Rapid relaxation pathway of the excited state of linear merocyanines in solutions

Excited‐state relaxation of linear merocyanine dyes in solution is investigated using time‐resolved spectroscopy techniques and quantum chemical calculations. The merocyanine L‐Mero4 and phenyl‐substituted P‐L‐Mero4 have a S‐trans and S‐cis structure, respectively, consisting of indole moiety as the donor, indandione as the acceptor, and the tetramethine as the bridge. The time‐correlated single‐photon counting (TCSPC) picosecond measurements after excitation at wavelength 515 nm to the ππ* state yield emission curves with a short component τ₁ in the range of 27–160 ps and a second component τ₂ of 200–780 ps for L‐Mero4. In P‐L‐Mero4, τ₁ lies in the range of 18–150 ps and τ₂ 220–520 ps. The subfemtosecond transient absorption measurements yield a short component around 0.4–1.4 ps, and the second/third components are similar to those in the TCPSC measurements. The analysis of the experimental data demonstrates that the ground state recovery exhibits a biexponential rise and rapidly indicates that the conversion back to the electronic ground state provides a fast, nonradiative pathway. Quantum chemical calculations on the electronic structures and their dependence on the molecular confirmation are performed. We identify the excited states and the relaxation path along the twist of the center double bonds in tetramethine that might be the nonradiative pathway. The C=C double bond is weakened in the ππ* state. The phenyl substitution in the conjugated double bond weakens this C=C bond, lowers the isomerization barrier, increases the nonradiative rate, and reduces the emission quantum yield. In polar solvents, the energy of the perpendicular conformer along the trans–cis isomerization path is increased to achieve less coupling to the ground state surface. Because of the small barrier to the trans form, these two conformers establish an equilibrium condition. The trans form, which lies at a lower energy, gains more population and thus has a higher emission yield.     

Solvent influence on photochemical isomerizations: Photophysics of DODCI

We have studied the photophysics of DODCI dissolved in a series of polar solvents. Through measurements of the temperature dependence of the photoisomer quantum yield, fluorescence lifetime and fluorescence quantum yields we clarify the kinetic mechanism for nonradiative decay. By measuring the isomerization rate as a function of temperature at constant viscosity we are able to separate the innuence of internal barriers and solvent viscous drag. The apparent activation energy observed in solutions is less than the sum of the internal and viscosity activation energies. This is shown to be consistent with the full Kramer's rate expression for diffusive barrier crossing. We also establish the temperature dependence of a second “direct” internal conversion process which does not lead to photoisomer formation and dominates the nonradiative decay of DODCI at low temperatures or in a rigid matrix.     

Photoswitchable elements within a peptide backbone-ultrafast spectroscopy of thioxylated amides

A series of thioxo compounds, thioacetamide, N-methylthioacetamide, a cyclic thioxoamide [(S)-5-thioxopyrrolidine-2-carboxylic acid ethyl ester], two thioxylated dipeptides (Ala-Psi[CS-NH]-Ala and Phe-Psi[CS-NH]-Ala) and a thioxylated dodecapeptide (Lys-Glu-Thr-Ala-Ala-Ala-Lys-Phe-Glu-Arg-Gln-His-Psi[CS-NH]-Nle-Asp-Ser-Ser-Thr-Ser-Ala-Ala, or [thioxo-His(12)]-S-peptide; Nle = norleucine) are investigated by ultrafast spectroscopy in the visible and near UV. The different molecules show very similar absorption dynamics featuring a rise of a strong visible absorption band on the subpicosecond and picosecond time scale. The decay of the visible absorption occurs within 150-600 ps. The observations are interpreted by the ultrafast formation of triplet states and their decay on the subnanosecond time scale. Comparison with published IR experiments on N-methylthioacetamide indicates that the cis-trans isomerization around the thioxopeptide bond is terminated within less than 1 ns.     

Photoconversion in the red fluorescent protein from the sea anemone Entacmaea quadricolor: is cis-trans isomerization involved?

