J. Phys. Chem. 1996, 100, 6879-6888
6879
ARTICLES Steady-State and Picosecond Spectroscopy of Li+ or Ca2+ Complexes with a Crowned Merocyanine. Reversible Photorelease of Cations Monique M. Martin,* Pascal Plaza, and Yves H. Meyer Laboratoire de Photophysique Mole´ culaire du CNRS (UPR 3361), Baˆ timent 213, UniVersite´ de Paris-Sud, 91405 Orsay, France
Fatima Badaoui, Jean Bourson, Jean-Pierre Lefe` vre, and Bernard Valeur* Laboratoire de Chimie Ge´ ne´ rale, CNAM, 75003 Paris, France, and Laboratoire PPSM (CNRS URA 1906), Ecole Normale Supe´ rieure, 94235 Cachan, France ReceiVed: September 21, 1995; In Final Form: January 3, 1996X
Reversible photorelease of Li+ and Ca2+ cations from complexes with a crowned derivative of the merocyanine dye DCM (4-(dicyanomethylene)-2-methyl-6-(p-(dimethylamino)styryl)-4H-pyran) in acetonitrile is investigated by steady-state and time-resolved spectroscopy. The results obtained by fluorescence spectroscopy reveal the presence of two emitting species in solutions containing DCM-crown complexes. One species with a 2 ns lifetime is attributed to the free ligand resulting from cation photorelease, with a maximum formation yield of 86% for Li+ and 41% for Ca2+. The other one with a 400 ps slightly blue-shifted emission for both cations is suggested to be due to a loose complex. The mechanism of cation photorelease is studied by transient absorption and gain spectroscopy within 2 ns after excitation with a 0.7 ps pulse at 425 nm. In the 360-660 nm spectral range, the main changes observed take place within 30 ps. For longer time delays the time-resolved differential spectra indicate a dominant decay of 1.5-2 ns in all spectral regions. From the analysis of the early spectral changes, it is concluded that the dominant excited-state mechanism includes the disruption of the binding between the nitrogen atom of the crown and the cation in the locally excited state of the complex, followed by intraligand charge transfer at a slower rate for Ca2+ than for Li+. In spite of the absence of experimental evidence for cation diffusion to the bulk, a significant increase in concentration of free cations should be obtained by using light pulses of long duration.
Introduction Photophysical changes resulting from cation binding to fluoroionophores (i.e., fluorescent dyes linked to ion-complexing units, like crown ethers, cryptands, etc.) have received much attention because these changes can be the basis of new methods of optical detection of cations.1-4 Another interesting application is the possibility of using these compounds for photorelease of cations to produce fast and spatially controllable concentration changes. This would offer an outstanding tool to study the response of biological systems to cation concentration jumps, which provides valuable insights into these systems. In fact, it has been established that many physiological functions (neurosynaptic transmission, muscle contraction, hormone secretion, etc.) are controlled by brief, localized fluctuations of intracellular free Ca2+ concentration. Photorelease of cations from a “cage” is possible by photoinduced irreversible changes in the chemical structure of a chelator.5 Jumps in concentration of intracellular free Ca2+ can be generated in this way, which allows one to study cellular responses, especially in nerve and muscle. Release of ions is also possible by photocleavage of chelators. The chelators designed by Kaplan and Ellis-Davies6-8 have an affinity for Ca2+ that decreases by 5 orders of magnitude upon illumination in the 350 nm range allowing successful studies of the X
Abstract published in AdVance ACS Abstracts, March 15, 1996.
