Femtosecond Time-Resolved Raman Spectroscopy Reveals

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A: Spectroscopy, Photochemistry, and Excited States

Femtosecond Time-Resolved Raman Spectroscopy Reveals Structural Evidence for meta Effect in Stilbenols Syed M. Bilal, Surajit Kayal, Krishnankutty S Sanju, and Yapamanu Adithya Lakshmanna J. Phys. Chem. A, Just Accepted Manuscript • DOI: 10.1021/acs.jpca.7b12339 • Publication Date (Web): 23 Apr 2018 Downloaded from http://pubs.acs.org on April 24, 2018

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The Journal of Physical Chemistry

Femtosecond Time-Resolved Raman Spectroscopy Reveals Structural Evidence for meta Effect in Stilbenols Syed M Bilal1, Surajit Kayal2, Krishnankutty S Sanju1 and Y Adithya Lakshmanna1,* 1

School of Chemistry, Indian Institute of Science Education and Research Thiruvananthapuram (IISER-TVM), Vithura, Thiruvananthapuram 695551, India 2

Department of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore, 560012, India E-mail: [email protected]

Abstract The meta effect in substituted aromatics plays a crucial role in their excited state photophysical properties. Meta-substituted hydroxyarenes such as naphthols, stilbenols, and chromophoric constituents of green fluorescent proteins show unusual photoacidity and enhanced fluorescence lifetime and quantum yield when compared to their para-derivatives. Variation in the excited states features of the meta-derivatives when compared to the para-derivatives in stilbenols has been attributed to the enhanced torsional barrier for interconversion between the planar and the twisted perpendicular forms. Herein, we employed femtosecond time-resolved Raman spectroscopy to provide the direct structural evidence for the enhanced torsional barrier in metastilbenol. The Raman band profiles of the olefinic C=C stretch related to the torsional motion are found to decay with time constants of ~750 ps and ~13 ps in meta-stilbenol and para-stilbenol respectively, unraveling the structural evidence for the observed enhanced photoacidity originating from enhanced rates of excited state proton transfer. Further, time-resolved fluorescence measurements are carried out to elucidate the relaxation pathways of the excited states of the stilbenols.

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1. Introduction Hydroxyarenes are known to exhibit acidity in the ground as well as electronically excited states.1-2 A competition between the decay rates of the excited singlet states and the excited state proton transfer (ESPT) rates dictates the extent of photoacidity in hydroxyarenes. Stilbenols, a class of hydroxyaromatic compounds are also expected to exhibit photoacidity. However, the photoacidity of stilbenols is found to be strongly dependent on the positioning of the hydroxyl groups in the stilbene moieties.3-8 Lewis et al. recently demonstrated exceptional photoacidity of m-hydroxystilbene (MHSB) and its cyano-derivative, while p-hydroxystilbene (PHSB) and its cyano-derivative showed weak photoacidity.9-11 The enhancement in photoacidity in the meta isomer is due to the favorable localization of electronic density at the appropriate site in its excited state leading to facile proton transfer, often referred to as the meta effect.7-8 Upon photoexcitation of the stilbene moiety, the trans form undergoes ultrafast trans-cis photoisomerization by initially populating an 1S* state (planar form) which evolves through a phantom state 1P* (perpendicular form), subsequently relaxing to the ground state with equal amounts of the cis and the trans forms.12-15 The challenge in the molecular design of photoacids is therefore to propose systems that can achieve longer excited state lifetimes, leading to facile proton transfer in the excited states. Substituent groups such as amino, hydroxyl and methoxy group, possessing large negative values of the Hammett parameter σp+ (an indicator of electron withdrawing/donating abilities of substituents) stabilize the excited states of the parent molecules effectively.16-17 Furthermore, the relative stabilization of the 1S* and the 1P* states by the substituent groups governs the energy barriers for transformation between the two states. The substituent groups located at meta positions are found to stabilize the 1S* states far more effectively than the 1P* states, leading to large torsional barriers for interconversion and longer excited state lifetimes for the stilbene-derivatives. As a consequence, ESPT process becomes facile, leading to enhanced photoacidity. m-aminostilbene and its derivatives were recently shown to exhibit longer excited singlet state lifetimes (~12 ns) when compared to paminostilbenes (~0.1 ns).18-19 Amino group at m-position could not stabilize the 1P* state effectively in comparison with the 1S* state, leading to large torsional barrier between these two states. The dipole moment of excited MHSB is higher than that of excited PHSB, although their ground state dipole moment values are comparable.10 Hence, a polar solvent such as acetonitrile 2 ACS Paragon Plus Environment

