J. Pkys. Chem. 1994, 98, 12478-12480
12478
Direct Measurement of Rotational Correlation Times of Luminescent Ruthenium(I1) Molecular Probes by Differential Polarized Phase Fluorimetry B. A. DeGraffi and J. N. Demas'9T Chemistry Department, James Madison University, Harrisonburg, Virginia 22807, and Chemistry Department, University of Virginia, Charlottesville, Virginia 22901 Received: October 14, 1994@
Direct measurements of rotational correlation times of luminescent ruthenium(I1) probes (RuL3*+, RuL~L'~', and RuL2(CN)2, L and L' = a-diimine) by differential polarization phase fluorimetry have been made. Correlation times and limiting anisotropies are related to structural parameters of the complexes. Implications for the use and design of metal complexes as dynamic probes of motions of macromolecular systems such as organic and biopolymers are given.
Introduction There is a continuing interest in the use of platinum metal complexes as sensors and moleculadenvironmental probes. Ru(11), Os(II), Re(I), and Ir(II1) complexes have proved particularly fruitful. These complexes have shown promise in revealing structural features and dynamics of macromolecular species.2 There have been no studies of the use of inorganic luminophores for studying dynamics of biomolecules, although organic probes have proved exceptionally valuable in unraveling the complex motions of macro-biomolecules through dynamic anisotropy measurements. To date, a wealth of organic fluorophores have been developed as probes. However, their useful time window is intrinsically limited by their short lifetimes (a few to tens of nanoseconds) since one cannot measure rotational correlation times that are much longer or much shorter than the lifetime of the probe. There is currently a growing appreciation that many of the more interesting processes such as protein folding occur on a much longer time scale than is accessible to these shortlived fluorescent organic probe^.^ While phosphorescent triplet organic probes have seen some utilization, their long lifetimes are generally achieved with low luminescence quantum yields. Inorganic complexes provide a potentially very useful new class of probes for studies of slow dynamics. The complexes can have very high quantum yields (0.01-0.8), long lifetimes (100's ns to 100 p),high anisotropies, and variable structures which allow for interactions with a variety of physical shapes and polarities and provide hooks for attaching to specific targets. All of these properties are amenable to structural control by the synthetic chemist, which further enhances their attractiveness as probe molecules. To use these complexes effectively as sensors and probes, one must understand their behavior independent of the substrate. Further, for rationally designed systems one must understand the alteration of these properties with structural changes of the
probe^.^ Of special relevance to understanding dynamic motion of probes in constrained systems is knowledge of their dynamic motion in isotropic fluid media. We report the first measurement of the rotational correlation times for a series of ruthenium(11) complexes and the control of this property by molecular modifications. Our results demonstrate that these molecules will
* To whom correspondence should be addressed. James Madison University. + University of Virginia. @
Abstract published in Advance ACS Abstracts, November 15, 1994.
0022-365419412098-12478$04.5010
make excellent probes for the slow dynamic processes that can occur in larger macromolecular systems such as synthetic and
biopolymer^.^ Experimental Section All measurements, including lifetime determinations, were made using an SLM 48000 Ultrasonic multifrequency phase/ modulation fluorimeter. Rotational correlation times were measured using differential polarization phase fl~orimetry.~ The long lifetimes of the complexes ('100 ns) required the Ultrasonic option since the standard model is limited to a minimum of 1 MHz. An Ar+ laser with intracavity prism for wavelength selection was used as the excitation source. All solutions were in dry glycerol, and the viscosity as a function of temperature was derived from literature data.6 Samples were thermostated with a cooled VWR 115 constant temperature circulator with glycerol water as the circulating media. Temperatures were measured at the sample with a Keithley 871 digital thermometer equipped with a type K thermocouple. Sample temperatures were stable to better than 0.1 "C during measurements. Data were fit using SLM's simplex routines. Parameters provided by these routines were 4, the rotational correlation time, and ro, the limiting anisotropy. The lifetimes were determined independently using both phase and modulation data. The lifetimes were then used as fixed parameters in the nonlinear least-squares fitting. This is essential because of the strong correlation among the fitted parameters in the dynamic polarization measurements. Static limiting anisotropy values were separately estimated using static polarization measurements on a Spex Fluorolog spectrofluorimeter. The complexes were dissolved in poly(methy1 methacrylate) to eliminate rotation. Complexes were either on hand or synthesized by standard techniques.' Optical densities were held below 0.2.
