Tomotsumi Fujisawa

Heavy Atom Substitution Effects in Non-Aromatic Ionic Liquids: Ultrafast Dynamics and Physical Properties

In this study, we have investigated the heavy atom substitution effects on the ultrafast dynamics in nonaromatic cation-based ionic liquids, as well as the static physical properties such as shear viscosity, surface tension, glass transition temperature, and melting point. Phosphonium-based ionic liquids show lower shear viscosities and lower glass transition temperatures than their corresponding ammonium-based ionic liquids. We have also examined the substitution of a (2-ethoxyethoxy)ethyl group for an octyl group in ammonium and phosphonium cations and found that the (2-ethoxyethoxy)ethyl group reduces the shear viscosity and increases the surface tension. From the results of the ultrafast dynamics, including intra- and interionic vibrations and reorientational relaxation in the ammonium- and phosphonium-based ionic liquids measured by means of femtosecond optically heterodyne-detected Raman-induced Kerr spectroscopy, we have found that the first moment of low-frequency Kerr spectrum, omitting the contributions of clear intraionic vibrational modes, correlates to the square root of surface tension divided by density. This fact indicates that heavy atom substitution in ionic liquids provides a weaker interionic interaction arising from the larger ionic volume. On the other hand, the ether group in the cations gives the stronger interionic interaction but with a more flexible and/or less segregated nature in the ILs than the alkyl group.

Comparison of interionic/intermolecular vibrational dynamics between ionic liquids and concentrated electrolyte solutions.

In this study, we have compared the interionic/intermolecular vibrational dynamics of ionic liquids (ILs) and concentrated electrolyte solutions measured by femtosecond optically heterodyne-detected Raman-induced Kerr effect spectroscopy. A typical anion in ILs, bis(trifluoromethanesulfonyl)amide ([NTf(2)](-)), has been chosen as the anion for the sample ILs and concentrated electrolyte solutions. ILs used in this study are 1-butyl-3-methylimidazolium, 1-butylpyridinium, N-butyl-N,N,N-triethylammonium, and 1-butyl-1-methylpyrrolidinium with [NTf(2)](-). Li[NTf(2)] solutions (approximately 3.3 M) of water, methanol, propylene carbonate, and poly(ethylene glycol) have been selected as control samples. Kerr transients of the ILs and electrolyte solutions show intra- and interionic/intermolecular vibrational dynamics followed by slow picosecond overdamped relaxation. Fourier transform Kerr spectra have shown a difference in the relative intensities of intraionic vibrational bands of [NTf(2)](-) (280-350 cm(-1)) between the ILs and electrolyte solutions. The origin of the difference is attributed to the change in the conformational equilibrium between cisoid and transoid forms of [NTf(2)](-), which is caused by a favorable stabilization of dipolar cisoid form due to Li(+) and dipolar solvent molecules in the electrolyte solutions. Low-frequency Kerr spectra (0-200 cm(-1)) exhibit unique features with the variation of cation and solvent species. The aromatic ILs have a prominent high-frequency librational motion at about 100 cm(-1) in contrast to the case for the nonaromatic ones. The common structure of the spectra observed at about 20 cm(-1) likely comes from an interionic motion of [NTf(2)](-). The nonaromatic ILs allow a fair comparison with the electrolyte solutions of propylene carbonate and poly(ethylene glycol) because of the structural similarities. The comparison based on the first moment of the interionic/intermolecular vibrational spectrum suggests the stronger interionic/intermolecular interaction in the concentrated electrolyte solutions than the ILs.

Solvent Effects on the Local Structure of p-Nitroaniline in Supercritical Water and Supercritical Alcohols

Raman spectra of p-nitroaniline in supercritical water and supercritical alcohols were measured, and the effects of solvents on the NO 2 and NH 2 stretching modes were investigated. The intensity and frequency of the NO 2 stretching mode significantly changed as a function of the solvent density and temperature. The frequency of the NO 2 stretching mode correlated with the absorption peak energy of the S 1<--S 0 transition. On the other hand, the vibrational frequency of the NH 2 stretching mode did not correlate with the absorption peak shift, although it had a large frequency shift as a function of the density. The correlation between the NO 2 frequency and absorption peak energy suggested that the solvent effects of supercritical water and supercritical alcohols were similar to those for nonpolar solvents. The density functional calculation using the polarizable continuum model and p-nitroaniline-water clusters qualitatively reproduced the density dependence of the NO 2 stretching mode as well as the solvent polarity dependence. Detailed vibrational analysis revealed that the coupling between the NO 2 and C-NH 2 vibrational motions at the harmonic level has an important effect on the intensity and frequency shift of the NO 2 stretching mode. The frequency shift of the NH 2 stretching mode correlated with the degree of hydrogen bonding between the solvent molecules estimated from NMR measurements [Hoffmann M. M.; Conradi, M. S. J. Phys. Chem. B. 1998, 102, 263]. The existence of intermolecular hydrogen bonding around the NH 2 group was demonstrated even at low-density conditions.