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(ACN) can induce an increase in the energy gap between the planar and the perpendicular forms in MHSB. The plausible mechanism for the excited m- and p-stilbenols in ACN is shown in Scheme 1. In m-stilbenol, the excited singlet state 1S* is more stabilized when compared to the 1

P* state. As the 1P* state is higher in energy than the 1S* state, excited species would prefer to

go through the relaxed 1S* state. However, in case of p-stilbenol, 1S* state is higher in energy, hence the excited species would have significant propensity to take the path through the 1P* state. Scheme 1: Schematic representation of the excited state evolution of m- and p-stilbenols in polar solvent, acetonitrile. Both the stilbenols are initially in their trans forms and the reaction coordinate is the torsional angle for motion around the C=C bond. Cet=Cet stretch and Cet-Cφ stretch are highlighted with red and green colors in the molecular structure. OH OH

OH

Cet=Cet (=) Cet-CΦ (-)

OH OH

OH

OH

1P* 1S*

1S*

1P*

MHSB 0o trans

90o

PHSB 180o cis

0o trans

90o

180o cis

Interesting parallels can be drawn between the nature of hydroxystilbenes and fluorescent chromophores in green fluorescent proteins (GFP). The chromophore responsible for the efficient fluorescence of GFP20-25 comprises of p-hydroxybenzolideneimidazolinone (p-HBI), which is formed autocatalytically from the residues 65-67 i.e. Ser-65, Tyr-66 and Gly-67. p-HBI has a p-phenol moiety with an efficient electron withdrawing imidazolinone ring bound to it. This chromophore can be viewed as a member of the family of hydroxyarenes, well known for their photoacidity. Generally, the p-phenol moiety is replaced with natural amino acids such as 3 ACS Paragon Plus Environment


tryptophan, phenylalanine and histidine to obtain modified GFPs. Recently, Dong et al.26 have demonstrated distinct photophysical properties of dimethyl derivative of HBI (HBDI) with hydroxyl groups at p- and m-positions. The lifetimes of m-HBDI and p-HBDI in excited state are found to be 90.0 ps and 1.5 ps, respectively. The observed fluorescence with 1.5 ps lifetime component in p-HBDI is a consequence of ultrafast cis-trans photoisomerization with respect to the C=C bond, while the 90.0 ps component in m-HBDI can be ascribed to the restricted rotation around the C=C bond with large torsional barrier. Similarly, m-amino derivative of benzolideneimidazolinone (m-ABDI) is shown to have an excited state lifetime of ~5.0 ns in non-polar solvents.24 However, excited lifetime is found to drastically decrease in polar protic environments due to the opening up of an efficient non-radiative decay channel. These results are in parallel with the observations made in case of MHSB and PHSB by Lewis et al.9-11 Recently, Mathies and co-workers, Tahara and co-workers have shown the importance of various skeletal motions during the excited state proton transfer in excited GFP by measuring the excited state vibrational features using ultrafast stimulated Raman spectroscopies.27-28 Photoacidity in general is estimated based on the measurement of fluorescence of the activated hydroxyarenes (ROH*) and their conjugate bases (RO-*). However, in some cases, non-fluorescent or weakly fluorescent nature of RO-* and ultrafast proton transfer in ROH* limit the use of fluorescence-based techniques for probing photoacidity. In such cases, employing ultrafast transient absorption spectroscopic methods has resulted in the successful characterization of the excited states. Thus far, femtosecond transient absorption and emission spectroscopic methods have been employed as invaluable tools for discerning the underlying reason for the unusually long lifetime of excited m-stilbenols by probing the time evolution of the excited electronic states. Such measurements revealed the crucial role of the torsional motion around the C=C bond. Although, they are still considered indirect evidences suggesting a plausible mechanism but are not adequate enough to interpret the crucial structural changes that take place in the excited states. Elucidating the time evolution of the vibrational signatures can provide a direct approach to monitor the changes that occur in molecular geometries in excited states. It is therefore very crucial to follow the central ethylenic C=C stretch frequency and other associated vibrational frequencies during the excited state evolution of stilbenols, an aspect that has not been explored so far in the literature. 4 ACS Paragon Plus Environment

