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The electronic spectroscopy of the amino acids tyrosine and phenylalanine in a supersonic jet
JOIJRNAL
OF MOLECULAR
SPECTROSCOPY
156,421-430
( 1992)
The Electronic Spectroscopy of the Amino Acids Tyrosine and Phenylalanine in a Supersonic Jet SELSOJ.MARTINEZIII, JOSEPH C. ALFANO,ANDDONALD The James
Fran&
Institute and The Department of Chemistry. Chicago. Illinois 60637
H. LEVY
The University qf Chicago.
The electronic spectra of the amino acids tyrosine and phenylalanine have been measured in a supersonic jet using laser-induced fluorescence spectroscopy. Through power-dependent studies we have shown that tyrosine and phenylalanine have multiple conformers, and we have assigned spectral features produced by the individual conformers. Dispersed emission spectra of the conformers following excitation of their respective electronic origins reveal that the major vibrational activity occurring in tyrosine and phenylalanine originates from the ring vibrational modes of their respective chromophores. In contrast with previous tryptophan studies, no evidence of intramolecular exciplex formation was observed in any of the tyrosine and phenylalanine dispersed emission spectra. The lack of broad, red-shifted emission characteristic of intramolecular exciplex formation was attributed to the large energy difference between the ‘L, and ‘Lb states. E 1992 Academx
Press, Inc.
INTRODUCTION
The amino acid tryptophan (shown in Fig. 1) dominates the near-ultraviolet absorption and fluorescence of many proteins ( 1, 2). However, this amino acid is often not present in large amounts in common proteins. Although tyrosine (see Fig. 1 ) is a weaker emitter, it contributes significantly because there is often more of it present. However, tyrosine usually is quenched by any nearby tryptophans because of energy transfer (3, 4). Phenylalanine (see Fig. 1) is a much weaker emitter. Consequently. it is nearly impossible to observe any optical contribution from phenylalanine in a protein if the other aromatic amino acids are present. Until recently, molecules of biological interest have been studied almost exclusively in solution. Many of these studies have examined the effect of conformation and solvent on the time- and wavelength-resolved fluorescence of amino acid residues so that the spectra of the aromatic amino acids could be used as optical probes of protein structure and dynamics (5- 7). However, poor spectral resolution in solution makes it impossible to resolve spectral features of individual conformers and to study their properties. To more completely understand the role of the solvent on the spectroscopy and dynamics of amino acids in solution, investigations of these molecules in supersonic jets were initiated. The cooling resulting from the supersonic expansion greatly reduces spectral congestion and facilitates the assignment of the remaining sharp structure. The cold, isolated environment of a molecular beam allows the study of dynamical processes intrinsic to the molecule of interest, in the absence of solvent. The amino acid tryptophan has recently been studied in detail. These studies have revealed the presence of six noninterconverting conformers, one of which exhibits a nearly harmonic 26-cm-’ vibrational progression which did not appear in the spectra of other indole derivatives (8). Analysis of the dispersed emission spectrum of this 421
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422
MARTINEZ. COOH I H-C-NH2 I
ALFANO,
AND
COOH I H-C-NH2 I C’H 2
LEVY COOH I H-C-NH2 I
3 0
OH
(4 FIG. 1. The molecular
structures
(b) of the amino acids (a) tryptophan,
(cl (b) tyrosine,
and (c) phenylalanine.
conformer revealed broad red-shifted fluorescence produced by the formation of an intramolecular exciplex (9). The fluorescence lifetime of this same conformer was found to be shorter than those of three other tryptophan conformers (10). To gain an understanding of the mechanism responsible for the formation of the intramolecular exciplex in tryptophan, Cable et al. (II) and Tubergen et al. ( 12) measured the electronic spectra of tryptophan peptides and tryptophan derivatives in a supersonic jet. They discovered that the existence of conformers with broad emission spectra appears to require the presence of an intramolecular hydrogen bond in the peptide or derivative backbone, which results in a large backbone dipole moment which strongly perturbs the electronic structure of the indole chromophore. Li and Lubman reported the observation of the electronic spectrum of isolated tyrosine cooled in a supersonic expansion (13). Like tryptophan’s spectrum, the spectrum of tyrosine exhibits the spectral complexity that results from a combination of vibrational structure and the existence of several conformers. In an effort to interpret this complicated spectrum, investigations of the spectroscopy of simpler analogs have been initiated. Song and Hayes measured fluorescence excitation spectra and dispersed fluorescence spectra for a series of seven 4-alkyl-substituted phenols ( 14). They assigned many vibrational bands and discussed the effect of multiple structural conformers on the spectra of these molecules. Through power dependent studies, Martinez et al. definitively identified the origin transitions of several molecular conformers (15). The study of 4-propylphenol revealed that two of its three origins resulted from a splitting of gauche structural conformers measured as 12 cm-’ and arising from the two different orientations of the hydroxyl group with respect to the gauche oriented propyl group. The third origin resulted from an anti structural conformer. Power-dependent fluorescence excitation spectra of 3-( 4-hydroxyphenyl) propionic acid, the acidic analog of tyrosine, revealed the existence of one conformer which had the propionic acid group in an anti orientation with respect to the plane perpendicular to the benzene ring and passing through carbon atoms 1 and 4 ( 16). Power dependent fluorescence excitation spectra of tyramine, the basic analog of tyrosine, revealed the existence of six conformers consisting of two distinct sets of gauche and anti conformers, each of which arises from a distinct orientation of the amino group ( 17).