Proteins from the family of the green fluorescent protein (GFP) are presently extensively used in molecular and cellular biology. Recent studies suggest that isomerization of the chromophore occurs upon excitation and is involved in nonradiative deactivation. Using Raman spectroscopy, we report on photoinduced cis-trans isomerization in the red fluorescent protein eqFP611 from the sea anemone Entacmaea quadricolor. The crystal structure of eqFP611 shows that the chemical structure of the chromophore, p-hydroxybenzylidene-imidazolinone with an extended -conjugated system, is nearly identical to the chromophore of other red fluorescent proteins such as DsRed and HcRed. However, the chromophore of eqFP611 has a trans configuration whereas the chromophore of DsRed has a cis configuration. Upon irradiation with 532-nm light, the absorption of eqFP611 peaking at 559 nm diminished, and concomitantly a drastic decrease in the quantum yield of fluorescence as well as more complex decay kinetics was observed. Upon irradiation, changes in the Raman spectrum of eqFP611 were observed, and the relative intensities and peak positions of the irradiated eqFP611 showed striking similarity with the peaks in the Raman spectrum of DsRed. These observations are tentatively interpreted as trans-to-cis isomerization of the chromophore taking place upon irradiation together with the opening of new, nonradiative pathways.     

An apparent angle dependence for the nonradiative deactivation of excited triplet states of sterically constrained, binuclear ruthenium(II) bis(2,2':6',2' '-terpyridine) complexes

The photophysical properties are reported for a series of binuclear ruthenium(II) bis(2,2':6',2"-terpyridine) complexes built around a geometrically constrained, biphenyl-based bridge. The luminescence quantum yield and lifetime increase progressively with decreasing temperature, but the derived rate constant for nonradiative decay of the lowest-energy triplet state depends on the length of a tethering strap attached at the 2,2'-positions of the biphenyl unit. Since the length of the strap determines the dihedral angle for the central C-C bond, the rate of nonradiative decay shows a pronounced dependence on angle. The minimum rate of nonradiative decay occurs when the dihedral angle is 90 degrees, but there is a maximum in the rate when the dihedral angle is about 45 degrees. This effect does not appear to be related to the extent of electron delocalization at the triplet level but can be explained in terms of variable coupling with a low-frequency vibrational mode associated with the strapped biphenyl unit.     

PHOTOCHEMISTRY OF MEROCYANINE 540

The photophysical properties of merocyanine 540 have been determined in methanol solution over a modest temperature range. Triplet state population is inefficient (the limiting triplet quantum yield being 0.25) due to rapid isomerization of the central double bond from the first excited singlet state. Activation energies have been measured for isomerization from the excited singlet state (20 kJ mol-1) and for conversion of the resultant cis-isomer back to the original trans-form (63 kJ mol-1), both processes involving formation of a twisted species. The dye is easily oxidized to give an unstable adduct which decomposes on the sub-ms timescale. Reversible redox chemistry occurs upon excitation in the presence of electron acceptors. These various observations are discussed in terms of the known chemotherapeutic activity of MC540 and it is concluded that the most probable mechanisms for cytotoxicity involve either local thermal disruption of cell membranes or in situ photogeneration of toxins derived from breakdown of the dye.     

Subcellular distributions and excited-state processes of hypericin in neurons

The photodynamic drug, hypericin, is studied in fetal rat neurons using fluorescence microscopy. Hypericin has an extremely high affinity for the cell membrane and is found to a smaller extent in the nucleus. Fluorescent excitation of hypericin is shown to cause irreversible damage to the cell membranes of living neurons. Fixed cells were used to make ultrafast time-resolved measurements to avoid the deleterious effects of long-term exposure to intense light and room temperatures. To our knowledge, these are the first ultrafast time-resolved measurements of the fluorescence lifetime of hypericin in a subcellular environment. Nonexponential fluorescence decay is observed in hypericin in the neurons. This nonexponential decay is discussed in terms of other examples where nonexponential decay is induced in hypericin upon its binding to biomolecules. The nonradiative processes giving rise to the nonexponential hypericin decay are attributed to excited-state electron transfer, excited-state proton transfer or both.     

