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J. Phys. Chem. B 1998, 102, 8336-8342
Time-Resolved Spectral Investigation of Bacteriochlorophyll a and Its Transmetalated Derivatives [Zn]-Bacteriochlorophyll a and [Pd]-Bacteriochlorophyll a Christian Musewald,† Gerhard Hartwich,† Florian Po1 llinger-Dammer,† Harald Lossau,† Hugo Scheer,‡ and Maria Elisabeth Michel-Beyerle*,† Institut fu¨ r Physikalische und Theoretische Chemie, Technische UniVersita¨ t Mu¨ nchen, Lichtenbergstrasse 4, D-85748 Garching, Germany, and Botanisches Institut der Ludwig-Maximilians-UniVersita¨ t Mu¨ nchen, Menzinger Strasse 67, D-80638 Mu¨ nchen, Germany ReceiVed: May 19, 1998; In Final Form: August 12, 1998
The lowest excited singlet (S1) and triplet (T1) states of bacteriochlorophyll a (BChl) and its central metal derivatives [Zn]- and [Pd]- BChl were investigated by femtosecond time-resolved absorption and fluorescence spectroscopy. In contrast to previously reported dynamic solvation effects on the picosecond time scale for BChl, no short kinetic components were observed for BChl and [Zn]-BChl provided that photochemical transformations of the pigments caused by multiple excitation are avoided. The S1 lifetimes of 2.6 ns, 2.1 ns, and 65 ps for BChl, [Zn]-BChl and [Pd]-BChl, respectively, are dominated by intersystem crossing (ISC) to the triplet state T1. The respective triplet quantum yields amount to 76%, 85%, and >99%. The data support that [Pd]-BChl is a highly efficient photosensitizer for photodynamic tumor therapy.
Introduction Pigments of the tetrapyrrole type play a central role in photosynthesis.1 They are responsible for the absorption of light and the transfer of excitation energy2-7 as well as for the initial steps in transmembrane charge separation8-10 in the reaction center of bacteria and the photosystems in plants. Apart from their key functions in photobiology, this class of pigments meets the requirements for photosensitizers in photodynamic tumor therapy (PDT).11-18 Recently, metal-substituted derivatives of bacteriochlorophyll a (BChl)19-21 differing in their redox properties19,22-26 have been introduced into the reaction center of Rhodobacter sphaeroides replacing the accessory pigments.26-32 These modifications, in particular the ones carrying a [Ni]-BChl, provide strong support for the sequential mechanism also in native reaction centers.10,33 Such studies on proteins containing metal-substituted BChls prompted us to investigate the photophysics of these pigments in solution focusing on the influence of the central metal ion on the excited-state dynamics. So far, complexes of cyclic tetrapyrroles with divalent metal ions other than Mg2+ were studied mainly in the chlorin (17,18dihydroporphyrin) and in the porphyrin series focusing on their electrochemical and photophysical properties.25,34-37 According to the four-orbital model of tetrapyrroles,38 the four lowest allowed one-electron π-π* promotions (a2u f egy, a1u f egx, y-polarized, and a1u f egy, a2u f egx, x-polarized) are linearly combined, giving rise to the By, Qy, Bx, and Qy absorption bands. They are pairwise degenerate in metalloporphyrins (E(Qx) ) E(Qy), E(Bx) ) E(By)) because of their D4h symmetry. Compared to the chlorins (chlorophylls), bacteriochlorins (7,8,17,18-tetrahydroporphyrins) and bacteriochlorophylls, in particular, are of potential advantage because all their four π-π* transitions are well separated.24,39-41 * To whom correspondence should be addressed. † Technische Universita ¨ t Mu¨nchen. ‡ Botanisches Institut der Ludwig-Maximilians-Universita ¨ t Mu¨nchen.
