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1La origin locations of methyl indoles in argon matrices
15November 1996
CHEMICAL PHYSICS LETTERS ELSEVIER
Chemical Physics Letters 262 (1996) 343-348
l L a origin locations of methyl indoles in argon matrices Bruce Fender, Patrik R. Callis Department of Chemistry and Biochemistry, Montana State University, Bozeman. MT 59717, USA
Received 3 June 1996; in final form 28 Augusl 1996
Abstract
Polarized fluorescence excitation and dispersed fluorescence spectra of 3-methylindole (3MI), 5-methylindole (5MI), and 2,3-dimethylindole (2,3DMI) in solid Ar at 20 K under site selective conditions have unambiguously revealed their t L a origin locations. For 3MI the IL a origin is 260 cm J above the ~L b origin and for 5MI it is 1800 cm-~ above IL b. For 2,3DMI, the IL a origin lies 190 cm-t below the ~L b origin. The latter assignment is made from details of the dispersed fluorescence in comparison to the phosphorescence spectrum of indole in argon at 20 K.
1. I n t r o d u c t i o n
Indole is the chromophore of the amino acid tryptophan, which is widely exploited as an intrinsic fluorescent probe of local protein environment [ 1-3]. Indole compounds have two low lying 7rTr * singlet excited states labeled tLa a n d ILb, which are nearly degenerate [4,5]. The ~L a state has a dipole about 6 debye larger than the ground a n d 1L b states [6], so its energy is quite sensitive to the local environment. In mobile polar environments, ~L a is always the fluorescent state of tryptophan, but in certain proteins which exhibit blue-shifted, structured tryptophan fluorescence, there exists uncertainty regarding the nature of the emitting state. This uncertainty is caused by the lack of precise knowledge concerning the location of the ~L a origin relative to that o f ILb, and by the lack of knowledge concerning the characteristic vibronic shapes of the two types of fluorescence. This is an important issue because the 1L~ state is easily stabilized by reorientation of a polar environment a n d / o r by local electric fields oriented with the excited state dipole. I f IL b fluorescence
could be confidently demonstrated for a tryptophan in a protein, a rigid environment with a strong local electric field opposing the dipole could be deduced. For the above reasons, it is desirable to establish with certainty the relative locations of the ~L:, and I L b origins of tryptophan in well defined environments, and to this end there has been extensive study of indole and its derivatives [4-18]. We have recently reported the polarized fluorescence and fluorescence excitation spectra of indole in solid argon [16], a study that revealed the existence of a f a l s e ~L a origin caused by Herzberg-Teller coupling of the Fermi doublet at 4 5 5 - 4 8 0 cm -~ , and that located the true L origin at 1100 cm -1 and 1300 cm -~ above the L b o n g m in solid Ar and in vacuum respectively. However, considerable uncertainty remained regarding the location of the ~L a origin of 3-methylindole (a close analog of tryptophan) and other indoles. (The indole ring and numbering system are displayed as part of Fig. 1.) In this Letter we report the extension of our study to 3-methylindote (3MI), 5-methylindole (5MI), and 2,3-dimethylindole (2,3DMI), with special emphasis 1
•
I
•
•
0009-2614/96/$12.00 Copyright © 1996 Published by Elsevier Science B.V. All rights reserved PII S0009-2614(96)01096-2
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344
B. Fender, P.R. Callis / Chemical Physics Letters 262 (1996) 343-348
placed on the location of the true ~L~ origins and the nature of the fluorescence spectra in solid argon under site selective conditions.
2. E x p e r i m e n t a l
All experiments were carried out with indole derivatives in Ar deposited on a sapphire window at 20 K within a Janis optical cryostat cooled with a CTI-Cryogenic closed cycle He refrigerator. The indoles were obtained from Aldrich and were used without further purification: 3MI (98%, lot # 11822CF), 5MI (99%, lot # 06701HX), and 2,3DMI (99%, lot # 092767). The Ar was ultrapure grade 99.999%. The various indoles were entrained in Ar by passing a portion of the Ar over the crystals maintained at 20-25°C. The rate of deposition Ar was about 4 millimole/h and the molar ratio of indole to Ar was about 10 -4. Slow deposition at 20 K provided the best (although mediocre) conditions for observing anisotropy. Excitation was with a Lumonics HyperDye dye laser pumped by a Lumonics HY200 Nd:YAG laser. The dye laser output was frequency doubled using an Inrad Autotracker II. The excitation linewidth was about 0.20 cm- ~. The sapphire window was tilted to attain a 45 ° angle of incidence relative with the laser beam. Fluorescence was collected at 90 ° through an f / 7 optical system and onto the slit of a Spex 1404 double monochromator with a 2400 grooves/mm grating. Slit widths were kept ~< 800 /zm for nonpolarized spectra, corresponding to a resolution of 20 cm-1. The laser beam was vertically polarized using a depolarizer followed by a Glan polarizer. The emitted light was passed through a Polacoat sheet polarizer, which could be oriented to pass vertically or horizontally polarized light. A quartz wedge depolarizer was mounted at the entrance slit of the monochromator to avoid spurious polarizations due to polarization dependent transmission. Fluorescence anisotropy ( r ) was measured as: r = (Ill-- l±)/(Iii
oriented, non-rotating sample, the theoretical limits are r = 0.4 for parallel excitation and emission transition moments, and r = - 0 . 2 for perpendicular excitation and emission moments [4,13]. These limits are expected to be approached in this study because numerous studies on indoles in crystals [17], stetched films [18], and glassy solvents [4,13] have shown the transition moment directions of the IL a and ]L b transitions to be nearly perpendicular. The experimental values reported here are low in comparison to the theoretical values due to depolarization of the exciting and emitting light by the Ar matrix, probably from scattering a n d / o r strain birefringence.
