The vibrational spectrum (800-1700 cm -1 region) of the J-625 intermediate, formed within 200-500 fs (3.5 ps decay time to K-590) in the room-temperature bacteriorhodopsin (BR) photocycle, is measured via picosecond time-resolved coherent anti-Stokes Raman spectroscopy (PTR/CARS). An examination of the excitation conditions and BR photocycle kinetics, as well as the vibrational CARS data, convincingly demonstrates that these PTR/CARS spectra can be quantitatively analyzed in terms of primarily BR-570 and J-625 by using third-order nonlinear susceptibility (χ (3) ) relationships. The resultant background-free (Lorentzian line shapes) CARS spectrum contains 24 distinct vibrational features which provide the most complete structural characterization of J-625 yet reported. Comparisons of the J-625 vibrational spectrum with those of groundstate BR-570 and the K-590 intermediate show that J-625 maintains some structural similarities with BR-570 while it has a significantly different structure than that of K-590. Specifically, J-625 has (i) an all-trans retinal configuration, (ii) increased electron density in the CdC stretching modes as manifested by increased CdC stretching frequencies relative to those in both BR-570 and K-590, (iii) significant delocalized hydrogen out-of-plane motion not observed in any other BR species, (iv) decreased C-CH 3 in-plane wagging motion, and (v) a Schiff-base bonding environment similar to that of BR-570 and distinctively different from that in K-590. Comparisons between the PTR/CARS spectra of J-625 and T5.12, an intermediate found in the photoreaction of the artificial BR pigment, BR5.12, containing a five-membered ring spanning the C 12 -C 13 dC 14 bonds (thereby blocking C 13 dC 14 isomerization), support the conclusion that the J-625 structure reflects the reaction coordinates in the BR photocycle that precede C 13 dC 14 isomerization. Since these PTR/ CARS data show J-625 to have an all-trans retinal, C 13 dC 14 isomerization cannot be the primary reaction coordinate described in numerous models for the BR photocycle. The all-trans to 13-cis isomerization must occur as J-625 transforms into K-590, and other changes in the retinal structural and/or retinal-protein interactions must comprise the primary reaction coordinates that precede C 13 dC 14 isomerization. These results require that significant changes in the mechanistic model describing the room-temperature BR photocycle be considered.

The vibrational spectrum of an intermediate, T5.12, in the photoreaction of an artificial bacteriorhodopsin (BR) pigment containing a five-membered carbon ring spanning the C12−C13C14 bonds (BR5.12) is measured by picosecond time-resolved coherent anti-Stokes Raman spectroscopy (PTR/CARS). Observed initially by picosecond transient absorption (PTA) measurements, T5.12 is the only intermediate in the BR5.12 photoreaction (i.e., T5.12 decays only to BR5.12). BR5.12 does not have a photocycle analogous to that in native BR, presumably because the five-membered ring blocks the reaction coordinate leading to C13C14 bond isomerization. Since T5.12 may therefore represent the molecular events (reaction coordinates) that precede C13C14 bond isomerization, its vibrational spectrum may aid in elucidating the primary reaction coordinate(s) in the BR photocycle. Although T5.12 is identified via a red-shifted absorption (660 nm maximum, <3 ps formation with 3 ps BR5.12 excitation and decay in 17 ± 1 ps), no spectroscopic data which directly characterize the retinal structure in T5.12, and thereby the role of bonding changes, have been available. The PTR/CARS vibrational data presented here show that T5.12 contains (i) an all-trans retinal configuration, (ii) significant hydrogen out-of-plane motion localized in specific normal modes, (iii) increased π-electron density in the CC stretching modes manifested by frequency increases, (iv) restricted in-plane C−CH3 rocking motion, and (v) a Schiff-base environment similar to that in BR5.12. These PTR/CARS data also confirm that T5.12 decays exclusively to BR5.12. The vibrational spectrum of T5.12 makes it evident that no complete CC isomerization nor C−C rotation in any retinal bond occurs upon excitation of BR5.12. The excellent agreement between the kinetic lifetime of T5.12 (from PTA and PTR/CARS data) and its stimulated emission lifetime suggests that T5.12 may be an excited electronic state. In such a case, the PTR/CARS data presented here are the first to be reported from an excited electronic state of a protein. Regardless of whether T5.12 is an excited or ground electronic state, the vibrational spectra of T5.12 reflect the retinal structure(s) that precedes C13C14 bond isomerization in the BR photocycle. The relevance of T5.12 PTR/CARS data to the native BR photocycle is discussed in terms of the intermediates K-590, J-625, and I-460. Direct analyses of the respective vibrational spectra of T5.12 and K-590 demonstrate that they contain distinctly different retinal structures, but since no vibrational data assignable directly to either I-460 or J-625 have been reported, comparisons of T5.12 with these intermediates are based only on analogy. Comparisons of the vibrational spectra of T5.12 and native BR intermediates independently provide insight into the structural changes in retinal that could occur prior to C13C14 bond isomerization in native BR.