Previous picosecond absorption studies of metalloporphyrins (Zn, Pt, Pd, Ni)42,43 revealed large differences in excited-state lifetimes depending on the energy of metal d-states42 and specific spin-orbit coupling.42,44 More recently, femtosecond spectroscopy on Ni porphyrins in two different coordination states revealed ultrafast photodissotiation of the axial ligands of the 6-fold-coordinated sample.45,46 In this paper we present a femtosecond time-resolved study of the excited states of [M]-BChl and its 132-hydroxy derivatives with the central ions Mg2+, Zn2+, and Pd2+ in toluene solution. Analogous measurements on [Ni]-BChl, where low lying metalcentered states are accessible during S1 deactivation, will be published in a forthcoming paper. The results support the potential of metal-substituted BChl as photosensitizers in photodynamic therapy.17,18 A picosecond, time-resolved, and nonlinear absorption study that complements this work and supports our conclusions on the excited-state dynamics of [M]-BChl has recently been conducted.47 Materials and Methods Sample Preparation. BChl was isolated from R. sphaeroides using standard methods.48-50 Hydroxylation at position C-132 to 132-OH-BChl was performed by storage of BChl in methanol for 5 days in the dark and subsequent silica gel chromatography.49,51 Demetalation of BChl was achieved by treatment with small amounts of glacial acetic acid to yield BPhe.52 [M]-BChl pigments were prepared by a transmetalation method described previously.19 First the precursor Cd complexes were prepared by refluxing BPhe in dimethylformamide with anhydrous Cd(OAc)2 and subsequent purification on silica gel. Addition of metal chlorides to a solution of the resulting [Cd]-BChl in acetone resulted in transmetalation to the respective Pd and Zn complexes. The samples were purified on silica gel. Toluene solutions of the pigments were degassed (flushed with N2), and the cuvettes were sealed in order to suppress photoinduced oxidation. The optical densities of the samples
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BChl and Its Derivatives [Zn]- and [Pd]-BChl were adjusted to OD ) 1 at 780 nm for transient absorption and to OD ) 0.1 at 780 nm for transient fluorescence measurements. Pump/Probe Absorption Spectroscopy. For the timeresolved absorption measurements we used a pump/probe laser system based on a 76 MHz Ti:sapphire oscillator and a regenerative amplifier with a variable repetition rate of 101000 Hz; 100 Hz and 1 kHz used for this experiment lead to excitation rates of 50 and 500 Hz. The self-mode-locked oscillator (Coherent MIRA Basic) pumped by an Ar+ laser (8 W, Coherent Innova 310, all lines) produces laser pulses at 780 nm with a duration of 100 fs (sech2 pulse shape) and an average power of 500 mW. The pulses with a spectral width of 15 nm were stretched to 150 ps before seeding the regenerative amplifier system (BMI Alpha 1000S) pumped by an 8 W YLF laser (BMI 621D) at 528 nm. After amplification (1.8 mJ) the output was split into pump and probe beam (9:1) and was recompressed separately to 100 fs in two identical compressor units. The more intense pulse was used for excitation of the sample at 780 nm, and the weaker one was sent over a delayline (0-1.5 ns, resolution 6.6 fs) before passing a white-light generation in a 10 mm flow cell containing ethanol. The whitelight continuum was sent through a chirp-compensated steppermotor controlled spectrometer to select sections of the spectrum with a bandwidth of 12 nm. The entire setup had an instrumental response function (IRF) of 120-180 fs. Pump and probe beam were focused weakly under an angle of 10° with magic angle polarization on a 1 mm cuvette with a spot diameter of 600 and 300 µm, respectively. In the spectral region of the fundamental wavelength (780 nm ( 15 nm) the experimental error is higher due to two effects: (I) The straylight of the pump beam cannot be eliminated by band-pass filters. This effect can partly be corrected for by subtraction of the straylight at negative delay. (II) Whitelight generation is a highly nonlinear process. This decreases the relative contribution of small pulses following the main probe pulse with a delay of some nanoseconds drastically in all spectral regions except for the generation wavelength. Transient absorption spectra of [Mg]- and [Zn]-BChl were performed at 100 ps and 1 ns after excitation at 780 nm by four different scan cycles (450-550 nm, 550-650 nm, 650750 nm, and 750-900 nm, respectively, to avoid sample degradation due to prolonged exposure to excitation light (see below). The higher stability of [Zn]-BChl allowed the measurement of transient absorption spectra in the examined spectral range (450-900 nm) in 20 nm steps with the same sample in one measurement cycle. Time-Resolved Fluorescence. Time-correlated single-photoncounting (TCSPC) results were obtained with an experimental setup described elsewhere.7 The IRF fwhm for those measurements was 35-40 ps. Femtosecond time-resolved fluorescence was measured with the upconversion technique as described in refs 7 and 28 and is briefly described in the following. The laser source was a Ti: sapphire oscillator (Coherent Mira Basic), pumped by 8 W of the output of an Ar+ laser (Coherent Innova 425). The laser delivered at least 450 mW of average power at the desired excitation wavelengths. Pulse repetition rate was 76 MHz. After compression with a four-prism (SF10) chirp compensation, we got pulse widths below 100 fs. The laser beam was split in two parts with equal power, one focusing onto the sample (pulse energy 0.7 nJ, spot diameter 50 µm), the other one (gate beam) passing a stepping motor driven delay stage (resolution 16.7 fs, range 1.3 ns). The latter was combined with the fluorescence
J. Phys. Chem. B, Vol. 102, No. 42, 1998 8337
Figure 1. Absorption spectra of metal-substituted BChl in toluene at 290 K, normalized to identical Qy absorption maximum: [Mg]-BChl (s), [Zn]-BChl (- - -), and [Pd]-BChl (‚‚‚). The inset shows the structural formula of the complexes (M ) Mg, Zn, Pd; R ) H, OH).