0022-3654/96/20100-6879$12.00/0
contraction of muscle fibers. Photorelease of alkali ions cannot be achieved with photolabile chelators. A more specific ionophore such as cryptands should be used. Lehn and coworkers9 have designed several photocleavable cryptands. Photorelease of K+ by this method can induce K+ binding to Na+, K+-ATPase, which allowed Grell and Warmuth10 to determine the rate constant for K+ binding which was found to be about 1000 times smaller than that observed for the K+ carrier valinomycin, thus implying slower structural rearrangements. The above-described photorelease processes are irreversible, and it is of interest to design reversible systems because repeated pulses of cation concentration permit improvement of the signalto-noise ratio by accumulation. The present paper deals with reversible photorelease of Li+ or Ca2+ cations from complexes with a crowned merocyanine, DCM-crown, which represents a first step toward this goal.
DCM-crown consists of DCM (4-(dicyanomethylene)-2methyl-6-(p-(dimethylamino)styryl)-4H-pyran), an often used laser dye, where the dimethylamino group has been replaced © 1996 American Chemical Society
6880 J. Phys. Chem., Vol. 100, No. 17, 1996 by a macrocycle (monoaza-15-crown-5) that can bind metal ions. The nitrogen atom of the azacrown plays a dual role: it participates in the conjugation of the dye as an electron-donating substituent, and it acts as a donor atom in the complex. The first paper devoted to DCM-crown11 reported the following effects of cation binding: (i) the absorption spectrum is drastically blue shifted, (ii) the position and shape of the fluorescence spectrum are only slightly changed, (iii) the fluorescence quantum yield is strongly reduced, and (iv) the fluorescence decay is hardly affected. Observation i can be interpreted in terms of interaction between the bound cation and the lone pair of the crown nitrogen atom resulting in reduction of the electron-donating character of the latter. On the other hand, observations ii and iv are quite surprising at first sight. They can be understood on the basis of photoinduced processes occurring in the parent molecule DCM. DCM has been the subject of various photophysical studies.12-18 This dye exhibits a strongly red-shifted fluorescence spectrum in polar solvents, and the dipole moment of the fluorescent state was estimated to be as large as 26 D.12 Photoinduced charge transfer from the nitrogen atom of the dimethylamino group to the dicyanomethylene group accompanied by internal twisting has been proposed to explain the photophysical behavior of this molecule.13 Two recent studies reported experimental observations in favor of a mechanism involving the fast formation of an emissive charge-transfer (CT) state from the emissive initially excited state of DCM.17,18 At the present time it is difficult to ascertain whether a twisted intramolecular charge-transfer (TICT) state is involved in the relaxation mechanism. In the case of DCM-crown, the photoinduced charge transfer process is expected to reduce considerably the electron density of the crown nitrogen atom so that the interaction with a cation would become much weaker in the excited state and could even be disrupted. Irreversible photoinduced changes in the chemical structure of a calcium chelator (EGTA type) with the effect of supressing the coordinating role of a nitrogen atom was shown to result in a decrease of the stability constant of the complex by a factor of 10-30.5 In previous papers,19,20 we provided evidence for cation photorelease from Li+- and Ca2+-DCMcrown complexes by picosecond pump-probe spectroscopy and suggested that photoejection occurs from the excited complex, once the latter has relaxed to its charge-transfer state.19 Lapouyade and co-workers21 reported a picosecond study of a crown-phenylstilbene derivative complexed with Ca2+ and proposed an excited-state mechanism including a reversible breaking of the nitrogen-calcium interaction followed by the reversible formation of a solvent-separated cation-probe pair. In the present paper, we report further measurements on DCMcrown complexes by steady-state and time-resolved fluorescence as well as picosecond transient absorption and gain spectroscopy, with the aim at further understanding the excited-state processes occurring in these complexes. Special attention is paid to the photoejection mechanism with the perspective of designing novel fast and reversible systems able to produce concentration jumps of cations. Experimental Section Materials. The synthesis of DCM-crown was reported in ref 11. Acetonitrile from Janssen (spectrometric grade) was used as a solvent for absorption and fluorescence measurements. Alkaline and alkaline-earth perchlorates purchased from Janssen and Alfa were of the highest quality available and vacuum dried over P2O5 prior to use. Tetraethylammonium perchlorate was purchased from Carlo Erba.
