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1La transitions of jet-cooled 3-methylindole
Volume 193, number 6
CHEMICAL PHYSICS LETTERS
12 June 1992
‘L, transitions of jet-cooled 3-methylindole David M. Sammeth,
Sonja S. Siewert, Lee H. Spangler and Patrik R: Callis
Department of Chemistry, Montana State University, Bozeman, MT 59717, USA
Received 2 1 February 1992
One-photon and polarized two-photon fluorescence excitation spectra of jet-cooled 3-methylindole (3MI) and 3-trideuteriomethylindole are reported at a resolution of ~0.2 cm-‘. On the basis of the two-photon intensity ratios for linear versus circular polarized excitation, strong lines with predominantly ‘L. character are identified in 3MI at 409,420,468,609,617, 739,820, and 9 18 cm-’ above the ‘L,, origin. The lowest-lying ‘L. line in 5-methylindole appears to be at 1424 cm-‘. Evidence for an avoided crossing of the IL, and ‘L, surfaces in 3MI is presented.
1. Introduction The methylindoles have been often studied as a model for the amino acid tryptophan (3-alanyl indole) to gain insight into the complex lowest UV absorption band (refs. [ l-41 provide pertinent examples). It is believed the lowest two excited singlet states S,( IL’,) and S,(‘L,) have nearly degenerate origin bands in vacuum, but that ‘L, becomes S, (and, therefore, the fluorescing state) in most condensed phase environments [ 45 1. On the basis of experimental [ 6 ] and theoretical studies [ 7 ] it is also believed that ‘L, has a larger dipole than either the ground or ‘Lb states, thus accounting for the diverse (and useful) variety of emission wavelengths and lifetimes displayed by tryptophan under different conditions [ 8-101. Because the ‘L, absorption and emissions are very broad in condensed phase, a number of investigations of indole derivatives have been carried out in cold expansion jets where the spectra show a relatively sparse array of sharp lines below 1000 cm- ’ from the origin [ 1 l- 17 1. UV excitation of these lines have not yielded a distinction between ‘Lb and ‘L, lines on the basis of dispersed emission or lifetimes. It is now established that polarized two-photon excitation (TPE) spectroscopy distinguishes ‘Lb and Correspondence to: P.R. Callis, Department of Chemistry, Montana State University, Bozeman, MT 597 17, USA.
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‘L, of indoles, since the latter are weaker in circular polarization relative to linear polarization [ 18-201. This technique has been used in solution [ 18 1, vapor [ 18,191, and most recently on jet-cooled indole [ 201, where the lowest ‘L, line was found only 455 cm-’ above the ‘Lb(SI) origin, Here, we report the extension of this study to 3-methylindole (3MI), its methyl deuterated analog 3-trideuterio-methylindole (3MI-d,), along with a few pertainent results from such a study on 5methylindole (5MI).
2. Experimental The details of the experimental apparatus are as described for the indole experiments [ 201. 3-methylindole (Sigma) and 5-methylindole (Aldrich) were used without further purification. The deuterated 3MI was synthesized following the procedure suggested in ref. [ 2 11. He gas at 4 atm was passed directly over the heated samples. The 3MI samples were kept at 85-100°C and the nozzle was maintained at 5°C higher than the sample. The emitted light was collected over more than a hemisphere of solid angle with an ellipsoidal collector, thereby increasing signal and avoiding unwanted photoselection effects. The two-photon polarization ratios were obtained by integrating the area under individual band contours. Each ratio involved lo-20 scans, alternating be-
0009-2614/92/$ 05.00 0 1992 Elsevier Science Publishers B.V. All
rights reserved.
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CHEMICAL PHYSICS LETTERS
Volume 193, number 6
tween linear and circular polarization, and required a total of 40-80 min per band.
/% \ OJ
3. Results
‘H
3methylindole. Figs. 1 and 2 show the one-photon fluorescence excitation spectra of 3MI and 3MI-d, extending from the respective origins to 1000 cm-’ above. The origin for 3MI is at 34876 cm-’ while that for the deuterated analog is at 34884 cm-‘, taking that of jet-cooled indole to be 35232 cm-’ [ 151. The origin is somewhat saturated and is off-scale whereas the other peak ratios are still in the linear response regime. The origin appears about three times stronger than the 739 cm-’ line when recorded in the linear response regime. The satellite lines occurring with 5%-10% relative intensity = 2.5 cm-’ to
,% \ CD
IOCQ
Relative Frequency (cm“)
‘H
Fig. 2. The same as fig. 1 except for 3-CD3 indole. The origin is at 34884 cm-’ and the weak line at - 8 cm-’ is from the undeuterated component.
the blue of the strong lines are due to a 1: 1 complex with He. The 1: 2 complex is also evident 5 cm-’ blue of origin. Fig. 3 shows an expanded view of the two-photon excitation spectrum of 3MI including the 420 and 427 cm-’ lines, using circular and linear polarization, plotted versus the energy of two visible photons. These two lines are assigned ‘L, and ‘Lb on the basis of the contour differences and integrated ratio of the circular and linear contours (Q). The splitting of the 420 cm-’ line is typical of most of the ‘L, lines, with the value varying from 0.9-l .3 cm-‘, except for the 468 cm-’ line which shows no measurable splitting. Tables 1 and 2 list the observed frequencies relative to the respective origins along with their relative one-photon intensities, their two-photon polarizations (8)) splitting (when observed), and electronic state assignment when possible. The two-photon po-
B1 oc loo0
Relative Frequency (cm-‘)
d
il
Fig. 1. One-photon fluorescence excitation spectra of jet-cooled 3 methylindole, corrected for laser power wavelength dependence. The frequencies are relative to the ‘Lt, origin at 34876 cm-‘. The origin line is truncated at about 30% of its maximum.