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Ultrafast vibrational spectroscopy can offer unique opportunities for elucidating the early-time structural events in excited states of chromophores. Ultrafast time-resolved Raman spectroscopy has evolved as one of the state-of-the-art spectroscopic techniques in recent times for probing the structural and dynamical aspects of short-lived species. Analysis of the time-dependence of the Raman peak positions and peak widths using femtosecond stimulated Raman spectroscopy (FSRS)27,

29-32

and ultrafast Raman loss spectroscopy (URLS)33-34 can yield rich information

about the structural and dynamical aspects of the excited species.35-39 Herein, we demonstrate the utility of femtosecond time-resolved Raman spectroscopy for probing the structural evolution of excited stilbenols (MHSB and PHSB) in ACN. Aprotic nature of ACN does not affect the molecular structure much unlike protic methanol or ethanol, although the extent of stabilization of the planar 1S* and the perpendicular 1P* states is similar to methanol or ethanol, indicating that ACN is a good choice for understanding the structural evolution of excited MHSB and PHSB. Our analysis of the Raman band integrals for the torsional modes in MHSB and PHSB provides direct experimental evidence for the existence of large torsional barriers and long excited state lifetimes in MHSB.

2. Experimental Methods 2.1 Steady-state measurements Steady-state absorption and emission measurements were carried out using Cary-300, UVVisible spectrophotometer and Horiba Jobin Yvon Fluorolog-3, spectrofluorimeter equipped with a 450 W Xe lamp and a Hamamatsu R928 photomultiplier tube, respectively. Steady state Raman measurements were carried out for the PHSB and MHSB crystals using confocal Raman microscope from Renishaw by exciting at 785 nm.

2.2 Time-resolved fluorescence measurements Time-resolved fluorescence measurements were carried out using time-correlated single photon counting (TCSPC) from Horiba JobinYvon-IBH (Fluorocube) system and femtosecond fluorescence up-conversion system from CDP systems. Briefly, in the fluorescence upconversion set up, the sample was excited by a pump pulse having central wavelength at 266 nm, 5 ACS Paragon Plus Environment


pulse width of 120 fs, which is obtained by a third harmonic generation of the fundamental light centered at 800 nm and pulse width of 100 fs, with a repetition rate of 80 MHz obtained from the Oscillator MaiTai-HP. The fluorescence from the system and the residual fundamental light are then focused onto the nonlinear BBO crystal to generate a sum frequency signal, which is essentially the fluorescence up-converted signal. The up-converted signal is effectively allowed to pass through a double monochromator and ultimately detected by a high-sensitive photomultiplier tube, which is coupled with a photon counter.

