J. Phys. Chem. 1992,96, 6204-6212
6204
Excited-State Properties of the Indole Chromophore. Electronic Transition Moment Directions from Linear Dichroism Measurements: Effect of Methyl and Methoxy Substltuents Bo Albinsson* and Bengt NordC Department of Physical Chemistry, Chalmers University of Technology, S-412 96 Gothenburg, Sweden (Received: February 12, 1992) From measurements of UV and IR linear dichroism on molecules partially oriented in stretched polyethylene host the transition moment directions for the first four T-T* transitions of indole and some indole derivatives were determined. Relative to the pseudosymmetry long axis of indole, the transitions were normally found to be polarized at (angles counted away from the ring nitrogen): +42 f 5' ('Al 'Lb at 287 nm), -46 5' ('Al 'La at 265 nm), 0 15' ('Al 'Bb at 220 nm), and for the 'A, 'B, transition occurring around 200 nm, at least at f30' away from this axis. In addition, indication for a weak, essentially short axis polarized transition was found at 235 nm, possibly due to the 'A, IC transition. An ambiguity problem regarding the sign of the angles was resolved by exploiting the change of orientation properties upon introduction of substituents. Orientation parameters (including diagonalizing angle) were determined by consideration of a large number of in-plane as well as out-of-plane polarized vibrational transitions. The question regarding effects on the excited states by the presence of methyl and methoxy substituents, at varied positions in the indole chromophore, was addressed in terms of the perturbations they caused on the transition moments. Whereas none of the four transitions was found to be very sensitive in this respect to methyl or methoxy groups introduced in 2-, 3-, 5-, or 7-position of indole, the directions of the weak 'A, 'Lb but also the strong 'A, 'Bb transition were found to become significantly altered by a methoxy group in 4- as well as 6-position. The conclusions are consistent with recent fluorescenceanisotropy data and semiempirical calculations.
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Introduction The excited-state properties of indole are important to understand for the interpretation of luminescence and absorption spectra of the amino acid tryptophan, in which indole is the side-chain chromophore. More specifically, the energetic distribution of the excited states and the associated transition moment directions of the absorbing and emitting electronic transitions are crucial parameters in techniques based on measurements of fluorescence anisotropy, fluorescence decay, and linear dichroism in studies of structure and dynamics of proteins and peptides. These methods provide information about, respectively, the mobility, the local environment, and the angular orientation of the chromophore in the protein.'v2 For example, time-resolved fluorescence anisotropy of tryptophan has been used to estimate the rotational mobility of indole residues in azurin3 and myelin basic proteins." A multiexponential fluorescence decay, frequently observed also for single tryptophans in proteins, indicates a complex photophysics of the indole chromophore that is most likely related to an effectively heterogeneous environment as a result of local conformational flexibility at the site of the residue. Linear dichroism, which can probe the orientation of the absorbing transition dipoles in a macroscopically oriented specimen, has been used, for example, to study the orientation of two tryptophan chromophores of RecA in its complex with single- and doublestranded DNAs.$ However, despite this variety of applications based on the indole chromophore, and most extensive research over the years on tryptophan's photophysics, the physical (quantum mechanical) background of the excited-state properties and their influence on the fluorescence decays are far from well understood. In this work we have studied indole and eight methyl and methoxy derivatives, with respect to polarization directions of the UV transitions, using linear dichroism in an anisotropic host of stretched polyethylene. From measurements of vibrational dichroism, conclusions about the orientational distribution of the solutes have been obtained. Such information is required for the interpretation of the UV dichroic spectra and for judging between electronic and orientational perturbations of substitution. Both kinds of perturbation effects are important and, in contrast to what is normally assumed, the direction of the transition moments may vary considerably upon substitution in the indole chromophore at certain positions. In order to explain the relevance of this work, let us put it into context by a brief review of today's knowledge on the excited states of indole (tryptophan).
The near-UV spectrum of indole consists of two absorption regions: two strongly overlapping bands centered around 270 nm due to the electronic transitions 'Al IL, and 'Al 'Lb (following the original notation of Platt),6 and one more intense absorption region beginning a t 220 nm assigned to another two transitions, 'Al 'B, and 'A, lBb. The extensive overlap between the vibronic transitions to the La and Lbstates, and the fact that the energetic order of their origins may even be reversed upon solvent interactions, lead to complex and solvent-sensitive absorption and emission For example, the emitting state changes from Lb in nonpolar solvents to La in polar solvents. In addition, the fluorescence quantum yields of indole and its derivatives are very dependent on temperature"-I3 which can be interpreted in terms of complex deexcitation pathways and, again, the sensitivity of the excited-state properties to environment; phot~ionization,'~ excited-state proton,ls and electron16J7transfer are effects that have been reported on. The photophysics of tryptophan is even more complex and is characterized by a double-exponential fluorescencedecay, attributed to a ground-state distribution of rotational conformers, with respect to the threesubstituent chain, which do not interconvert during the lifetime of the excited state.'* Time-resolved supersonic jet experiments show that the individual rotamers have monoexponential decaysIg-their different lifetimes are believed to be a result of quenching by intramolecular proton- or electron-transfer react i o n ~ . ~ *A~simplified ~ decay behavior of a conformationally constrained cyclic tryptophan derivative also lends strong support to this ground-state rotamer h y p ~ t h e s i s .At ~ ~ the same time, however, there is experimental evidence for the occurrence of excited-state reactions, side-chain and solvent complexes lowering the strongly dipolar La state energyF6 In this context, to be able to assess the effects of conformation of side chain, and in particular of the interaction with medium, detailed knowledge of the charge distribution of the excited state is req ~ i r e d , information ~' that involves complete determination of transition moment vectors An important question is thus how perturbations such as substituent moieties and solvent fields may perturb ground-state and excited-state wave functions and, as a result, the appearing transition energies and transition moment directions. In a recent study from this laboratory based on measurement of linear dichroism in an anisotropic host (stretched polyethylene), combined with fluorescence anisotropy measurements, transition
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0022-3654/92/2096-6204%03.00f 0 0 1992 American Chemical Society
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Properties of the Indole Chromophore
The Journal of Physical Chemistry, Vol. 96, NO. 15, 1992 6205 films were stretched to 5 times their original length and thereafter exposed to the sample substance in a sealed cell either at room temperature or at slightly elevated temperature (70 "C). Because of the high volatility of the indole derivatives no carrier solvent such as chloroform was needed to introduce any of the substances into the films. Linear Dichroism. LD is defined as LD = All - A ,
Indole Figure 1. Indole with substituent positions and definition of an in-plane angle relative to the pseudosymmetry long axis (broken line).