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~~~ I
-_--__’
,-
420
---T-T--
428
424
Relative Frequency
(cm’)
Fig. 3. Two-photon fluorescence excitation spectrum in the region of the 420 and 427 cm- ’ lines of 3-methylindole with circularly (upper trace) and linearly (lower trace) polarized excitation. The 419-420 cm-’ doublet, attributed to the difference in (a)-(e) methyl rotor tunneling splitting in the ground and ‘L, states, is collapsed to a single line in the 3-methylindole -dr spectrum.
larization is measured in separate experiments visible light such that AE=2hv. Q is defined 9=&irc/d1in
3
using as (1)
the ratio of two-photon signals generated from circular and linear polarized light [ 22 1, integrated over the band. The uncertainty in these ratios is + 0.1 except for the origins, where it is + 0.0 1. It is seen that, as in the case of indole [ 201, the Q values fall mainly into two distinct classes: those above about 1.O, which are assigned as ‘Lb and those below 0.8 which are assigned as ‘L,. Compared to indole [ 201 there are more ‘L, lines and much more ‘L, intensity below 900 cm-‘. The strong peaks at 468,609-617, 736-749 and 820-829 cm-’ are distinctly of ‘L, character. Those at 427, 527, and 71 l7 19 cm- ’ are clearly of ‘Lb character. The peaks at 409 and 420 cm- ’ have 52 values that fall between the above stated ranges but have band contours representative of ‘L,. Several weak lines at lower frequencies are identified whose character is uncertain at this time. Another aspect of the 3MI spectrum which contrasts with that of indole is the striking clustering of lines, particularly evident at 609, 7 12, 534
12 June 1992
740, and 825 cm-‘, where several lines spaced by a few cm-’ are seen. Deuteration of the methyl group causes several interesting effects: ( 1) The clustering of many of the lines is largely collapsed. In the case of the 409-427 cm-’ triplet there appear changes in mixing with resultant intensity redistribution. (2) The x 1 cm-’ doubling of the ‘L, lines collapses to an unmeasurable value ( < 0.1 cm). This accounts for some apparent intensity redistribution. (3) The pair of lines near 190 cm-’ appears to collapse and shift to 145 cm-‘. 5methylindole. From similar experiments on 5MI done in series with the 3MI experiments we find that the polarized TPE of 5MI shows the lowest lying line with obvious ‘L, character to be at 1424 cm- ’ above the ‘Lb origin. Further details of the 5MI investigation will be reported in another paper [ 23 1.
4. Discussion General. The first reported jet excitation spectrum of 3MI was by Hays et al. [ 121 with which our spectrum agrees very well, both in terms of relative intensities and frequencies relative to the ‘Lb origin. Our spectrum also agrees reasonably with the less resolved one-color photoionization spectrum of Hager et al. [ 141, but is rather different from that of Bersohn et al. [ 131 in terms of intensity ratios. In our attempt to locate the ‘L, origin we have documented new lines at 188, 196.5, and 251 cm-‘. We also verify lines at 217 and 334 cm-’ reported by Bersohn et al. [ 13 ] #‘. Comparison with vapor absorption spectra [ 1 ] reveals that these excitation spectra, which are normalized for laser intensity, exhibit the usual strong loss of fluorescence quantum yield for isolated molecules as the excess vibrational energy is increased. The clusters of lines noted at 609 cm-’ and above exposed by our somewhat higher resolution are suggestive of the onset of intermediate coupling between individual S2 vibronic states and several quasidegenerate S, states. In contrast to cases where the #’ The frequencies listed in ref. [ 131 are systematically 10 cm-’ or so lower than found by ref. [ 121 and this work.
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Table 1 One-photon fluorescence excitation frequencies, relative one-photon intensities, and two-photon polarization ratios for 3-methylindole Au ” 0.0
188.1 196.5 216.6 251.3 334.1 408.5 420.0 427.2 451.9 461.8 512.2 526.1 602.1 609.2 611.2 620.4 658.6 111.4 115.1 118.9 136.1 138.9 744.1 148.5 152.1 158.2
State
Au p)
Intensity b,
1.13
‘Lb
0.9 0.8 0.9 1.2 0.8 0.5
?
162.2 782.9 196.9 814.1 819.5 823.5 828.7 832.9 839.3 859.2 865.3 812.4 819.7 893.2 891.2 906.3 914.1 918.1 931.5 946.1 950.1 952.9 961.1 961.1 911.3 911.4 1000
0.8 1.3 0.8 0.1 8.5 6.6 4.2 1.1 1.2 1.0 4.9 2.5 0.9 2.1 I.3 4.4 1.5 16.1 1.9 6.1 5.0 2.6 1.1 1.1 1.3 2.5 1.3
Intensity b,
QC’
100 0.8 0.1 1.9 1.2 1.9 6.1 1.9 12.1 1.2 18.6 I.0 3.3 1.4 16.2 15.1 1.5 1.0 4.0 13.0 2.9 5.0 21.4 1.6 14.9 0.1 3.6
A d,
0.5 1.1 0.9
‘L. ‘La ‘Lb ‘L. ‘L.
I.4
‘Lb
0.6 0.5
‘Ll ‘L.
1.2 1.3 1.3 0.1 0.6 0.6 0.1
Q ‘)
A *)
State
0.6 0.1 0.6
‘L.
0.5
‘L.
0.6
‘L
0.1
‘L.
