Excited State Vibrational Dynamics Reveals a Photocycle That Enhances the Photostability of the TagRFP-T Fluorescent Protein.

High photostability is a desirable property of fluorescent proteins (FPs) for imaging, yet its molecular basis is poorly understood. We performed ultrafast spectroscopy on TagRFP and its 9-fold more photostable variant TagRFP-T (TagRFP S158T) to characterize their initial photoreactions. We find significant differences in their electronic and vibrational dynamics, including faster excited-state proton transfer and transient changes in the frequency of the v520 mode in the excited electronic state of TagRFP-T. The frequency of v520, which is sensitive to chromophore planarity, downshifts within 0.58 ps and recovers within 0.87 ps. This vibrational mode modulates the distance from the chromophore phenoxy to the side chain of residue N143, which we suggest can trigger cis/trans photoisomerization. In TagRFP, the dynamics of v520 is missing, and this FP therefore lacks an important channel for chromophore isomerization. These dynamics are likely to be a key mechanism differentiating the photostability of the two FPs.

Formation of thioglucoside single crystals by coherent molecular vibrational excitation using a 10-fs laser pulse.

Compound crystallization is typically achieved from supersaturated solutions over time, through melting, or via sublimation. Here a new method to generate a single crystal of thioglucoside using a sub-10-fs pulse laser is presented. By focusing the laser pulse on a solution in a glass cell, a single crystal is deposited at the edge of the ceiling of the glass cell. This finding contrasts other non-photochemical laser-induced nucleation studies, which report that the nucleation sites are in the solution or at the air-solution interface, implying the present crystallization mechanism is different. Irradiation with the sub-10-fs laser pulse does not heat the solution but excites coherent molecular vibrations that evaporate the solution. Then, the evaporated solution is thought to be deposited on the glass wall. This method can form crystals even from unsaturated solutions, and the formed crystal does not include any solvent, allowing the formation of a pure crystal suitable for structural analysis, even from a minute amount of sample solution.

Transient process spectroscopy for the direct observation of inter-molecular photo-dissociation.

Transient process spectroscopy has previously been thought to be applicable only to the analysis of intra-molecular processes. Two metal ion bridges used in the present work have allowed us to visualize real-time variations of the molecular vibration frequencies during photo-disproportionation inside bimolecule aggregates, which directly shows transient inter-molecular reactions.

Pulsed Nd:YAG laser-induced photoreaction of cis,cis-1,3-cyclooctadiene at 266 nm: selective cyclization to cis-bicyclo[4.2.0]oct-7-ene.

Nanosecond-scale pulsed laser irradiation of cis,cis-1,3-cyclooctadiene via an Nd:YAG laser at 266 nm induced highly selective cyclization of the 1,3-diene moiety to afford cis-bicyclo[4.2.0]oct-7-ene in high yield. The pulsed Nd:YAG laser light is highly monochromatic thereby allowing efficient control of the photoreaction selectivity by controlling the photostationary state.

The reaction mechanism of Claisen rearrangement obtained by transition state spectroscopy and single direct-dynamics trajectory.

Chemical bond breaking and formation during chemical reactions can be observed using "transition state spectroscopy". Comparing the measurement result of the transition state spectroscopy with the simulation result of single direct-dynamics trajectory, we have elucidated the reaction dynamics of Claisen rearrangement of allyl vinyl ether. Observed the reaction of the neat sample liquid, we have estimated the time constants of transformation from straight-chain structure to aromatic-like six-membered ring structure forming the C¹-C6 bond. The result clarifies that the reaction proceeds via three steps taking longer time than expected from the gas phase calculation. This finding provides new hypothesis and discussions, helping the development of the field of reaction mechanism analysis.

Triazoyl-phenyl linker system enhancing the aqueous solubility of a molecular probe and its efficiency in affinity labeling of a target protein for jasmonate glucoside.