SPECTROSCOPY
OF TYR
AND PHE
423
The spectroscopy of the simpler analogs of phenylalanine has also been investigated. Breen et al. measured one-color two-photon ionization spectra of ethylbenzene and n-propylbenzene (18, 19). The spectrum of ethylbenzene contains a single origin transition at 37 587.8 cm-‘, with weaker features at 28.6 and 98.0 cm-’ to higher wavenumber of the origin, presumably due to torsions of the ethyl group. The spectrum of n-propylbenzene contains two origin transitions at 37 533.9 and 37 583.1 cm-‘. These two features arise from the existence of two stable and distinct conformations of the propyl group. The former was assigned as a gauche conformer origin, while the latter was assigned as an anti conformer origin. Power-dependent fluorescence excitation spectra of hydrocinnamic acid, the acidic analog of phenylalanine, revealed the existence of two conformers whose structures are presently undetermined ( 16). Power-dependent fluorescence excitation spectra of phenethylamine, the basic analog of phenylalanine, revealed the existence of four conformers consisting of two distinct sets of gauche and anti conformers, each of which arises from a distinct orientation of the amino group ( 17). The techniques developed in earlier tryptophan studies are used in this study to examine the properties of isolated tyrosine and phenylalanine molecules. The powerdependent fluorescence excitation spectra and the dispersed emission spectra of tyrosine and phenylalanine have been measured to determine the number of noninterconverting conformers and to determine whether any of these conformers exhibit intramolecular exciplex formation. The present study indicates that although the spectroscopy of tyrosine and phenylalanine is similar to that of tryptophan in some respects, significant differences exist which limit the generalizations which can be drawn from the tryptophan studies. EXPERIMENTAL
DETAILS
The compounds DL-tyrosine, DL-phenylalanine, and uracil were purchased from Sigma Chemical Company and used without further purification. Like tryptophan, the amino acids tyrosine and phenylalanine have low vapor pressures and are thermally unstable. Volatilizing tyrosine and phenylalanine with virtually no thermal decomposition was accomplished using the same type of continuous supersonic jet source that was developed to produce gas-phase samples of tryptophan ( 10). That is, a continuous supersonic jet source of tyrosine or phenylalanine was formed by passing helium over an equally weighted mixture of the amino acid and uracil heated in a standard oven. Specifically, 0.25 g of DL-tyrosine or DL-phenylalanine and 0.25 g of uracil were weighed and mixed before being loaded behind a 0.2-mm-diameter nozzle. The mixture was heated and a continuous jet was produced with 3.4 atm of helium. If the temperature was controlled to t15”C, suitable amounts of gas-phase sample could be maintained for up to 3 hr. Fluctuations in temperature ( f 1“C) were minimized by heating the sample reservoir with a I “-diameter by 1“-width mica band heater (Watlow Electric Mfg. Co.) regulated by a single setpoint proportional controller ( Model 400 1A, OMEGA Engineering, Inc.). The output of a Nd:YAG laser excited dye laser operating on Rhodamine 590 laser dye was frequency doubled in a KDP crystal, producing about 1 mJ of ultraviolet light. This was used to excite the tyrosine sample. The frequency doubled output of the same dye laser operating on Coumarin 500 laser dye was used to excite the phenylalanine sample. Total fluorescence and dispersed emission were detected at right angles to the jet propagation direction and the laser light path. Total fluorescence was
424
MARTINEZ,
ALFANO,
AND
LEVY