Role of Coherent Low-Frequency Motion in Excited-State Proton Transfer of Green Fluorescent Protein Studied by Time-Resolved Impulsive Stimulated Raman Spectroscopy

Green fluorescent protein (GFP) from jellyfish Aequorea victoria, an essential bioimaging tool, luminesces via excited-state proton transfer (ESPT) in which the phenolic proton of the p-hydroxybenzylideneimidazolinone chromophore is transferred to Glu222 through a hydrogen-bond network. In this process, the ESPT mediated by the low-frequency motion of the chromophore has been proposed. We address this issue using femtosecond time-resolved impulsive stimulated Raman spectroscopy. After coherently exciting low-frequency modes (<300 cm(-1)) in the excited state of GFP, we examined the excited-state structural evolution and the ESPT dynamics within the dephasing time of the low-frequency vibration. A clear anharmonic vibrational coupling is found between one high-frequency mode of the chromophore (phenolic CH bend) and a low-frequency mode at ∼104 cm(-1). However, the data show that this low-frequency motion does not substantially affect the ESPT dynamics.

Structural Dynamics of a Noncovalent Charge Transfer Complex from Femtosecond Stimulated Raman Spectroscopy

Femtosecond stimulated Raman spectroscopy is used to examine the structural dynamics of photoinduced charge transfer within a noncovalent electron acceptor/donor complex of pyromellitic dianhydride (PMDA, electron acceptor) and hexamethylbenzene (HMB, electron donor) in ethylacetate and acetonitrile. The evolution of the vibrational spectrum reveals the ultrafast structural changes that occur during the charge separation (Franck-Condon excited state complex → contact ion pair) and the subsequent charge recombination (contact ion pair → ground state complex). The Franck-Condon excited state is shown to have significant charge-separated character because its vibrational spectrum is similar to that of the ion pair. The charge separation rate (2.5 ps in ethylacetate and ∼0.5 ps in acetonitrile) is comparable to solvation dynamics and is unaffected by the perdeuteration of HMB, supporting the dominant role of solvent rearrangement in charge separation. On the other hand, the charge recombination slows by a factor of ∼1.4 when using perdeuterated HMB, indicating that methyl hydrogen motions of HMB mediate the charge recombination process. Resonance Raman enhancement of the HMB vibrations in the complex reveals that the ring stretches of HMB, and especially the C-CH(3) deformations are the primary acceptor modes promoting charge recombination.

Development of an Azo-Based Photosensitizer Activated under Mild Hypoxia for Photodynamic Therapy

Photodynamic therapy (PDT) utilizes photoirradiation in the presence of photosensitizers to ablate cancer cells via generation of singlet oxygen (1O2), but it is important to minimize concomitant injury to normal tissues. One approach for achieving this is to use activatable photosensitizers that can generate 1O2 only under specific conditions. Here, we report a novel photosensitizer that is selectively activated under hypoxia, a common condition in solid tumors. We found that introducing an azo moiety into the conjugated system of a seleno-rosamine dye effectively hinders the intersystem crossing process that leads to 1O2 generation. We show that the azo group is reductively cleaved in cells under hypoxia, enabling production of 1O2 to occur. In PDT in vitro, cells under mild hypoxia, within the range typically found in solid tumors (up to about 5% O2), were selectively ablated, leaving adjacent normoxic cells intact. This simple and practical azo-based strategy should be widely applicable to design a range of activatable photosensitizers.