Martin et al. Steady-State and Fluorescence Lifetime Measurements. Absorption spectra were recorded on a Kontron Uvikon 940 spectrophotometer. Corrected emission spectra were obtained on an SLM-Aminco 8000C spectrofluorometer. The fluorescence lifetimes were measured on a multifrequency phase/modulation fluorometer equipped with a He-Cd laser (442 nm) and a Pockels cell operating at frequencies ranging from 0.1 to 200 MHz.22 Subpicosecond Pump-Probe Experiments. Transient absorption and gain spectra were measured by the pump-probe technique using a 0.7 ps, 15-30 µJ pump pulse at 425 nm and a continuum probe. The white-light probe was produced by focusing a 0.3 ps, up to 200 µJ, 710 nm pulse in a 1 cm H2O cell. The pump-probe experiments were carried out with a two-beam probe arrangement in which the two beams are sent respectively onto the sample and onto a reference cell containing either the solvent or the unexcited sample. Sample and reference cell paths were 1 mm long. Pump and probe beams had a diameter of about 1 mm on the sample and crossed at an angle of ∼10°. The transmitted probe beams were then guided through PCS600, 2.50 m long, optical fibers to the 150 µm entrance slit of a polychromator (Jarrell-Ash). The spectra of the two probe beams were simultaneously recorded by a computer-controlled double diode array detector (Princeton Instrument Inc.). A 270 nm spectral range was analyzed in a single experiment. The pump-probe delay time was adjusted by means of a stepper motor translation. Pump and probe beam polarizations were set at the magic angle. Data were accumulated over 500 laser shots without the pulse intensity discrimination. The 425 and 710 nm subpicosecond beams used in the pump-probe experiments were generated by a cheap all-dye laser system, according to unconventional methods described in ref 23. The whole system is driven by a standard seeded 10 Hz Q-switched Nd:YAG laser delivering smooth 6 ns pulses at 532 and 355 nm. High-power 500 fs pulses at 600 nm are produced by a two-step shortening process and used to generate a continuum which is divided into two beams. One beam is filtered at 710 nm and amplified in a rhodamine 700 dyeamplifier chain pumped by the second harmonic of the Nd: YAG laser, and the other one is filtered at 425 nm and amplified in a stibene 420 dye-amplifier chain pumped by the third harmonic of the same Nd:YAG laser. All experiments were performed at room temperature. Results Steady-State Experiments. The absorption and fluorescence spectra of DCM-crown in the presence of an excess of lithium or calcium perchlorate in acetonitrile were previously reported11 and are recalled in Figure 1. In the present work, the effect of gradual addition of Li+ or Ca2+ on the fluorescence of DCMcrown was carefully examined. The concentration of DCMcrown was 4.2 × 10-6 M. Figures 2 and 3 show the fluorescence spectra recorded upon excitation at the wavelength of the isosbestic point between the ligand and complex absorption spectra. When the concentration of cation is increased, a slight blue-shift of the fluorescence spectrum is observed together with a decrease in intensity, and the spectra exhibit an isoemissive point in both cases. The fluorescence spectrum obtained after addition of enough metal salt so that the amount of free ligand is negligible in the ground state will be called fluorescence spectrum “in the presence of cation”. Fluorescence Decay Measurements. Fluorescence decays of DCM-crown (4.2 × 10-6 M in acetonitrile) without cation and in the presence of Li+ and Ca2+ were measured at different
Reversible Photorelease of Cations
Figure 1. Absorption and fluorescence spectra of DCM-crown and its complexes with Li+ (0.1 M) and Ca2+ (0.1 M) in acetonitrile.
Figure 2. Change in the fluorescence spectrum of DCM-crown with increasing concentration of Li+, up to 0.02 M in acetonitrile. The isoemissive point is enlarged in the insert.
