Photochemistry of Singlet Oxygen Sensor Green

Transcription

1 Supporting Information Photochemistry of Singlet Oxygen Sensor Green Sooyeon Kim, Mamoru Fujitsuka, and Tetsuro Majima* The Institute of Scientific and Industrial (SANKEN), Osaka University, Mihogaoka 8-1, Ibaraki, Osaka (Japan)

2 Experimental Details Materials Singlet Oxygen Sensor Green (Fl-An, Invitrogen/Molecular Probes ), 5(6)-carboxy-2,7 - dichlorofluorescein (Fl-Cl, 21882, Sigma), 5(6)-carboxyfluorescein (21877, Sigma), 9,10- dimethylanthracene (DMA, D146706, Aldrich), and α,β,γ,δ-tetrakis(1-methylpyridinium-4- yl)porphyrin (TMPyP4, A5014, TCI) were used without further purification. Anthracene (AC495, TCI) and p-pheynylenediamine (P0170, TCI) were exploited as triplet state energy donor and one electron reduction agents, respectively. Furthermore, we prepared endoperoxide form of Fl-An (Fl- EP) either by continuous irradiation of Fl-An [1, 2] or chemical method that used Na 2MoO 4 2H 2O ( , WAKO) and H 2O 2 ( , WAKO) as reported previously. [3] Using either a 50 mw cm -2 Hg lamp (REX-250, Asahi Spectra) with a bandpass filter (BP , Olympus) or 100 mw cm -2 Xe lamp (LAX-C100, Asahi Spectra) with a bandpass filter (BA , Olympus), 2 ml of Fl-An solution was irradiated in the wavelength range of or nm, respectively, with a magnetic stirring. Optical properties of fluorescein derivatives, including Fl-An and Fl-Cl, are known to be sensitive to ph because its conjugation and energy level become differed by (de)protonation of xanthene moiety (Xa) or forming a transparent lactone form. Therefore, we chose an aqueous buffer with ph 8.0 in order to maintain Fl-An and Fl-Cl as a trianionic form. Throughout this study except nanosecond laser flash photolysis, we used ph 8.0 Tris-K + buffer, which was prepared with 200 mm KCl, 4 mm MgCl 2, and 28 mm Tris-HCl. HCl and NaOH solution were added to adjust ph. Measurement of fluorescence lifetime (τ fl) and quantum yield of fluorescence (Φ fl) τ fls of Fl-An and Fl-Cl were measured using a time-resolved fluorescence microscope with a confocal optics (MicroTime 200; PicoQuant, Berlin-Adlershof, Germany). In order to measure τ fl, 70 µl of Fl-An and Fl-Cl solution in ph 8.0 Tris-K + buffer was loaded into the micro-chamber made by a 1-inch glass cover slip and Secure-Seal (S24733, Invitrogen). The samples were excited through an oil objective (Olympus, UAPON 150XOTIRF; 1.45 NA, 150x) with a 485-nm pulsed laser (PicoQuant, full width at half-maximum 120 ps) controlled by a PDL-800B driver (PicoQuant). The excitation power of ~ 0.15 µw was used. The emission was collected with the same objective and detected by a single photon avalanche photodiode (Micro Photon Devices, PDM 50CT and 100CT) through a 75-µm pinhole for spatial filtering to reject out-of-focus signals. The data collected using the PicoHarp 300 TCSPC module (PicoQuant) were stored in the timetagged time-resolved mode (TTTR), recording every detected photon with its individual timing. To determine Φ fl of Fl-An and Fl-EP, 5(6)-carboxyfluorescein (Φ fl = 0.92) was used as a reference. For the preparation of Fl-EP for Φ fl determination, a chemical method to generate 1 O 2