2.3 Time-resolved Raman measurements We have adopted the URLS method33-34 to probe the early events of excited stilbenols by employing the following protocol: the photo-pump or actinic pump is set at 305 nm, with 100 fs pulse-width and energy of ~ 1 µJ; Raman pump is set at 580 nm, with ~ 20 cm-1 bandwidth and energy of ~ 300 nJ. Raman probe, a white light continuum is generated by focusing ~790 nm fundamental light from the amplifier onto a ~2 mm thick CaF2 crystal with repetition rate of 1 kHz. Choppers have been placed in the paths of the actinic pump and the Raman pump and are operated at appropriate frequencies to measure the pump on/pump off signals. The molecules under investigation, MHSB and PHSB are prepared by following the reported procedures.9-11

3. Results and Discussion 3.1 Steady-state measurements The absorption and emission spectra of PHSB and MHSB in ACN are shown in Figure 1. It is clear from the figure that the absorption and emission features of both the isomers are similar. The spectral positions of PHSB however are red-shifted with respect to those of MHSB. The maxima of absorption and emission are found to occur at ~300 nm and ~380 nm, respectively for the isomers. The observed spectral features are in agreement with the reported data.9


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MHSB P HSB

1.0

1.0

0.8

0.8

0.6

0.6

0.4

0.4

0.2

0.2

0.0

Normalized Intensity (a.u)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Normalized Absorbance (OD)

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0.0

300

400

500

Wavelength (nm)

Figure 1: Steady state absorption (solid lines) and emission spectra (dashed lines) of PHSB and MHSB in ACN.

3.2 Time-resolved measurements Initially, ultrafast transient absorption spectroscopic measurements are carried out on MHSB and PHSB. The femtosecond transient absorption (TA) spectra of these molecules in acetonitrile (ACN), a polar aprotic solvent are presented in Figure 2. PHSB initially has a TA band centered at ~480 nm, which exhibits a nominal blue shift with time. A second order scattered light of ~308 nm appears at ~620 nm in TA. Therefore, this portion has been avoided during TA analysis of PHSB. It is noticed that TA in PHSB decays significantly beyond a time delay of 50 ps, clearly indicating that the excited PHSB has a short lifetime. In contrast, in the case of MHSB, TA initially peaks at 550 nm and shifts to ~525 nm. The band at 525 nm however maintains significant amplitude even after a time delay of 1 ns, evidencing the existence of a long-lived excited species. The spectral features and the kinetic decay parameters resulting from these studies are listed in Table 1. The elucidated spectral features are also in reasonable agreement with the reported results.9-11



Figure 2: Transient absorption spectra of (a) MHSB and (b) PHSB in ACN, at various time delays after photo-excitation at 305 nm. We have carried out time-resolved emission studies of PHSB and MHSB in ACN using femtosecond fluorescence up-conversion and TCSPC methods, respectively. The fluorescence decay profiles for MHSB and PHSB are shown in Figure 3. Figure 3a shows the fluorescence decay profiles for MHSB at 410, 430 and 450 nm obtained using TCSPC while exciting at 377 nm. The fluorescence decay profiles are best fitted with bi-exponential decay having time constants of ~0.8 ns and ~2.0 ns. These time constants represent the lifetimes of the emissive states. The ~0.8 ns is ascribed due to the radiative lifetime of the emissive state 1S*, while ~2.0 ns component could be due to the other decay channels from the 1S* state. Figure 3b shows the fluorescence decay profiles for PHSB in ACN at 460, 470 and 490 nm, obtained by exciting it at 266 nm. The decay profiles are best fitted with bi-exponential decay having time constants of ~ 5.0 ps and ~ 12.0 ps. The 12.0 ps time component is assigned to the lifetime of the emissive state i.e. the planar state, 1S*. The ~5.0 ps component can be attributed to the vibrational relaxation. It is ideal to have kinetics of the full emission spectra in order to follow the emissive states. However, we could not obtain the kinetics at shorter wavelengths due to limitations in our upconversion experimental set up. We could not find an appropriate fluorescent system as a standard to optimize for the up-converted signal in this region. Hence, we restricted the experiments only to the red-edge of the fluorescence emission spectrum. In case of femtosecond fluorescence up-conversion experiment, we could optimize the set up with the standard fluorescent dye, Coumarin 343 in methanol, whose emission spectrum covers the red-edge of the emission spectrum of PHSB. For MHSB, up-conversion measurements were not successful, due 8 ACS Paragon Plus Environment