In methods employing molecular probes to explore the targets of bioactive small molecules, long or rigid linker moieties are thought to be critical factors for efficient tagging of target protein. We previously reported the synthesis of a jasmonate glucoside probe with a highly rigid linker consisting of a triazoyl-phenyl (TAzP) moiety, and this probe demonstrated effective target tagging. Here we compare the TAzP probe with other rigid or flexible probes with respect to target tagging efficiency, hydrophobic parameters, aqueous solubility, and dihedral angles around the biaryl linkage by a combination of empirical and calculation methods. The rigid biaryl linkage of the TAzP probe has a skewed conformation that influences its aqueous solubility. Such features that include rigidness and good aqueous solubility resulted in highly efficient target tagging. These findings provide a promising guideline toward designing of better linkers for improving molecular probe performance.

Photo-impulsive reactions in the electronic ground state without electronic excitation: non-photo, non-thermal chemical reactions.

Allyl phenyl ether has an absorption band in the ultraviolet region (λ < 400 nm); therefore, irradiation with few-optical-cycle ultraviolet pulses (λ = 360-440 nm) causes a transition to the ultraviolet band, which leads to an electronic state and a photo-Claisen rearrangement (radical reaction) in the electronic excited state. However, the reaction scheme of allyl phenyl ether under irradiation with few-optical-cycle visible pulses (λ = 525-725 nm) was determined to be same as that of the thermal Claisen rearrangement ([3,3]-sigmatropic rearrangement), which is symmetry-allowed in the electronic ground state. Photo-excitation with few-optical cycle visible pulses below the absorption band induces a photo-impulsive reaction in the electronic ground state without electronic excitation, of which the trigger scheme is different from that of photoreaction or thermal-reaction. The photo-impulsive reaction in the electronic ground state is highly possible as a novel reaction scheme.

The experimental visualisation of molecular structural changes during both photochemical and thermal reactions by real-time vibrational spectroscopy.

Have you ever hoped to observe transition states? Chemists have long desired to monitor the deformation of molecular structures via transition states to understand the mechanisms of complicated reactions. Detailed knowledge of transition states helps find strategies to develop novel reaction schemes for introducing new functionalities to chemicals. Molecular structural changes via transition states can be observed by real-time vibrational spectroscopy using sub-5 fs laser pulses. In this paper, I report the direct observation of time-dependent frequency shifts of relevant molecular vibrational modes, which allowed for the clear visualization of ultrafast structural changes in molecules during bond breaking and bond reformation steps. Various mechanisms for photochemical reactions were clarified using sub-5 fs laser pulses. Moreover, a non-thermal vibrational excitation method for efficiently driving chemical reactions in the electronic ground state in solution with the use of broadband visible sub-5 fs laser pulses has been developed. The respective chemical reaction processes were directly observed, including transition states during not only "photochemical" but also "thermal" reactions. Time-resolved spectroscopy with a time resolution of a few femtoseconds enables observation of real-time vibrational amplitudes of complicated molecules and opens up new ways for clarifying reaction mechanisms and developing new chemical transformations.

"Click-made" biaryl-linker improving efficiency in protein labelling for the membrane target protein of a bioactive compound.

We report on the design, synthesis and assessment of a novel biaryl-linked (BArL) molecular probe for the exploration of low-abundant target proteins for bioactive compounds based on the activity based protein profiling (ABPP) approach. Surprisingly, the performance of the BArL probe was better than that of the stepwise tagging approach that is considered to be the most effective method used in ABPP study.

Direct observation of molecular structural change during intersystem crossing by real-time spectroscopy with a few optical cycle laser.

Ultrafast spectroscopy by a sub-5 fs pulse laser was applied to the simultaneous study of electronic relaxation and vibrational dynamics in Ru(II)(TPP)(CO). The electronic lifetimes of (1)Q(x(1,0))(pi,pi*) and (1)Q(x(0,0))(pi,pi*) were determined to be 230 +/- 70 fs and 1150 +/- 260 fs, respectively. The spectrogram shows the time dependent changes in the vibrational spectrum associated with the spin state change from the Franck-Condon state in the excited singlet state to the triplet state via the curve crossing point between the singlet and triplet potential surfaces. The time constant of the intersystem crossing process was determined to be about 1.0 ps from observed electronic relaxation and vibrational dynamics reflecting the transition from the singlet to triplet electronic excited state.