3 was chosen because the direct irradiation of Fl-An causes the irreversible photobleaching of fluorescein moiety (Fl), leading to the lower Φ fl than the actual value. In order to decrease the error and prevent the intermolecular interactions, we prepared the dye samples with absorbance of 0.005, 0.01, and at 465 nm. Next, the integrated areas of fluorescence spectra of the samples were calculated and linearly fitted against the responsive absorbance. Finally, Φ fl was determined by comparing the slope of the linear plots with that for 5(6)-carboxyfluorescein. As summarized in Table S1, the Φ fl of Fl-An and Fl-EP were found to be more than 20 times different, which was large enough to distinguish between Fl-An and Fl-EP. In terms of the brightness, Fl-EP was not as bright as carboxyfluorescein (Φ fl = 0.92), but still moderately bright (Φ fl 0.4) suitable for the various experiments including single-molecule fluorescence spectroscopy which requires higher Φ fl than 0.1. [4] Measurement of femtosecond laser flash photolysis (fs-lfp) The sub-picosecond transient absorption spectra were measured by the pump and probe method using a regenerative amplified titanium sapphire laser (Spectra Physics, Spitfire Pro F, 1 khz) pumped by a Nd:YLF laser (Spectra Physics, Empower 15) for each dyes dissolved by ph 8.0 Tris- K + buffer. The samples of Fl-An and Fl-Cl were prepared at the concentration where they have the same absorbance of approximately 1.5 with 0.2-cm path length ([Fl-An] = 120 μm and [Fl-Cl] = 108 μm). An excitation pulse at 500 nm was generated by optical parametric amplifier (Spectra Physics, OPA-800CF). A white continuum pulse, which was generated by focusing the residual of the fundamental light to a sapphire plate after a computer controlled optical delay, was divided into two parts and used as the probe and the reference lights, of which the latter was used to compensate the laser fluctuation. Both probe and reference lights were directed to a rotating sample cell with 0.1 cm of path length and were detected with a charge-coupled device detector equipped with a polychromator (Solar, MS3504). The pump pulse was chopped by a mechanical chopper synchronized to one-half of the laser repetition rate, resulting in a pair of the spectra with and without the pump, from which an absorption change induced by the pump pulse was estimated. Measurement of nanosecond laser flash photolysis (ns-lfp) The nanosecond transient absorption spectra were measured by the pump and probe method using the third-harmonic oscillation (355 nm, 4 ns fwhm) and second harmonic oscillation (532 nm, 4 ns fwhm) from a Q-switched Nd:YAG laser (Continuum, Surelite II-10) as the excitation sources. The light from a Xe flash lamp (Osram, XBO-450) was focused into the sample solution for the transient absorption measurement. Time profiles of the transient absorption in the UV Vis region were measured with a monochromator (Nikon, G250) equipped with a photomultiplier (Hamamatsu Photonics, R928) and digital oscilloscope (Tektronics, DPO 3054). Each decay

4 profile was collected after averaging 16 and 64 times for the 355- and 532-nm excitation, respectively. During the ns-lfp measurements, it should be noted that 45 volume% (v%) of acetonitrile (ACN) mixed ph 8.0 Tris-K + buffer was used because the absorption of Fl-An at 532 nm in ph 8.0 buffer was too small to obtain the acceptable transient absorption signal for the 532-nm excitation. In the ACN mixed buffer, approximately 5 nm red-shift occurred in Fl-An, resulting in approximately twice increase in absorbance at 532 nm. Meanwhile, the absorption spectral shape of Fl-An in the ground state did not change, which indicated that the trianionic form of Fl was well maintained. In addition, 355-nm excitation caused significant photodecomposition of dyes during the repetitive scanning. Due to the photo-instability of Fl-An under the 355-nm excitation, it was impossible to obtain the whole transient absorption spectrum with good signal-to-noise ratio. Instead, we measured only decay profiles at the peak position of the transient absorption (430 nm) with 16 averaging times as shown in Figure 5a. On the other hand, in the case of transient absorption spectrum of Fl-Cl (Figure 4b), most of the photons were absorbed by anthracene molecules, triplet sensitizers. Thus, photodecomposition of Fl-Cl by direct excitation was largely prevented. Time-resolved phosphorescence measurement The samples of Fl-An, Fl-EP, and TMPyP4 were prepared in ph 8.0 Tris-K + buffer in a 1 1 cm 2 quartz cell ([Fl-An] = 74.6 μm, [Fl-Cl] = μm, and [TMPyP4] = 48.8 μm where they have the same absorbance of 0.5 at 532 nm). The second-harmonic oscillation (532 nm, 4 ns fwhm, mj cm -1 pulse -1 ) from a Q-switched Nd:YAG laser (Continuum, Surelite II-10) was used for the excitation light. The photoinduced luminescence from the sample cell was collected with quartz lenses, passed through a monochromator, and then introduced into a near IR photomultiplier tube module (Hamamatsu Photonics, H10330A-75). After amplified by 350 MHz amplifier unit (Stanford Research, SR445A), the output of the photomultiplier was sent to a gated photon counter (Stanford Research, SR400) under direct control from a PC via GPIB interface. To measure the lifetime of 1 O 2, the signals were accumulated (10 repetitions) by changing the delay time from 0 to 70 µs with a gate width of 0.3 µs. Calculation of ionization quantum yield (Φ ion) In light of our previous report, [5] Φ ion of one-electron oxidized Fl (X) was determined with a reference compound, benzophenone (BP). Briefly, Φ ion can be determined by Φ ion = [X]/[ 3 BP*] (Eq. 1) where [X] and [ 3 BP*] are the concentrations of X and BP in the triplet excited state ( 3 BP*), respectively. [ 3 BP*] is equal to the photon concentration of the laser flash at 355 nm, determined