Results and Discussion Figure 1 shows a typical data set of differential phase and modulation data plotted versus modulation frequency. The solid lines are derived by simultaneous fits to phase and amplitude data. This result is typical and shows the noise level of the system, although we occasionally get deviations in the phase data at the higher modulation frequencies (Le., past the peak). This is not surprising given that at the higher frequencies there is virtually no amplitude and modulation for making reliable differential phase measurements. 0 1994 American Chemical Society
J. Phys. Chem., Vol. 98, No. 48, I994 12479
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TABLE 1: Rotational Correlation Tim& complex A,,,(nm) 4 (ns) rhyd (hb ro Complex Type:' RuLs2+ [RU@PY )SI 458 571 4.52 0.084 [Ru(phen)3I2+ 458 677 4.77 0.081 [Ru(5-Mephen)3Izf 458 882 5.21 0.086 [Ru(5-Phphen)31z+ 476 1690 6.48 0.086 [Ru(4,7-Ph2phen)3I2+ 476 3577 8.32 0.075 Complex Type: [RuLzL']*+ (L = phen, L' = substituted phen) 488 768 4.98 0.080 L' = 5-Mephen L' = 5-Phphen 458 1,165 5.72 0.092 L' = 4,7-Phzphen 476 1,814 6.63 0.148 Complex Type: [RuLL'J2+ (L = phen, L' = substituted phen) 458 1064 5.55 0.091 L' = 5-Phphen L' = 4,7-Phzphen 476 2540 7.42 0.133 Complex Type: RuL,(CN), Ru(phen)dCN)t 476 472 4.23 0.188 Temperature = 6.4 "C in glycerol. Based on an isotropic, spherical rotator, V = (4/3)d. Abbreviations used are bpy = 2,2'-bipyridine, phen = 1,lO-phenanthroline, 5-Mephen = 5-methyl-1,lO-phenanthroline, 5-Phphen = 5-phenyl-l,lO-phenanthroline, and 4,7-Ph~phen= 4,7-
'+
I
11.1
0.10
0.01
Modulation Frequency (MHz)
Figure 1. Differential phase (triangles) and modulation (diamonds) responses of [Ru(4,7-Ph2phen)(phen)2I2+ at 6.4 "C in glycerol as a function of modulation frequency. The solid lines are the best fits to a single isotropic rotator model. 2000 7
-I
~
b 6 9 . 6K
285.8 K
,
O I 0.20
I
0.40
0.60
q I T (PIK)
Figure 2. Plot of the rotational correlation time as a function of the in glycerol (diaviscosity/temperature (q/T) ratio for [Ru(bp~)3]~+ monds). The best linear fit is the solid line. The temperature of each measurement is indicated beside the point.
Figure 2 shows the tempeiature dependence of rotational correlation time as a function of v/T where 7 is the viscosity and T is the absolute temperature. For a spherical rotator in a continuous media, theory predicts a linear dependen~e.~ As shown in Figure 2, 4 is a linear function of the vIT. In this experiment, the 4's varied from 240 to over 1900 ns (269.6285.8 K). These results suggest that no unusual interaction occurs between solvent and solute within this temperature range. Table 1 shows @s, and estimated hydrodynamic radii, rhyd, based on the isotropic modela
rhyd
V = 4RTlr,1
(1)
= [(314)v/n] 1'3
(2)
where R is the gas constant, V is the hydrodynamic volume, and T and 17 are as defined earlier. The 4's are based on the assumption of a single correlation time. All attempts to fit the data to a multicorrelation time
diphenyl-1,lO-phenanthroline.