3. R e s u l t s
Figs. 1-3 show fluorescence excitation spectra and the corresponding anisotropy in argon matrices at 20 K for 3MI, 2,3DMI, and 5MI respectively. For ease of comparison, the bottom panel shows previously published jet-cooled fluorescence excitation
0.10- ~
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. ..,
~/9111
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i
, A ~ ,7",
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--
ffl ~
,
m
1 i0 l~
°;I
0.6
0 "S ~ /
0 6
~
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1,3't°' I 500
+ 21_c).
With vertical excitation, 111 is the intensity of the vertically polarized emission while I~_ is the horizontally polarized emission intensity. For a randomly
,
I._
cm q from the
1000 Lb
1500
origin.
Fig. l. Comparison of jet-cooled (a) and Ar matrix isolated (b) fluorescence cence
excitation
spectra of 3-methylindole.
T h e fluores-
anisotropyin the Ar matrix is shown in panel (c).
345
B. Fender, P.R. Callis / Chemical Physics Letters 262 (1996) 343-348
00 t\/ 010~
o.oo
V
-0 05-
Anisotropy '
'
i
. . . .
i
. . . .
i
. . . .
i
. . . .
I
. . . .
....
i
a
_~" C
c ~2
o
ongm Ar Matrix, 20K
:3 u_ '
'
i
. . . .
0
500
1000
....
i
1500
2000
c m q f r o m t h e L a origin.
Fig. 2. Fluorescence excitation spectra (a) and corresponding fluorescence anisotropy (b) of 2,3-dimethylindole in Ar matrix.
spectra in the case of 3MI and 5MI, with the t L b origin aligned with that of the matrix spectrum. The two-photon "polarization ratio" [19], /2, is noted for the stronger lines of the jet spectra..O for a
given line is the ratio of integrated two-photon excited intensities obtained with circularly and linearly polarized light, respectively. It has been found that ~L b lines have ,(2 = 1.3-1.4, whereas ~L~ lines have $2= 0.5-0.8 [11,12,14,20]. The jet-cooled fluorescence excitation spectrum of 2,3DMI is extremely weak above the origin, and is not shown. The S~ origins were found to be shifted to lower frequencies relative to vacuum by 254 c m - i for 3M1, 270 c m for 2,3DMI, and 80 cm ~ for 5MI. The anisotropy maximum and minimum magnitudes were found to vary from sample to sample, but their ratios remained constant. In a number of cases the minimum value approaches minus one half the maximum value, independent of the extent of depolarization. We take this to be the signature of perpendicularly polarized transitions. The emission monochromator was tuned to the prominent 26~ ( = 760 c m - ~ ) line in the fluorescence for the argon matrix excitation spectra of 3MI and 5MI, and to the peak at 1600 cm ~ for 2,3DMI. For the jet spectra, all of the fluorescence was collected.
0 1 0 -
c~ 0.05
-
£
~
H
0 O0 -
P
Y o.
d
-005 -
c/) O) fo t"{/} O O
13
L.L
1.3
0 0
500
1000
1500
2000
2500
500
1000
1500
2000
2500
3000
cm -~ from the Lb origin. Fig. 3. Comparison of jet-cooled (a) and Ar matrix isolated (b) fluorescence excitation spectra of 5-methylindole. The fluorescence anisotropy in the Ar matrix is shown in panel (c).
cm q
Fig. 4. Comparison of the Ar matrix isolated (20 K) dispersed fluorescence spectra of indole (a), 3-methylindole(b), 5-methylindole (c), 2,3-dimethylindole (d), with the dispersed pho,~7~horescenee of indole (e).
346
B. Fender, P.R. Callis / Chemical Physics Letters 262 (1996) 343-348
Fig. 4 compares dispersed fluorescence spectra of indole, 3MI, 5MI, and 2,3DMI with the phosphorescence spectrum of indole, all in argon matrices at 20 K. Again, the origins are aligned.
4. Discussion
4.1. Expected IL a shift T h e I L a - ] L b state spacing is known to be affected by substituent and solvent effects. Of the derivatives studied here, it is known that methyl substitution at the 5 position lowers ~L b, thereby increasing the gap relative to indole, whereas a methyl at the 3 position lowers ~La more than JL b, thereby decreasing the gap [7,10,11,21]. It has been found from polarized two-photon fluorescence excitation spectra of indole, 5MI, 3MI, and 2,3DMI [11] that the ~La origin clearly lies b e l o w IL b prior to excitation in hexane at 25°C for 2,3DMI, and that it is clearly above the IL b origin for indole and 5MI. For 3MI it appears that the two origins are superimposed in hexane solution, prior to excitation. It is known that the ~La state of aromatic hydrocarbons is red-shifted more by solvents than is the ~L b state [22]. For indole in argon at 20 K we found that the ~La origin (actually a cluster of lines) redshifts about 200 c m - ] more than that o f ILb, relative to their position in vacuum [16]. Similar behavior is expected for the compounds studied here, because methylation does not severely alter the permanent dipole. Because hexane has a higher refractive index than argon, we expect the two orgins to be very close for both 3MI and 2,3DMI in argon.