5 using chemical actinometry of the T 1 T n absorption of BP as the standard according to the method described. [X] was calculated using the previously reported molar absorption coefficient (ε) of X, M -1 cm -1 at 428 nm. [6] Since X is generated by the two-photon absorption, Φ ion increases as the laser intensity is raised (Table S5). Calculation of rate constant for bimolecular triplet-triplet energy transfer (k q) from Fl-An in the triplet excited state ( 3 Fl*-An) to O 2 From the decay profile of transient absorption of 3 Fl-An* excited at 532 nm (Figure 5a, below), k q can be obtained by k T = k 0 T + k q[o 2] (Eq. 2) where k T, k 0 T, and k q are reciprocals of the decay time of 3 Fl*-An in the air- and Ar-saturated conditions, and the bimolecular quenching rate constant between 3 Fl*-An and O 2, respectively. [7] Considering that 45 v% ACN mixed ph 8.0 Tris-K + buffer solution was used during the ns-lfp measurement and the dissolved oxygen concentrations were different between ACN and water ([O 2] = 1.9 and 0.27 mm at the room temperature, respectively), [8] k q = M -1 s -1 was obtained.

6 Supporting Tables and Figures Table S1. Φ fl and τ fl of Fl-An, Fl-EP, fluorescein, and Fl-Cl. Compound Φ fl τ fl / ns Fl-An Fl-EP [a] [a] [b] n.d. [c] Fluorescein 0.92 [d] 3.6 [e] Fl-Cl [a] Ref. 2. [b] τ fl of Fl-An is considered to be originated from Fl impurities without anthracene moiety (An) and/or preformed Fl-EP. [c] n.d. stands for no data available. [d] Ref. 9. [e] Ref. 10. Table S2. Energy levels of Fl, An, and EP in the singlet and triplet excited states, and oxidation and reduction potentials of Fl and An from the previous studies. Moiety (reference compound) Singlet excitation energy (kj mol -1 ) Triplet excitation energy (kj mol -1 ) E (D + /D) (V vs NHE) E (A/A - ) (V vs NHE) Fl (Fl-Cl) [a] [b] 1.66 [c] [c] An (DMA) 301 [d] 168 [d] 1.91 [d] [e] EP (DMA with endoperoxide) [f] [f] n.d. [g] n.d. [g] [a] Calculated from the 0-0 absorption of Fl-An. [b] Ref. 11. [c] Ref. 12. [d] Ref. 13. [e] Ref. 6. [f] Ref. 14. [g] n.d. stands for no data available.

7 Fl-An Fl-EP Figure S1. Chemical structures of Fl-An and Fl-EP analyzed by Gollmer et al. [2]

8 Xa Carboxyfluorescein Fl-Cl One-electron oxidized Fl (X) One-electron reduced Fl (R) Figure S2. Chemical structures and abbreviations of the chromophores of Fl-An, reference compounds, and radical species of Fl. The chromophore of Fl is Xa whose exact name is 6- hydroxy xanthen-3-one. Since Fl is trianion in ph 8.0 Tris-K + buffer, the net charges of oneelectron oxidized and reduced Fl are -2 and -4, respectively. The expected chemical structure of one-electron oxidized and reduced Fl are as shown above and named as X and R, respectively.

9 λ ex = 350 nm λ ex = 375 nm λ ex = 485 nm Figure S3. Fluorescence spectra of Fl-An excited at 350 nm (cyan), 375 nm (red), and 485 nm (blue). Inset: absorption spectra of Fl-An in ph 8.0 Tris-K + buffer (black) and in ACN (magenta), and Fl-Cl in ph 8.0 Tris-K + buffer (green). Cyan, red, and blue arrows indicate the excitation wavelength of 350, 375, and 485 nm, respectively, used for the measurements of fluorescence spectra. Since the absorption peaks at nm and nm correspond to An and Fl, respectively (Figure S3, inset), fluorescence of Fl-An would have the excitation-wavelength (λ ex) dependence if energy transfer from An in the singlet excited state ( 1 An*) to Fl does not occur. As shown in Figure S3, however, neither An fluorescence nor enhancement of Fl fluorescence was observed when Fl-An was excited at 350 and 375 nm (cyan and red, respectively). The weak Fl fluorescence was resulted from the direct excitation of Fl because Fl also has weak absorption in nm wavelength regions (inset, green line). The sharp emission peak observed in nm range was water Raman scattering signal. Overall, the Fl-An fluorescence centered at 520 nm was mainly induced by Fl, while the An fluorescence was so weak regardless of λ ex that its intensity was weaker than water Raman scattering signal. This result indirectly suggests that 1 An* is mostly deactivated by the nonradiative processes since neither the intramolecular singlet-singlet energy transfer from 1 An* to the Fl nor An fluorescence were observed.