moment directions and transition band envelopes were determined for the L, and Lb transitions of indole and a few The directions for indole, which should display very little solvent and exciton effects in this medium, were found to be -54" (La) and +45' (Lb) where the angle is counted relative to the pseudosymmetry long axis (with positive sign in the 2-3 direction, see Figure 1). A number of earlier experimental studies, such as linear dichrosim on ~rystals,2~ in poly(viny1 alcohol)30and polyethylene3' hosts on different substituted indole derivatives, have yielded scattered results for the transition moment directions. Also, rotationally resolved spectra on supersonic jet expanded indole32 and tryptamine33have given information on the direction of the 'Lb transition moment. In the crystal study on 3'Al indolylacetic acid29the directions were concluded to be -38' and +54", respectively, thus suggesting only some minor perturbation by the 3-aceto substituent and by exciton interactions with adjacent indole chromophores in the crystal. Not unexpectedly, the introduction of weak perturbers such as methyl substituents was found not to markedly change the polarization directions (generally less than 10") whereas, as we shall see, stronger perturbation are obtained with methoxy substituents at certain positions. The observation of approximately orthogonal La and Lb transitions was also in agreement with inference from fluorescence anisotropy results.3e36 More recent fluorescence anisotropy measurements by Eftink et al.37support the conclusions that methyl substitution in the 2- or 3-position causes little change on the angle between the Laand Lb transition moments. However, these authors found that methyl substituents at the imino nitrogen, or in the benzoid ring, cause variable photophysical changes, with the La and Lb levels being nearly degenerate in some cases. The angle between the moments was concluded to remain around 90" for substitution at position 5, but to be less than 50" for 4-, 6-, and 7-methoxyindoles. Semiempirical molecular orbital calculations gave transition moment directions that were in qualitative agreement with the experimental relative polarizations. The present study extends earlier transition moment determinations on the low-energy spectrum of the indole chromophore to a series of methoxy and methyl derivatives and also to higher energy transitions. Whereas fluorescence anisotropy only provides the angle between the absorbing and emitting transition moments, UV linear dichroism measurements, assisted by IR linear dichroism, can give information about the individual moments in the molecular frame. In this way we hope to gain better understanding of the perturbation mechanisms recently reported from fluorescence anisotropy studies on indole derivatives and to obtain results that should be of general usefulness in the biophysical techniques exploiting fluorescence and dichroism properties of tryptophan.
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Materials and Methods Chemicals. Indole, 3-methylindole (3MI), 5-methylindole (5MI), 4-methoxyindole (4MOI), 5-methoxyindole (5MOI), 6-methoxyindole (6MOI), and 7-methoxyindole (7MOI) purchased from SIGMA were used without further purification. 2-Methylindole (2MI) and 7-methylindole (7MI) were a gift from Professor Erik W. Thulstrup. The polyethylene films were manufactured by pressing pure polyethylene pellets between two polished aluminum blocks kept at 120 OC using a hydraulic press and then rapidly quenching the melt to room temperature. The
(1)
where Ailand A, are the absorbances measured with plane polarized light parallel and perpendicular to the unique sample axis (the stretching direction of the film). The reduced linear dichroism, LD' is defined as LD' = LD/Ai,
(2)
where A,, is the absorbance of a corresponding isotropic sample. For a uniaxial sample such as the stretched polyethylene film, the isotropic absorbance can be calculated from the polarized components a~~~
= y3(All + 2AJ.)
(3)
The LD' of a pure in-plane polarized transition i in a chromophore like indole with C, symmetry (having only a plane of symmetry) is related to the angle (ei) between a transition moment, polarized in the plane of the molecule, and the preferred molecular orientation axis (z) according t~~~ LDri = 3(S,, cos2 Bi
+ SYysin2 ei)
(4)
where S,,and S, are respectively the Saupe orientation parameters for the in-plane (diagonal) axes z and y.@ The LD' of any out-of-plane polarized transition is equal to the orientation parameter for the out-of-plane axis x multiplied by 3 LD', = 3S,,
(5)
and the orientation parameters are related according to
s, + syy+ s, = 0
(6)
For overlapping transitions the observed LD' is a weighted average of the LDri values of the contributing t r a n ~ i t i o n s ~ ~ EAi(A)LDri
where Ai@) is the molar absorption (at wavelength A) associated with transition i. The "pure" reduced linear dichroism LDrI is obtained from the experiments by a trial and error method ("TEM")41q42based on the stepwise formation of linear combinations of the polarized spectra (e.g., All- dJ,). The subtraction coefficient (di) for which a specific spectral feature disappears in the linear combination is related to the LDri of the transition containing that feature43
Polarized IR measurementswere performed on a Perkin-Elmer 1800 Fourier transform spectrometer equipped with a KRS-5 polarizer (IGP228 Cambridge Physical Science). The nominal resolution was 2 cm-I and each spectrum was an average over 100 scans. A baseline for polyethylene was recorded similarly and subtracted on an interfaced computer. The LD', values were evaluated with respect to peak heights and peak areas. The All and A, s p r a were also linearly combined according to the TEM method which proved particularly useful to resolve solute peaks in presence of a strongly wavelength dependent polyethylene baseline. Polarized UV measurements were performed on a CARY 2300 spectrophotometer equipped with two rotatable Glan air-space