4.2.3-Methylindole The fluorescence excitation spectrum of 3MI in argon shown in Fig. lb is considerably different from its jet spectrum shown in Fig. la. This contrasts with the behavior of indole, where we found a close correspondence of the jet and argon spectra, except for the red-shifted ~L a origin grouping near 1200 c m - ~. Whereas in the jet spectrum of 3MI the ILa origin is apparently split into 4 or 5 major components involving lines at 356, 409, 467(?), 609-617, and 739-749 cm - l , in solid argon the obvious can-
didate for the ~L a origin is the single line lying only 260 c m - ~ above the JL b origin. The strong doublets seen in the jet near 610 and 745 cm -I are not identifiable in the matrix; they, along with the other ~L a " o r i g i n " lines, have apparently shifted and coalesced into a single origin line. This may be because at only 260 cm -) above the IL b origin there exists an insufficient number of nearby ~L b vibronic states to cause the splitting. The IL a assignment is based on the fluorescence (see Section 4.5), which is very similar to that of indole. The integrated intensities of the ~La and IL b origins in argon are nearly identical, as is to be expected on theoretical grounds [7,23]. In the jetcooled excitation spectrum, the sum of the intensities of the JL a lines between 356 and 740 c m - ~ is close to the intensity of the IL b origin. The intense line at 467.5 cm-~ was not counted because it is believed to be a ~L b line that steals its IL a character by Herzberg-Teller vibronic coupling, similar to the 471 c m - ~ line of indole in Ar at 20 K [16]. This line is stronger than for indole, probably because of the close vicinity of the ~L~ origin, and is distinguished by its two-photon excitation contour; unlike the other lines with ~L~ type signature, there is no a~-e splitting [ 14]. The two lines in the jet spectrum at 427 and 715 cm-~ have a I L b signature ( ~ = 1.2 and 1.3, respectively), and they also appear in the Ar spectrum at the same respective positions. They can be identified as IL b lines by their identical shift in the matrix relative to the i L b origin and by their high anisotropy. The 427 cm-~ line lies on a ~L~ background but the anisotropy jumps from r = - 0 . 0 4 to r = 0.0 over the peak and the 715 cm -~ line has r = +0.075. The line at 980 cm -~, where r drops from +0.075 to 0.0, is 720 cm -~ above the ~L~ origin and may reasonably be assigned as the 26~ transition for ~L~, corresponding to the 715 cm -~ line for the tL b transition.
4.3. 2,3-Dimethylindole Parallel to what was found in solution, it is expected that the IL a origin of 2,3DMI will be redshifted relative to 1L b by the addition of the 2-methyl to 3MI. This is what is seen in the excitation spectrum and anisotropy of Fig. 2, where the origin
B. Fender, P.R. Callis / Chemical Physics Letters 262 (1996)343-348
region is composed of two overlapping bands separated by only 190 c m - t and the anisotropy drops from + 0.08 to - 0 . 0 5 across the pair. However, the anisotropy only says that the two transition dipoles are perpendicular, and does not tell which of the two origins is I L a. The dispersed fluorescence characteristics point strongly to the lower one as being ~L a (see Fig. 4 and Section 4.5). 4.4. 5-Methylindole Unlike 3MI, the methyl group of 5MI undergoes a 60 ° conformational change of the methyl rotor on excitation [24,25], which results in methyl rotor Franck-Condon (FC) activity. The jet-cooled excitation spectrum is therefore similar to that of indole except that each line is split into a group of five major lines and additional minor ones spanning about 200 cm-~, as can be seen in Fig. 3. The argon matrix spectrum of 5MI shown in Fig. 3b matches the jet spectrum quite closely if one assumes that this basic envelope is broadened sufficiently to fuse the methyl rotor lines into one broad peak. Above 1000 c m - t the Ar spectrum seems to have more intensity than the jet spectrum. This is due to a high density of lines in this region of the jet spectrum - which constitutes more intensity than is evident until the lines are broadened by the Ar matrix [23]. It is known from previous spectroscopic studies [7,10,11,21] that substitution at the 5 position with methyl increases the separation of the IL a and IL b origins, but a precise location has not been given. The anisotropy goes sharply and permanently negative at a point 1800 cm-~ above the ~L b origin. We assign the peaks extending from 1800 to 2600 c m as constituting the true ~La origin, which appears to be split into several components, as it is for indole and 3MI (in vacuum). As in the case of indole [16], the u28 HerzbergTeller activity accounts for the low anisotropy (r = 0.01) between 400 and 600 cm -1 in Fig. 3c. Other dips in the anisotropy between 700 and 1500 c m are also likely due to this. At this time we are not sure why the corresponding lines in the jet-cooled • I spectrum failed to reveal L a character that was seen • l • . . for mdole [25]. The strong L b l.,26 hne ns evident at ils usual position of 720 cm -~ and has high anisotropy, r = +0.05.
347
4.5. Fluorescence spectra In Fig. 4 it is seen that the fluorescence spectra of 3MI and 5MI in solid argon are quite similar to that of indole, although somewhat broader. It appears that emission from 3MI is coming from two different sites, in spite of many attempts to avoid this. On the other hand, the broadness of the 5MI spectrum is almost certainly because of the methyl rotor structure discussed in the previous section. The similarity to the indole and 5MI spectra is the basis for the assignment of S~ for 3MI as ~L b, because the origins of the former are firmly assigned i I as L b [11,16]. L b fluorescence is characterized by a dominant u26 line at 760 cm-~ and two relatively strong lines at 1230 and 1350 cm -~. The in-plane bending vibrations u:8 and u27 in the 500-600 cm-~ region are much weaker than the 760 c m line. In contrast, the fluorescence from 2,3DMI (Fig. 4d) looks like a broader version of the phosphorescence from indole in solid argon (Fig. 4e), known to come from the 3L a state. The FC pattern for the latter differs from that of ~L b by having 27 o (600 cm ~) stronger than 26 o (760 cm- ~) and by having a peak in the 1600 c m - l region. The differences between ~L b and ~L a emission just noted are in accord with spectra computed from the FC factors based on vibrational modes and geometry differences obtained using ab initio electronic structure calculations [23]. The nature of the bond order changes for the two excitations dictates what type of modes will be most FC active• For ~L b the more or less uniform expansion of the benzene ring favors the breathing mode, ~'26. In contrast. ~L, excitation causes large bond order decreases in the 2-3, 6 - 7 , mad 4 - 9 bonds [5,26], causing the higher frequency C -C stretching vibrations, u s - t, lo (15001600 cm -~) to be active [23]. The calculations also invariably predict that Uz7 near 600 c m - I is more active than 1,26 (760 cm-~). Although we have not yet measured the anisotropy as a function of fluorescence wavelength for the methyl indoles, we would expect Herzberg-Teller mixing to cause reduced anisotropy in the vicinity of the 281~ (540 cm ~) lines of 3MI and 5MI, as we observed for indole [16]. Care should therefore be taken when interpreting the cause of reduced
348
B. Fender, P.R. Callis / Chemical Physics Letters 262 (1996) 343-348
anisotropy in the fluorescence of these compounds in non-polar environments; not all reduced anisotropy in the fluorescence spectrum can be attributed to inversion of the ~L b and IL a origins. However, in the more common case of IL a emission, the Herzberg-Teller contribution is expected to be less important because the oscillator strength of the ~L a transition is about three times that of ~L b.