10 Figure S4. a) Absorption and b) fluorescence (λ ex = 465 nm) spectral changes during the irradiation of Fl-An with the continuous visible light in the range 510 to 550 nm (dark blue to green). Black arrows show spectral change occurred with a conversion of Fl-An into Fl-EP.

11 Figure S5. Transient absorption spectrum of one-electron reduced Fl-Cl (analogues to R) observed at 70 μs after a pulse during the ns-lfp of Fl-Cl at λ ex = 532 nm (20 mj pulse -1 ) with 1-cm path length, [Fl-Cl] = 100 μm, and [p-phenylenediamine] = 2 mm.

12 Figure S6. Transient absorption spectra of 1 Fl* hot and relaxed 1 Fl*, calculated from the global fitting of 56 time profiles monitored during the fs-lfp of Fl-Cl ( nm) with λ ex = 500 nm. The fitting equation used for the global fitting of the spectra above is y(x) = A1exp(-k cx) + A2{-exp(-k cx) + exp(-k d1x)} + A3exp(-k d2x) + A4 (Eq. 3) where A1, A2, A3, and A4 are the amplitudes correlated to the maximum concentration of 1 Fl* hot, relaxed 1 Fl*, Fl in the ground state, and a constant for the fitting, respectively. The detailed descriptions and rate constants for each process are summarized in Table S3.

13 Table S3. Descriptions and rate constants for the relaxation pathways of Fl-Cl in the excited states as shown in Figure S6. Processes Description k / s -1 1 Fl* hot-an 1 Fl*-An Vibrational cooling of 1 Fl* hot k c = Fl*-An Fl-An + heat and/or hν Radiative and/or non-radiative decay of 1 Fl* k d1 = Recovery of Fl in the ground state k d2 =

14 a) b) Figure S7. a) Diagram to illustrate relaxation processes of Fl-An in the excited states. k c, k CS1, k CS2, k CR, and k d indicate the rate constants for vibrational cooling including solvation, intramolecular charge separation between An and either 1 Fl* in a Franck-Condon state ( 1 Fl* hot) or relaxed 1 Fl*, charge recombination of R-An +, and other deactivation processes of 1 Fl*, respectively. b) Time profiles of transient absorption at 550, 560, and 570 nm where the stimulated emission of 1 Fl*-An and An + formation can be observed (open circles) during the fs-lfp of Fl-An with λ ex = 500 nm. Results of multi-exponential fitting using Eq. 3 are shown as black lines. After the excitation at 500 nm, 1 Fl*-An in a Franck-Condon state ( 1 Fl* hot-an) is instantly populated, followed by photoinduced electron transfer (PET, used as the same meaning of charge separation in this report) and/or vibrational cooling processes to be relaxed via its charge separated state and/or 1 Fl*-An, respectively. In order to obtain rate constants accurately from the fitting of transient absorption at 420 nm (absorption of 1 Fl*) or around 550 nm (stimulated emission of 1 Fl*), a careful consideration is required because the signs for rise and decay in the fitting equation become inverse if (k CS2 + k d) is larger than k c. [15] From the simple double exponential fitting of the transient absorption at 720 nm, the formation of An + ( s -1, Figure 3c) was found to be faster than vibrational cooling of 1 Fl* ( s -1, see Figure S6). At the same time, non-exponential decay curves of 1 Fl* absorption and stimulated emission necessitate at least one slow component (< s -1 and/or a constant indicating longer than the time limit, 2 ns) for the accurate fitting. Taken together, under the present experimental condition (ph 8.0 Tris-K + buffer and λ ex = 500 nm), charge separation takes place mainly in 1 Fl* hot-an rather than as a sequential process after vibrational cooling. Time profiles of transient absorption for the stimulated emission of 1 Fl*-An and An + formation observed at 550, 560, and 570 nm (Figure S7b) were globally fitted by the equation as shown below y(x) = A1{-exp(-k CS1x) + exp(-k CRx)} + A2{-exp(-(k CS1+k c)x) + exp(-k dx)} + A3 (Eq. 4) where A1, A2, and A3 are the coefficients correlated to the maximum concentration of An +, stimulated emission from 1 Fl*-An, and a constant for the fitting, respectively. Here, A1 and A2 are