6206 The Journal of Physical Chemistry, Vol. 96, No. 15, 1992 calcite polarizers in sample and reference beams. The spectrophotometer was interfaced with a computer and five data points per nanometer were collected and the spectral bandwidth was 1 nm. Orientation Information from Polarid IR Measmments. The calculation of transition moment directions from LD‘ values by eq 4 requires first a determination of the orientation parameters and the direction of the orientation axis z in the molecular framework (Le., of the diagonal system yz). A molecule such as indole where the only symmetry element is the molecular plane has its average orientation properties fully described (to second moments) by either three orientation parameters, Sf$, S, , and S,, for an arbitrary in-plane system z’y’, or equivalently, i y two orientation parameters in the diagonal system and the direction of the diagonalizing axis z (the orientation axis).38 In order to determine these entities we measured the polarized IR spectra of the indole derivatives in the polyethylene films. In total as many as 171 vibrational transitions were observed. For a given molecule the number of transitions that could be observed depends on its solubility in the polymeric host and the tendency for the indole derivative to form hydrogen-bonded aggregates (this limits the maximum concentration to be used) as well as intrinsic spectroscopic properties such as strengths and wavenumber position of the peaks. With three unknown quantities we need at least three IR transitions with known moment directions. As expected, all out-of-plane polarized transitions are found to have approximately the same LD‘ for a given substance providing the orientation parameter for the out-of-plane axis, Sxx. Hence, two in-plane transitions with different and known dipole directions are needed to settle the orientation. Unfortunately, only the N-H stretching vibration fulfills the latter criterion of being known as to its polarization. Therefore, we have to rely on an approximate, statistical method. For each substance we observe as many as 10-23 in-plane polarized transitions. Since these transitions do not cluster into any preferred directions we can on purely statistical grounds expect one of them to be relatively close to the direction of orientation axis or the direction perpendicular to it. The LD‘ value for the transition closest to either of these directions, which is the extreme in-plane value, @vesan estimation of the appropriate orientation parameter (Szzor Syy)and the other in-plane orientation parameter may then be calculated from eq 6. This method has been used for all of the indole derivatives. In addition, the inclusion of the ether moiety in the methoxy indoles provides us with another possibility for the orientation parameter determination, briefly described as follows. The methoxy group has a strong asymmetricalC-O stretching band near 1250 cm-’.” This transition is polarized along a direction joining the end atoms in the COC group or, expressed otherwise, the transition moment for the asymmetrical C-O stretch is directed perpendicular to the bisector of the COC angle. This is true only if this vibrational motion does not couple significantly with any of the ring modes. Together with the N-H stretching mode and the out-of-plane polarized modes the asymmetrical C-0 stretch provides enough information to resolve the orientation problem. In order to use the asymmetrical C-O stretch we must therefore know the orientation of the methoxy group in relation to the indole moiety. The two main conformers where the COC part is coplanar with the indole ring, so as to maximize the pz overlap, seem to be more likely than any in which the exocyclic group is not in the plane of the ring. The two methoxy group conformations give the asymmetrical C-0 stretch transition moment two quite different directions. For the assignment of orientation axis directions we calculated the orientation parameters for both these conformations and assumed both are possible. In 4methoxy- and 7-methoxyindole the calculations yield reasonable values for the orientation parameters and orientation axis directions only if the methoxy groups are oriented “trans” to the pyrole ring. Such a geometry is actually expected for steric reasons since the distances between the methoxy hydrogens and the ring hydrogens are much smaller in the “trans” than in the “cis” configuration. In 5-methoxy- and 6-methoxyindole the alternative in-plane
Albinsson and Norden orientations of the methoxy group gave similar values on the orientation parameters. However, the orientation parameters obtained by this analysis for 5-methoxyindole were considered less reliable than those obtained by the alternative “statistical” method based on maximum and minimum LDr values. Electronic Transition Moments from Polarized UV Spectra. The LDri values provide measures of the orientation of the transition moments relative to the molecular axis that is on the average best aligned, Le., relative the orientation axis z. For a particular molecule the L D can, for in-plane polarized transitions, vary between 3Syy and 3Sz, according to eq 4. An in-plane transition moment oriented perpendicular to the orientation axis shows a small LD‘ value and one polarized close to the orientation axis shows a large LD‘ value. Given the orientation parameters and the direction of the orientation axis z the polarizations of the UV transition moments were calculated by use of eq 4. The angle 0, is transformed into an angle 6, within the molecular framework by adding or subtracting to the angle (a)that specifies the direction of the orientation axis 6i =
* leil
(9)
where ai and a are the angles from the pseudosymmetry long axis of the indole chromophore to the transition dipole moment and the orientation axis, respectively. Results The present extended study of indole chromophores was performed in order to further explore the nature of the near-ultraviolet transitions and, more specifcally, how their polarization properties are affected by methyl and methoxy substitutions. Indole is a hetereocyclic compound with 10 T electrons distributed over 9 centers and is iso T electronic with naphthalene. We shall below adopt the commonly used Platt nomenclature for the electronic states, with the lowest excited states called Lb and La and the higher Bb and B,, although strictly speaking these terms have distinct meanings for alternant hydrocarbons only. While this work concems primarily electronic transitions, it reports as well, as a byproduct, moment directions of a large number of IR transitions that can be quite valuable, e.g., for testing ab initio calculated force field^.