Transition states and nonlinear excitations in chloroform observed with a sub-5 fs pulse laser.

Sub-5 fs pulse dynamics observed in neat chloroform (CHCl(3)) and the CHCl(3)...O(2) charge-transfer complex in oxygen-saturated chloroform (O(2)-CHCl(3)) were found to be dominated by wave packet motions in the excited state and the ground state, respectively. The time-resolved signal of CHCl(3) exhibits dynamics in the electronic excited state generated by a three-photon absorption process, and that of O(2)-CHCl(3) is explained in terms of the dynamics of the electronic ground state excited by the stimulated Raman process. In addition, we found that the oxidation reaction of chloroform in the charge-transfer complex of chloroform and oxygen easily proceeds via a C-H insertion process triggered by the stimulated Raman process under the irradiation of a visible laser pulse. The spectrogram analysis enabled direct observation of the real-time dynamics of the Raman-triggered oxidation process. These results demonstrate that observation of transition states by sub-5 fs time-resolved spectroscopy is applicable to "ground-state reactions" as well as "excited-state reactions" via stimulated Raman excitation in a wide variety of chemical reactions.

The reactive intermediate of catalytic borohydride reduction by Schiff base-cobalt complexes.

The key reactive intermediate of borohydride reduction catalyzed by Schiff base-cobalt complexes is proposed to be the dichloromethylcobalt hydride with a sodium cation, based on experimental and theoretical studies. It was revealed that chloroform is not the solvent but the reactant that activates the cobalt catalyst. The substrate carbonyl compounds are fixed and activated by the alkali cation, which is captured by the oxygen atoms of the planar ligand and the chlorine atom of the axial ligand, and attacked by the hydride on the cobalt atom via a six-membered-like transition state to afford the corresponding alcohol.

Proposal for the metallacycle pathway during the cyclopropanation catalyzed by cobalt-Schiff base complexes.

[reaction: see text] The density functional study of the cobalt(II) complex-catalyzed cyclopropanation revealed that the cobalt(II)-Schiff base complex without an ethylene bridge would be flexible enough to transform into the cis-beta conformer while approaching the olefin; consequently, the metallacycle pathway would be preferred, while the ethylene bridge would stabilize the planar conformer to allow reaction via the concerted mechanism.

Enantioselective borodeuteride reduction of aldimines catalyzed by cobalt complexes: preparation of optically active deuterated primary amines.

[reaction: see text] The enantioselective borodeuteride reduction catalyzed by optically active beta-ketoiminato cobalt complexes was applied to N-(di(o-tolyl)phosphinyl)aldimines to afford the corresponding optically active deuterated primary amines in high yields with high enantiomeric excesses after simple deprotection. The present deuteride reduction of aldimines is in the opposite sense of the enantioselective for the previously reported borohydride reduction of ketones or diphenylphosphinyl aldimines. The stereochemical course in these enantioselective reductions is discussed.

Cobalt-carbene complex with single-bond character: intermediate for the cobalt complex-catalyzed cyclopropanation.

It is generally considered that metal-carbene carbon bonds in carbene complexes for cyclopropanation should be double-bonded; however, the theoretical and FT-IR analyses revealed that the cobalt-carbene carbon bond of the 3-oxobutylideneaminato or the salen-cobalt-carbene complexes was characterized as a single bond.

Theoretical analysis of the reaction pathway and the effect of the axial ligand for 3-oxobutylideneaminatocobalt(II)-catalyzed cyclopropanation.

[structure: see text] The reaction pathway of the cyclopropanation catalyzed by the 3-oxobutylideneaminatocobalt(II) complex was analyzed by the density functional method to reveal that the axial donor ligand produced two prominent effects. One is that the activation energy for the formation of the cobalt carbene complex was reduced and that the activation energy for the cyclopropanation step was increased. The other is that the distance of the carbene carbon above the ligand plane was shortened during the cyclopropanation step.