model failed to give fits that were superior to the single 4 model and generally gave physically unrealistic values for the extra parameters. There are interesting variations of the parameters with complex structure. We turn now to explore some of the trends. First, as expected, the 4's and rhyd's are a strong function of the size of the complexes as determined by their physical size. Compare the trends in rhyd for the homochelated R u L ~ ~ + complexes. These physical sizes reflect the number of ligands, and their substituents and are comparable to and follow the same size trend as determined from physical models. Measurements using CPK models gave average radii of [Ru(bp~)3]~+ 6.2 A, [Ru(phen)3I2+6.3 A, [Ru(5-Mephen)3I2+7.0 A, [Ru(5Phphen)3I2+8.1 A, and [Ru(Phzphen)3I2+8.7 A. These are the average radii determined from three approximately perpendicular molecular axes (N-metal-N). Because of the asymmetry of the complexes, these averages should be taken as approximations to the values for a spherical system. This method of approximations should tend to overestimate the rotational size of the complexes. The structures are more open than spheres, having a propeller-like character. Thus, the effective rotational size should be smaller than the calculated, which is what we observe. However, since most of the complexes have a similar structure, we would expect the observed trend to monotonically mirror the computed trend, which is what is observed. Beginning with [Ru(phen)3I2+, sequentially replacing the phen's with a bulkier ligand also results in a monotonic increase of both 4 and rhyd. In particular, compare the results for the 4,7-Ph2phen series. lhyd increases monotonically through the series [Ru(phen)3I2+,[Ru(phen)2(4,7-Ph2phen)lZ+, [Ru(phen)(4,7-Ph2phen)2l2+,and finally [Ru(4,7-Phzphen)3I2+,where rhyd increases by 74%. The effect, though monotonic, is not linear. The greatest incremental change (about 39%) occurs with the addition of the first 4,7-Phzphen ligand. The next two substitutions increase fiyd about 17% each. This suggests that, whatever rotational axis is involved in anisotropy loss, the motion includes a significant drag component from the phenyls. From the absence of a second detectable rotational correlation time, it appears either that these complexes are behaving as isotropic rotators or that the other correlation times differ from the fitst by too little to be detected by our fitting methods. Presently, we favor the latter explanation, especially since the asymmetry of the complexes is not too great and all rotational
Letters
12480 J. Phys. Chem., Vol. 98, No. 48, 1994 constants will not be too different. Thus, unlike the well-studied planar organic systems such as perylene? there is no low-friction rotation available that also causes anisotropy loss. As expected, substitution of phen’s by smaller ligands can dramatically lower both 4 and Ryd, as illustrated by the bis cyano complex. This complex has the lowest 4 observed in this series since the two CN’s are smaller than even a bpy. Before considering the rotational anisotropies, we review the expected results for different combinations of absorbing and emitting axes and the basic spectroscopy of Ru(I1) complexes. If the absorption is exclusively along a single molecular axis (z) and the emission is only on the same axis (z), then ro is 0.4. If the absorption is in the xy plane and emission is also allowed only in the xy plane, then the limiting ro is 0.1. For a z axis absorption and xy axis emission or for an xy absorption and z emission, ro is -0.2.1° Examples of molecules approaching these limiting ro’s are known in a variety of system^.^ In practice, measured ro’s generally are smaller in magnitude than the theoretical limits. This is frequently caused by overlapping absorption spectra that have different polarizations. The observed ro is then a superposition of the different polarizations. The spectroscopy of Ru(I1) complexes is quite complicated.” We present here a simplified description for explaining our results. The homochelated complexes are trigonal with D3 symmetry where we assign the 3-fold axis as being z. In contrast, the mixed-chelate complexes, RuL~L’,have the very low symmetry of C2, with only a single CZ axis of rotation through the metal and the center of the unique ligand. Further, if even one of the L’s has an asymmetric substituent (e.g., 5-Phphen), then there are no symmetry elements and the complexes have C1 symmetry. However, functionally, the formal symmetry and the apparent symmetry are not always the same. For example, if L and L’ are not radically different, then RuLzL’ may behave spectroscopically very similarly to an RuL3 complex. That is, it behaves as if it has 0 3 microsymmetry even though the true symmetry is much lower. Of course, an apparently high microsymmetry will eventually be broken as the spectroscopic properties of L’ deviate enough from those of L. The lowest absorptions and emissions in our complexes are metal-to-ligand charge transfer (MLCT) where an electron is moved radially from the metal d