‘L. ‘L
a) cm-’ relative to the ‘La origin at 34816 cm-‘. b, One-photon intensity as % of the ‘L,, origin intensity and corrected for laser power. ‘) The uncertainfy is f 0.1 except for the origin where it is + 0.0 1. d, Splitting of line (cm-‘) seen in linear TPA spectrum. S2 origin is a few thousand cm-’ above that of S’, e.g. ovaline [ 241 and pyrene [ 251, here there is at most a few hundred cm-’ gap. It is interesting that the clusters are largely collapsed upon deuterating the methyl. The x 1 cm-’ doublets seen for most of the ‘L, lines (table 1, fig. 3) are attributed to the difference in methyl rotor tunnelling splitting in the ground and ‘L, states. This doubling is not seen for ‘L’, states, and it is unmeasurably small for the 3MI-d,. This aspect of the spectra will be more fully addressed in another paper [ 231. Where is the ‘L, origin? Data from several sources indicates that methyl substitution at the 3 position red-shifts the ‘L, band significantly more than it redshifts the ‘Lb band [ 1,4]. The vapor absorption
spectra of Strickland [ 1 ] show the broad ‘L, maximum at 260 nm in indole shifting to 270 nm in 3MI while the ‘Lb origin shifts only from 283 to 287 nm. One therefore might expect the ‘L, origin to shift about 1000 cm-’ to the red relative to the ‘L, origin. On the other hand, a methyl at the 5 position redshifts the ‘Lb origin by 950 cm-’ while leaving the ‘L, band unshifted [ 31. Resolutions from fluorescence polarization in propylene glycol glasses are in qualitative agreement with these shifts [ 45 1. The expectation for 5MI that the ‘L,-‘L, gap widens by x 950 cm- ’ [ 3,4,18] is in accord with our observation of what appears to be the ‘L, origin at 1424 cm- ’ above the ‘Lb origin in the jet [ 23 1, since the lowest ‘L, lines observed for jet-cooled indole by TPE were at 455 and 480 cm-’ above the ‘Lb origin 535
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CHEMICAL PHYSICS LETTERS
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Table 2 One-photon fluorescence excitation frequencies, relative one-photon intensities, and two-photon polarization ratios for 3-trideuteriomethylindole Av a’
Intensity b,
QC’
State
Au”’
Intensity b,
.C’
State
0.0 146.2 194.5 239.8 332.6 400.5 408.5 414.3 448.1 464.8 515.5 570.0 584.2 590.6 608.6 698.5 723.1 728.6 732.5 737.8 741.9
100 0.9 0.9 0.6 1.2 5.4 12.0 2.7 1.8 11.0 1.9 0.8 2.4 11.5 1.3 11.3 0.7 3.1 3.8 23.3 3.6
1.19
‘Lb
1.0 0.9 0.9 1.1 0.6 0.5 1.2 0.7 0.6 0.5 0.5 1.4 0.5 0.5 0.5 0.6 0.5
? ‘L, ‘La
755.8 808.7 812.6 816.0 818.1 820.3 832.8 871.6 881.3 884.6 902.5 915.0 918.0 925.1 939.5 943.8 948.1 958.6 971.5 995.3 1017.7
3.5 3.0 4.8 11.6 1.6 1.8 7.1 1.3 1.6 4.8 1.2 10.9 3.1 2.1 5.6 2.2 0.7 0.7 1.0 1.8 1.7
0.6 1.3 1.3 1.1 0.7 0.7 0.5 0.5 0.6 0.5
‘L. ‘I+ ‘Lb ‘Lb ‘L. ‘L. ‘L, ‘L. ‘L. ‘L.
1.1
‘Lb
0.8 0.7 0.8
‘L. ‘L, ‘L,
0.9 1.2
‘L, ‘Lb
ILb
‘L, ‘L. ‘Lb ‘L. ‘L. ‘L, ‘L. ILb
‘L, ‘L, ‘La ‘La ‘L.
‘) cm-’ relative to the ‘Lb origin at 34884 cm-‘. b, One-photon intensity as 96of the ‘Lb origin intensity and corrected for laser power. ‘) The uncertainty is k 0.1 except for the origin where it is k 0.01
[ 201. This result solidifies the assignment of the 455480 cm- I pair of indole as a split origin by removing the 480 cm-’ line from consideration as a ‘Lb line with Herzberg-Teller derived ‘L, or ‘B, intensity. On the other hand, the expectation that the ‘L, origin in 3MI is several hundred cm-’ to the red of the ‘Lb origin was not met. We carefully searched the region 700 cm-’ to the red of the strong origin peak and found no one-photon excitation intensities above 0.1% of that of the origin, except those of the 1: 1 water complex [ 13,171. Apparently the ‘L, origin does not shift below that of ‘L,, in response to the methyl at the 3 position. Since we were unable to get definitive polarizations of the weak lines below 400 cm-‘, we next examine these from other perspectives. The effect of deuterating the methyl might be used to help identify the ‘L, origin. The ‘Lb origin is blueshifted by 8 cm-’ in 3MI-d, (which is the direction expected on the basis of zero-point vibrational energy differences when the excited state frequencies are less than those of the ground state), whereas most
536
other vibronic lines are red-shifted. One expects the ‘L, origin to be blue-shifted by at least as much. By this criterion only the 334 and 468 cm-’ lines are candidates for the ‘L, origin. The 334 cm-’ line does appear to have considerable ‘L, character, but its weakness causes a large uncertainty in its Q value. The large apparent shift and collapse of the 188197 cm-’ pair to 146 cm-’ upon deuteration requires that these lines be associated with weak methyl rotor Franck-Condon activity built on the ‘Lb origin. Model calculations of the frequency shifts and intensities are in accord with this preliminary assignment [ 2 3 1. The appearance of a ‘Lb line at only 2 17 cm-’ requires an out-of-plane vibration of only 108 cm-‘, assuming the same selection rules as for indole, where onequantum changes in a” modes are forbidden. The lowest frequency fundamental observed in ‘Lb indole is 158 cm-’ [ 151. Model calculations using MOPAC [ 261 suggest that the 3MI ground state has only two more vibrations with frequency below 700
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CHEMICAL PHYSICS LETTERS
cm-l than that of indole. The lowest is predicted to be 40 cm-’ lower than the lowest frequency in indole, so that a 108 cm-’ fundamental for the ‘Lb state of 3MI is not unreasonable. The isotope effect of about 10% is consistent with this being a nearly pure methyl out-of-plane wag. It is extremely awkward to assign any of the weak lines, 217, 25 1, or 334 cm-‘, as the ‘L, origin. No harmonic-based assignments can be made for the ‘L, lines as was possible in the beautiful work by Bickel et al. for the ‘Lb state of indole [ 15 1. If the ‘L, origin were one of these weak lines, the strong lines at 409420, 468, and 609-617 cm-’ should exhibit long Franck-Condon progressions of combinations and overtones, the first few members of which should be easily seen in spite of the fall off of quantum yield. The 251 cm-’ line (240 cm-’ in 3MI-d,) has been tentatively assigned as ‘L,, because of a barely detectable peak at 125 cm-‘, moving to 120 cm-’ in 3MI-d, [ 23 1. Avoided crossing. The absence of harmonic progressions, even though the spectrum is quite sparse below 700 cm-‘, and the apparent absence of ‘L, lines more than about 140 cm- ’ red of those seen for indole [ 201 suggests that the ‘L, and ‘Lb surfaces exhibit an avoided crossing. Such an avoided crossing was proposed along the N-H stretch before there was direct evidence for ‘L, excitations [ 14,16 1, but here we propose that the anharmonicity and apparent splitting of the ‘L, origin (possibly into the peaks including 334,409,420,452, and 468 cm-‘) arise from an avoided crossing along a direction in normal coordinate space recently suggested [ 71 on the basis of bond order changes. calculated from INDO/S-CI wavefunctions, and shown here in fig. 4. We have now done INDO/S computations using geometries and parameters designed to make ‘L, and ‘Lb precisely degenerate which exhibit precisely such an avoided crossing. As the geometry is changed along the vibrational displacement shown in fig. 4, the lower state gradually takes on ‘L, character, while the upper state becomes more ‘Lb-like. At the point of maximum mixing the surfaces remain z 200 cm-’ apart. Thus, the low Q value of the ‘Lb origin in 3MI (only 1.13 versus 1.41 for indole) may be a reflection of such a mixing connected with an avoided crossing. Supporting the idea that the origin may have mixed character is the fact that values of Q z 1.3, i.e.