Figure 3. Change in the fluorescence spectrum of DCM-crown with increasing concentration of Ca2+, up to 0.03 M in acetonitrile. The isoemissive point is enlarged in the insert.
wavelengths. The results are reported in Table 1. The fluorescence decay of the free ligand is a single exponential with a lifetime of 2.2 ns. In the presence of cation, the fluorescence decay observed at high wavelengths is still a single exponential with a decay time of 2.0 ns, i.e., close to the lifetime of the free ligand. In contrast, no satisfactory fit with a single exponential can be obtained at the lower observation wavelengths (544 and 581 nm). Two examples of experimental curves are given in Figures 4 and 5, and it is shown that for a fit with a single exponential the weighted residuals are not randomly distributed; moreover, the reduced chi-squared (χ2) value is abnormally high. When trying a biexponential fit, the reduced χ2 value decreases by a factor of about 10 (Table 1) and the weighted residuals are randomly distributed (see Figures 4 and 5). Therefore, there is no doubt about the existence of (at least) two emitting species. In these biexponential decays, one of the decay times is very slightly shorter than that of the
J. Phys. Chem., Vol. 100, No. 17, 1996 6881 free ligand while the other one is much shorter, i.e., (0.45 ( 0.05) ns in the case of Li+ and (0.40 ( 0.05) ns in the case of Ca2+ (Table 1). Subpicosecond Pump-Probe Experiments. Transient absorption and gain spectra of DCM-crown (1 × 10-4-1.3 × 10-4 M), free and fully complexed with Li+ (0.1 M) and Ca2+ (0.05 M), were measured in the wide 360-660 nm wavelength range, within 2 ns after excitation with a 0.7 ps pulse. The spectral changes taking place within the initial 30 ps are analyzed in detail. Time-ResolVed Differential Spectra of the Free Ligand. The differential spectra (∆D ) D - D0) measured for the free ligand within 1-4 and 4-31 ps delays are shown in Figure 6a and b, respectively. The absorption spectrum of the unexcited sample is given in optical density (D0). A “transient absorption band” (i.e., ∆D > 0, D0 not necessarily ) 0) showing three maxima around 410, 470, and 530 nm and a “net gain band” above 570 nm (i.e., ∆D < 0, no ground state absorption) are observed immediately after excitation and keep growing within a few picoseconds (Figure 6a). It is interesting to note that, meanwhile, the time-resolved gain spectra show a temporary isosbestic point (constant ∆D) at 585 nm. Another striking observation is that the net gain band shifts to the red for the 1-4 ps delays then slightly shifts back to the blue between 4 and 31 ps and keeps increasing (Figure 6b). During the blueshift, the 410 and 470 nm ∆D maxima further grow and a dip occurs around 500 nm. For longer delays, we did not find further changes in shape of the transient spectra. Typical kinetics at selected wavelengths are given in Figures 9 and 10 for picosecond and nanosecond ranges. Comparable nanosecond decays are observed. Time-ResolVed Differential Spectra of the DCM-CrownLi+ Complex. The differential spectra of the DCM-crownLi+ complex are presented in Figure 7. Immediately after excitation, the spectra show an absorption band between 450 and 550 nm with a maximum around 480 nm (Figure 7a) and a gain band above 550 nm. Some bleaching can be seen below 440 nm in the short-wavelength edge of the ground-state absorption. Within 4 ps after excitation, transient absorption increases around 530 nm while the gain band increases and shifts to the red and a temporary isosbestic point comes out at 570 nm. ∆D also increases in the short-wavelength range, and the initial bleaching is no longer observed for delays larger than 4 ps. For 4-21 ps delays (Figure 7b), the transient absorption band keeps growing below 500 nm while the gain band increases and shifts back to the blue. No appreciable change in the spectra was observed for longer delays. Picosecond and nanosecond decays are compared to those obtained for the free ligand in Figures 9 and 10. Here again comparable nanosecond decays are observed at the selected wavelengths, but the decay is found to be slightly more rapid than that of the free ligand. Time-ResolVed Differential Spectra of the DCM-CrownCa2+ Complex. In the same way as for DCM-crown-Li+, the differential spectra of the Ca2+ complex show first (Figure 8) transient absorption between 420 and 550 nm with a peak around 470 nm, gain above 550 nm, and some bleaching around 390 nm. Within 30 ps after excitation, transient absorption increases below 430 nm and around 530 nm, and gain increases in the red. The absorption peak at 470 nm decreases during a few picoseconds then remain constant. For the whole range of delays the time-resolved spectra give evidence for two temporary simultaneous isosbestic points: at 510 nm in the absorption band and at 570 nm in the net gain band. No further spectral change could be noticed for longer delays. Picosecond and nanosecond kinetics are shown in Figures 9 and 10.