5. Conclusions The polarized fluorescence excitation spectra and dispersed fluorescence spectra in Ar matrices, in conjunction with both one- and two-photon jet-cooled spectra, have provided a unified picture of the overlapping I L b - l L a manifold of indole and methyl indoles, thereby providing a f i n n baseline for comparison to theory and for predicting tryptophan spectra in proteins. The matrix-induced differential shift of the JL a vibronic states relative to ~L b has played a key role, as well as the absence of quantum yield problems. In general, the anisotropies are in accord with two-photon based assignments. When the I L a origin is well above the ~L b origin, as in the case of indole and 5MI in vacuum and matrix, and for 3MI in vacuum, it is split into several components covering a 2 0 0 - 3 0 0 cm-1 range. For 3MI and 2,3DMI in solid argon, where the two origins are within 250 cm -~ of each other, the IL a origin appears to be a single line, probably because it is not degenerate with a number of 1L b vibronic states. That the two origins can approach each other this closely, is testimony to the weak non-adiabatic coupling between the two electronic states, a result also in accord with recent ab initio calculations [26]. The large difference between the fluorescence from 2,3DMI and 3MI indicates that the additional methyl causes a crossing of the IL a and JL b potential surfaces, with IL a being the lower for 2,3DMI. These results have afforded a rare observation of the difference between ~L b and JL a fluorescence spectra under vibronically resolved conditions, again a picture supported by ab initio calculations [23].
Acknowledgement This work was supported by US PHS NIH Grant No. GM31824. We acknowledge Dr. David Sammeth for contributing the jet-cooled spectra.
References [1] A.P. Demchenko, Ultraviolet spectroscopy of proteins (Springer, Berlin, 1986). [2] S.V. Konev, Fluorescence and phosporescence of proteins and nucleic acids (Plenum Press, New York, 1967). [3] M.R. Eftink, Methods Biochem. Anal. 35 (1991) 127. [4] B. Valeur and G. Weber, Photochem. Photobiol. 25 (1977) 441. [5] P.R. Callis, J. Chem. Phys. 95 (1991) 4230. [6] D.W. Pierce and S.G. Boxer, Biophys. J. 68 (1995) 1583. [7] E.H. Stricklandand C. Billups,Biopolymers 12 (1973) 1989. [8] L.J. Andrews and L.S. Forster, Photochem. Pbotobiol. 19 (1974) 353. [9] S.R. Meech and D. Phillips, Chem. Phys. 80 (1983) 317. [10] H. Lami and N. Glasser, J. Chem. Phys. 84 (1986) 597. [11] A.A. Rehms and P.R. Callis, Chem. Phys. Lett. 140 (1987) 83. [12] J.R. Cable, J. Chem. Phys. 92 (1990) 1627. [13] M.R. Eftink, L.A. Selvidge, P.R. Callis and A.A. Rebms, J. Phys. Chem. 94 (1990) 3469. [14] D.M. Sammeth, S.S. Siewert, L.H. Spanglerand P.R. Callis, Chem. Phys. Lett. 193 (1992) 532. [15] D.R. Detainer, G.W. Leach and S.C. Wallace, J. Phys. Chem. 98 (1994) 12834. [16] B.J. Fender, D.M. Sammeth and P.R. Callis, Chem. Phys. Letc 239 (1995) 31. [17] Y. Yamamoto and J. Tanaka, Bull. Chem. Soc. Jpn. 45 ( t 972) 1362. [18] B. Albinssonand B. Norden, J. Phys. Chem. 96 (1992) 6204. [19] W.M. McClain, Acc. Chem. Res. 7 (1974) 129. [20] D.M. Sammeth, S. Yan, L.H. Spangler and P.R. Callis, J. Phys. Chem. 94 (1990) 7340. [21] E.H. Strickland, J. Horwitz and C. Billups, Biochemistry 9 (1970) 4914. [22] J.N. Murrell, The theory of the electronic spectra of organic molecules (Wiley, New York, 1963). [23] P.R. Callis, J.T. Vivian and L.S. Slater, Chem. Phys. Lett. 244 (1995) 53. [24] G.A. Bickel, G.W. Leach, D.R. Demmer, J.W. Hager and S.C. Wallace, J. Chem. Phys. 88 (1988) 1. [25] D.M. Sammeth, S.S. Siewert, P.R. Callis and L.H. Spangler, J. Phys. Chem. 96 (1992) 5771. [26] L.S. Slater and P.R. Callis, J. Phys. Chem. 99 (1995) 8572.