from the sample in a β-barium borate (BBO) crystal (thickness 1 mm) under an angle of 11°, thus allowing noncritical type I phase matching. The geometry was chosen to upconvert fluorescence being polarized parallel to the excitation. The sample cell was rotating with up to 3000 rpm to reduce sample degradation by distributing the excitation energy over a larger sample volume. The intensity of sum frequency light is detected with a photomultiplier (Hamamatsu R4220P) after passing a double monochromator (Jobin Yvon HD20). The sum frequency photons were separated from the remaining background photons by counting them synchronously to the phase of light choppers in the excitation and gate pathways. To reduce the influence of long-term drifts of the laser and degradation of the samples upon the decay curves, we collected up to 32 individual time traces and altered the direction of acquisition after each run. The IRF was taken before each fluorescence measurement using scattered light from the sample. The alignment was not changed except for adjusting the phase-matching angle of the crystal and the detection wavelength in order to achieve an optimized signal-to-noise ratio. The full width at half-maximum (fwhm) of the IRF was about 140 fs. Fitting Procedure. Time constants were extracted from the experimental transient absorption and fluorescence data by fitting a convolution of the experimental IRF and exponential decay functions to the data using the Levenberg-Marquardt method. This deconvolution method the resolved lifetimes down to a quarter of the IRF fwhm. The IRF of the absorption measurements was a free Gaussian parameter. The quality of fit was judged by observing the residuals and values of the reduced χν2. Results and Discussion Steady-State Absorption and Fluorescence. The groundstate absorption spectra of [Mg]-, [Zn]-, and [Pd]-BChl dissolved in toluene are shown in Figure 1. All spectra exhibit the expected four electronic transitions in the UV to NIR region (the By, Bx, Qx, and Qy transitions in order of decreasing energy). The respective maxima are located at 361, 395, 581, and 781 nm for [Mg]-BChl, at 355, 393, 555, and 773 nm for [Zn]BChl, and at 334, 388, 535, and 762 nm for [Pd]-BChl. The corresponding 132-hydroxy derivatives exhibit identical absorption properties except for a systematical blue shift of the Qx band of ∼5 nm. The absorption maxima compare well to literature data for these complexes dissolved in diethyl ether.19
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Figure 2. Spontaneous fluorescence decay of [Mg]-BChl (λem ) 860 nm), [Zn]-BChl (λem ) 830 nm), and [Pd]-BChl (λem ) 780 nm) in toluene at 290 K measured by TCSPC.
[Mg]- and [Zn]-BChl fluoresce 20 nm shifted to the Qy absorption at 800 and 790 nm, whereas [Pd]-BChl shows no fluorescence under these conditions. The lowest excited states of all examined [M]-BChl are located in the near-infrared, exhibiting a singlet-triplet splitting of about 4550 cm-1,17,19,53 somewhat smaller than that of chlorins and porphyrins. Thus, increased intersystem crossing rates from S1 to T1 can be expected. The low lying S1 states in [M]-BChl provide that empty (d,d) states of most transition metals (including Pd2+ 42) are higher and metal-induced spinorbit coupling effects on the S1 (π-π*) excitation can be studied without interference of transient populations of metal-centered states. Time-resolved fluorescence of [Mg]-, [Zn]-, and [Pd]-BChl probed at 860, 830, and 780 nm, respectively, is depicted in Figure 2. The detection wavelength is kept at the red wing of the steady-state fluorescence maxima (788, 782, and 764 nm, respectively19) to avoid stray light. All decay traces can be fitted sufficiently with a single-exponential component. The S1 decay lifetimes are 2.6 ns, 2.1 ns, and 65 ps for [Mg]-BChl, [Zn]BChl, and [Pd]-BChl, respectively. A negligible contribution (