Theoretical Insights into the Substituent Effects on the Fluorescence of Boron Complexes of 2-(Benzothiazol-2-yl)phenols: a Quantum Mechanics Study

The performance of organic fluorescent materials can be improved by chemical substitutions with auxochrome groups such as amino to increase solubility, alter emitting color, or modify film quality. The complex 6,6-difluoro-6-bora-5-oxa-11-thia-6 a-aza-benzo[a]fluorine(BOBTP) and its derivatives, which possess excellent luminescent property at room temperature, were theoretically simulated by density functional theory. The geometries of the ground state and the first excited state of BOBTPs complexes were investigated and their bond parameters were obtained. Further, these bond parameters are compared with each other, and the computational wavelengths of maximum absorption and emission of studied complexes match up with the experimental values. It was found that amino substituent bonding to appropriate positions of BOBTP can reduce the reorganization energy significantly, which is ascribed to electron-donating effect of the amino group. The reorganization energy also plays an important role in the fluorescence quantum yield of all the BOBTPs. In particular, the radiative decay of complexes 3 and 4 is dominant due to the smaller reorganization energies, so their fluorescence quantum yield is almost 1, on the contrary the non-radiative decay and intersystem crossing rate of both the 1 and 2 can not be ignored for the larger reorganization energies, and the corresponding fluorescence quantum yields were calculated when the radiative decay rate(kr) and nonradiative decay rate(k(nr)) were taken into account.     

Enhanced fluorescence of epicocconone in surfactant assemblies as a consequence of depth-dependent microviscosity

The extents of fluorescence enhancement of epicocconone are found to be different in the micelles of the surfactants sodium dodecyl sulfate (SDS) and Triton X100 (TX 100). A decrease in fluorescence, observed in the cationic cetyltrimethylammonium bromide (CTAB) micelles, is rationalized by the formation of anions of the fluorophore at the Stern layer. To understand the difference in the effects of SDS and TX 100, the nature of the excited-state process in the fluorophore has been investigated by fluorescence spectroscopy, supported by complementary quantum chemical calculations. The excited-state dynamics of epicocconone is found to depend on polarity and viscosity of the medium, with a more pronounced dependence on viscosity. An inspection of the molecular orbitals involved in the electronic absorption of the molecule reveals the possibility of photoisomerization, which conforms to the observed solvent dependence of the fluorescence spectral properties. An apparent mismatch between trends observed in steady-state spectra and those in temporal decays indicates a significant contribution of an ultrafast component, which cannot be detected in the time resolution of our instrument. The viscosity dependence of the fluorescence quantum yields provides an explanation for the difference in the extents of fluorescence enhancement in the two micelles, in the light of location of the fluorophore at different depths of the micelle. The enhancement of fluorescence, with an unchanged fluorescence maximum, opens up the possibility that the fluorophore could be a useful dual emitting marker for fluorescence microscopy of heterogeneous systems, as the fluorescence of protein-bound epicocconone has been previously reported to be significantly red-shifted.     

Highly effective quenching of the ultrafast radiationless decay of photoexcited pyrimidine bases by covalent modification: photophysics of 5,6-trimethylenecytosine and 5,6-trimethyleneuracil

5,6-Trimethylenecytosine (TMC) and 5,6-trimethyleneuracil (TMU), in which the twist of the C5-C6 bond (or the pyrimidalization of C5) is strongly hindered, do not exhibit the subpicosecond excited-state lifetime characteristic of the naturally occurring pyrimidine bases. This result demonstrates the important role the out-of-plane deformation of the six-membered ring plays in the ultrafast (subpicosecond) internal conversion of photoexcited nucleobases. The dramatically shorter fluorescence lifetime of TMU ( approximately 30 ps) relative to TMC ( approximately 1.2 ns), in aqueous solution at room temperature, is attributed to the presence in TMU of an efficient, secondary nonradiative decay channel of S(1)(pipi*) involving a low-lying (1)npi* state.     