CHEMICAL PHYSICS LETTERS ELSEVIER
Chemical Physics Letters 262 (1996) 343-348
l L a origin locations of methyl indoles in argon matrices Bruce Fender, Patrik R. Callis Department of Chemistry and Biochemistry, Montana State University, Bozeman. MT 59717, USA
Received 3 June 1996; in final form 28 Augusl 1996
Abstract
Polarized fluorescence excitation and dispersed fluorescence spectra of 3-methylindole (3MI), 5-methylindole (5MI), and 2,3-dimethylindole (2,3DMI) in solid Ar at 20 K under site selective conditions have unambiguously revealed their t L a origin locations. For 3MI the IL a origin is 260 cm J above the ~L b origin and for 5MI it is 1800 cm-~ above IL b. For 2,3DMI, the IL a origin lies 190 cm-t below the ~L b origin. The latter assignment is made from details of the dispersed fluorescence in comparison to the phosphorescence spectrum of indole in argon at 20 K.
1. I n t r o d u c t i o n
Indole is the chromophore of the amino acid tryptophan, which is widely exploited as an intrinsic fluorescent probe of local protein environment [ 1-3]. Indole compounds have two low lying 7rTr * singlet excited states labeled tLa a n d ILb, which are nearly degenerate [4,5]. The ~L a state has a dipole about 6 debye larger than the ground a n d 1L b states [6], so its energy is quite sensitive to the local environment. In mobile polar environments, ~L a is always the fluorescent state of tryptophan, but in certain proteins which exhibit blue-shifted, structured tryptophan fluorescence, there exists uncertainty regarding the nature of the emitting state. This uncertainty is caused by the lack of precise knowledge concerning the location of the ~L a origin relative to that o f ILb, and by the lack of knowledge concerning the characteristic vibronic shapes of the two types of fluorescence. This is an important issue because the 1L~ state is easily stabilized by reorientation of a polar environment a n d / o r by local electric fields oriented with the excited state dipole. I f IL b fluorescence
could be confidently demonstrated for a tryptophan in a protein, a rigid environment with a strong local electric field opposing the dipole could be deduced. For the above reasons, it is desirable to establish with certainty the relative locations of the ~L:, and I L b origins of tryptophan in well defined environments, and to this end there has been extensive study of indole and its derivatives [4-18]. We have recently reported the polarized fluorescence and fluorescence excitation spectra of indole in solid argon [16], a study that revealed the existence of a f a l s e ~L a origin caused by Herzberg-Teller coupling of the Fermi doublet at 4 5 5 - 4 8 0 cm -~ , and that located the true L origin at 1100 cm -1 and 1300 cm -~ above the L b o n g m in solid Ar and in vacuum respectively. However, considerable uncertainty remained regarding the location of the ~L a origin of 3-methylindole (a close analog of tryptophan) and other indoles. (The indole ring and numbering system are displayed as part of Fig. 1.) In this Letter we report the extension of our study to 3-methylindote (3MI), 5-methylindole (5MI), and 2,3-dimethylindole (2,3DMI), with special emphasis 1
•
I
•
•
0009-2614/96/$12.00 Copyright © 1996 Published by Elsevier Science B.V. All rights reserved PII S0009-2614(96)01096-2
-
344
B. Fender, P.R. Callis / Chemical Physics Letters 262 (1996) 343-348
placed on the location of the true ~L~ origins and the nature of the fluorescence spectra in solid argon under site selective conditions.
2. E x p e r i m e n t a l
All experiments were carried out with indole derivatives in Ar deposited on a sapphire window at 20 K within a Janis optical cryostat cooled with a CTI-Cryogenic closed cycle He refrigerator. The indoles were obtained from Aldrich and were used without further purification: 3MI (98%, lot # 11822CF), 5MI (99%, lot # 06701HX), and 2,3DMI (99%, lot # 092767). The Ar was ultrapure grade 99.999%. The various indoles were entrained in Ar by passing a portion of the Ar over the crystals maintained at 20-25°C. The rate of deposition Ar was about 4 millimole/h and the molar ratio of indole to Ar was about 10 -4. Slow deposition at 20 K provided the best (although mediocre) conditions for observing anisotropy. Excitation was with a Lumonics HyperDye dye laser pumped by a Lumonics HY200 Nd:YAG laser. The dye laser output was frequency doubled using an Inrad Autotracker II. The excitation linewidth was about 0.20 cm- ~. The sapphire window was tilted to attain a 45 ° angle of incidence relative with the laser beam. Fluorescence was collected at 90 ° through an f / 7 optical system and onto the slit of a Spex 1404 double monochromator with a 2400 grooves/mm grating. Slit widths were kept ~< 800 /zm for nonpolarized spectra, corresponding to a resolution of 20 cm-1. The laser beam was vertically polarized using a depolarizer followed by a Glan polarizer. The emitted light was passed through a Polacoat sheet polarizer, which could be oriented to pass vertically or horizontally polarized light. A quartz wedge depolarizer was mounted at the entrance slit of the monochromator to avoid spurious polarizations due to polarization dependent transmission. Fluorescence anisotropy ( r ) was measured as: r = (Ill-- l±)/(Iii
oriented, non-rotating sample, the theoretical limits are r = 0.4 for parallel excitation and emission transition moments, and r = - 0 . 2 for perpendicular excitation and emission moments [4,13]. These limits are expected to be approached in this study because numerous studies on indoles in crystals [17], stetched films [18], and glassy solvents [4,13] have shown the transition moment directions of the IL a and ]L b transitions to be nearly perpendicular. The experimental values reported here are low in comparison to the theoretical values due to depolarization of the exciting and emitting light by the Ar matrix, probably from scattering a n d / o r strain birefringence.