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to the photobleaching in the sample. We believe that it is due to the long excited state lifetime and high repetition rate of the excitation source (80 MHz) i.e., excited molecules have not reached their ground state completely prior to the arrival of the next pulse (after ~12 ns). In case of TCSPC, the instrument response function (IRF) or temporal resolution for the 377 nm excitation is ~200 ps. Excitation sources having wavelengths below 377 nm possess IRF of ~800 ps, hence these sources were not chosen for the measurements. A summary of the analysis of the fluorescence decay profiles is presented in Table 1.

a

410 nm 430 nm 450 nm Prompt

4

10

3

b

2

10

460 nm 470 nm 490 nm

0.9

Counts

10

Counts

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.6

0.3 1

10

0.0

0

10

0

10

20

30

40

-10

50

0

10

20

30

40

50

60

70

time (ps)

Time (ns)

Figure 3: Time-resolved fluorescence measured at various wavelengths as labeled for (a) MHSB and (b) PHSB in ACN by exciting at 377 nm and 266 nm, respectively. Table 1: Kinetic parameters for the time-resolved absorption and emission spectra of MHSB and PHSB in ACN. Fluorescence

MHSB

TA (Global Analysis)

410 nm

430 nm

450 nm

τ1 (ps)

830.00 (54%)

850.00 (54 %)

980.00 (58%)

0.50

τ2 (ps)

1980.00 (46 %)

2000.00 (46 %)

2240.00 (42 %)

32.50 1500.00

τ3 (ps) 460 nm

470 nm

490 nm

τ1 (ps)

4.60 (66 %)

4.50 (60 %)

4.80 (64 %)

0.20

τ2 (ps)

12.60 (34 %)

10.50 (40 %)

10.20 (36 %)

0.75

PHSB 14.0

τ3 (ps)



Recently, Han et al. demonstrated the changes in vibrational spectral features of pyranine, a commonly used photoacid, in water from ground state (protonated form) to excited state (deprotonated form) using femtosecond stimulated Raman spectroscopy.40 In order to validate the Raman signatures of the excited states of stilbenols, we have also carried out the ground state Raman measurements of the stilbenols. Figure 4 shows the Raman spectra of PHSB and MHSB in the crystalline form. Both the molecules exhibit intense Raman peaks at 1635, 1595 and 1192 cm-1, in agreement with the reported Raman bands.41 The Raman bands at 1635, 1595 and 1192 cm-1 correspond to the Cet=Cet and Cet-CΦ stretch frequencies, respectively. The steady-state Raman measurements of molecules in crystalline form essentially provide their vibrational signatures in the ground state. When the molecules are photo-excited to higher energy levels, the incident light causes a perturbation. Time-resolved Raman measurements of photo-excited molecules provide the vibrational signatures of the excited molecules and can be used to assess the perturbation on the molecules caused by the incident light. The emphasis here is to monitor the changes in the vibrational frequencies in the excited state as these modes have a strong correlation with the torsional motion around the C=C bond.

1635

MHSB PHSB 1595

1.0

0.8

1192

N orm alizedIntensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

0.6

0.4

0.2

0.0 800

1000

1200

1400

1600

1800

-1

Raman shift (cm )

Figure 4: Steady state Raman spectra of MHSB and PHSB in crystalline form. Spectra were recorded by exciting at 785 nm. A π-π* electronic excitation (~ 305 nm) in stilbenols causes an excitation to the Franck-Condon (FC) active modes. Among the FC modes, olefinic C=C (Cet=Cet) stretch is majorly activated during the π-π* excitation. Transient Raman spectra of MHSB and PHSB in ACN, obtained 10 ACS Paragon Plus Environment