^^,^^ Below, the UV results will be first presented, then briefly the IR measurements and the orientation characteristics, and in the last section these parts are brought together in the evaluation of transition moment directions for the first 3-4 electronic transitions of indole. Polarized UV Spectra of Indoles. The two lowest transitions in indoles are nearly degenerateand constitute the first absorption band. The ‘Al ‘Lb is the lowest energy transition responsible for most of the vibronic fine structure of the first absorption band and is best recognized by its distinct 0,Opeak at 287 nm for indole in polyethylene host. The ‘A, lL, is the second lowest transition, dominating the broad absorption maximum around 265 nm. The energy separation between these two transitions is a key parameter in many instances and, as can be seen in Figure 2, is sensitive to substitution site. Putting a substituent in the 3-position lowers the energy of the La transition whereas substituting in the 5- or 6-position lowers the Lb transition energy. The third electronic transition in the simple Platt scheme is the strong, and in benzene fully allowed, ‘A, ‘Bb transition. In Figure 2 all indole derivatives (except 5-methoxyindole) show a maxinum around 220 nm which may be ascribed to this transition. In this wavelength region, however, contributionsfrom other transitions are possible as well, and the simple four-orbital picture, which rather accurately describes the L bands,47is likely to break down in this higher energy region because of contributions from other excitations. In gas-phase spectra of indoles48a second strong band at 195 nm is resolved. It is of similar magnitude as the B,,band and has been assigned to the ’Al IB, transition.49 The reduced linear dichroism (LD’) spectra of the nine indole derivatives in stretched polyethylene are also shown in Figure 2. The LDrspectra show strong variations both in shape and relative amplitude between the different compounds because of different modes of orientation and/or different directions of the transition +
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Properties of the Indole Chromophore
The Journal of Physical Chemistry, Vol. 96, No. 15, I992 6207
1
0.8
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0.04 5MOI
ID
5MOI
0.4
0.2
0
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200
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200
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0.00
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Wavelength (nm)
Figure 2. Reduced linear dichroism (LD') and absorption (Aiw)spectra of indole, 3-methylindole, Smethylindole, 2-methylindole, 7-methylindole, 4-methoxyindole, 5-methoxyindole, 6-methoxyindole, and 7methoxyindole in stretched polyethylene host. LD' (scale) and Ai, (arbitrary units) calculated from measured All and A, spectra (see text). 1.5
1
A.U. 0.5
0
-0.5 200
250
300
350
Wavelength (nm)
Figure 3. Stepwise spectral reduction All - diA, for 5-methoxyindole. The coefficient d, is varied between 0 and 2 in steps of 0.2. The subtraction coefficients related to each electronic band are indicated.
dipoles. Below we shall show that most of the indole derivatives have transition moments with approximately conserved directions in relation to the unsubstituted molecule. Only in two cases (4methoxy- and 6-methoxyindole) it was found necessary to take into account considerable changes in the transition moment directions in order to explain the LD' spectra. The LDrivalues evaluated from the spectral reduction method are presented in Table I (IR data) and Table I1 (W data). Figure 3 shows an example of this spectral analysis for the UV spectrum of 5-methoxyindole. A rough estimate of the intrinsic LDrivalues can also be obtained from the limiting values of the presented LD' spectra. In Table I1 it is seen that the orientation parameters and orientation axes vary with substitution pattern. The more elongated molecules (2MI,5MI, 5MOI,6MOI) show larger span in S values than those in which the substituent points in the short-axis direction (7MI,4MOI, 7MOI). Also the orientation axis changes in a reasonable way according to the molecular shape. Polarized IR Spectra of Indoles. Figure 4 shows an excerpt from the polarized IR spectrum of 5-methoxyindole in which several in-plane polarized transitions and one prominent out-ofplane polarized transition (at 830 cm-') are seen. The LDr data evaluated from the IR spectra are collected in Table I together
Wovenumber (cm- 1)
Figure 4. Polarized vibrational spectrum of 5-methoxyindole in stretched polyethylene. (Top) LD = All - A,; (bottom) All and A, (parallel component offset by 0.025 absorbance units for clarity). Filled circles show in-plane polarized transitions; unfilled circle shows out-of-plane transition.
with vibrational assignment^.^^ The out-of-plane polarized transitions are easily recognized by their large negative LIY values. In order to more thoroughly analyze the in-plane assignments, a normal-coordinate calculation would be necessary. In Table I those transitions which show matching frequencies, between the derivatives, are put on the same line, without claiming any other relationship (the assignments are for unsubstituted indole). Transition Moment Directions. The electronic transition moment directions relative to the respective orientation axes, for the nine studied compounds, are calculated from the LDrivalues and the orientation parameters according to eq 4. This angle is transformed into the angle 6 in the molecular framework by adding or subtracting to the angle defining the orientation axis direction (eq 9) and is presented for the four pronounced near-UV transitions in Table I1 and Figure 5. In Table I11 the angular difference between the directions of the Lb and La transition moments are calculated and compared to the corresponding angles obtained from fluorescence anisotropy mea~urements.~~J' The Lb transition is found to remain polarized between + 3 5 O and +44O from the long axis in indole, 3-methylindole, 5methylindole, 2-methylindole, and 5-methoxyindole. We conclude that in this set of molecules the 'Al 'Lb transition is essentially conserved with respect to its direction whereas the energy shows some variations (e.g., 5-methylindole with a 9-nm red shift). This may be true also for 7-methyl- and 7-methoxyindolewhere in each case at least one solution (+28O and +38O, respectively) is not far away from the +45O for the parent compound and no energy shift is observed. For 4-methoxy- and 6-methoxyindole large deviations in this transition moment direction are found despite the absence of strong shifts. For the 'Al 'La transition a direction between -31O and -53' is found for all derivatives except 2-methylindolewhich has a more long axis polarized La band (-15O). The 'Al 'Bb transition shows local maxima in the LIY spectra for all compounds in Figure 2 except for 4-methoxyindole. This is because the Bb transition is polarized close to the long axis in indole and most of its derivatives. As seen in Table 11, for most of the compounds, one solution falls within &IOo along the long axis. Such an invariance is not surprising for a strong transition and, therefore, the exception in 4methoxyindole which has a much more short axis polarized Bb band (+52O or -74') is notable. To
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6208 The Journal of Physical Chemistry, Vol. 96, No. 15, 1992
Albinsson and Nord6n
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Properties of the Indole Chromophore
The Journal of Physical Chemistry, Vol. 96, No. 15, I992 6209
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TABLE III: Moment Directions for the 'Al 1band IAl 'L, Transitions Comparisons of Differences Obtained from LD and FPA +
Measurements
8.dLD) = 16.