Photoisomerization mechanism of 4-methylpyridine explored by electronic structure calculations and nonadiabatic dynamics simulations

In the present paper, different electronic structure methods have been used to determine stationary and intersection structures on the ground (S(0)) and (1)ππ? (S(2)) states of 4-methylpyridine, which is followed by adiabatic and nonadiabatic dynamics simulations to explore the mechanistic photoisomerization of 4-methylpyridine. Photoisomerization starts from the S(2)((1)ππ?) state and overcomes a small barrier, leading to formation of the prefulvene isomer in the S(0) state via a S(2)∕S(0) conical intersection. The ultrafast S(2) → S(0) nonradiative decay and low quantum yield for the photoisomerization reaction were well reproduced by the combined electronic structure calculation and dynamics simulation. The prefulvene isomer was assigned as a long-lived intermediate and suggested to isomerize to 4-methylpyridine directly in the previous study, which is not supported by the present calculation. The nonadiabatic dynamics simulation and electronic structure calculation reveal that the prefulvene isomer is a short-lived intermediate and isomerizes to benzvalene form very easily. The benzvalene form was predicted as the stable isomer in the present study and is probably the long-lived intermediate observed experimentally. A consecutive light and thermal isomerization cycle via Dewar isomer was determined and this cycle mechanism is different from that reported in the previous study. It should be pointed out that formation of Dewar isomer from the S(2)((1)ππ?) state is not in competition with the isomerization to the prefulvene form. The Dewar structure observed experimentally may originate from other excited states.     

The N‐Arylamino Conjugation Effect in the Photochemistry of Fluorescent Protein Chromophores and Aminostilbenes

To understand the nonradiative decay mechanism of fluorescent protein chromophores in solutions, a systematic comparison of a series of (Z)‐4‐(N‐arylamino)benzylidene‐2,3‐imidazolinones (ABDIs: 2P , 2PP , 2OM , and 2OMB ) and the corresponding trans‐4‐(N‐arylamino)‐4′‐cyanostilbenes (ACSs: 1P , 1PP , 1OM , and 1OMB ) was performed. We have previously shown that the parameter Φ_f+2 Φ_tc, in which Φ_f and Φ_tc are the quantum yields of fluorescence and trans→cis photoisomerization, respectively, is an effective probe for evaluating the contribution of twisted intramolecular charge transfer (TICT) states in the excited decays of trans‐aminostilbenes, including the push–pull ACSs. One of the criteria for postulating the presence of a TICT state is Φ_f+2 Φ_tc?1.0, because its formation is decoupled with the C?C bond (τ) torsion pathway and its decay is generally nonradiative. Our results show that the same concept also applies to ABDIs 2 with the parameter Φ_f+2 Φ_ZE in which Φ_ZE is the quantum yield of Z→E photoisomerization. We conclude that the τ torsion rather than the C? C bond (φ) torsion is responsible for the nonradiative decays of ABDIs 2 in aprotic solvents (hexane, THF, acetonitrile). The phenyl‐arylamino C? N bond (ω) torsion that leads to a nonradiative TICT state is important only for 2OM in THF and acetonitrile. If the solvent is protic (methanol and 10–20 % H₂O in THF), a new nonradiative decay channel is present for ABDIs 2 , but not for ACSs 1 . It is attributed to internal conversion (IC) induced by solvent (donor)–solute (acceptor) hydrogen‐bonding (HB) interactions. The possible HB modes and the concept of τ torsion‐coupled proton transfer are also discussed.     