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using the URLS method are shown in Figures 5a and 5b. It is evident from Figure 5 that MHSB (PHSB) shows prominent FC-active Raman bands centered at ~1145 (1165), 1220 (1235) and 1535 (1515) cm-1, which are assigned to the Cet-CΦ stretch, Cet-H in-plane bend and Cet=Cet stretch vibrations, respectively. The Raman band positions observed in URLS are red shifted when compared to the ground state positions, clearly indicating the weakening of the corresponding bonds in the excited states. Recently, Weigel et al.39 have reported a wavepacket analysis of the Raman modes of excited stilbene, with a detailed analysis of the 1550 cm-1 mode, showing an anharmonic coupling between the C=C stretch and the phenyl/ethylenic torsional modes. Hence, it is clear that the dynamics of the ~1520 cm-1 mode in both PHSB and MHSB represents a direct measure of the nature of the barrier for torsional motion at the Cet=Cet bond. In contrast, transient absorption and fluorescence measurements can only provide indirect evidences for the structural changes in excited states, leading to the meta effect. URLS spectra of MHSB and PHSB are obtained by using Raman pump centered at 580 nm, which is onresonance with the excited state absorption. Therefore, it is clear that the Raman signatures in URLS essentially originate from the excited trans-forms of PHSB and MHSB. The contributions from the ground and excited cis-forms to the URLS spectra are thus ruled out.



Figure 5. Time-resolved URLS spectra of (a) MHSB and (b) PHSB in ACN; Kinetics of the torsional motion around the Cet=Cet bond in (c) MHSB and (d) PHSB. We therefore further monitored the band integrals of the ~1520 cm-1 modes in MHSB and PHSB. The peak heights and peak widths get affected as the excited states evolve over time due to various solute-solvent interactions. However, the band integral (which is essentially area under the peak) is a better indicator for representing the populations of the states. The kinetics of the band integrals are depicted in Figures 5c and 5d. The kinetics is best fitted with multiexponentials, whose values are summarized in Table 2. The shortest lifetime of the band integrals in MHSB and PHSB is ~ 0.4 ps. This could be attributed to the intramolecular vibrational redistribution arising due to dynamic solvation in ACN. The longest timescale for the decay of the Cet=Cet stretch in case of PHSB is ~ 13 ps, which is a typical signature for the decay of trans-stilbene and p-aminostilbene in polar solvents.18-19 However, in case of MHSB, the decay of the band integral of the Cet=Cet stretch shows ~ 40 ps and ~ 750 ps time constants. The longest time constant measured for MHSB is a clear evidence for the existence of a long-lived


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excited state. The observed time constants are in accordance with the values obtained from the time-resolved emission measurements. Table 2: Parameters for the kinetics of the band integral decay and peak blue shifts for the Cet=Cet and the Cet-CΦ modes in MHSB and PHSB. Time constants are represented in ps. Relative amplitudes are given in parentheses.

Band Integral

MHSB

PHSB

Peak Position

(Cet=Cet)

(Cet-CΦ)

(Cet=Cet)

(Cet-CΦ)

1535 cm-1

1145 cm-1

1535 cm-1

1145 cm-1

τ1(ps)

0.40 (26%)

0.56 (32%)

0.90 (66%)

0.50 (73%)

τ2(ps)

42.0 (29%)

32.0 (26%)

13.00 (34%)

5.40 (27%)

τ3(ps)

750.0 (45%)

770.0 (42%)

1515 cm-1

1165 cm-1

1515 cm-1

1165 cm-1

τ1(ps)

0.40 (42%)

0.44 (37%)

0.30 (55%)

0.33 (67%)

τ2(ps)

13.00 (58%)

16.00 (63%)

5.00 (45%)

6.00 (33%)