substance indole
888
o m -
*-w
+h +h +h
- 6kl'
MFPA) 70-90: 83' 6+90,b 6oC 60-90: 86c 60-90b 50-90b 20-30b 70-90: 7ff 30-4ob 40-50b
88 85 I9 50 61 21 or 62 85 19 or 81 85
3MI 5MI 2MI 7MI 4MOI 5MOI 6MOI 7MOI
'From this study. bReference37. 'Reference 28.
our knowledge no systematiccalculations of the directions for this higher energy transition have been made to study its substitution sensitivity. It would be highly relevant to see if this dramatic change in direction for this strongly allowed transition may be accounted for by a quantum mechanical approach. In Figure 2 a drop in the LD' spectra is seen for all compounds at the blue end of the second, strong absorption band indicating 'Ba) is more short axis polarized. that the next transition ('Al Around 235 nm a weak feature is seen in all LD' spectra suggesting the existence of another, so far unidentified, weak transition.
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Discussion This work reports on the one hand transition moments for the high-energy 'Al 'Bband 'Al '€3, transitions of indole and its methyl and methoxy derivatives and, on the other, how these 'Lb and transitions, as well as the low-energy transitions 'Al 'Al lLa,respond to the perturbations from substituents. The lack of information about the former transitions from fluorescence studies can be ascribed to a strongly decreased quantum yield, possibly due to photoelectron e j e c t i ~ n , obviating ~ ' ~ ~ ~ reliable fluorescence polarization measurements. Such information is anticipated to become useful, however, in techniques based on absorption anisotropy (linear dichroism); indeed, our transition moment data for the Bb band has already been exploited in one study to aid determination of the intrinsic orientation of a t r y p tophan chromophore in a macroscopically aligned protein complex, R ~ c A - D N A . Below ~ we shall discuss the implications of our findings to the understanding of the excited states of indole and how these are perturbed by the various substituents. UV Transition Moments. Since the absorption probability depends quadratically on the transition moment all experimental determinations will lack sign information about the dipole direction. This difficulty may be circumvented by three different approaches: (1) by inference from orientation effects of substituents in derivatives, (2) by comparison with fluorescence anisotropy providing angles between transition moments, and (3) by correlation with quantum chemical calculations. To use the first method the dipole direction must be essentially independent of substitution. This will be our initial trial approach. Two indole derivatives substituted differently will possess different orientation axes and thereby show different LD' spectra. Each LD' value yields two possible moment directions and these are compared painvise between the molecules. The solutions that show strong variation with substitution are rejected. This p r d u r e is extended to include all derivatives. In some cases no reasonable substitution-invariant direction is found and it is then concluded that the transition moment direction must be significantly changed. For example, compare the LD' spectra for 5-methylindole and 3methylindole. In 5MI the La band has a large positive LD' value (0.64,Table 11) and the Lb band a smaller LDrvalue (0.14). In 3MI the relative magnitudes are switched with Lb being high (0.47)and La low (0.20). This is found to be due entirely to a change of orientation axis direction and the 'Al 'Lband 'Al 'Latransitions are concluded to have approximately unchanged directions (+44O, -35O) and (+41°,- 4 4 O ) , respectively, compared to the discarded solution (-72, +7) and (-19, +66). The
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6210 The Journal of Physical Chemistry, Vol. 96, No. 15, 1992 Indole
Albinsson and Norden 5-methox yindole
2-methylindole
4-methoxyindole
5-methylindole
7-methoxyindole +SZ
f
Lb
+-
-. -32
'
La - 4 7
-S? Ilh(illl)
-74
Finun 5. Transition moment directions concluded from the present dichroic study. The dashed lines represent the preferred molecular orientation a& z. The lengths of the arrows are arbitrary.
fluorescence anisotropy provides the angle between the transition moments as compared to, in the dichroism, their projections on the orientation axis. This property is particularly useful in the indole systems where the directions of the L, and Lb transition dipoles lie almost symmetrical with respect to the orientation axis and are, therefore, hard to discriminateon purely LD' basis. The dipole directions for the 'Al 'Lb and 'Al 'La transitions concluded in this study are in fair agreement with the fluorescence anisotropy results of Eftink et al.37 except for the case of 6methoxyindole and 7-methoxyindole (Table 111). The disagreement for 6MOI may be as small as 1 1 O if the smaller angle difference is preferred, Le., for the more parallel fashion of the La and Lb moments. On the other hand, for 7MOI we advocate the larger difference (85O) in favour of the other possibility (29O) because the LD' spectrum of this compound could be explained without requiring a drastic change of the IA, 'Lb transition dipole direction (Table 11). It should be noted that the LD' values in Table I1 for the 'A, 'La transition are obtained at the absorption band maximum around 265 nm in order to compare all derivatives on an equal basis. For some of the compounds (indole, 3M1, 2MI) the 0.0 region is measurable: LD' values from this region generally gave transition moment angles relative to the orientation axis which were 5-loo larger (Le., more negative values) than those for the higher vibronic transitions. This behavior is expected since vibronic coupling with the more long axis polarized B transitions would alter the transition moments to make the higher vibronic La components more long axis polarized. Please note that the L,