Raman Spectroscopic Study on the Solvation of p-Aminobenzonitrile in Supercritical Water and Methanol

Raman spectra of the C[triple bond]N stretching vibration of p-aminobenzonitrile (ABN) have been investigated in water, methanol, and cyclohexane under sub- and supercritical conditions, and in acetonitrile under subcritical condition. In all solvent fluids covering the supercritical region, the vibrational frequency of the C[triple bond]N stretching mode decreased with increasing solvent density from the gaseous region to the medium density region rho(r) approximately = 2, where rho(r) is the reduced density by the critical density of the solvent. However, from the medium density region to the higher density region, the vibrational frequency turned to increase with the solvent density. The temperature-induced low frequency shift of the C[triple bond]N stretching Raman band was also ascertained by the measurement of the temperature dependence of Raman spectrum of ABN vapor above 543 K. The electronic absorption spectra in the UV region of ABN were also measured under the same experimental conditions. The absorption peak energies decreased with an increase of the solvent density, except in water above rho(r) = 2.8. The vibrational frequency shift in cyclohexane was explained by a sum of contributions of the repulsive interaction, the mean field attractive interaction, and the pure temperature effect probably due to the hot-band contribution. The residual frequency shift after the subtraction of the repulsive and temperature effects in water and methanol showed the low frequency shift with increasing solvent density from rho(r) congruent with 0 to 2.8. However, above rho(r) congruent with 2.8 in water, the residual shift showed a high frequency shift with increasing solvent density. The electronic state calculations based on the PCM model using the density functional theory (DFT) indicated that the solvent polarity change caused the low frequency shift of the C[triple bond]N stretching mode, which was also correlated with the shift of the electronic absorption spectrum. The results of the DFT calculations on the cluster of ABN with water molecules and the molecular dynamics simulations indicated that the high frequency shift of the C[triple bond]N stretching mode in water above rho(r) congruent with 2.8 could be due to the hydrogen bonding between water and ABN.

Signaling-State Formation Mechanism of a BLUF Protein PapB from the Purple Bacterium Rhodopseudomonas palustris Studied by Femtosecond Time-Resolved Absorption Spectroscopy.

We studied the signaling-state formation of a BLUF (blue light using FAD) protein, PapB, from the purple bacterium Rhodopseudomonas palustris, using femtosecond time-resolved absorption spectroscopy. Upon photoexcitation of the dark state, FADH(•) (neutral flavin semiquinone FADH radical) was observed as the intermediate before the formation of the signaling state. The kinetic analysis based on singular value decomposition showed that FADH(•) mediates the signaling-state formation, showing that PapB is the second example of FADH(•)-mediated formation of the signaling state after Slr1694 (M. Gauden et al. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 10895-10900). The mechanism of the signaling-state formation is discussed on the basis of the comparison between femtosecond time-resolved absorption spectra of the dark state and those obtained by exciting the signaling state. FADH(•) was observed also with excitation of the signaling state, and surprisingly, the kinetics of FADH(•) was indistinguishable from the case of exciting the dark state. This result suggests that the hydrogen bond environment in the signaling state is realized before the formation of FADH(•) in the photocycle of PapB.

Excitation wavelength dependence of the Raman-Stokes shift of N,N-dimethyl-p-nitroaniline

Raman spectra of N,N-dimethly-p-nitroaniline have been measured in various solvents. The Raman-Stokes shift of the band assigned to the NO2 stretching mode excited at 488 nm was found to be linearly dependent on the pi-pi* absorption band center. Furthermore, it is found that the Raman-Stokes shift of the NO2 stretching mode is dependent upon the excitation wavelength. The extent of the shift when excited at 355 versus 488 nm is almost linearly dependent on the vibrational bandwidth of the NO2 mode. The phenomenon is interpreted as the result of the solvation state selective excitation of the vibrational mode as in the case of phenol blue [Yamaguchi et al., J. Chem. Phys. 109, 9075 (1998); 109, 9084 (1998)].

Development of an Azoreductase-based Reporter System with Synthetic Fluorogenic Substrates.

Enzyme/substrate pairs, such as β-galactosidase with chromogenic x-gal substrate, are widely used as reporters to monitor biological events, but there is still a requirement for new reporter systems, which may be orthogonal to existing systems. Here, we focused on azoreductase (AzoR). We designed and synthesized a library of azo-rhodamine derivatives as candidate fluorogenic substrates. These derivatives were nonfluorescent, probably due to ultrafast conformational change around the N═N bond after photoexcitation. We found that AzoR-mediated reduction of the azo bond of derivatives bearing an electron-donating group on the azobenzene moiety was followed by nonenzymatic cleavage to afford highly fluorescent 2-methyl-rhodamine green (2-Me RG), which was well retained in cells. We show that the AzoR/compound 9 reporter system can detect azoreductase-expressing live cells at the single cell level.