We also extended the analysis of kinetics of band integrals of the 1145 (1165) cm-1 and the 1220 (1235) cm-1 modes in MHSB (PHSB) that are shown in Figure 6 and kinetics parameters are summarized in Table 2. It is evident from the figure that, these bands in MHSB undergo a significant decay at initial times but they continue to stay on for longer time delays. The bands do not completely reach base level even after a long time delay of 1400 ps. However, in case of PHSB, band integrals decay completely in ~50 ps time window. The observed kinetics clearly show an evidence for a long-lived planar structure of the excited state of MHSB. In case of 13 ACS Paragon Plus Environment


PHSB, the band integrals decay with a time constant of ~13.0 ps, which indicates the dominance of an ultrafast trans-cis isomerization process within the lifetime of the excited state. The kinetic traces as given in Figure 6b for the Raman modes in PHSB appear to have rise components when compared to their counterparts in Figure 6a. The reason for such an appearance is the difference in the time scales presented in Figures 6a and 6b. The x-axis in Figure 6a is given up to ~1400 ps, whereas in Figure 6b, it is given only up to ~50 ps. The kinetic parameters as given in Table 2 clearly show that none of the Raman modes show any rise components. However, the difference in the appearance of the kinetics of the 1235 cm-1 and 1165 cm-1 modes in Figure 6b could be due to mode-dependent response, unlike in TA measurements, wherein it represents cumulative signature. The excited energy is initially localized into the Cet=Cet stretch, and subsequently gets redistributed among other vibrational modes. The timescales for attaining the maximum amplitude in case of each mode may be different. We also attempted to analyze the band integrals for the other low frequency modes. However, we note that the analysis becomes rather complex due to the dispersive lineshapes in the spectral region. It is well known that the low frequency modes in URLS measurements often exhibit dispersive lineshapes.34 Deconvolution of the complex dispersive lineshapes into Lorentzian profiles is rather non-trivial and is not pursued. Thus, we restricted the analysis of the Raman bands to frequencies above 1000 cm-1.

Figure 6: Kinetics of the Raman band integrals of the Cet-CΦ and the Cet-H modes in (a) MHSB and (b) PHSB in ACN. 14 ACS Paragon Plus Environment

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We also analyzed the time evolution of the peak positions for the 1535 (1515) and the 1145 (1165) cm-1 modes of MHSB (PHSB) that are given in Figure 7 and kinetic parameters are summarized in Table 2. The Raman bands reach their equilibrium positions with longest time constants of ~5.0 ps for PHSB and ~12.0 ps in case of MHSB in ACN. The time constants are in accordance with the earlier reports on picosecond time-resolved Raman studies of the parent molecule, stilbene. The time constants observed in the kinetics of the Raman peak positions of PHSB and MHSB can be attributed to the vibrational relaxation in the excited state. The time constants obtained from the kinetics of the band integrals and the peak positions are different. The reason for such difference can be traced to their origin. In case of band integrals, the time constants represent the decay of the population in the excited state, in agreement with the kinetics of TA. However, in case of peak shifts, the time constants majorly represent vibrational cooling or relaxation from higher vibrational levels to lower vibrational levels within an electronic state and conformational changes as a result of molecules reaching nearby electronic states (due to varying extent of rotation as these molecules are not so rigid in nature) possessing different vibrational frequencies. This is also clearly evident from the parameters obtained from the kinetics of the fluorescence emission in case of PHSB. However, emission studies of MHSB could not provide such a time constant due to lower time-resolution of TCSPC i.e., ~200 ps.

Figure 7: Time evolution of the peak positions for the Cet=Cet and the Cet-CΦ stretch modes in MHSB ((a) and (b)) and PHSB ((c) and (d)) in ACN. 15 ACS Paragon Plus Environment