3. R e s u l t s
Figs. 1-3 show fluorescence excitation spectra and the corresponding anisotropy in argon matrices at 20 K for 3MI, 2,3DMI, and 5MI respectively. For ease of comparison, the bottom panel shows previously published jet-cooled fluorescence excitation
0.10- ~
'
~o.oo -0.054
. ..,
~/9111
I1~-"' i
Anisotropy
i
, A ~ ,7",
bl ¢-
~
'
0
--
ffl ~
,
m
1 i0 l~
°;I
0.6
0 "S ~ /
0 6
~
(
1,3't°' I 500
+ 21_c).
With vertical excitation, 111 is the intensity of the vertically polarized emission while I~_ is the horizontally polarized emission intensity. For a randomly
,
I._
cm q from the
1000 Lb
1500
origin.
Fig. l. Comparison of jet-cooled (a) and Ar matrix isolated (b) fluorescence cence
excitation
spectra of 3-methylindole.
T h e fluores-
anisotropyin the Ar matrix is shown in panel (c).
345
B. Fender, P.R. Callis / Chemical Physics Letters 262 (1996) 343-348
00 t\/ 010~
o.oo
V
-0 05-
Anisotropy '
'
i
. . . .
i
. . . .
i
. . . .
i
. . . .
I
. . . .
....
i
a
_~" C
c ~2
o
ongm Ar Matrix, 20K
:3 u_ '
'
i
. . . .
0
500
1000
....
i
1500
2000
c m q f r o m t h e L a origin.
Fig. 2. Fluorescence excitation spectra (a) and corresponding fluorescence anisotropy (b) of 2,3-dimethylindole in Ar matrix.
spectra in the case of 3MI and 5MI, with the t L b origin aligned with that of the matrix spectrum. The two-photon "polarization ratio" [19], /2, is noted for the stronger lines of the jet spectra..O for a
given line is the ratio of integrated two-photon excited intensities obtained with circularly and linearly polarized light, respectively. It has been found that ~L b lines have ,(2 = 1.3-1.4, whereas ~L~ lines have $2= 0.5-0.8 [11,12,14,20]. The jet-cooled fluorescence excitation spectrum of 2,3DMI is extremely weak above the origin, and is not shown. The S~ origins were found to be shifted to lower frequencies relative to vacuum by 254 c m - i for 3M1, 270 c m for 2,3DMI, and 80 cm ~ for 5MI. The anisotropy maximum and minimum magnitudes were found to vary from sample to sample, but their ratios remained constant. In a number of cases the minimum value approaches minus one half the maximum value, independent of the extent of depolarization. We take this to be the signature of perpendicularly polarized transitions. The emission monochromator was tuned to the prominent 26~ ( = 760 c m - ~ ) line in the fluorescence for the argon matrix excitation spectra of 3MI and 5MI, and to the peak at 1600 cm ~ for 2,3DMI. For the jet spectra, all of the fluorescence was collected.
0 1 0 -
c~ 0.05
-
£
~
H
0 O0 -
P
Y o.
d
-005 -
c/) O) fo t"{/} O O
13
L.L
1.3
0 0
500
1000
1500
2000
2500
500
1000
1500
2000
2500
3000
cm -~ from the Lb origin. Fig. 3. Comparison of jet-cooled (a) and Ar matrix isolated (b) fluorescence excitation spectra of 5-methylindole. The fluorescence anisotropy in the Ar matrix is shown in panel (c).
cm q
Fig. 4. Comparison of the Ar matrix isolated (20 K) dispersed fluorescence spectra of indole (a), 3-methylindole(b), 5-methylindole (c), 2,3-dimethylindole (d), with the dispersed pho,~7~horescenee of indole (e).
346
B. Fender, P.R. Callis / Chemical Physics Letters 262 (1996) 343-348
Fig. 4 compares dispersed fluorescence spectra of indole, 3MI, 5MI, and 2,3DMI with the phosphorescence spectrum of indole, all in argon matrices at 20 K. Again, the origins are aligned.
4. Discussion
4.1. Expected IL a shift T h e I L a - ] L b state spacing is known to be affected by substituent and solvent effects. Of the derivatives studied here, it is known that methyl substitution at the 5 position lowers ~L b, thereby increasing the gap relative to indole, whereas a methyl at the 3 position lowers ~La more than JL b, thereby decreasing the gap [7,10,11,21]. It has been found from polarized two-photon fluorescence excitation spectra of indole, 5MI, 3MI, and 2,3DMI [11] that the ~La origin clearly lies b e l o w IL b prior to excitation in hexane at 25°C for 2,3DMI, and that it is clearly above the IL b origin for indole and 5MI. For 3MI it appears that the two origins are superimposed in hexane solution, prior to excitation. It is known that the ~La state of aromatic hydrocarbons is red-shifted more by solvents than is the ~L b state [22]. For indole in argon at 20 K we found that the ~La origin (actually a cluster of lines) redshifts about 200 c m - ] more than that o f ILb, relative to their position in vacuum [16]. Similar behavior is expected for the compounds studied here, because methylation does not severely alter the permanent dipole. Because hexane has a higher refractive index than argon, we expect the two orgins to be very close for both 3MI and 2,3DMI in argon.