15 positive and negative, respectively. The detailed descriptions and rate constants for each process are summarized in Table S4. As a result, k CS1 is determined to be s -1, and hence the yields for the deactivation processes of 1 Fl* hot-an, vibrational cooling and charge separation, are 0.2 and 0.8, respectively. Thus, R-An + mainly originated from 1 Fl* hot-an, and we denoted the k CS1 as the representative time constant for intramolecular PET (k PET) in the manuscript. It should be noted that the charge separation of relaxed 1 Fl*-An is still possible and may become the major process to induce charge separated state provided that the different solvent and excitation wavelength are used, resulting in the same consequence, effective fluorescence quenching.

16 Table S4. Descriptions and rate constants for the relaxation pathways of Fl-An in the excited states as shown in Figure S7. Processes Description k / s -1 1 Fl* hot-an R-An + Charge separation to form R and An + (k PET in the manuscript) k CS1 = R-An + Fl-An Charge recombination between R and An + k CR = Fl* hot-an 1 Fl*-An Vibrational cooling of 1 Fl* hot (see Figure S7) 1 Fl*-An Fl-An + heat and/or hν k c = (Fixed) Radiative and/or non-radiative decay of 1 Fl* k d =

17 Figure S8. Time profiles of normalized fluorescence decay of Fl-Cl (black) and Fl-An (blue) (λ ex = 485 nm).

18 Figure S9. Transient absorption spectrum of DMA in the triplet excited state observed at 10 μs after a laser pulse during the ns-lfp of DMA in Ar-saturated benzene (λ ex = 355 nm).

19 TMPyP4, Φ Δ = 0.74 Fl-Cl, Φ Δ = 0.06 Figure S10. a) Time profiles of 1 O 2 phosphorescence decay of TMPyP4 (blue), Fl-Cl (red), and Fl- An (blue) observed at 1280 nm (λ ex = 532 nm at 6.5 mj cm -2 ) in ph 8.0 Tris-K + solution. P. I. stands for phosphorescence intensity. b) Determination of the singlet oxygen generation quantum yield (Φ Δ) of Fl-Cl (red circles) as compared with TMPyP4 (black circles, Φ Δ = 0.74). By comparing the slope of the two linear plots between TMPyP4 and Fl-Cl (black and red lines, respectively), Φ Δ of Fl-Cl is calculated to be O 2 is generated by the bimolecular triplet-triplet energy transfer from a Sens in the triplet excited state ( 3 Sens*) to 3 O 2. Thus, the detection of 1 O 2 phosphorescence at 1280 nm indirectly indicates the presence of 3 Sens* in the photoreaction. Weaker phosphorescence of Fl-An than that of Fl-Cl can be explained by (1) the presence of ultrafast deactivation pathway via the intramolecular PET and (2) chemical quenching of 1 O 2 by An. Phosphorescence observed for Fl-Cl in the triplet excited state ( 3 Fl*-Cl) is in accordance with our suggested relaxation pathway as shown in Figure 6. Furthermore, Φ Δ of Fl-Cl was determined as compared to a reference compound, TMPyP4 (Φ Δ = 0.06 and 0.74, respectively) (Figure S10b). The same calculation method to that for Φ fl was used (see Experimental Details). Here, Φ Δ is the term containing the quantum yield of triplet formation (Φ T) and fraction of triplet excited state quenched by O 2 which gives singlet oxygen (f T Δ), Φ Δ = Φ Tf T Δ. [13] In addition, Φ Δ of Fl-An and Fl-EP could not be determined because of their negligible phosphorescence signal. Taken together, Φ Δ of Fl in Fl-An and Fl-EP is smaller than that of Fl-Cl (Φ Δ = 0.06), and it is tentatively assumed that smaller Φ Δ of Fl-An and Fl-EP is related to high internal conversion quantum yield (Φ IC) than that of Fl-Cl. Detailed discussion on Φ IC of Fl-An can be found in the manuscript.

20 Figure S11. Laser intensity dependences of the absorbance at 430 nm observed at 100 ns and 5 μs after a laser pulse during the ns-lfp of Fl-An with the excitation at λ ex = 355 (red) and 532 nm (black), respectively, in the air-saturated 45 v% ACN ph 8.0 Tris-K + buffer solution with 1-cm path length.

21 Table S5. Φ ion of Fl-An at various laser pulse intensities (I). I / mj cm -2 Φ ion /

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