Article pubs.acs.org/JPCB
Vibrationally Coherent Preparation of the Transition State for Photoisomerization of the Cyanine Dye Cy5 in Water Michael M. Bishop,† Jerome D. Roscioli, Soumen Ghosh, Jenny Jo Mueller,‡ Nolan C. Shepherd,§ and Warren F. Beck* Department of Chemistry, Michigan State University, East Lansing, Michigan 48824-1322, United States S Supporting Information *
ABSTRACT: Femtosecond pump−continuum probe spectroscopy with impulsive excitation was employed to observe coherent wavepacket motions of the cyanine dye Cy5 in water that promote photoisomerization after optical preparation of the first excited singlet state, S1. The chief component in the excited-state vibrational coherence is a resonance Raman-inactive, 273 cm−1 mode of mixed carbon−carbon bond length alternation and out-of-plane or twisting character. The ultrafast (30 fs) damping of these motions arises from trajectories that irreversibly cross the transition state barrier; after several recurrences to the transition state region, vibrational cooling traps a significant fraction of the excited-state molecules in the planar, Franck−Condon region of the potential energy surface. Motion in the 273 cm−1 promoting mode is apparently launched by a change in conformation of the conjugated polyene backbone during the first few vibrations of the high-frequency C−C and CC bond length alternation coordinates that principally contribute to the initial displacement from the Franck−Condon structure. To our knowledge, this work provides the first direct observations of the intramolecular redistribution of excited-state potential energy into reactive motions that are rapidly damped by transition state barrier-crossing events leading to photoisomerization in a conjugated polyene. These results provide insight into the vibrational dynamics that contribute to the photoisomerization of retinal protonated Schiff bases in the rhodopsins and to the formation of intramolecular charge transfer character in carotenoids in photosynthetic light-harvesting proteins.
■
INTRODUCTION Like the protonated Schiff bases (PSBs) and carotenoids involved in vision and photosynthetic light harvesting, respectively, the cyanines1 (Figure 1) exhibit excited-state torsional dynamics of a central conjugated polyene backbone after π → π* excitation. As indicated by an inverse dependence on temperature and solvent viscosity for the fluorescence quantum yield2 and fluorescence lifetime,3 large-amplitude torsional motions of the conjugated polyene mediate fluorescence quenching via trans−cis photochemistry or intersystem crossing to the first excited triplet state, T1. These processes are related to fluorescence blinking phenomena in single-molecule experiments4 and to photoswitching or photochromism. Nevertheless, certain cyanines are widely employed as fluorescent probes in imaging applications,5−7 single-molecule spectroscopy,8 or as sensitizers in solar cells.9,10 © 2015 American Chemical Society
The key feature in the excited-state potential energy surfaces of polyenes that controls photochemistry and nonradiative decay is a conjugation-length dependent transition state barrier that divides planar and twisted conformations.11 Cyanines with a single conjugated pair of carbon−carbon bonds, such as 1122C or 1144-C, exhibit an effectively barrierless potential energy surface.12−18 The vibrational modes that promote the torsional photophysics in these molecules are resonance Raman active; a potential energy gradient along torsional coordinates leads directly from the Franck−Condon geometry in the S1 state to a conical intersection with the ground state, S0, at 90° twisted conformations.19 Near the conical intersection, the biradicaloid Received: March 11, 2015 Revised: May 17, 2015 Published: May 20, 2015 6905
DOI: 10.1021/acs.jpcb.5b02391 J. Phys. Chem. B 2015, 119, 6905−6915
Article
The Journal of Physical Chemistry B
Figure 2. Potential energy curves and scheme for nonradiative decay and photoisomerization of cyanines and protonated Schiff bases with medium to long conjugation lengths, after similar diagrams by Sanchez-Galvez et al.11 The reaction coordinate corresponds to sequential activity principally along bond length alternation and torsional (ϕ) coordinates on the S1 state potential surface, starting from the Franck−Condon (FC) geometry and leading to the ϕ = 90° twisted minimum and a conical intersection with the ground state, S0. The transition state potential energy barrier (‡) on the S1 state surface marks a division of the potential energy surface between planar and twisted conformations. The character of the motion is depicted by planar and twisted ethylenic structures, the latter emphasizing the biradical character of the twisted structures past the transition state.