12June1992
Fig. 4. The “Franck-Condon” mode, showing the direction of proposed displacement of ring atoms upon excitation to the ‘L, state. This direction stabilizes ‘L, much more than ‘Lband is predicted by INDO/S-CI calculations to bring about an avoided crossing of the two surfaces.
significantly higher than the “‘Lb” origin, are seen for several of the higher frequency lines. Comparison with rotational structure-based assignments. Wu [ 27 ] and Levy [ 28 ] have carried out a high-resolution UV fluorescence excitation study of jet-cooled 3MI. From the rotational band structure they have determined the square of the projection made by the transition dipole for 24 lines in the O900 cm-’ range. They found a wide variation of projections, indicating that the transitions are somewhat mixed. Their assignments for ‘L, and IL,, are often the same as ours. Several cases of apparent disagreement invariably correspond to projections which are ambiguous and could be assigned to either state.
5. Conclusions As with indole the ‘L, origin is apparently split, possibly over the set of lines including 334,409,420, 452, and 468 cm-’ (or perhaps is too weak for us to observe). This splitting, the lack of harmonic behavior, the fact that the origin region has shifted only about 140 cm- ’ red of its position in indole, and the apparent contamination of the ‘L,, origin with ‘L, character, all point to an avoided crossing of the ‘L, and IL,, surfaces, presumably along a coordinate involving a high amplitude of C2-Cs double bond stretch. We are presently tracing the relative shifts of ‘L, and ‘Lb lines due to van der Waals complexation with various small molecules and employing semi537
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CHEMICAL PHYSICS LETTERS
empirical calculations to gain a clearer picture of the avoided crossing.
Acknowledgement
The authors thank Dr. C.J. Lee and Professor B.P. Mundy for synthesis of the deuterated 3M1, and Professor Don Levy and Dr. Ruth Wu for communicating part of their study to us prior to publication. This work was supported by NIH grant GM3 1824 to PRC and a MONTS grant to LHS.
References [ 11 E.H. Strickland, J. Horwitz and C. Billups, Biochemistry 25 (1970) 4914. [2] E.H. Strickland, C. Billups and E. Kay, Biochemistry 11 (1972) 3657. [3] E.H. Strickland and C. Billups, Biopolymers 12 ( 1973) 1989. ]4 M.R. Eftink, L.A. Selvidge, P.R. Callis and A.A. Rehms, J. Phys. Chem. 94 ( 1990) 3469. 15 B. Valeur and G. Weber, Photochem. Photobiol. 25 ( 1977) 441. ]6 H. Lami and N.J. Glasser, Chem. Phys. 84 ( 1986) 597; S.R. Meech, D. Phillips and A.G. Lee, Chem. Phys. 80 (1983) 317. P.R. Callis, J. Chem. Phys. 95 (1991) 4230. ‘IJ.R. Lakowicz, Principles of fluorescence spectroscopy (Plenum Press, New York, 1983). ‘IJ.R. Beecham and L. Brand, Ann. Rev. Biochem. 54 ( 1985) 43.
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[ 101 A.P. Demchenko, Ultraviolet spectroscopy of proteins (Springer, Berlin, 1986). [ 111 Y. Nibu, H. Abe, N. Mikami and M. Ito, J. Phys. Chem. 87 (1983) 3898; C.K. Teh, A. Garavi and M. Sulkes, Chem. Phys. Letters 165 (1990) 460. [ 12] J.R. Hays, W.E. Henke, H.L. Selzle and E.W. Schlag, Chem. Phys. Letters 97 (1983) 347. [ 131 R. Bersohn, U. Even and J. Jortner, J. Chem. Phys. 80 (1984) 1050. [ 141 J.W. Hager, D.R. Demmer and SC. Wallace, J. Phys. Chem. 91 (1987) 1375. [ 15 ] G.A. Bickel, D.R. Demmer, E.A. Outhouse and S.C. Wallace, J. Chem. Phys. 91 (1989) 6013. [ 161 D.R. Demmer, G.W. Leach, E.A. Outhouse, J.W. Hager and S.C. Wallace, J. Phys. Chem. 94 ( 1990) 582. [ 171 M.J. Tubergen and D.H. Levy, J. Phys. Chem. 95 (1991) 2175. [ 181 A.A. Rehms and P.R. Callis, Chem. Phys. Letters 140 (1987) 83. [ 191 J.R. Cable, J. Chem. Phys. 92 (1990) 1627. [ 201D.M. Sammeth, S. Yan, L.H. Spangler and P.R. Callis, J. Phys. Chem. 94 (1990) 7340. [21] J.C. Huijzer, J.D. Adams Jr. and G.S. Yost, Toxicol. Appl. Pharamacol. 90 ( 1987) 60. [22] W.M. McClain, J. Chem. Phys. 57 (1972) 2264. [23] D.M. Sammeth, S. Siewert, P.R. Callis and L.W. Spangler, submitted for publication. [24] A. Amirav, U. Even and J. Jortner, Chem. Phys. Letters 71 (1980) 12. [ 25 ] E.A. Mangle and M.R. Topp, J. Chem. Phys. 90 ( 1986) 802. [26] J.J.P. Stewart, QCPE Bulletin 10 (1990) 86; M.J.S. Dewar, E.G. Zoebisch, E.F. Healy and J.J.P. Stewart, J. Am. Chem. Sot. (1985) 3902. [27] R. Wu, Ph.D. Thesis, University ofchicago (1989). [ 281 D.H. Levy, private communication.