Bonded excimer in stacked adenines:Semiclassical simulations

The nonradiative decay of a π-stacked pair of adenine molecules,one of which was excited by an ultrafast laser pulse,is studied by semiclassical dynamics simulations.This simulation investigation is focused on the effect of the formation of bonded excimer in stacked adenines on the mechanism of ultrafast decay.The simulation finds that the formation of the bonded excimer significantly lowers the energy gap between the LUMO and HOMO and consequently facilitates the deactivation of the electronically excited molecule.On the other hand,the formation of the chemical bond between two stacked adenines restricts the deformation vibration of the pyrimidine of the excited molecule due to the steric effect.This slows down the formation of the coupling between the HOMO and LUMO energy levels and therefore delays the deactivation process of the excited adenine molecule to the electronic ground state.     

Radiationless decay of red fluorescent protein chromophore models via twisted intramolecular charge-transfer states

We use CASSCF and MRPT2 calculations to characterize the bridge photoisomerization pathways of a model red fluorescent protein (RFP) chromophore model. RFPs are homologues of the green fluorescent protein (GFP). The RFP chromophore differs from the GFP chromophore via the addition of an N-acylimine substitution to a common hydroxybenzylidene-imidazolinone (HBI) motif. We examine the substituent effects on the manifold of twisted intramolecular charge-transfer (TICT) states which mediates radiationless decay via bridge isomerization in fluorescent protein chromophore anions. We find that the substitution destabilizes states associated with isomerization about the imidazolinone-bridge bond and stabilizes states associated with phenoxy-bridge bond isomerization. We discuss the results in the context of chromophore conformation and quantum yield trends in the RFP subfamily, as well as recent studies on synthetic models where the acylimine has been replaced with an olefin.     

Ultrafast excited-state dynamics in photochromic N-salicylideneaniline studied by femtosecond time-resolved REMPI spectroscopy

Ultrafast processes in photoexcited N-salicylideneaniline have been investigated with femtosecond time-resolved resonance-enhanced multiphoton ionization spectroscopy. The ion signals via the S(1)(n,pi( *)) state of the enol form as well as the proton-transferred cis-keto form emerge within a few hundred femtoseconds after photoexcitation to the first S(1)(pi,pi( *)) state of the enol form. This reveals that two ultrafast processes, excited-state intramolecular proton transfer (ESIPT) reaction and an internal conversion (IC) to the S(1)(n,pi( *)) state, occur on a time scale less than a few hundred femtoseconds from the S(1)(pi,pi( *)) state of the enol form. The rise time of the transient corresponding to the production of the proton-transferred cis-keto form is within 750 fs when near the red edge of the absorption is excited, indicating that the ESIPT reaction occurs within 750 fs. The decay time of the S(1)(pi,pi( *)) state of the cis-keto form is 8.9 ps by exciting the enol form at 370 nm, but it dramatically decreases to be 1.5-1.6 ps for the excitation at 365-320 nm. The decrease in the decay time has been attributed to the opening of an efficient nonradiative channel; an IC from S(1)(pi,pi( *)) to S(1)(n,pi( *)) of the cis-keto form promotes the production of the trans-keto form as the final photochromic products. The two IC processes may provide opposite effect on the quantum yield of photochromic products: IC in the enol form may substantially reduce the quantum yield, but IC in the cis-keto form increase it.     