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transition moment directions for indole and 3MI in our earlier paperz8refer to the 0,Oregion and thus deviates by approximately loo from the directions presented in this article. The third pronounced transition (lA1 lBb) is found between 215 and 224 nm (Table 11) for all of the compounds. The directions evaluated from the LD',values for this transition falls in the interval between -loo and +IOo for all derivatives except 4-methoxyindole. The somewhat larger uncertainty (Table 11) for this long-axis-polarizedtransition is due to the insensitivity of the cosz B function to angular variations for angles close to B = 0. CMethoxyindole has undoubtedly a more short axis polarized & band ( + 5 2 O or -74O). Since no fluorescence anisotropy results have been published for this wavelength region it is hard to favor one solution over the other. The reason for the repolarization is unclear, however, since the 'Al 'Lb direction is markedly perturbed too but not the !Al IL,direction,the same arguments that explain the changes for the Lb moment,37by analogy with substituted benzenes, might apply also for this transition. The 'Al 'Bb transition in 6-methoxyindole should by similar arguments show repolarization as well. Indeed, an observation that should be taken for what it is worth is that one possible solution for the transition in 6MOI is +49O, a value close to one of the solutions obtained for 4MOI (+52O). Comparisoawith Quantum Mechanical cI.lculetioaa A number of quantum chemical calculations of the excited states for the indole chromophore have been reported.29,U),36,37,47,53-56 Although properties of the lowest excited states, such as energies, are fairly well described by more simple calculations considering only the
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The Journal of Physical Chemistry, Vol. 96, No. 15, 1992 6211
Properties of the Indole Chromophore two highest occupied and two lowest unoccupied 7r molecular orbitals, a number of higher excited configurations appear to be needed to "stabilize" the lowest excited states. This seems par'Lb transition which at the ticularly important for the 'Al four-orbital approximation level is dominated by an asymmetrical combination of HOMO (LUMO 1) and (HOMO - 1) LUMO excitations. In naphthalene and other alternant hydrocarbons the transition moments for these two excitations exactly cancel giving a forbidden 'Al 'Lb transition in these molecules within the zero differential overlap approximation. This occurs in the indole system as well but to less extent. Due to the cancellation, the 'Al 'Lb transition can be expected to be sensitive to perturbations and this is indeed the case also in the quantum chemical calculations which become unstable. If, for example, the amount of the HOMO LUMO excitation (which dominates the 'Al 'La transition) is increased in the 'Al lL,, transition, the Lb transition moment direction changes dramatically (unpublished results). The La transition moment seems much less easy to perturb. In spite of these properties of the indole chromophore, a virtually reliable parameter set for an INDO/S program, which is able to describe the 'Al 'Lb and 'Al 'La transitions appropriately for a number of indole derivatives, has been developed by Callis and co-worker~.~'The indole transition moment directions were calculated by these authors to be +59" and -50" for the Lb and La transitions, respectively. Small changes are predicted for indole derivatives having substituents in the pyrrole ring or at the 5-position of the phenyl moiety, whereas substitution at the 4-, 6-, and T-position with an electron-donating group like methoxy considerably perturbs the L,,transition moment direction. In these derivatives, the 'Al 'Lb transition moment is according to the calculations predicted to be almost parallel to the La transition, a proposal that for the 4-methoxyindole and 6-methoxyindole, but not quite for 7-methoxyindole, has support from the fluorescence anisotropy measurements (Table 111). Our linear dichroism results are compatible with the calculations and we thus conclude that a significant change in the Lb transition moment direction is needed to explain the LD' spectra, at least for 4- and 6-methoxyindole. The LD' spectra for 7-methoxyindole is possible to explain without changing the Lb moment direction dramatically, as discussed earlier. Thus, with the exception of 4- and 6-methoxyindole, the experimental data (Le., linear dichroism and fluorescence anisotropy) indicate that the transition moment direction for the 'Al 'Lb transition is conserved in all studied compounds. An 'Al 'C Transition in indole. The main features in the near-UV spectrum of the indole chromophore are well described by four prominent electronic transitions in the studied wavelength region (200-340 nm). However, this is the minimum number of transitions and there could exist other, weaker transitions that are covered by the stronger bands. Around 230-240 nm the LD' spectra in Figure 2 for all of the compounds show a local feature, either a minimum (indole, 3M1, 5M1, 2M1, 7M1, 4MO1, and 7MOI) or a plateau value (5MOI and 6MOI) indicating a weak short-axis-polarized transition not evident in the normal absorption spectra. There have been suggestions in the literature about an extra transition occurring in this region on the basis of circular dichroism spectra of tryptophan49and other chiral indole deriva t i v e ~ .These ~ ~ authors suggested that it was the "forbidden" 'Al 'C transition. Also in quantum chemical calculations for the indole chromophore (CNDO/S and PPP, unpublished results), evidence for an extra transition above 200 nm has been obtained.
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Conclusions The near-UV spectrum of the indole chromophore originates mainly from four electronic transitions; 'Al 'Lb at 287 nm, 'Al 'La at 265 nm, 'Al at 220 nm,and 'Al 'B, around 200 nm which are polarized at, respectively, +42", 46",OD, and more than 30" to the indole pseudosymmetry long axis (Figure 5 ) . In addition, there is evidence for a weak, roughly shortaxis-polarizedtransition around 235 nm. The transition moment directions for the main four transitions are insensitive to methyl and methoxy substituents at the 2-, 3-, 5-, and 7-position in the
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indole chromophore. By contrast, both the 'Al 'Lb and 'A, 'Bb transition moments are found to become significantly altered in 4- and 6-methoxyindole.
Acknowledgment. This work is supported by the Swedish Natural Science Research Council. Registry No. Indole, 120-72-9; 3-methylindole, 83-34-1; 5-methylindole, 614-96-0; 4-methoxyindole, 4837-90-5; 5-methoxyindole, 100694-6; 6-methoxyindole, 3 189-13-7; 7-methoxyindole, 3 189-22-8; 2methylindole, 95-20-5; 7-methylindole, 933-67-5; polyethylene, 900288-4.
