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H. G. Brittain and F. S. Richardson
Solvent Induced Circularly Polarized Emission from Fluorescein H. G. Brittain and F. S. Richardson” Deparfment of Chemistry, University of Virginia, Charlottesville, Virginia 2290 7 (Received July 6, 1976) Publication costs assisted by the Petroleum Research Fund
Circularly polarized emission (CPE) from fluorescein dissolved in an optically active solvent (a-phenylethylamine) is reported. The relatively large emission anisotropy factors (gem)observed for the fluorescein/aphenylethylamine system suggest strong solute-solvent interactions which are highly specific. Variations of gemwith X across the emission band further suggest either the presence of several emitting species in solution or different vibronic coupling mechanisms operative in total emission vs. CPE intensity processes. No induced circular dichroism (CD) was observed for the fluorescein/a-phenylethylaminesystem, indicating that the solute-solvent interactions differ significantly in the ground and emitting states of fluorescein.
I. Introduction Circularly polarized emission (CPE) spectroscopy is the emission analogue to circular dichroism (CD) and as such is capable of providing stereochemical and electronic structural information about the luminescent states of molecules just as CD provides similar information on molecular ground states. To exhibit CPE or CD a molecule must either be chiral or reside in a chiral environment. Optical activity of achiral molecules dissolved in an optically active solvent depends critically upon the strength and specificity of the solute-solvent interactions since the chirality sensed by the chromophoric group of the solute molecule derives entirely (directly or indirectly) from the solvent molecules. It is well known that optical activity may be induced in achiral compounds or in racemic mixtures of chiral compounds by dissolution in an optically active solvent. In the former instance optical activity in the achiral solute molecules is generated by chiral perturbations upon the solute chromophoric group by the solvent molecules. The effects of these perturbations may be: (1)to chirally distort the nuclear configuration of the solute molecule making it inherently optically active; (2) to induce optical activity in chromophoric transitions of the solute molecule through direct (chiral) electrostatic interactions between the solute molecules and the electronic charge distributions on the inherently achiral chromophoric group of the solute molecule; or, (3) a combination of the above. The induction of optical activity in achiral solute molecules by chiral solvent environments has been the subject of a number of recent experimental and theoretical st~dies.l-~ The optical activity observed for racemic mixtures of chiral solute molecules dissolved in an optically active solvent is presumed to arise from differential d(solute)-d(solvent) and 1(solute)-d(so1vent) interactions (in the case where a dextrorotatory solvent is used) which produce an equilibrium mixture of nonenantiomeric solute-solvent pairs. The net chirality generated in the formation of these nonenantiomeric solute-solvent pairs can lead to observable optical activity in transitions of the solute molecules.1° Induced optical activity arising from solute-solvent interactions in solution media is a potentially valuable source of information on the nature of solute-solvent interactions and on the structure of solute-solvent “complexes” formed under various physical and chemical conditions. Unlike solventinduced frequency shifts and intensity changes in ordinary The Journal of Physical Chemistry, Vol. 80, No. 23, 1976
absorption spectra, induction of optical activity requires that a rather specific set of structural and interactive conditions be met by the solute-solvent pairs (or clusters). CPE spectroscopy is a relatively new spectroscopic technique for probing the stereochemical and electronic structural details of molecular systems. In all the CPE studies reported to date the luminescent species (molecules or ions) were either inherently chiral or were coordinated directly to chiral ligands or to a chiral ligand environment (such as luminescent ions bound to a protein m ~ l e c u l e ) . ~In~ the - ~ ~present study we report the first observation of solvent-induced CPE from an achiral molecule dissolved in an optically active solvent. The achiral solute molecule is the highly luminescent fluorescein system and the optically active solvent is “neat” a-phenylethylamine. Solvent-induced CD is not observable for this solute-solvent system, so the induced optical activity (in the fluorescein dye) appears to be unique to the luminescent state of the solute molecules.
11. Experimental Section (I?)- and (S)-a-phenylethylaminewere obtained from Norse Laboratories and were used without further purification. Fluorescein (Aldrich) was recrystallized once and its purity was verified by thin-layer chromatography. All emission/CPE and absorption/CD measurements were carried out a t room temperature in neat a-phenylethylamine solvent. The dye (fluorescein) concentration was 2 X M. The emission/ CPE spectra were recorded on a high-sensitivity, high-resolution emission spectrophotometer constructed in this laboratory,lZband the 458-nm output of an argon ion laser (Coherent Radiation) was used as an excitation source. Emission/CPE spectra were measured using both 90 and 180’ excitation-emission geometries to look for spurious details due to linear polarization, photoselection, and inner-filter effects. The emission/CPE results reported here were obtained by using a 90’ excitation-emission geometry in which the excitation beam was focused onto the forward edge of the sample cuvette. This procedure was effective in eliminating innerfilter effects. The total emission (TE) and CPE spectra are reported in relatiue intensity units, whereas emission anisotropy factors, gem(see eq 11,are reported in absolute units.