torsional coordinates that contribute to photoisomerization, however, the Franck−Condon structure lies at a potential energy minimum.11,27,28 How the torsional motions that lead to formation of the transition state are activated in cyanines and PSBs is accordingly an important dynamical question. Further, in bacteriorhodopsin and rhodopsin, where relatively high product quantum yields for photoisomerization are observed, the steric and electrostatic environment of the binding site for the retinal chromophore impacts the minimum-energy path taken on the S1-state potential energy surface. The presence of charges and dipoles in the surrounding medium is especially important owing to the development of a charge-transfer character that accompanies twisting and the assumption of a biradicaloid character. Hynes and co-workers29 have recently discussed the coupling of excited-state torsions in cyanines and PSBs with intramolecular charge transfer and the associated role of polar solvation, which increases the rate of nonradiative decay through the conical intersection and favors formation of ground-state photoisomers. For intermediate-length cyanines, such as Cy3 and Cy5, the activation barrier between planar and torsionally displaced conformations on the S1 potential surface is low enough to permit a fraction of the molecules to escape the Franck− Condon minimum. These systems accordingly provide interesting examples in which the dynamics that precede photoisomerization or intersystem crossing can be studied optimally, and the results are potentially relevant to an understanding of the factors that control the quantum yield of isomerization of PSBs in solution and in proteins.30−34 In this contribution, we report the use of femtosecond spectroscopy to monitor directly the evolution of the excited-state structure of Cy5 in water prior to the crossing of the activation barrier. For the first time in a system of this type, we directly
Figure 1. Example structures of cyanine dyes.
character of the twisted polyene20 enables intersystem crossing to the first excited triplet state, T1.21,22 The activation energy required to access twisted conformations increases with the length of the conjugated region because the π* character of the S1 state decreases. The impact of the transition state barrier on the dynamics in cyanines can be qualitatively judged from the line shape of the fluorescence spectrum. For example, the fluorescence of 1144-C is weak and its spectrum is broadened because emission competes with the escape of excited-state molecules from the Franck−Condon region along torsional coordinates.17 In contrast, the absorption and fluorescence spectra of HDITCP are essentially mirror symmetric;23 most of the excited-state ensemble is confined to a planar region of the S1-state potential energy surface by a significant barrier. The barrier height has a commensurate effect on the photoisomerization quantum yield, which can be assessed by fluorescence correlation spectroscopy. As an example, the photoisomerization yield for Cy3 is three times larger than that of Cy5, which has only one additional CC bond.24 Figure 2 describes the potential energy surfaces of the S1 state of cyanines and PSBs with medium to long conjugation lengths. Upon optical excitation, these molecules evolve by compressing the C−C bonds and stretching the CC bonds. The resulting displacement from the Franck−Condon structure is essentially an inversion of the pattern of bond length alternation in the conjugated polyene.25,26 With respect to the 6906
DOI: 10.1021/acs.jpcb.5b02391 J. Phys. Chem. B 2015, 119, 6905−6915
Article
The Journal of Physical Chemistry B