The Raman bands around ~1160, ~1220 and ~1520 cm-1 in both MHSB and PHSB represent vibrational motions corresponding to the, Cet-CΦ stretch, Cet-H bend motions and ethylenic Cet=Cet stretch. The higher frequency of the Cet=Cet stretch in MHSB when compared to PHSB indicates higher double bond strength in MHSB and in effect governs the relative barrier for rotation around this bond. The time evolution features of these vibrational modes and the band integrals, as represented in Figures 5 and 6 are therefore indicative of the extent of planarity and a measure of the torsional barriers in the excited states of stilbenols. The long time constants arising out of the band integrals of the Cet-CΦ and the Cet=Cet modes in MHSB provide a direct evidence for the existence of a stable planar structure and a large torsional barrier in the excited state. However, we cannot exclude the possibility of photoisomerization, although the excited state lifetime is drastically increased. Murohoshi et al. have demonstrated that both PHSB and MHSB in ACN show significant photoisomerization after UV irradiation with quantum yields of 0.35 and 0.25, respectively.42 The quantum yields of fluorescence of PHSB and MHSB in ACN are found to be 0.0054 and 0.32, respectively.42 Thus, we infer that the unusually long lifetime of the excited state of MHSB essentially decelerates the isomerization process, rather than completely blocking the pathway of photoisomerization. The time constants for the URLS peak frequency shifts (vibrational cooling; ~5 ps and ~13 ps) and band integrals (population decay, ~40 ps and ~750 ps) in MHSB clearly indicate that excited molecules undergo vibrational relaxation prior to reaching twisted intermediates. Upon taking into account the kinetic parameters from time-resolved absorption, time-resolved fluorescence and time-resolved Raman studies, we summarize the pathways for the time evolution of photo-excited MHSB and PHSB in Scheme 2. The present analysis revealed from the vibrational features in stilbenols using ultrafast timeresolved Raman spectroscopy could be further seamlessly adopted to describe vibrational dynamics in chromophoric constituents of biological systems like GFP. Here, we note that femtosecond time-resolved infrared spectroscopy has been utilized in the past to understand the underlying reasons for enhanced fluorescence quantum yield in m-ABDI.24 In such contexts, Raman techniques offer enormous advantages while dealing with biological systems in aqueous media and can be effectively used in synergy with the infrared techniques. We believe that the 16 ACS Paragon Plus Environment

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current study involving stilbenols can serve as a prototype system and a starting point for unraveling the structural dynamics of chromophores responsible for the biological activities of various proteins such as GFP.

Scheme 2: Schematic representation of the potential energy surfaces for the ground and the excited states of MHSB and PHSB. URLS process: blue refers to actinic pump; orange refers to Raman pump; green refers to probe. Excited state lifetimes for MHSB and PHSB in ACN are also shown. OH

OH

OH OH

1S*

1S*

12 ps

1P*

800 ps

OH

0

1P*

OH

0

90 180 Torsional angle (degrees)

90

180

Torsional angle (degrees)

4. Conclusions In summary, by a careful investigation of the torsional motion around the olefinic C=C bonds using the femtosecond time-resolved Raman measurements, we demonstrate the structural evidence for the meta effect that is responsible for the different photoacidity of stilbenols. The effective stabilization of the 1S* state in comparison with the 1P* state leads to a large torsional barrier between the two states in case of the meta isomer. Femtosecond vibrational spectroscopy, in conjunction with transient absorption and time-resolved emission spectroscopic techniques, can unravel the structural and dynamical aspects of the excited state features in chromophoric 17 ACS Paragon Plus Environment


systems. Such comprehensive analysis can lend appropriate control over the chromophoric features, enabling better design of organic chromophores for various applications. Vibrational measurements on selective chromophoric groups as demonstrated here can also be extended to reveal signatures of structure-activity relationships in biological systems such as proteins.

Acknowledgements YAL is grateful to Prof. S. Umapathy for generously granting access to the laser facility and for his constant support. YAL acknowledges the Department of Science and Technology (DST Nanomission Project; SR/NM/NS-23/2016), Government of India for financial support. SMB and SK acknowledge the Department of Science and Technology (DST) and Council of Scientific and Industrial Research (CSIR), Government of India, respectively for the fellowships. KSS thanks IISER-TVM for financial support.


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