CHEMICAL PHYSICS LETTERS
12 June 1992
‘L, transitions of jet-cooled 3-methylindole David M. Sammeth,
Sonja S. Siewert, Lee H. Spangler and Patrik R: Callis
Department of Chemistry, Montana State University, Bozeman, MT 59717, USA
Received 2 1 February 1992
One-photon and polarized two-photon fluorescence excitation spectra of jet-cooled 3-methylindole (3MI) and 3-trideuteriomethylindole are reported at a resolution of ~0.2 cm-‘. On the basis of the two-photon intensity ratios for linear versus circular polarized excitation, strong lines with predominantly ‘L. character are identified in 3MI at 409,420,468,609,617, 739,820, and 9 18 cm-’ above the ‘L,, origin. The lowest-lying ‘L. line in 5-methylindole appears to be at 1424 cm-‘. Evidence for an avoided crossing of the IL, and ‘L, surfaces in 3MI is presented.
1. Introduction The methylindoles have been often studied as a model for the amino acid tryptophan (3-alanyl indole) to gain insight into the complex lowest UV absorption band (refs. [ l-41 provide pertinent examples). It is believed the lowest two excited singlet states S,( IL’,) and S,(‘L,) have nearly degenerate origin bands in vacuum, but that ‘L, becomes S, (and, therefore, the fluorescing state) in most condensed phase environments [ 45 1. On the basis of experimental [ 6 ] and theoretical studies [ 7 ] it is also believed that ‘L, has a larger dipole than either the ground or ‘Lb states, thus accounting for the diverse (and useful) variety of emission wavelengths and lifetimes displayed by tryptophan under different conditions [ 8-101. Because the ‘L, absorption and emissions are very broad in condensed phase, a number of investigations of indole derivatives have been carried out in cold expansion jets where the spectra show a relatively sparse array of sharp lines below 1000 cm- ’ from the origin [ 1 l- 17 1. UV excitation of these lines have not yielded a distinction between ‘Lb and ‘L, lines on the basis of dispersed emission or lifetimes. It is now established that polarized two-photon excitation (TPE) spectroscopy distinguishes ‘Lb and Correspondence to: P.R. Callis, Department of Chemistry, Montana State University, Bozeman, MT 597 17, USA.
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‘L, of indoles, since the latter are weaker in circular polarization relative to linear polarization [ 18-201. This technique has been used in solution [ 18 1, vapor [ 18,191, and most recently on jet-cooled indole [ 201, where the lowest ‘L, line was found only 455 cm-’ above the ‘Lb(SI) origin, Here, we report the extension of this study to 3-methylindole (3MI), its methyl deuterated analog 3-trideuterio-methylindole (3MI-d,), along with a few pertainent results from such a study on 5methylindole (5MI).
2. Experimental The details of the experimental apparatus are as described for the indole experiments [ 201. 3-methylindole (Sigma) and 5-methylindole (Aldrich) were used without further purification. The deuterated 3MI was synthesized following the procedure suggested in ref. [ 2 11. He gas at 4 atm was passed directly over the heated samples. The 3MI samples were kept at 85-100°C and the nozzle was maintained at 5°C higher than the sample. The emitted light was collected over more than a hemisphere of solid angle with an ellipsoidal collector, thereby increasing signal and avoiding unwanted photoselection effects. The two-photon polarization ratios were obtained by integrating the area under individual band contours. Each ratio involved lo-20 scans, alternating be-
0009-2614/92/$ 05.00 0 1992 Elsevier Science Publishers B.V. All
rights reserved.
12 June 1992
CHEMICAL PHYSICS LETTERS
Volume 193, number 6
tween linear and circular polarization, and required a total of 40-80 min per band.
/% \ OJ
3. Results
‘H
3methylindole. Figs. 1 and 2 show the one-photon fluorescence excitation spectra of 3MI and 3MI-d, extending from the respective origins to 1000 cm-’ above. The origin for 3MI is at 34876 cm-’ while that for the deuterated analog is at 34884 cm-‘, taking that of jet-cooled indole to be 35232 cm-’ [ 151. The origin is somewhat saturated and is off-scale whereas the other peak ratios are still in the linear response regime. The origin appears about three times stronger than the 739 cm-’ line when recorded in the linear response regime. The satellite lines occurring with 5%-10% relative intensity = 2.5 cm-’ to
,% \ CD
IOCQ
Relative Frequency (cm“)
‘H
Fig. 2. The same as fig. 1 except for 3-CD3 indole. The origin is at 34884 cm-’ and the weak line at - 8 cm-’ is from the undeuterated component.
the blue of the strong lines are due to a 1: 1 complex with He. The 1: 2 complex is also evident 5 cm-’ blue of origin. Fig. 3 shows an expanded view of the two-photon excitation spectrum of 3MI including the 420 and 427 cm-’ lines, using circular and linear polarization, plotted versus the energy of two visible photons. These two lines are assigned ‘L, and ‘Lb on the basis of the contour differences and integrated ratio of the circular and linear contours (Q). The splitting of the 420 cm-’ line is typical of most of the ‘L, lines, with the value varying from 0.9-l .3 cm-‘, except for the 468 cm-’ line which shows no measurable splitting. Tables 1 and 2 list the observed frequencies relative to the respective origins along with their relative one-photon intensities, their two-photon polarizations (8)) splitting (when observed), and electronic state assignment when possible. The two-photon po-
B1 oc loo0
Relative Frequency (cm-‘)
d
il
Fig. 1. One-photon fluorescence excitation spectra of jet-cooled 3 methylindole, corrected for laser power wavelength dependence. The frequencies are relative to the ‘Lt, origin at 34876 cm-‘. The origin line is truncated at about 30% of its maximum.