6882 J. Phys. Chem., Vol. 100, No. 17, 1996
Martin et al.
TABLE 1: Decay Times of Acetonitrile Solutionsa of DCM-Crown in the Absence and in the Presence of Cations at Different Observation Wavelengths (Excitation Wavelength: 442 nm) λobs (nm) DCM-crown +Li+ (2 × 10-2 M)
+Ca2+ (3 × 10-2 M)
R1d
f1e
581b 619b >630c 544b 544b 581b 581b >630c 544b 544b 581b 581b >630c
0.67
0.91
0.71
0.91
0.58
0.85
0.65
0.91
τ1 (ns) 2.14 ( 0.05 2.08 ( 0.05 2.20 ( 0.05 1.8 ( 0.1 1.96 ( 0.05 1.7 ( 0.15 2.04 ( 0.05 2.01 ( 0.05 1.7 ( 0.1 1.89 ( 0.04 1.7 ( 0.1 1.87 ( 0.04 2.01 ( 0.03
R2d
f2e
τ2 (ns)
0.33 ( 0.04
0.09 ( 0.01
0.40 ( 0.07
0.29 ( 0.03
0.09 ( 0.01
0.50 ( 0.05
0.42 ( 0.03
0.15 ( 0.01
0.45 ( 0.07
0.35 ( 0.04
0.09 ( 0.01
0.35 ( 0.09
χr2 f 1.13 1.98 1.48 11.1 0.89 31.4 2.60 0.75 15.5 1.45 10.2 1.01 1.24
a In the presence of 0.1 M tetraethylammonium perchlorate. b Observation through an interferential Balzers filter (10 nm bandpass). c Observation through a Schott cut-off filter (RG630). d Preexponential factors (normalized to R1 + R2 ) 1). e Fractional intensities (f1 ) R1τ1/(R1τ1 + R2τ2); f2 ) R2τ2/(R1τ1 + R2τ2)). f Reduced chi-squared values obtained with standard deviations of 0.5° and 0.005 for phase shift and modulation ratio, respectively.
Figure 4. Phase and modulation data for a solution of DCM-crown in acetonitrile in the presence of an excess of Li+ (3 × 10-2 M). Excitation wavelength: 544 nm. The solid line corresponds to the best fit with a biexponential decay. Weighted residuals are shown for the best fit with one and two exponentials.
Differential Spectra at 20 ns Pump-Probe Delay. The main goal of the present study is to examine whether DCM-crown could be a promising candidate for photorelease of cations to produce fast and spatially controllable cation concentration changes. To test whether some photoreleased cations have diffused to the bulk, differential spectra were measured for a pump-probe delay long enough to detect the residual free-ligand concentration, once the excited state has relaxed and before diffusion-controlled recombination. The differential spectrum of a sample of 3 × 10-4 M DCM-crown in the presence of 10-3 M Ca2+, measured at 20 ns pump-probe delay, is shown in Figure 11. At this concentration of Ca2+, a fraction of 80% of DCM-crown is complexed and both the free ligand and the complex are excited at the pump wavelength. Figure 11 shows that a bleaching signal remains 20 ns after excitation. We obtained similar results for the free ligand as well as for the parent molecule DCM. Discussion Fluorescence of DCM-Crown Complexes. The presence of an isoemissive point in the fluorescence spectra of DCM-
Figure 5. Phase and modulation data for a solution of DCM-crown in acetonitrile in the presence of an excess of Ca2+ (2 × 10-2 M). Excitation wavelength: 581 nm. The solid line corresponds to the best fit with a biexponential decay. Weighted residuals are shown for the best fit with one and two exponentials.