References and Notes (1) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Plenum Press: New York, 1983. Rev. Biophys. 1992,25, (2) Norden, B.; Kubista, M.; Kurucsev, T. Quart. 5 1-1 70. (3) Petrich, J. W.; Longworth, J. W.; Flemming, G. R. Biochemistry 1987, 26. 271 1-2722. (4) Lakowicz, J. R.; Mailwal, B. P.;Cherek, H.; Baker, A. Biochemistry 1983, 22, 1741-1752. ( 5 ) Hagmar, P.; Norden, B.; Baty, D.; Chartier, M.; Takahashi, M. J. Mol. Biol., in press. (6) Platt, J. R. J. Chem. Phys. 1949, 17, 484-495. (7) Martinaud, M.; Kadiri, A. Chem. Phys. 1978, 28, 473-485. (8) Rehms, A. A.; Callis, P. R. Chem. Phys. Leu. 1987, 140, 83-89. (9) Illich, P.;Haydock C.; Rendergast, F. G. Chem. Phys. Lett. 1989,158, 129-134. (10) Demmer, D. R.; Leach, G. W.; Outhouse, E. A.; Hager, J. W.; Wallace, S. C. J. Phys. Chem. 1990, 94, 582-591. (1 1) Eisinger, J.; Navon, G. J . Chem. Phys. 1969, 50, 2069-2077. (12) Glasser, N. J.; Lami, H. J. Chem. Phys. 1978,68, 3317-3319. (13) Kirby, E. P.; Steiner, R. F. J. Phys. Chem. 1970, 74, 4480-4490. (14) Bent, D. V.; Hayon, E. J. J. Am. Chem. SOC.1975,97,2612-2619. (1 5 ) Stryer, L. J. J. Am. Chem. SOC.1966,88, 5708-57 12. (16) Steiner, R. F.; Kirby, E. P. J. Phys. Chem. 1969, 73, 4130-4135. (17) Ricci, R. W.; Nesta, J. M. J . Phys. Chem. 1976, 80, 974-980. (18) Szabo,A. G.; Rayner, D. M. J. Am. Chem. Soc. 1980,102,554-563. (19) Phillips, I. A.; Webb, S. P.; Martinez, S.J. 111; Flemming, G. R.; Levy, D. J. Am. Chem.Soc. 1988, 110, 1352-1355. (20) Shizuka, H.; Serizawa, M.; Shimo, T.; Saito, I.; Matsuura, T. J . Am. Chem. Soc. 1988, 110, 1930-1934. (21) Chang, M. C.; Petrich, J. W.; McDonald, D. B.; Flemming, G. R. J. Am. Chem. Soc. 1983, 105, 3819-3824. (22) Petrich, J. W.; Chang, M. C.; McDonald, D. B.; Flemming, G. R. J. Am. Chem. SOC.1983, 105, 3824-3832. (23) Engh, R. A.; Chen, L. X.-Q.; Flemming, G . R. Chem. Phys. Lerr. 1986. 126. 365-372. -. - ., . -. . .. . (24) Tilstra, L.; Sattler, M. C.; Cherry, W. R.; Barkley, M. D. J. Am. Chem. SOC.1990, 112,9176-9182. (25) Willis, K. J.; Szabo,A. G.; Kracjanki, D. T. Chem. Phys. Len. 1991, 182.614-616. (26) Tubergen, M. J.; Levy, D. H. J. Phys. Chem. 1991,95,2175-2181. (27) Levy, R. M.; Westbrook, J. D.; Kitchen, D. B.; Krogh-Jespersen, K. J . Phys. Chem. 1991, 95, 6756-6758. (28) Albinsson, B.; Kubista, M.; Norden, B.; Thulstrup, E. W. J. Phys. Chem. 1989, 93, 6646-6654. (29) Yamamoto, Y.; Tanaka, J. Bull. Chem. SOC.Jpn. 1972, 45, 1362-1366. (30) Maki, I.; Nishimoto, K.; Sugiyama, M.; Hiratsuka, H.; Tanizaki, Y. Bull. Chem. SOC.Jpn. 1981, 54, 8-12. (31) Suwaiyan, A. Spectrochim. Acta 1986,42A, 1021-1025. 1321 Phillios. L. A.: Levv. D. H. J . Chem. Phvs. 1986. 85. 1327-1332. (33) Phillips; L. A.; Levy; D. H. J. Phys. Chdm. 1986; 90; 4921-4923. (34) Weber, G. Biochem. J. 1960, 75, 335-352. (35) Valeur, B.; Weber, G. Photochem. Phorobiol. 1977, 25, 441-444. (36) Song, PA.;Kurtin, W. E. J. Am. Chem. Soc. 1%9.91,4892-4906. (37) Eftink, M. R.; Selvidge, L. A.; Callis, P.R.; Rehms, A. A. J. Phys. Chem. 1990, 94, 3469-3479. (38) Michl, J.; Thulstrup, E. W. Spectroscopy with Polarized Light; VCH Publishers: New York, 1986. (39) Norden, B. Appl. Specrrosc. Rev. 1978, 14, 157-248. (40) Saupe, A. Mol. Cryst. 1966, 1, 527-540. (41) Thulstrup, E. W.; Michl, J.; Eggers, J. H. J. Phys. Chem. 1970, 74, 3868-3878. (42) Michl, J.; Thulstrup, E. W.; Eggers, J. H. J . Phys. Chem. 1970, 74, 3878-3884. (43) Albinsson, B.; Kubista, M.; Sandros, K.; Norden, B. J . Phys. Chem. 1990,94,4006-4011 (44) Silverstein, R. M.; Bassler, G. C.; Morrill, T. C. Spectrometric Idenrification of Organic Compounds, 3rd ed.;John Wiley t Sons,Inc.: New York, 1974; p 94. (45) Bradley, R. A.; Balaji, V.; Downing, J. W.; Radziszewski, J. G.; Fisher, J. J.; Michl, J. J . Am. Chem. SOC.1991, 113, 2910-2919. (46) Holmen, A.; Albinsson, B.; Norden, B. Unpublished results (Holmen, A. MSc. Thesis).
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(47) Callis, P. R. J . Chem. Phys. 1991, 95, 4230-4240. (48) Illich, P. Can. J. Spectrosc. 1987, 32, 19-27. (49) Auer, H.E. J . Am. Chem. SOC.1973, 95, 3003-3011. (50) Lautie, A.; Lautie, M. F.; Gruger, A.; Fakhri, S. A. Spectrochim. Acta 1980, 36A, 85-94. (51) Feitelson, J. Photochem. Photobiol. 1971, 13, 87-96. (52) Tatischeff, I.; Klein, R.; Zemb, T.; Duquesne, M.Chem. Phys. Lert. 1978, 54, 394-398.