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~~~ I
-_--__’
,-
420
---T-T--
428
424
Relative Frequency
(cm’)
Fig. 3. Two-photon fluorescence excitation spectrum in the region of the 420 and 427 cm- ’ lines of 3-methylindole with circularly (upper trace) and linearly (lower trace) polarized excitation. The 419-420 cm-’ doublet, attributed to the difference in (a)-(e) methyl rotor tunneling splitting in the ground and ‘L, states, is collapsed to a single line in the 3-methylindole -dr spectrum.
larization is measured in separate experiments visible light such that AE=2hv. Q is defined 9=&irc/d1in
3
using as (1)
the ratio of two-photon signals generated from circular and linear polarized light [ 22 1, integrated over the band. The uncertainty in these ratios is + 0.1 except for the origins, where it is + 0.0 1. It is seen that, as in the case of indole [ 201, the Q values fall mainly into two distinct classes: those above about 1.O, which are assigned as ‘Lb and those below 0.8 which are assigned as ‘L,. Compared to indole [ 201 there are more ‘L, lines and much more ‘L, intensity below 900 cm-‘. The strong peaks at 468,609-617, 736-749 and 820-829 cm-’ are distinctly of ‘L, character. Those at 427, 527, and 71 l7 19 cm- ’ are clearly of ‘Lb character. The peaks at 409 and 420 cm- ’ have 52 values that fall between the above stated ranges but have band contours representative of ‘L,. Several weak lines at lower frequencies are identified whose character is uncertain at this time. Another aspect of the 3MI spectrum which contrasts with that of indole is the striking clustering of lines, particularly evident at 609, 7 12, 534
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740, and 825 cm-‘, where several lines spaced by a few cm-’ are seen. Deuteration of the methyl group causes several interesting effects: ( 1) The clustering of many of the lines is largely collapsed. In the case of the 409-427 cm-’ triplet there appear changes in mixing with resultant intensity redistribution. (2) The x 1 cm-’ doubling of the ‘L, lines collapses to an unmeasurable value ( < 0.1 cm). This accounts for some apparent intensity redistribution. (3) The pair of lines near 190 cm-’ appears to collapse and shift to 145 cm-‘. 5methylindole. From similar experiments on 5MI done in series with the 3MI experiments we find that the polarized TPE of 5MI shows the lowest lying line with obvious ‘L, character to be at 1424 cm- ’ above the ‘Lb origin. Further details of the 5MI investigation will be reported in another paper [ 23 1.
4. Discussion General. The first reported jet excitation spectrum of 3MI was by Hays et al. [ 121 with which our spectrum agrees very well, both in terms of relative intensities and frequencies relative to the ‘Lb origin. Our spectrum also agrees reasonably with the less resolved one-color photoionization spectrum of Hager et al. [ 141, but is rather different from that of Bersohn et al. [ 131 in terms of intensity ratios. In our attempt to locate the ‘L, origin we have documented new lines at 188, 196.5, and 251 cm-‘. We also verify lines at 217 and 334 cm-’ reported by Bersohn et al. [ 13 ] #‘. Comparison with vapor absorption spectra [ 1 ] reveals that these excitation spectra, which are normalized for laser intensity, exhibit the usual strong loss of fluorescence quantum yield for isolated molecules as the excess vibrational energy is increased. The clusters of lines noted at 609 cm-’ and above exposed by our somewhat higher resolution are suggestive of the onset of intermediate coupling between individual S2 vibronic states and several quasidegenerate S, states. In contrast to cases where the #’ The frequencies listed in ref. [ 131 are systematically 10 cm-’ or so lower than found by ref. [ 121 and this work.
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Table 1 One-photon fluorescence excitation frequencies, relative one-photon intensities, and two-photon polarization ratios for 3-methylindole Au ” 0.0
188.1 196.5 216.6 251.3 334.1 408.5 420.0 427.2 451.9 461.8 512.2 526.1 602.1 609.2 611.2 620.4 658.6 111.4 115.1 118.9 136.1 138.9 744.1 148.5 152.1 158.2
State
Au p)
Intensity b,
1.13
‘Lb
0.9 0.8 0.9 1.2 0.8 0.5
?
162.2 782.9 196.9 814.1 819.5 823.5 828.7 832.9 839.3 859.2 865.3 812.4 819.7 893.2 891.2 906.3 914.1 918.1 931.5 946.1 950.1 952.9 961.1 961.1 911.3 911.4 1000
0.8 1.3 0.8 0.1 8.5 6.6 4.2 1.1 1.2 1.0 4.9 2.5 0.9 2.1 I.3 4.4 1.5 16.1 1.9 6.1 5.0 2.6 1.1 1.1 1.3 2.5 1.3
Intensity b,
QC’
100 0.8 0.1 1.9 1.2 1.9 6.1 1.9 12.1 1.2 18.6 I.0 3.3 1.4 16.2 15.1 1.5 1.0 4.0 13.0 2.9 5.0 21.4 1.6 14.9 0.1 3.6
A d,
0.5 1.1 0.9
‘L. ‘La ‘Lb ‘L. ‘L.
I.4
‘Lb
0.6 0.5
‘Ll ‘L.
1.2 1.3 1.3 0.1 0.6 0.6 0.1
Q ‘)
A *)
State
0.6 0.1 0.6
‘L.
0.5
‘L.
0.6
‘L
0.1
‘L.