level to an antibonding n* level on the complex. There are two common descriptions of the MLCT excited states of these metal complexes. In one the electron is delocalized over all equivalent ligands, which for a tris complex gives rise to a planar-planar model. More recently, this has been challenged with evidence that suggests that the excitation is largely localized in a single ligand.I2 This second model predicts a linear result with any deviation ascribable to mixing of the excitation on the other ligands. DeArmond’s model is based on results obtained in rigid lowtemperature glasses.12 It is not clear whether his localized model applies to fluid solutions, such as used here. Our measurements do not allow us to distinguish between the two models. In the absence of any direct evidence for DeArmond’s model, we will interpret our results on the basis of the delocalized model. This has a direct physical interpretation in terms of the molecular axes and does not contradict any of our experimental results. In the limit of a highly asymmetric molecule, there is no distinction between either of the prevalent models. The TO’S for the D3 homochelated tris complexes are in reasonable agreement with the 0.1 value expected for a planar oscillator/emitter (xy, xy). In 0 3 symmetry the x and y axes are equivalent. Thus, this result is reasonable if the absorbing and emitting axes are both in the xy plane. In the lower C2
symmetry RuLzL’ systems, the behavior depends on the differences between the L and L‘ ligands. Similar L and L’ give RuL3 behavior. If there is a large difference between the MLCT states of L and L’, we would expect behavior of a C2 system. The excitation in this case will be localized in the ligand with the lowest energy MLCT state. We turn now to the more complex asymmetric systems. We had hoped that by changing one of the ligands we would introduce sufficient asymmetry to produce a linear-linear oscillator model. This was only the case if the perturbing ligand was substantially different from the parent phen. Thus, methyls and even one phenyl substituent on a phen had a negligible effect. We attribute this to the fact that the MLCT states of both ligands in these cases are very similar in energy. Only when the MLCT states of the ligands differ significantly in energy do we observe an increase in ro. In keeping with this model, the MLCT state of 4,7-Phzphen ligand is substantially lower in energy than that of phen. Thus, the excitation localizes on the 4,7-Ph2phen, and we observe an increase in ro to well above the theoretical limit for an xy absorber-xy emitter system. Ru(phen)z(CN)z has a still higher ro, since there is no low-energy MLCT component involving electron movement toward the CN’s. However, all of the complexes have limiting values well below the 0.4 value expected for a linear-linear oscillator. The most likely cause is the presence of overlapping absorptions of different polarizations. These results demonstrate that the ro’s and z’s for Ru(I1) complexes are in an excellent range for probing dynamics of synthetic and biopolymers, especially systems with long correlation times. Further, these properties lend themselves to the rational design of transition metal complexes for probes of dynamic anisotropy. We are examining a number of these complexes and will report on the detailed spectroscopy and applications to microheterogeneous media including vesicles and DNA.
Acknowledgment. We gratefully acknowledge support by the National Science Foundation (91-18034) and an NIH Biomedical Research Support Grant BRS-5-36187. Also, B.A.D. is pleased to acknowledge sabbatical support from James Madison University. References and Notes ( 1) Kalyanasundaram, K. Photochemistry in Microheterogeneous Systems; Academic Press: New York, 1987. (2) (a) Pyle, A. M.; Barton, J. K. Prog. Inorg. Chem. 1990, 38, 413. (b) Murphy, C. J.; Barton, J. K. Methods EnTymol. 1993, 226, 576. (c) Tysoe, S. A.; Morgan, R.; Baker, A. D.; Strekas, T. C. J . Phys. Chem. 1993, 97, 1707. (3) (a) Ludescher, R. D. Spectroscopy 1989, 5, 20. (b) Cantor, C. R.; Schimmel, P. R. Biophysical Chemistry: The Conformation of Biological Macromolecules; W. H. Freeman: San Francisco, 1980. (c) Huber, R. Trends Biochem. Sei. 1979, 4 , 41. (4) Demas, J. N.; DeGraff, B. A. Anal. Chem. 1991, 63, 829A. (5) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Plenum Press: New York, 1984. ( 6 ) Lide, D. R., Ed. Handbood of Chemistry and Physics, 71st ed.; CRC Press: Boca Raton, E, 1991; p 6-144. (7) Bacon, R. J.; Demas, J. N. Anal. Chem. 1987, 59, 2780. (8) Kawski, A. Crit. Rev. Anal. Chem. 1993, 23, 459. (9) Piston, D. W.; Bilash, T.; Gratton, E. J . Phys. Chem. 1989, 93, 3963. (10) Fujita, I.; Kobayashi, H. Inorg. Chem. 1973, 12, 2758. (11) Krausz, E.; Ferguson, J. In Progress in Inorganic Chemistry; Lippard, S. J., Ed.; John Wiley & Sons: New York, (1989); Vol. 37, pp 293-390. (12) (a) Myrick, M. L.; Bakley, R. L.; DeArmond, M. K.; Arthur, M. L. J . Am. Chem. Soc. 1988, 110, 1325-1336. (b) DeArmond, K. D.; Myrick, M. L. Arc. Chem. Res. 1989,22, 364-370.