4.2.3-Methylindole The fluorescence excitation spectrum of 3MI in argon shown in Fig. lb is considerably different from its jet spectrum shown in Fig. la. This contrasts with the behavior of indole, where we found a close correspondence of the jet and argon spectra, except for the red-shifted ~L a origin grouping near 1200 c m - ~. Whereas in the jet spectrum of 3MI the ILa origin is apparently split into 4 or 5 major components involving lines at 356, 409, 467(?), 609-617, and 739-749 cm - l , in solid argon the obvious can-
didate for the ~L a origin is the single line lying only 260 c m - ~ above the JL b origin. The strong doublets seen in the jet near 610 and 745 cm -I are not identifiable in the matrix; they, along with the other ~L a " o r i g i n " lines, have apparently shifted and coalesced into a single origin line. This may be because at only 260 cm -) above the IL b origin there exists an insufficient number of nearby ~L b vibronic states to cause the splitting. The IL a assignment is based on the fluorescence (see Section 4.5), which is very similar to that of indole. The integrated intensities of the ~La and IL b origins in argon are nearly identical, as is to be expected on theoretical grounds [7,23]. In the jetcooled excitation spectrum, the sum of the intensities of the JL a lines between 356 and 740 c m - ~ is close to the intensity of the IL b origin. The intense line at 467.5 cm-~ was not counted because it is believed to be a ~L b line that steals its IL a character by Herzberg-Teller vibronic coupling, similar to the 471 c m - ~ line of indole in Ar at 20 K [16]. This line is stronger than for indole, probably because of the close vicinity of the ~L~ origin, and is distinguished by its two-photon excitation contour; unlike the other lines with ~L~ type signature, there is no a~-e splitting [ 14]. The two lines in the jet spectrum at 427 and 715 cm-~ have a I L b signature ( ~ = 1.2 and 1.3, respectively), and they also appear in the Ar spectrum at the same respective positions. They can be identified as IL b lines by their identical shift in the matrix relative to the i L b origin and by their high anisotropy. The 427 cm-~ line lies on a ~L~ background but the anisotropy jumps from r = - 0 . 0 4 to r = 0.0 over the peak and the 715 cm -~ line has r = +0.075. The line at 980 cm -~, where r drops from +0.075 to 0.0, is 720 cm -~ above the ~L~ origin and may reasonably be assigned as the 26~ transition for ~L~, corresponding to the 715 cm -~ line for the tL b transition.
4.3. 2,3-Dimethylindole Parallel to what was found in solution, it is expected that the IL a origin of 2,3DMI will be redshifted relative to 1L b by the addition of the 2-methyl to 3MI. This is what is seen in the excitation spectrum and anisotropy of Fig. 2, where the origin
B. Fender, P.R. Callis / Chemical Physics Letters 262 (1996)343-348
region is composed of two overlapping bands separated by only 190 c m - t and the anisotropy drops from + 0.08 to - 0 . 0 5 across the pair. However, the anisotropy only says that the two transition dipoles are perpendicular, and does not tell which of the two origins is I L a. The dispersed fluorescence characteristics point strongly to the lower one as being ~L a (see Fig. 4 and Section 4.5). 4.4. 5-Methylindole Unlike 3MI, the methyl group of 5MI undergoes a 60 ° conformational change of the methyl rotor on excitation [24,25], which results in methyl rotor Franck-Condon (FC) activity. The jet-cooled excitation spectrum is therefore similar to that of indole except that each line is split into a group of five major lines and additional minor ones spanning about 200 cm-~, as can be seen in Fig. 3. The argon matrix spectrum of 5MI shown in Fig. 3b matches the jet spectrum quite closely if one assumes that this basic envelope is broadened sufficiently to fuse the methyl rotor lines into one broad peak. Above 1000 c m - t the Ar spectrum seems to have more intensity than the jet spectrum. This is due to a high density of lines in this region of the jet spectrum - which constitutes more intensity than is evident until the lines are broadened by the Ar matrix [23]. It is known from previous spectroscopic studies [7,10,11,21] that substitution at the 5 position with methyl increases the separation of the IL a and IL b origins, but a precise location has not been given. The anisotropy goes sharply and permanently negative at a point 1800 cm-~ above the ~L b origin. We assign the peaks extending from 1800 to 2600 c m as constituting the true ~La origin, which appears to be split into several components, as it is for indole and 3MI (in vacuum). As in the case of indole [16], the u28 HerzbergTeller activity accounts for the low anisotropy (r = 0.01) between 400 and 600 cm -1 in Fig. 3c. Other dips in the anisotropy between 700 and 1500 c m are also likely due to this. At this time we are not sure why the corresponding lines in the jet-cooled • I spectrum failed to reveal L a character that was seen • l • . . for mdole [25]. The strong L b l.,26 hne ns evident at ils usual position of 720 cm -~ and has high anisotropy, r = +0.05.
347
4.5. Fluorescence spectra In Fig. 4 it is seen that the fluorescence spectra of 3MI and 5MI in solid argon are quite similar to that of indole, although somewhat broader. It appears that emission from 3MI is coming from two different sites, in spite of many attempts to avoid this. On the other hand, the broadness of the 5MI spectrum is almost certainly because of the methyl rotor structure discussed in the previous section. The similarity to the indole and 5MI spectra is the basis for the assignment of S~ for 3MI as ~L b, because the origins of the former are firmly assigned i I as L b [11,16]. L b fluorescence is characterized by a dominant u26 line at 760 cm-~ and two relatively strong lines at 1230 and 1350 cm -~. The in-plane bending vibrations u:8 and u27 in the 500-600 cm-~ region are much weaker than the 760 c m line. In contrast, the fluorescence from 2,3DMI (Fig. 4d) looks like a broader version of the phosphorescence from indole in solid argon (Fig. 4e), known to come from the 3L a state. The FC pattern for the latter differs from that of ~L b by having 27 o (600 cm ~) stronger than 26 o (760 cm- ~) and by having a peak in the 1600 c m - l region. The differences between ~L b and ~L a emission just noted are in accord with spectra computed from the FC factors based on vibrational modes and geometry differences obtained using ab initio electronic structure calculations [23]. The nature of the bond order changes for the two excitations dictates what type of modes will be most FC active• For ~L b the more or less uniform expansion of the benzene ring favors the breathing mode, ~'26. In contrast. ~L, excitation causes large bond order decreases in the 2-3, 6 - 7 , mad 4 - 9 bonds [5,26], causing the higher frequency C -C stretching vibrations, u s - t, lo (15001600 cm -~) to be active [23]. The calculations also invariably predict that Uz7 near 600 c m - I is more active than 1,26 (760 cm-~). Although we have not yet measured the anisotropy as a function of fluorescence wavelength for the methyl indoles, we would expect Herzberg-Teller mixing to cause reduced anisotropy in the vicinity of the 281~ (540 cm ~) lines of 3MI and 5MI, as we observed for indole [16]. Care should therefore be taken when interpreting the cause of reduced
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B. Fender, P.R. Callis / Chemical Physics Letters 262 (1996) 343-348
anisotropy in the fluorescence of these compounds in non-polar environments; not all reduced anisotropy in the fluorescence spectrum can be attributed to inversion of the ~L b and IL a origins. However, in the more common case of IL a emission, the Herzberg-Teller contribution is expected to be less important because the oscillator strength of the ~L a transition is about three times that of ~L b.