crown with gradual addition of Li+ or Ca2+ (Figures 2 and 3) provides evidence for the presence of at least two emitting species in the solutions containing cations. A fluorescent species other than the free ligand is thus produced when excited solutions contain DCM-crown complexes. Time-resolved fluorescence experiments with solutions containing only complexes (in the presence of an excess of cations) show instrument-limited rises and wavelength-dependent decays. A monoexponential decay with a 2 ns lifetime is found in the red-edge part of the fluorescence spectrum, whereas an extra short component of 400-450 ps is found in the short-wavelength edge, for both Li+ and Ca2+ complexes. These results further indicate the presence of two emitting species after excitation of the complex. We attribute the species with a lifetime of about 2 ns to the ligand, either the free ligand formed through cation photorelease or a complex in which the cation is no longer in interaction with the nitrogen of the crown. But it is less easy to attribute the species with a shorter lifetime. It was not obvious at first sight11 that the fluorescence spectrum of DCM-crown fully complexed in the ground state is due to two emitting species. The spectrum is slightly blue shifted with respect to that of the free ligand (Figure 1). The
Reversible Photorelease of Cations
J. Phys. Chem., Vol. 100, No. 17, 1996 6883
Figure 6. Change in optical density ∆D(λ,t) (left scale) of DCMcrown in acetonitrile (a) within 4 ps and (b) within 4-31 ps, after excitation with a 0.7 ps pulse at 425 nm. Unexcited sample optical density D0(λ) is presented on the right scale.
Figure 9. Typical kinetics of the change in optical density ∆D(λ,t) within 30 ps after subpicosecond excitation of DCM-crown (top) and its complexes with Li+ (0.1 M) (middle) and Ca2+ (0.05 M) (bottom) in acetonitrile, at selected wavelengths. The time zero has been chosen at half of the initial rise of ∆D at the shortest wavelength given in the figure. The plotted curves are just artist smooths.
Figure 7. Change in optical density ∆D(λ,t) (left scale) of DCMcrown in acetonitrile in the presence of 0.1 M Li+ (a) within 4 ps and (b) within 4-21 ps, after excitation with a 0.7 ps pulse at 425 nm. Unexcited sample optical density D0(λ) is presented on the right scale.
Figure 8. Change in optical density ∆D(λ,t) (left scale) of DCMcrown in acetonitrile in the presence of 0.05 M Ca2+ within 32 ps after excitation with a 0.7 ps pulse at 425 nm. Unexcited sample optical density D0(λ) is presented on the right scale.
presence of an extra fluorescence band at shorter wavelengths is thus one possible cause of the blue shift. If the two decay components found at 544 and 581 nm (Table 1) are due to independent species, the ratio of the respective fractional intensities f1/f2 (with f1 ) R1τ1/(R1τ1 + R2τ2) and f2 ) R2τ2/ (R1τ1 + R2τ2) where R1 and R2 are the preexponential factors and τ1 and τ2 are the decay times) gives directly the ratio of the
steady-state fluorescence intensities of these species. One thus expects the steady-state intensity of the extra band to be only about 9% of the total fluorescence intensity for Li+ at 544 and 581 nm and respectively 15% and 9% for Ca2+. The maximum values for the free ligand formation yields can be estimated from the ratio of the maximum fluorescence intensity in the presence and in the absence of cation, that is
