand.

Small luminescent molecular probes based on the iridium(III) complex BTP, (btp)2Ir(acac) (btp = benzothienylpyridine, acac = acetylacetone) have been developed for sensing intracellular and in vivo O2. These compounds are BTPSA (containing an anionic carboxyl group), BTPNH2 (containing a cationic amino group), and BTPDM1 (containing a cationic dimethylamino group); all substituents are incorporated into the ancillary acetylacetonato ligand of BTP. Introduction of the cationic dimethylamino group resulted in an almost 20-fold increase in cellular uptake efficiency of BTPDM1 by HeLa cells compared with BTP. The phosphorescence intensity of BTPDM1 internalized in living cells provided a visual representation of the O2 gradient produced by placing a coverslip over cultured monolayer cells. The intracellular O2 levels (pO2) inside and outside the edge of the coverslip could be evaluated by measuring the phosphorescence lifetime of BTPDM1. Furthermore, intravenous administration of 25 nmol BTPDM1 to tumor-bearing mice allowed the tumor region to be visualized by BTPDM1 phosphorescence. The lifetime of BTPDM1 phosphorescence from tumor regions was much longer than that from extratumor regions, thereby demonstrating tumor hypoxia (pO2 = 6.1 mmHg for tumor and 50 mmHg for extratumor epidermal tissue). Tissue distribution studies showed that 2 h after injection of BTPDM1 into a mouse, the highest distribution was in liver and kidney, while after 24 h, BTPDM1 was excreted in the feces. These results demonstrate that BTPDM1 can be used as a small molecular probe for measuring intracellular O2 levels in both cultured cells and specific tissues and organs.

Internal conversion of o-aminoacetophenone in solution

The photophysical properties of o-aminoacetophenone (o-AAP) in solution have been studied by using a femtosecond laser–single photon counting system and time-resolved thermal lensing (TRTL) method. The fluorescence quantum yield (Φf) and lifetime (τf) of o-AAP depend strongly on the nature of the solvent. In nonpolar solvents, o-AAP gives very small Φf values (Φf = 2.4 × 10−4 in n-hexane) and remarkably short fluorescence lifetimes (τf = 9.4 ps in n-hexane), suggesting the presence of very fast nonradiative deactivation processes. The measurements of the quantum yield (Φisc) of intersystem crossing based on the energy transfer and TRTL methods clearly show that the fast radiationless processes in nonpolar solvents are due to internal conversion. In aprotic solvents, the rate (kic) of internal conversion for o-AAP decreases significantly with increasing solvent polarity (kic = 1.0 × 1011 s−1 in n-hexane, kic = 2.4 × 109 s−1 in acetonitrile). In protic solvents, the kic value tends to increase with an increase of hydrogen-bonding donor ability of the solvent. The internal conversion rate in aprotic solvents is scarcely affected by deuterium substitution of the NH2 group in o-AAP, while a large isotope effect is found for o-AAP in deuteriated protic solvents. It is concluded that the efficient S1 → S0 internal conversion in nonpolar aprotic solvents arises from vibronic interactions between close-lying 1(π,π*) and 1(n,π*) states (the proximity effect), and in protic solvents intermolecular hydrogen-bonding interactions with solvent molecules also facilitate the nonradiative process.

Systematic Structure–Property Investigations on a Series of Alternating Carbazole–Thiophene Oligomers

A series of alternating carbazole-thiophene oligomers, namely 2,7-linked carbazole-thiophene oligomers 1, 3, 5, 7, and 9 and 3,6-linked ones 2, 4, 6, 8, and 10, in which the molecular length was systematically elongated, were synthesized by Suzuki-Miyaura coupling reactions. The effects of the conjugation connectivity between the carbazole and thiophene moieties and the molecular length on the electronic, photophysical, and electrochemical properties of 1-10 were comprehensively investigated. In the present oligomer architectures, the connection with thiophene at the 2,7-positions of carbazole ensures π-conjugation to a high extent and high fluorescence quantum yields, while that at the 3,6-positions enhances the donor ability. The increase in the molecular length of the 2,7-linked oligomers effectively extends π-conjugation. The relationship between structural variations and photophysical properties was examined by fluorescence lifetime measurements in detail. The X-ray crystal structure of 6 was also disclosed.

π-Extended thiadiazoles fused with thienopyrrole or indole moieties: synthesis, structures, and properties.

We report the syntheses, structures, photophysical properties, and redox characteristics of donor-acceptor-fused π-systems, namely π-extended thiadiazoles 1-5 fused with thienopyrrole or indole moieties. They were synthesized by the Stille coupling reactions followed by the PPh(3)-mediated reductive cyclizations as key steps. X-Ray crystallographic studies showed that isomeric 1b and 2b form significantly different packing from each other, and 1a and 4a afford supramolecular networks via multiple hydrogen bonding with water molecules. Thienopyrrole-fused compounds 1b and 2b displayed bathochromically shifted intramolecular charge-transfer (CT) bands and low oxidation potentials as compared to indole-fused analog 3b and showed moderate to good fluorescence quantum yields (Φ(f)) up to 0.73. In 3b-5b, the introduction of electron-donating substituents in the indole moieties substantially shifts the intramolecular CT absorption maxima bathochromically and leads to the elevation of the HOMO levels. The Φ(f) values of 3-5 (0.04-0.50) were found to be significantly dependent on the substituents in the indole moieties. The OFET properties with 1b and 2b as an active layer were also disclosed.

Intracellular and in vivo oxygen sensing using phosphorescent iridium(III) complexes

Molecular oxygen plays an indispensable role as a terminal electron acceptor in the electron transport chain in mitochondria. Acute or chronic oxygen deprivation (hypoxia) in organisms results in various diseases, and the elucidation of the pathogenic mechanism of hypoxia-related diseases and various cellular responses to hypoxia is an urgent issue. Optical oxygen imaging methods using phosphorescent probes have opened up techniques for noninvasive imaging of the intracellular and tissue oxygen status, and oxygen-sensitive probes play a key role in the development of this approach. We expect that phosphorescent Ir(III) complexes can serve as new oxygen-sensing probes for intracellular and intravascular oxygen imaging in vivo.