bandpass. In order to obtain dichroism-free signals, the planes of linear polarization of the pump and probe beams were set to be 54.7° apart at the sample using calcite polarizers and λ/2 − retarding wave plates. The probe beam was detected by an amplified photodiode (Thorlabs PDA-55) after it passed through the sample and then through a monochromator (Spectral Products CM112) with a 4 nm bandpass. Prior to the sample, the pump and probe beams were simultaneously modulated by a dual-frequency wheel mounted on a Palo Alto Research model 300 chopper. The pump−probe signal was obtained as ΔT/T, the normalized pump-induced change in probe transmission; ΔT was obtained with an SRS SR-830 lock-in amplifier referenced to the sum of the pump and probe modulation frequencies (4.1 kHz), and T was obtained with an SRS SR-850 lock-in amplifier referenced to the probe modulation frequency (2.5 kHz). The ΔT/T signal exhibited a flat baseline at negative probe delays far from the pump− probe zero of time and was completely free of background signals from pump and probe light scattering. One-color transient grating and stimulated photon-echo peak-shift (3PEPS) measurements36−38 were performed with a diffractive-optic based, passively phase-stabilized, three-pulse photon-echo spectrometer. The layout of the spectrometer was based on designs discussed by Brixner et al.39 and by Moran and Scherer.40 The results reported here were obtained with homodyne detection, without the use of a local oscillator beam, and with integration over the spectrum of the third-order signal. The coherence delay, τ, between the first two pulses was controlled by translating wedge-shaped prisms across the beams.39 The pump−probe population delay T between the second and third pulses was controlled by a time-of-flight delay. Both time axes were driven by Melles-Griot Nanomover actuators. For the 3PEPS experiment, τ was scanned from negative (nonrephasing) to positive (rephasing) values while maintaining a fixed delay T between the second and third pulses. For the transient grating experiments, τ was set to zero and T was scanned. In both experiments, the third-order signal radiated in the −k1 + k2 + k3 direction, as defined by the three incident pulse directions ki in the forward boxcars arrangement, was isolated by a set of apertures. The signal was detected directly with a photodiode and an SRS SR-850 lock-in amplifier, which was referenced to the sum of the pump and probe modulation frequencies. The pump−probe and photonecho detection systems were controlled by LabVIEW routines.
observe the molecular vibrations that promote formation of the transition state, and in this case, we find that the barrier crossing events occur in a vibrationally coherent regime. Especially strong activity is observed in a very rapidly damped 273 cm−1 mode that combines stretching and torsional motions of the conjugated polyene.
■
EXPERIMENTAL SECTION Sample Preparation. Solutions of the sulfonated, aminemonoreactive, N-hydroxysuccinimidyl (NHS) ester of Cy5 (GE Healthcare, PA25001), used as received, were prepared in a 25 mM sodium phosphate buffer solution at pH 7.0. The NHS ester moiety, at the end of a (CH2)5 tether, hydrolyzes rapidly in aqueous solution to yield a carboxylate.3 For femtosecond spectroscopy, the Cy5 samples were held at room temperature in a stirred quartz cuvette (1 mm path length), with the absorbance adjusted to 0.3 at the center of the spectrum of the 665 nm laser pulses used in femtosecond spectroscopic experiments. Linear Spectroscopy. Absorption spectra were acquired with a Hitachi U-4001 spectrophotometer (2 nm bandpass). Fluorescence spectra were recorded using a home-built fluorescence spectrometer consisting of a Jobin-Yvon AH10 100 W tungsten-halogen light source, a Jobin-Yvon H10 excitation