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The dynamics over the initial 100 ns (3 ps time resolution) of the room-temperature photocycle of the E46Q mutant of photoactive yellow protein (PYP E46Q ) are measured using picosecond transient absorption (PTA) spectroscopy. Three intermediates, I 0 E46Q , I 0 qE46Q , and I 1 E46Q , are observed in the room-temperature PYP E46Q photocycle. Although their respective formation and decay rates differ, I 0 E46Q , I 0 qE46Q , and I 1 E46Q correspond to analogous intermediates (i.e., I 0 , I 0 q , and I 1 ) observed by PTA in the room-temperature photocycle of the wild type (WT) photoactive yellow protein (PYP). These PTA data show that the replacement of glutamic acid 46 with glutamine influences the kinetic properties of the PYP photocycle, but does not alter the general photochemical mechanism itself. The influence of the E46Q mutation on the PYP chromophore can be independently obtained by measuring changes in the vibrational degrees of freedom of ground-state PYP and PYP E46Q . Vibrational spectra (1100-1700 cm -1 ) of both PYP and PYP E46Q are measured under the same experimental conditions (i.e., ω 1 ) 490 nm and ω s ) 518-535 nm) using picosecond resonance coherent anti-Stokes Raman scattering (PR/CARS). Although the 14 vibrational bands observed in the PR/CARS spectrum of PYP E46Q are generally analogous to those found in the PR/CARS spectrum of PYP, detailed comparisons reveal significant differences in both the positions and relative intensities of vibrational bands assigned to the phenolate part of the cinnamyl chromophore. These PR/CARS results demonstrate that while the chromophore within both PYP and PYP E46Q have similar vibrational degrees of freedom, the E46Q mutation selectively alters the structure of the phenolate ring, apparently through differences in the hydrogen bonding network involving glutamic acid 46 and the negatively charged oxygen in the phenolate ring. When considered together, the changes in the kinetic rate constants for the photocycle (PTA data) and in the vibrational spectra (PR/CARS data) caused by the E46Q mutation suggest that the I 0 and I 0 q intermediates involve structural and/or electronic energy changes localized on the phenolate ring of the PYP chromophore. † Part of the special issue "Edward W. Schlag Festschrift".

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A triazole-containing 8-hydroxyquinoline (8-HQ) ether 2 was efficiently synthesized in two steps from the "click" strategy. Compound 2 gave a strong fluorescence (Φ = 0.21) in nonprotic solvent like CH(3)CN, and a weak fluorescence (Φ = 0.06) in protic solvent like water. In water, a more than 100 nm red shift of the fluorescence maximum was observed for compound 2 in comparison with that in CH(3)CN. This fluorescence difference may be attributed to the intermolecular photoinduced proton transfer (PPT) process involving the protic solvent water molecules. Similarly, this intermolecular PPT process was also observed in the high-water-content CH(3)CN aqueous solution (e.g., CH(3)CN/H(2)O = 5/95, v/v). The water content in the CH(3)CN/H(2)O binary solvent mixture greatly affected the fluorescence intensity (e.g., Φ = 0.06 and 0.25 when CH(3)CN/H(2)O = 5/95 and 95/5, v/v, respectively) and emission wavelength. Using this interesting property, by simple variation of the water content in the CH(3)CN aqueous solution, compound 2 was tuned from a selective "turn-on" fluorescent sensor for Zn(2+) (CH(3)CN/H(2)O = 5/95, v/v) to a ratiometric one for Zn(2+) and a selective "turn-off" one for Fe(3+) (CH(3)CN/H(2)O = 95/5, v/v) over a wide range of pH value. In high-water-content (CH(3)CN/H(2)O = 5/95, v/v) aqueous solution compound 2 shows a selective "turn-on" response toward Zn(2+), with a 10-fold enhancement in the fluorescence intensity at 428 nm and a 62 nm blue shift of the emission maximum (490 to 428 nm) due to the inhibition of intermolecular PPT process upon chelating with Zn(2+). However, in a less polar solvent (CH(3)CN/H(2)O = 95/5, v/v) in which compound 2 has high fluorescence (quantum yield =0.25), it shows a ratiometric response toward Zn(2+), with a continuous decrease of the fluorescence intensity at 399 nm and an increase at 423 nm. More interestingly, in this case, it also exhibits a very sensitive, selective, and ratiometric fluorescence quenching in the presence of Fe(3+), with an 81 nm red shift of the emission maximum (399 to 480 nm) in a wide range of pH through a metal ligand charge transfer (MLCT) effect.

A phase I/II clinical trial for treating malignant melanoma by boron neutron capture therapy (BNCT) was designed to evaluate whether the world’s first in-hospital neutron irradiator (IHNI) was qualified for BNCT. In this clinical trial planning to enroll 30 patients, the first case was treated on August 19, 2014. We present the protocol of this clinical trial, the treating procedure, and the clinical outcome of this first case. Only grade 2 acute radiation injury was observed during the first four weeks after BNCT and the injury healed after treatment. No late radiation injury was found during the 24-month follow-up. Based on positron emission tomography-computed tomography (PET/CT) scan, pathological analysis and gross examination, the patient showed a complete response to BNCT, indicating that BNCT is a potent therapy against malignant melanoma and IHNI has the potential to enable the delivery of BNCT in hospitals.