5. Conclusions The polarized fluorescence excitation spectra and dispersed fluorescence spectra in Ar matrices, in conjunction with both one- and two-photon jet-cooled spectra, have provided a unified picture of the overlapping I L b - l L a manifold of indole and methyl indoles, thereby providing a f i n n baseline for comparison to theory and for predicting tryptophan spectra in proteins. The matrix-induced differential shift of the JL a vibronic states relative to ~L b has played a key role, as well as the absence of quantum yield problems. In general, the anisotropies are in accord with two-photon based assignments. When the I L a origin is well above the ~L b origin, as in the case of indole and 5MI in vacuum and matrix, and for 3MI in vacuum, it is split into several components covering a 2 0 0 - 3 0 0 cm-1 range. For 3MI and 2,3DMI in solid argon, where the two origins are within 250 cm -~ of each other, the IL a origin appears to be a single line, probably because it is not degenerate with a number of 1L b vibronic states. That the two origins can approach each other this closely, is testimony to the weak non-adiabatic coupling between the two electronic states, a result also in accord with recent ab initio calculations [26]. The large difference between the fluorescence from 2,3DMI and 3MI indicates that the additional methyl causes a crossing of the IL a and JL b potential surfaces, with IL a being the lower for 2,3DMI. These results have afforded a rare observation of the difference between ~L b and JL a fluorescence spectra under vibronically resolved conditions, again a picture supported by ab initio calculations [23].
Acknowledgement This work was supported by US PHS NIH Grant No. GM31824. We acknowledge Dr. David Sammeth for contributing the jet-cooled spectra.
References [1] A.P. Demchenko, Ultraviolet spectroscopy of proteins (Springer, Berlin, 1986). [2] S.V. Konev, Fluorescence and phosporescence of proteins and nucleic acids (Plenum Press, New York, 1967). [3] M.R. Eftink, Methods Biochem. Anal. 35 (1991) 127. [4] B. Valeur and G. Weber, Photochem. Photobiol. 25 (1977) 441. [5] P.R. Callis, J. Chem. Phys. 95 (1991) 4230. [6] D.W. Pierce and S.G. Boxer, Biophys. J. 68 (1995) 1583. [7] E.H. Stricklandand C. Billups,Biopolymers 12 (1973) 1989. [8] L.J. Andrews and L.S. Forster, Photochem. Pbotobiol. 19 (1974) 353. [9] S.R. Meech and D. Phillips, Chem. Phys. 80 (1983) 317. [10] H. Lami and N. Glasser, J. Chem. Phys. 84 (1986) 597. [11] A.A. Rehms and P.R. Callis, Chem. Phys. Lett. 140 (1987) 83. [12] J.R. Cable, J. Chem. Phys. 92 (1990) 1627. [13] M.R. Eftink, L.A. Selvidge, P.R. Callis and A.A. Rebms, J. Phys. Chem. 94 (1990) 3469. [14] D.M. Sammeth, S.S. Siewert, L.H. Spanglerand P.R. Callis, Chem. Phys. Lett. 193 (1992) 532. [15] D.R. Detainer, G.W. Leach and S.C. Wallace, J. Phys. Chem. 98 (1994) 12834. [16] B.J. Fender, D.M. Sammeth and P.R. Callis, Chem. Phys. Letc 239 (1995) 31. [17] Y. Yamamoto and J. Tanaka, Bull. Chem. Soc. Jpn. 45 ( t 972) 1362. [18] B. Albinssonand B. Norden, J. Phys. Chem. 96 (1992) 6204. [19] W.M. McClain, Acc. Chem. Res. 7 (1974) 129. [20] D.M. Sammeth, S. Yan, L.H. Spangler and P.R. Callis, J. Phys. Chem. 94 (1990) 7340. [21] E.H. Strickland, J. Horwitz and C. Billups, Biochemistry 9 (1970) 4914. [22] J.N. Murrell, The theory of the electronic spectra of organic molecules (Wiley, New York, 1963). [23] P.R. Callis, J.T. Vivian and L.S. Slater, Chem. Phys. Lett. 244 (1995) 53. [24] G.A. Bickel, G.W. Leach, D.R. Demmer, J.W. Hager and S.C. Wallace, J. Chem. Phys. 88 (1988) 1. [25] D.M. Sammeth, S.S. Siewert, P.R. Callis and L.H. Spangler, J. Phys. Chem. 96 (1992) 5771. [26] L.S. Slater and P.R. Callis, J. Phys. Chem. 99 (1995) 8572.