Solvent Induced Circularly Polarized Emlsslon
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I
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A
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i
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the emission bands of the different species do not have exactly the same wavelength dependence). Differences in the delayed and prompt fluorescence spectra of fluorescein in boric acid glasses have been previously attributed to the presence of more than one emitting species.l6 However, fluorescence lifetime studies on fluorescein in H20/NaOH solution suggest the existence of a single emitting species.17 Although the variation of gem with X is rather striking, our data do not permit a detailed analysis or definitive comment regarding its origin. Steric crowding caused by the carboxyl moiety in the fluorescein molecule quite likely leads to nonplanarity between the o -carboxyphenyl and 6-hydroxy-3-isoxanthenone fragments of the system. This nonplanar distortion between the two fragments renders the structure inherently chiral. In a nonoptically active medium, however, the fluorescein would
600
X(nrn)
Flgure 1. Total emission (I), circularly polarized emlsslon (A/), and emission anisotropy factors (gem)for fluorescein dlssolved In (@aphenylethylamine (2 X M).
where IL and IR refer, respectively, to the intensities of leftand right-circularly polarized emission. We shall refer to A I as CPE and to I as TE. The CD spectra measurements were performed on a modified Durrum-Jasco J-1OB CD spectrophotometer. Fluorescein emission was excluded from the photomultiplier detector by placing a standard black glass filter (opaque from 400 to 650 nm) after the sample. N o CD was observed below 400 nm even on the most sensitive signal detection/amplification scales.
111. Results and Discussion The T E ( I ) and CPE (hl)observed for a dilute (2 X M) solution of fluorescein dissolved in (R)-a-phenylethylamine are shown in Figure 1. The TE spectrum resembles closely (in bandshape and wavelength maximum) that reported for fluorescein dissolved in H20-NaOH solution (at a pH of Il).ljThe spectra shown in Figure 1were obtained using a goo excitation-emission geometry with illumination concentrated near the forward edge of the sample. The CPE spectrum of Figure 1 is that induced by (R)-aphenylethylamine. The S isomer induces exactly the same CPE spectrum except for a reversal of sign. The TE spectra of fluorescein in ( R ) -and (S)-a-phenylethylamine are identical. Emission anisotropy factors (gem) calculated at various wavelengths within the emission region are also plotted in Figure 1. We note that while the T E and CPE bandshapes appear to be nearly identical, the emission anisotropy vs. wavelength plot reveals significant variations. Variation of gem within a given emission band can arise in several ways. The most significant of these are: (1)different intensity mechanisms for the CPE vs. TE processes; (2) presence of several emitting species which have different chiroptical properties; (3) dual emission from two different emitting states which have different chiroptical properties and whose emission bands are superimposed (overlapping). If the CPE intensity has a dependence upon vibronic coupling mechanisms which is different from the TE intensity dependence upon vibronic couplings, then one may expect gem to vary with A. Likewise, if several different emitting species are present and these species interact differently with the optically solvent, then one may expect variation of gemwith X (assuming, of course, that
HO fluorescein be racemic. In an optically active solvent (such as a-phenylethylamine) it is possible that one enantiomeric form will be preferentially stabilized through specific solute-solvent interactions thus effecting a partial resolution. This is one mechanism whereby CPE may be induced in fluorescein by a-phenylethylamine solvent. One may expect that a-phenylethylamine will interact rather strongly and specifically with fluorescein and that the strongest interaction site will be a t the carboxyl moiety. Given the relatively large gemvalues observed for the system it is unlikely that the dominant source of induced CPE originates with chiral dispersive interactions between the solute and solvent molecules. Due to interference from fluorescein emission, the CD spectrum of the fluoresceinla-phenylethylaminesolution could not be measured through the 525-400-nm region. However, CD was measured from 400 to 210 nm and no signal was observed. The absorption spectrum of fluorescein/aphenylethylamine in this region shows a moderately intense (6 4 0 0 0 ) band centered a t 275 nm and the onset of a much more intense band below 230 nm. If induced CD is generated, the associated gabs (aA€/€) value must be less than 5 X The observation of induced CPE but no induced CD indicates significantly different solute-solvent interactions in the emitting state vs. the ground state of fluorescein. This may be due to different electronic factors involved in ground state vs. excited state solute-solvent interactions or to stereochemical factors reflecting significant structural differences between the ground state and emitting state fluorescein species. Fluorescein dissolved in “neat” optically active a-phenylethanol and in diethyltartrate solvents did not exhibit any induced CPE. Acknowledgments. This work was supported by the National Science Foundation, the donors of the Petroleum Research Fund, administrated by the American Chemical Society, and the Camille and Henry Dreyfus Foundation (through a Teacher-Scholar Award to F.R.). The Journal of Physical Chemistry, Vol. 80, No. 23, 1976