monochromator (4 nm bandpass), an Acton Research SP-150 emission spectrograph (2 nm bandpass), and a Jobin-Yvon Symphony back-illuminated CCD detector. Compensation for the wavelength dependence of the efficiency of the detector and spectrograph was performed with a reference spectrum from a quartz-halogen source. As presented as a function of wavenumber, the fluorescence intensities are multiplied by the square of the wavelength in order to compensate for the fixed (in wavelength units) spectral bandpass of the emission spectrograph.35 The fluorescence spectrometer was controlled by LabVIEW (National Instruments) programs. Femtosecond Spectroscopy. Femtosecond pump−continuum-probe experiments were conducted with excitation pulses from an optical parametric amplifier (OPA, Coherent OPA 9450), which was driven by a 250 kHz regeneratively amplified Ti:sapphire laser (Coherent RegA 9050 amplifier and Coherent Mira-Seed oscillator). The oscillator and amplifier were continuously pumped by Coherent Verdi V5 and V10 Nd3+:YVO4 lasers, respectively. The signal-beam output of the OPA was compensated for group-delay dispersion by an SF10 Brewster prism pair and variably delayed by a retroreflector on an optical delay line driven by a Melles-Griot Nanomover actuator. The pump−pulse spectrum was determined to be 19 nm in width (430 cm−1, fwhm) and centered at 665.3 nm with an Ocean Optics U2000 spectrometer (0.5 nm bandpass). The pump-pulse duration was estimated to be ≤40 fs in duration (fwhm, Gaussian shapes assumed) from a zero-background autocorrelation trace obtained with a 100 μm KDP crystal. Probe pulses for pump−probe experiments were obtained from a single-filament femtosecond continuum, which was generated in a sapphire plate in the OPA. The chirp on the probe beam was determined from heterodyne-detected optical Kerr-effect (OKE) measurements in a CS2 sample. Timeresolved spectra were recorded point-by-point by scanning the probe wavelength at a given pump−probe delay, so the chirp could be directly compensated by adjusting the pump−probe delay as a function of the probe wavelength. The rise time for the pump−probe signal was typically 75 fs for a 4 nm probe
■
RESULTS Linear Spectroscopy. Figure 3 shows the continuous-wave absorption and fluorescence spectra obtained from Cy5 in water. Both spectra exhibit a vibronic progression in an 1173 cm−1 mode. The most intense peaks in both spectra correspond to the 0−0 vibronic transition; the 0−1 and 0−2 peaks are observed in both as partially resolved satellites. The mode frequency identifies activity in the bond length alternation coordinates of the conjugated polyene backbone. The vibronic peaks are fit well by log-normal (asymmetric Gaussian) lineshapes,41 with 1760 cm−1 line widths (fwhm) and skews (asymmmetry factors) of 1.2. Using the relative areas of the 0− 0 and 0−1 peaks in the absorption spectrum as estimates for the Franck−Condon factors, the vibronic progression can be described with a Huang−Rhys factor, S = 0.315, which corresponds to a dimensionless ground-to-excited-state displacement, Δ, of 0.78.42 The solvation reorganization energy, λ = 220 cm−1, is estimated as one-half of the Stokes shift from the 6907
DOI: 10.1021/acs.jpcb.5b02391 J. Phys. Chem. B 2015, 119, 6905−6915
Article
The Journal of Physical Chemistry B
Figure 3. Continuous-wave absorption (blue) and fluorescence (red) spectra from Cy5 in water at room temperature, the latter with excitation at 660 nm (15152 cm−1). The spectra are plotted as relative dipole strengths, ε(ν)/ν and F(ν)/ν3, respectively, with normalization at peak intensity. Superimposed on the fluorescence spectrum is the mirror image (red dotted curve) of a fitted model for the absorption dipole-strength spectrum.