(53) Callis, P. R. Int. Quantum Chem., Quantum Chem. Symp. 1984,18, 579-588. (54) Nilsson, I.; Berg, U.; Sandstrh, J. Acto Chem. S c a d . 1986, B40, 652-651. (55) Tomas-Vert, F.; Ponce, C. A.; Estrada, M. R.; Silber, J.; Singh, J.; Anunciatta, J. J . Mol. Struct. 1991, 246, 203-215. (56) Souto, M. A.; Wallace, S . L.; Michl, J. Tetrahedron 1980, 36, 1521-1530.
Piezoelectric Detection of High Vibrational Overtones of Liquid (CH3),Si, (CH,),Ge,
and
(CH3),Sn Carlos Manzanares I.,* Jingping Peng, and Victor M. Blunt Department of Chemistry, Baylor University, Waco, Texas 76798 (Received: March 23, 1992)
The liquid-phasehigh vibrational overtone spectra of (CH3)Si,(CH3)4Ge, and (CH3)4Snhave been recorded using piezoelectric detection. The overtones have been measured in the spectral regions of the C-H stretching modes corresponding to Av = 6 and 7. A technique is presented which uses a piezoelectric detector, lock-in amplification, and a continuous wave dye laser modulated at frequencies from 10 to 120 kHz with an acousto-opticmodulator. Acoustic resonance frequencies for a quartz cuvette and a cylindrical cell are observed experimentally using liquid (CH3)4Mcompounds (M = Si, Ge, and Sn) as the sample. The speeds of sound in the three liquids are determined using the acoustic resonant frequencies of a quartz cuvette. The peak cross sections of the C-H (Au = 7) overtones are determined. The overtone bands have been computer deconvoluted to obtain peak positions and bandwidths of the individual transitions. Assignments for the overtones of the C-H stretch and local mode-normal mode combination bands are presented. The possible relationship between the bandwidths of the transitions and energy localization is discussed.
1. Introduction
Pulsed lasers have been preferred to modulated continuous wave (CW) lasers in studies of piezoelectric detection of weak absorptions in condensed phases.'-3 In the pulsed experiments, the advantages afforded by the high output power of the laser and gated extraction of the acoustic signal are high sensitivity coupled with discrimination against window absorption and laboratory noise. The CW technique uses a piezoelectric detector, lock-in amplification, a beam modulator, and a CW laser. It has been shown that the CW technique can afford similar advantages to the pulsed technique despite the lower output power of CW lasers compared to pulsed laser^.^,^ In the CW technique, enhanced sensitivity can be achieved by modulating the laser at or near the resonant frequencies of the piezoelectn'.cmaterial. In the resonance experiment, the range of modulating frequencies (10-120 kHz) is well separated from typical laboratory noise below 10 kHz and, as in the case of pulsed experiments, preamplifiers with bandpass filters can be used to reduce this noise. Although nonresonant piezoelectric detection of weak absorptions with CW lasers has and in analytical been used in studies of solids and application^,'^^^ the resonant technique has rarely been used in condensed phases. In contrast, resonant photoacoustic spectroscopy in the gas phase has been successfully applied to trace gas analysis, determination of thermophysical properties, kinetic processes, and s p e c t r o s c ~ p y . ' It ~ ~has ~ ~also been used as a very sensitive technique in laser intracavity experiments of gas phase overtone absorptions.16 The liquid-phase C-H stretching overtone spectra of (CH3)4Si have been investigated" using a conventional spectrophotometer (Av = 2-5) and the thermal lens technique (hu = 6). The overtone spectra of tetramethylsilicon, -germanium, and -tin have been measured in the spectral regions of the C-H stretching overtones corresponding to Av = 2-7 in the gas phase18J9and Av = 2-6 in the liquid phase.19 The liquid experiment around the Au = 6 region was done with a conventional spectrophotometerand cells of 8 or 10 cm path length. The spectra showed weak absorption profiles. The observed spectral features were assigned on the basis
of the local mode model. The local mode frequencies and anharmonicities were determined. Narrow bandwidths were also observed in both phases. The spectra around the Av = 7 region have not been measured in the liquid phase. In this paper we report the use of the resonant CW technique on the molecules (CH3),Si, (CH3)4Ge,and (CH3)4Sn.This group of molecules was chosen to observe the weak Av = 6 and 7 transitions of the tetramethylmetalcompounds. The spectra were obtained using both a cylindrical cell and a cuvette. This work shows that the technique can be used to detect weak absorptions using a quartz cuvette as the cell, modulating the laser at a resonant frequency of the cuvette, and is not limited to a cylindrical cell with submersed piezoelectric detector. The cross section of the Av = 7 transition of (CH3)$i was measured in a cuvette. In addition, the observed spectra are computer deconvoluted, the observed peak positions are assigned, and the bandwidths of the absorptions are reported and compared with previous studies in the liquid and gas phase. 2. Experimental Section
A detailed description of the photoacoustic apparatus used for the study of weak absorptions in liquid samples has been presented in a previous paper.4 The output beam of a Laser Ionics 544A CW argon ion laser operating in the all lines mode passes through an acousto-optic modulator. The amustooptic modulator (AOM) system (Newport modulator and driver) is used to modulate the laser beam from 10 to 200 kHz as a square wave. The strongest (514 run)diffracted beam is used to pump a CW Coherent 599-01 dye laser. The power of the pump beam is 3 W. Wavelength tuning of the (1 cm-l bandwidth) dye laser is accomplished with a birefringent filter driven by a stepper motor. The stepper motor is controlled with a microcomputer. The modulated output of the dye laser is directed along the length of a photoacoustic cell. The cylindrical photoacoustic cell is 12.7 mm in diameter and 15.0 mm in length. The cell is bored out of a brass cube. The transducer, similar to the one described by Pate1 and Tam,' is inserted radially at the center of one side of the cube. Only a
0022-3654/92/2096-62 12S03.OO/O 0 1992 American Chemical Society