Isomerization mechanism of the HcRed fluorescent protein chromophore

To understand how the protein achieves fluorescence, the isomerization mechanism of the HcRed chromophore is studied both under vacuum and in the solvated red fluorescent protein. Quantum mechanical (QM) and quantum mechanical/molecular mechanical (QM/MM) methods are applied both for the ground and the first excited state. The photoinduced processes in the chromophore mainly involve torsions around the imidazolinone-bridge bond (τ) and the phenoxy-bridge bond (φ). Under vacuum, the isomerization of the cis-trans chromophore essentially proceeds by τ twisting, while the radiationless decay requires φ torsion. By contrast, the isomerization of the cis-trans chromophore in HcRed occurs via simultaneous τ and φ twisting. The protein environment significantly reduces the barrier of this hula twist motion compared with vacuum. The excited-state isomerization barrier via the φ rotation of the cis-coplanar conformer in HcRed is computed to be significantly higher than that of the trans-non-coplanar conformer. This is consistent with the experimental observation that the cis-coplanar-conformation of the chromophore is related to the fluorescent properties of HcRed, while the trans-non-planar conformation is weakly fluorescent or non-fluorescent. Our study shows how the protein modifies the isomerization mechanism, notably by interactions involving the nearby residue Ile197, which keeps the chromophore coplanar and blocks the twisting motion that leads to photoinduced radiationless decay.     

Spin sublevel-selective and position-dependent isotope effects in T1(ππ*) → S0 intersystem crossing of polycyclic para-diazines

ODMR measurements are combined with phosphorescence quantum yield data to show that deuterium isotope effects on the nonradiative decay rates of quinoxaline and dibenzoquinoxaline are spin sublevel-selective as well as position-dependent.     

On the origin of the ultrafast internal conversion of electronically excited pyrimidine bases

The ultrafast radiationless decay of photoexcited uracil and cytosine has been investigated by ab initio quantum chemical methods based on CIS and CR-EOM-CCSD(T) electronic energy calculations at optimized CIS geometries. The calculated potential energy profiles indicate that the S(1) --> S(0) internal conversion of the pyrimidine bases occurs through a barrierless state switch from the initially excited (1)pipi state to the out-of-plane deformed excited state of biradical character, which intersects the ground state at a lower energy. This three-state nonradiative decay mechanism predicts that replacement of the C5 hydrogen by fluorine introduces an energy barrier for the initial state switch, whereas replacement of the C6 hydrogen by fluorine does not. These predictions are borne out by the very different fluorescence yields of 5-fluorinated bases relative to the corresponding 6-fluorinated bases. It is concluded from these results that the origin of the ultrafast radiationless decay is the same for the two pyrimidine bases.     

Primary reactions of bacteriophytochrome observed with ultrafast mid-infrared spectroscopy

Phytochromes are red-light photoreceptor proteins that regulate a variety of responses and cellular processes in plants, bacteria, and fungi. The phytochrome light activation mechanism involves isomerization around the C(15)═C(16) double bond of an open-chain tetrapyrrole chromophore, resulting in a flip of its D-ring. In an important recent development, bacteriophytochrome (Bph) has been engineered for use as a fluorescent marker in mammalian tissues. Bphs covalently bind a biliverdin (BV) chromophore, naturally abundant in mammalian cells. Here, we report an ultrafast time-resolved mid-infrared spectroscopic study on the Pr state of two highly related Bphs from Rps. palustris , RpBphP2 (P2) and RpBphP3 (P3) with distinct photoconversion and fluorescence properties. We observed that the BV excited state of P2 decays in 58 ps, while the BV excited state of P3 decays in 362 ps. By combining ultrafast mid-IR spectroscopy with FTIR spectroscopy on P2 and P3 wild type and mutant proteins, we demonstrate that the hydrogen bond strength at the ring D carbonyl of the BV chromophore is significantly stronger in P3 as compared to P2. This result is consistent with the X-ray structures of Bph, which indicate one hydrogen bond from a conserved histidine to the BV ring D carbonyl for classical bacteriophytochromes such as P2, and one or two additional hydrogen bonds from a serine and a lysine side chain to the BV ring D carbonyl for P3. We conclude that the hydrogen-bond strength at BV ring D is a key determinant of excited-state lifetime and fluorescence quantum yield. Excited-state decay is followed by the formation of a primary intermediate that does not decay on the nanosecond time scale of the experiment, which shows a narrow absorption band at ～1540 cm(-1). Possible origins of this product band are discussed. This work may aid in rational structure- and mechanism-based conversion of BPh into an efficient near-IR fluorescent marker.