E. Hsi, R. Mason, and R. G. Bryant
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ferences a n d Notes D. P. Craig, E. A. Power, and T. Thirunamachandran, Chem. Phys. Lett., 27,
149 (1974). B. Bosnich, J. Am. Chem. SOC.,89, 6143 (1967). L.D. Hayward and R. N. Totty, Can. J. Chem., 49,623 (1971). S . Claesson and L.D. Hayward, Chem. Phys. Lett., 20,85 (1973). P. E. Schipper, lnorg. Chim. Acta, 14, 161 (1975). P. E. Schipper, Chem. Phys. Lett., 30, 323 (1975). L.D. Hayward and S. Claesson, Chem. Script., 9, 21 (1976). S. F. Mason, Chem. Phys. Lett., 32, 201 (1975). P. E. Schipper, Mol. Phys., 29, 1705 (1975). P. E. Schipper, lnorg. Chim. Acta, 12, 199 (1975). See, for example: I. Steinberg in “Concepts in Biochemical Fluorescence”, Vol. I, R. F. Chen and H. Edelhoch, Ed., Marcel Dekker, New York, N.Y.,
1975, Chapter 3. (a) C. K. Luk and F. S. Richardson, J. Am. Chem. Soc., 96,2006 (1974); (b) C. K. Luk and F. S. Richardson, /bid., 97,6666 (1975); (c) H. G. Brittain and F. S . Richardson, ibid., in press; (d) H. G. Brittain and F. S. Richardson, lnorg. Chem., 15, 1507 (1976). (a) T. L. Miller, D. J. Nelson, H. G. Brittain, F. S. Richardson, R. B. Martin, and C. M. Kay, FEBS Lett., 58, 262 (1975); (b) H. G. Brittain, F. S.Richardson, R. B. Martin, L. D. Burtnick, and C. M. Kay, Blochem. Blophys. Res. Commun.. 66. 1013 119761. (a) H. P. J. M. Dekkers‘and L: E. Closs, J. Am. Chem. Soc., 98,2210 (1976); (b) C. A. Emeis and L. J. Oosterhoff, J. Chem. Phys., 54,4809 (1971); (c) C. A. Emeis and L. J. Oosterhoff, Chem. Phys. Lett., 1, 129, 266 (1967). C. A. Parker and W. T. Rees, Analyst, 87, 83 (1962). J. B. Birks and J. Grzywacz, Chem. Phys. Lett., 1, 187 (1967). S. Strickler and R. Berg, J. Chem. Phys., 37, 814 (1962).
Magnetic Resonance Studies of a-Chymotrypsin Crystals E. Hsi,la R. Mason,lb and R. G. Bryant+la*C Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455 and Veterans Administration Hospital, Minneapolis, Minnesota 55455 (Received February 18, 1976) Publication costs assisted by the National lnstitutes of Health
Nuclear magnetic resonance relaxation times and ESR nitroxide radical line shapes are measured to study the dynamics of water and solute motion inside crystals of a-chymotrypsin, It is concluded that a major fraction of the water within the crystal has rotational correlation times which are sufficiently short to be characteristic of liquid rather than solid behavior. The motional characteristics of some of the water inside the crystal is modified so that motional freedom is somewhat restricted although relatively rapid motion persists well below the usual freezing temperatures. The motion of the free-radical solute in the crystal is isotropic although somewhat hindered and the effects of salt on the thermodynamic and dynamic properties of water in the protein crystal are complex.
Introduction In spite of its clearly central position in the chemistry of biological processes, water and its many interactions are incompletely understood. Recently there has been increased effort to understand the several ways that water molecules acting singly and in concert may determine in part the properties of macromolecules or macromolecular aggregates such as enzymes and whole tissues. Nuclear magnetic resonance (NMR) and electron spin resonance (ESR) investigations are very promising approaches to this problem because the several possible measurements imply both structural and dynamic information. Interpretation of NMR measurements made on such complex systems as muscle, blood cells, and malignant and benign tumors28 must ultimately zest on understanding separately the possible interactions of water with the several constituents of these systems. The interpretation of ESR investigations of spin labels within the cytoplasm of whole cells would also benefit from an understanding of simpler systems. The present study of protein crystals was undertaken because the protein crystal provides a structurally well-characterized protein matrix in which protein motion is minimal. The crystal thus represents a limiting model for proteins in a constrained environment. The Journal of Physical Chemistry, Vol. 80. No. 23, 1976
Experimental Section a-Chymotrypsin used in this study was obtained from Worthington Biochemical Corp. Crystals were obtainedzbby treating a 2% protein solution at pH 3.5 with saturated ammonium sulfate to the cloud point. The solution was back titrated to clear with deionized water and allowed to stand in 10 mm test tubes. Excellent crystals were obtained in about 1week.3 Nuclear magnetic resonance measurements were made on a 30-MHz spectrometer built in this l a b ~ r a t o r y T2 . ~ measurements were made using the Gill-Meiboom modification of the Carr-Purcell pulse ~ e q u e n c eIn . ~most cases the signal was enhanced by signal averaging on a Varian C-1024 CAT and use of a sample and hold circuit eliminated the problems often caused by the radiofrequency transients associated with the successive 180° pulses. T I measurements were made using a 18Oo-9O0pulse sequence and the transient following the second pulse was often averaged for the weak samples. At low temperatures when Tz became less than 100 p s the free induction decay or a two pulse spin echo experiment was taken as a measure of Ta. There is no discontinuity apparent in the data from making this change in experimental detail. The 90’ pulse widths were less than 5 p s and the receiver recovery time is 5 p s . Errors in single component T I and T2 values are esti-