‘L. ‘L
a) cm-’ relative to the ‘La origin at 34816 cm-‘. b, One-photon intensity as % of the ‘L,, origin intensity and corrected for laser power. ‘) The uncertainfy is f 0.1 except for the origin where it is + 0.0 1. d, Splitting of line (cm-‘) seen in linear TPA spectrum. S2 origin is a few thousand cm-’ above that of S’, e.g. ovaline [ 241 and pyrene [ 251, here there is at most a few hundred cm-’ gap. It is interesting that the clusters are largely collapsed upon deuterating the methyl. The x 1 cm-’ doublets seen for most of the ‘L, lines (table 1, fig. 3) are attributed to the difference in methyl rotor tunnelling splitting in the ground and ‘L, states. This doubling is not seen for ‘L’, states, and it is unmeasurably small for the 3MI-d,. This aspect of the spectra will be more fully addressed in another paper [ 231. Where is the ‘L, origin? Data from several sources indicates that methyl substitution at the 3 position red-shifts the ‘L, band significantly more than it redshifts the ‘Lb band [ 1,4]. The vapor absorption
spectra of Strickland [ 1 ] show the broad ‘L, maximum at 260 nm in indole shifting to 270 nm in 3MI while the ‘Lb origin shifts only from 283 to 287 nm. One therefore might expect the ‘L, origin to shift about 1000 cm-’ to the red relative to the ‘L, origin. On the other hand, a methyl at the 5 position redshifts the ‘Lb origin by 950 cm-’ while leaving the ‘L, band unshifted [ 31. Resolutions from fluorescence polarization in propylene glycol glasses are in qualitative agreement with these shifts [ 45 1. The expectation for 5MI that the ‘L,-‘L, gap widens by x 950 cm- ’ [ 3,4,18] is in accord with our observation of what appears to be the ‘L, origin at 1424 cm- ’ above the ‘Lb origin in the jet [ 23 1, since the lowest ‘L, lines observed for jet-cooled indole by TPE were at 455 and 480 cm-’ above the ‘Lb origin 535
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Table 2 One-photon fluorescence excitation frequencies, relative one-photon intensities, and two-photon polarization ratios for 3-trideuteriomethylindole Av a’
Intensity b,
QC’
State
Au”’
Intensity b,
.C’
State
0.0 146.2 194.5 239.8 332.6 400.5 408.5 414.3 448.1 464.8 515.5 570.0 584.2 590.6 608.6 698.5 723.1 728.6 732.5 737.8 741.9
100 0.9 0.9 0.6 1.2 5.4 12.0 2.7 1.8 11.0 1.9 0.8 2.4 11.5 1.3 11.3 0.7 3.1 3.8 23.3 3.6
1.19
‘Lb
1.0 0.9 0.9 1.1 0.6 0.5 1.2 0.7 0.6 0.5 0.5 1.4 0.5 0.5 0.5 0.6 0.5
? ‘L, ‘La
755.8 808.7 812.6 816.0 818.1 820.3 832.8 871.6 881.3 884.6 902.5 915.0 918.0 925.1 939.5 943.8 948.1 958.6 971.5 995.3 1017.7
3.5 3.0 4.8 11.6 1.6 1.8 7.1 1.3 1.6 4.8 1.2 10.9 3.1 2.1 5.6 2.2 0.7 0.7 1.0 1.8 1.7
0.6 1.3 1.3 1.1 0.7 0.7 0.5 0.5 0.6 0.5
‘L. ‘I+ ‘Lb ‘Lb ‘L. ‘L. ‘L, ‘L. ‘L. ‘L.
1.1
‘Lb
0.8 0.7 0.8
‘L. ‘L, ‘L,
0.9 1.2
‘L, ‘Lb
ILb
‘L, ‘L. ‘Lb ‘L. ‘L. ‘L, ‘L. ILb
‘L, ‘L, ‘La ‘La ‘L.
‘) cm-’ relative to the ‘Lb origin at 34884 cm-‘. b, One-photon intensity as 96of the ‘Lb origin intensity and corrected for laser power. ‘) The uncertainty is k 0.1 except for the origin where it is k 0.01
[ 201. This result solidifies the assignment of the 455480 cm- I pair of indole as a split origin by removing the 480 cm-’ line from consideration as a ‘Lb line with Herzberg-Teller derived ‘L, or ‘B, intensity. On the other hand, the expectation that the ‘L, origin in 3MI is several hundred cm-’ to the red of the ‘Lb origin was not met. We carefully searched the region 700 cm-’ to the red of the strong origin peak and found no one-photon excitation intensities above 0.1% of that of the origin, except those of the 1: 1 water complex [ 13,171. Apparently the ‘L, origin does not shift below that of ‘L,, in response to the methyl at the 3 position. Since we were unable to get definitive polarizations of the weak lines below 400 cm-‘, we next examine these from other perspectives. The effect of deuterating the methyl might be used to help identify the ‘L, origin. The ‘Lb origin is blueshifted by 8 cm-’ in 3MI-d, (which is the direction expected on the basis of zero-point vibrational energy differences when the excited state frequencies are less than those of the ground state), whereas most
536
other vibronic lines are red-shifted. One expects the ‘L, origin to be blue-shifted by at least as much. By this criterion only the 334 and 468 cm-’ lines are candidates for the ‘L, origin. The 334 cm-’ line does appear to have considerable ‘L, character, but its weakness causes a large uncertainty in its Q value. The large apparent shift and collapse of the 188197 cm-’ pair to 146 cm-’ upon deuteration requires that these lines be associated with weak methyl rotor Franck-Condon activity built on the ‘Lb origin. Model calculations of the frequency shifts and intensities are in accord with this preliminary assignment [ 2 3 1. The appearance of a ‘Lb line at only 2 17 cm-’ requires an out-of-plane vibration of only 108 cm-‘, assuming the same selection rules as for indole, where onequantum changes in a” modes are forbidden. The lowest frequency fundamental observed in ‘Lb indole is 158 cm-’ [ 151. Model calculations using MOPAC [ 261 suggest that the 3MI ground state has only two more vibrations with frequency below 700
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cm-l than that of indole. The lowest is predicted to be 40 cm-’ lower than the lowest frequency in indole, so that a 108 cm-’ fundamental for the ‘Lb state of 3MI is not unreasonable. The isotope effect of about 10% is consistent with this being a nearly pure methyl out-of-plane wag. It is extremely awkward to assign any of the weak lines, 217, 25 1, or 334 cm-‘, as the ‘L, origin. No harmonic-based assignments can be made for the ‘L, lines as was possible in the beautiful work by Bickel et al. for the ‘Lb state of indole [ 15 1. If the ‘L, origin were one of these weak lines, the strong lines at 409420, 468, and 609-617 cm-’ should exhibit long Franck-Condon progressions of combinations and overtones, the first few members of which should be easily seen in spite of the fall off of quantum yield. The 251 cm-’ line (240 cm-’ in 3MI-d,) has been tentatively assigned as ‘L,, because of a barely detectable peak at 125 cm-‘, moving to 120 cm-’ in 3MI-d, [ 23 1. Avoided crossing. The absence of harmonic progressions, even though the spectrum is quite sparse below 700 cm-‘, and the apparent absence of ‘L, lines more than about 140 cm- ’ red of those seen for indole [ 201 suggests that the ‘L, and ‘Lb surfaces exhibit an avoided crossing. Such an avoided crossing was proposed along the N-H stretch before there was direct evidence for ‘L, excitations [ 14,16 1, but here we propose that the anharmonicity and apparent splitting of the ‘L, origin (possibly into the peaks including 334,409,420,452, and 468 cm-‘) arise from an avoided crossing along a direction in normal coordinate space recently suggested [ 71 on the basis of bond order changes. calculated from INDO/S-CI wavefunctions, and shown here in fig. 4. We have now done INDO/S computations using geometries and parameters designed to make ‘L, and ‘Lb precisely degenerate which exhibit precisely such an avoided crossing. As the geometry is changed along the vibrational displacement shown in fig. 4, the lower state gradually takes on ‘L, character, while the upper state becomes more ‘Lb-like. At the point of maximum mixing the surfaces remain z 200 cm-’ apart. Thus, the low Q value of the ‘Lb origin in 3MI (only 1.13 versus 1.41 for indole) may be a reflection of such a mixing connected with an avoided crossing. Supporting the idea that the origin may have mixed character is the fact that values of Q z 1.3, i.e.