0−0 absorbance peak to the 0−0 fluorescence peak. The Stokes shift arises in this case from a nonpolar solvation response.43 The vertical change in permanent dipole moment that accompanies the π → π* transition is very small because the conjugated polyene has a 2-fold mirror plane of symmetry.23 The fluorescence spectrum of Cy5 deviates from mirror symmetry with the absorption spectrum owing to an excitedstate change in structure. The mirror reflection of the model for the absorption spectrum shown in Figure 3 is with respect to the wavenumber for the 0−0 transition, 15206 cm−1, where the absorption and fluorescence spectra cross. The region of the 0− 1 peak in the fluorescence is significantly more intense than the 0−1 peak in the absorption spectrum. The perturbed fluorescence spectrum indicates that an excited-state change in structure that affects the symmetry of the conjugated polyene backbone occurs on a short timescale after optical preparation of the S1 state. Femtosecond Spectroscopy. In all the experiments reported in the following, the OPA was tuned to 665.3 nm (15030 cm−1, 430 cm−1 fwhm, ≤40 fs duration pulses from autocorrelation measurements), so the excited spectral region includes the wavenumber of the 0−0 transition and spans almost 400 cm−1 to the red of it. These pulses prepare the S1 state near to the v = 0 vibrational level for the high-frequency modes, including the bond length alternation coordinates. In one-color transient-grating and 3PEPS experiments, these OPA pulses were split into pump and delayed probe pulses. In the pump−continuum-probe experiments, which are discussed first, the probe pulses were derived from a femtosecond continuum spanning the 480−770 nm region. Figure 4 shows a series of time-resolved pump−continuum probe spectra from Cy5 in water at room temperature over the 500−760 nm probe region. The spectra are composed of two overlapping contributions, from photobleaching (PB) and stimulated emission (SE), which resemble the absorption and fluorescence dipole-strength spectra, respectively. An excitedstate absorption (ESA) signal is located over the 500−580 nm region, to the blue of the PB/SE peak. The time evolution of the pump−probe spectra was characterized by a global analysis using the software tools and approach previously described by
Figure 4. Time-resolved 665 nm pump−continuum−probe spectra from Cy5 in water at room temperature. The spectra are plotted as the pump-induced change in transmittance, ΔT/T; net photobleaching or stimulated emission is shaded red, whereas net excited-state absorption is shaded blue. The top panel (PB, gray hatched shading) shows the instantaneous photobleaching spectrum, the product of the pumppulse spectrum and the absorption dipole-strength spectrum from Cy5 in water (see Figure 2). The left panel plots the 500−600 nm excitedstate absorption region with an expanded abscissa and a 3.6× expansion of the ordinate to show the detail in the excited-state absorption region.
van Stokkum et al.44−46 The results of this analysis describe the time evolution of the pump−probe spectrum in terms of a model composed of a set of principal spectral/kinetic components and timescales; the model response is convoluted with the pump−probe instrument-response function. Singular value decomposition of the pump−probe spectral data spanning the −60 fs−141 ps range indicates that three components are required at minimum to describe the spectral response by Cy5 in water. The decay-associated spectra (DAS) and evolution-associated spectra (EAS) determined by a global analysis for a three component model are shown in Figure 5. A probe dispersion component was not included in the model because the chirp on the femtosecond continuum was characterized independently using OKE signals and then compensated point-by-point to maintain a fixed pump−probe delay as the probe monochromator was scanned. Further, the fit to the pump−probe spectra was not improved by inclusion of a “coherence-spike” feature, here implemented as a Gaussian centered at the 665 nm peak of the pump spectrum. The spectral evolution reported by the global model is best interpreted using the EAS, which characterize the sequential formation of two intermediate species associated with the time evolution of the structure of the Cy5 chromophore and the reorganization of the surrounding solvent. The first and second EAS shown in Figure 5 indicate that significant changes occur 6908
DOI: 10.1021/acs.jpcb.5b02391 J. Phys. Chem. B 2015, 119, 6905−6915
Article
The Journal of Physical Chemistry B
and sharpening of the ESA to the blue and a shift of the SE to the red. The second intermediate decays back to the ground state with an estimated time constant of 446 ps. The distortion of Cy5’s conjugated polyene backbone on the 1 ps damping times that are usually characteristic of resonance Raman active modes in π → π* chromophores such as porphyrins,54 perhaps because of interference between modes of comparable frequency in the low-frequency regime (see the normal mode calculation results discussed in Figure S4 of the Supporting Information). The 3PEPS response (Figure 8) shows that a majority fraction of the nonpolar solvation of Cy5 occurs on the 30 fs timescale. Only the 2.7 ps component in the 3PEPS response describes the characteristic timescale for motions of the surrounding aqueous medium that are slow enough to contribute to frictional damping of the 273 cm−1 mode. A blue shift of Cy5’s ESA spectrum on the