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Fig. 4. The “Franck-Condon” mode, showing the direction of proposed displacement of ring atoms upon excitation to the ‘L, state. This direction stabilizes ‘L, much more than ‘Lband is predicted by INDO/S-CI calculations to bring about an avoided crossing of the two surfaces.
significantly higher than the “‘Lb” origin, are seen for several of the higher frequency lines. Comparison with rotational structure-based assignments. Wu [ 27 ] and Levy [ 28 ] have carried out a high-resolution UV fluorescence excitation study of jet-cooled 3MI. From the rotational band structure they have determined the square of the projection made by the transition dipole for 24 lines in the O900 cm-’ range. They found a wide variation of projections, indicating that the transitions are somewhat mixed. Their assignments for ‘L, and IL,, are often the same as ours. Several cases of apparent disagreement invariably correspond to projections which are ambiguous and could be assigned to either state.
5. Conclusions As with indole the ‘L, origin is apparently split, possibly over the set of lines including 334,409,420, 452, and 468 cm-’ (or perhaps is too weak for us to observe). This splitting, the lack of harmonic behavior, the fact that the origin region has shifted only about 140 cm- ’ red of its position in indole, and the apparent contamination of the ‘L,, origin with ‘L, character, all point to an avoided crossing of the ‘L, and IL,, surfaces, presumably along a coordinate involving a high amplitude of C2-Cs double bond stretch. We are presently tracing the relative shifts of ‘L, and ‘Lb lines due to van der Waals complexation with various small molecules and employing semi537
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empirical calculations to gain a clearer picture of the avoided crossing.
Acknowledgement
The authors thank Dr. C.J. Lee and Professor B.P. Mundy for synthesis of the deuterated 3M1, and Professor Don Levy and Dr. Ruth Wu for communicating part of their study to us prior to publication. This work was supported by NIH grant GM3 1824 to PRC and a MONTS grant to LHS.
References [ 11 E.H. Strickland, J. Horwitz and C. Billups, Biochemistry 25 (1970) 4914. [2] E.H. Strickland, C. Billups and E. Kay, Biochemistry 11 (1972) 3657. [3] E.H. Strickland and C. Billups, Biopolymers 12 ( 1973) 1989. ]4 M.R. Eftink, L.A. Selvidge, P.R. Callis and A.A. Rehms, J. Phys. Chem. 94 ( 1990) 3469. 15 B. Valeur and G. Weber, Photochem. Photobiol. 25 ( 1977) 441. ]6 H. Lami and N.J. Glasser, Chem. Phys. 84 ( 1986) 597; S.R. Meech, D. Phillips and A.G. Lee, Chem. Phys. 80 (1983) 317. P.R. Callis, J. Chem. Phys. 95 (1991) 4230. ‘IJ.R. Lakowicz, Principles of fluorescence spectroscopy (Plenum Press, New York, 1983). ‘IJ.R. Beecham and L. Brand, Ann. Rev. Biochem. 54 ( 1985) 43.
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[ 101 A.P. Demchenko, Ultraviolet spectroscopy of proteins (Springer, Berlin, 1986). [ 111 Y. Nibu, H. Abe, N. Mikami and M. Ito, J. Phys. Chem. 87 (1983) 3898; C.K. Teh, A. Garavi and M. Sulkes, Chem. Phys. Letters 165 (1990) 460. [ 12] J.R. Hays, W.E. Henke, H.L. Selzle and E.W. Schlag, Chem. Phys. Letters 97 (1983) 347. [ 131 R. Bersohn, U. Even and J. Jortner, J. Chem. Phys. 80 (1984) 1050. [ 141 J.W. Hager, D.R. Demmer and SC. Wallace, J. Phys. Chem. 91 (1987) 1375. [ 15 ] G.A. Bickel, D.R. Demmer, E.A. Outhouse and S.C. Wallace, J. Chem. Phys. 91 (1989) 6013. [ 161 D.R. Demmer, G.W. Leach, E.A. Outhouse, J.W. Hager and S.C. Wallace, J. Phys. Chem. 94 ( 1990) 582. [ 171 M.J. Tubergen and D.H. Levy, J. Phys. Chem. 95 (1991) 2175. [ 181 A.A. Rehms and P.R. Callis, Chem. Phys. Letters 140 (1987) 83. [ 191 J.R. Cable, J. Chem. Phys. 92 (1990) 1627. [ 201D.M. Sammeth, S. Yan, L.H. Spangler and P.R. Callis, J. Phys. Chem. 94 (1990) 7340. [21] J.C. Huijzer, J.D. Adams Jr. and G.S. Yost, Toxicol. Appl. Pharamacol. 90 ( 1987) 60. [22] W.M. McClain, J. Chem. Phys. 57 (1972) 2264. [23] D.M. Sammeth, S. Siewert, P.R. Callis and L.W. Spangler, submitted for publication. [24] A. Amirav, U. Even and J. Jortner, Chem. Phys. Letters 71 (1980) 12. [ 25 ] E.A. Mangle and M.R. Topp, J. Chem. Phys. 90 ( 1986) 802. [26] J.J.P. Stewart, QCPE Bulletin 10 (1990) 86; M.J.S. Dewar, E.G. Zoebisch, E.F. Healy and J.J.P. Stewart, J. Am. Chem. Sot. (1985) 3902. [27] R. Wu, Ph.D. Thesis, University ofchicago (1989). [ 281 D.H. Levy, private communication.