collected with a fluorescence collection optics system similar to that described elsewhere (20). Dispersed emission was collected by f/1.O optics, imaged onto the entrance slit of a 0.275-m monochromator (SpectraPro 275, Acton Research Corporation) and detected by an optical multichannel analyzer system (OMA III, EG & G Princeton Applied Research). The mass spectra and resonantly enhanced two-photon ionization spectra of these molecules were measured prior to collecting their fluorescence, and the usual diagnostics were performed to ensure that all spectral features arose from cold isolated molecules and not from vibrationally hot molecules or from impurities or decomposition products. In our power-dependence studies a variable attenuator (Newport Corporation ) was used to reduce the laser power before the laser beam was directed into the vacuum chamber. When saturation of the spectral transitions was desired, the attenuator was replaced by a 50-cm focal length lens which focused the excitation beam into the vacuum chamber. RESULTS
AND
DISCUSSION
(A) Total Fluorescence Data Fluorescence excitation spectra of tyrosine in the region 35 455 to 35 800 cm-’ taken at low and high power densities are shown in Fig. 2. As in previous studies. the power saturation behavior of these spectra is used to identify different molecular conformers (8). The features labeled A through J are assigned as origin transitions of ten distinct conformers of tyrosine, and their positions are listed in Table I. Our spectrum of tyrosine exhibits significant differences from that obtained by Lubman et al. using a pulsed laser desorption method for sample introduction into the jet expansion (13). The high-wavenumber region of 35 600 to 35 700 cm-’ is similar
(a)
I
I
35500
I
35600
WAVENUMBER
35700
(CM-‘)
(b)
I
35500
-’
, 35600
WAVENUMBER
I 35700
(CM-‘>
FIG. 2. Fluorescence excitation spectra of tyrosine in the region 35 455 to 35 800 cm-’ taken at different laser power densities. The spectrum in (b) is taken at about 100 times the power density of that in (a), The oven temperature is 23O”C, the carrier gas is helium at a backing pressure of 3.4 atm, and the nozzle diameter is 0.2 mm.
SPECTROSCOPY
OF TYR
AND
425
PHE
TABLE I Origin Transitions
of Tyrosine
A
35
492
B
35
519
C
35
524
D
35
540
E
35
615
F
35
624
G
35
637
H
35
644
35
646
35
660
J
a
Typical
uncertainty
1s 21
cm-’
in both spectra, but the low-wavenumber region of 35 455 to 35 600 cm-’ is quite different. This low-wavenumber region has no intense features in Lubman’s spectrum; however, our spectrum has features of comparable intensity to those observed in the high-wavenumber region. These were observed whether we used the thermospraypulsed jet technique (8), or the tyrosine/uracil continuous jet technique as the sample source. We attribute these differences to both different source preparation techniques. Fluorescence excitation spectra of phenylalanine in the region 37 520 to 37 625 cm-’ taken at low and high power densities are shown in Fig. 3. The features labeled
d’
(O) 37550
WAVENUMBER
(b)
37600
37650
KM-‘)
A(J_
I 37600
37550
WAVENUMBER
37650
(CM“)
FIG. 3. Fluorescence excitation spectra of phenylalanine in the region 37 520 to 37 625 cm-’ taken at different laser power densities. The spectrum in (b) is taken at about 100 times the power density of that in (a). The oven temperature is 183°C. the carrier gas is helium at a backing pressure of 3.4 atm. and the nozzle diameter is 0.2 mm.
426
MARTINEZ, ALFANO, AND LEVY
A through E are assigned as origin transitions of five conformers of phenylalanine, and their positions are listed in Table II. In both amino acids the redmost origin transition occurs fairly close to the origin transition of their respective chromophores. The redmost origin transition of tyrosine occurs at 35 492 cm-’ while the origin transition of its chromophore, p-cresol, occurs at 35 329 cm-’ ( 14. 21). The redmost origin transition of phenylalanine occurs at 37 537 cm-’ while the origin transition of its chromophore, toluene, occurs at 37 477 cm-’ (22, 23). Evidently, the amino acid side chain exerts only a minor perturbation on the electronic states of tyrosine and phenylalanine. The spectra of tyrosine and phenylalanine are too complex to assign conformer structures to the observed origin transitions using our modular approach. However. the determination of IO tyrosine conformers is consistent with the determination of five phenylalanine conformers. Tyrosine is simply phenylalanine with a hydroxyl group aura-substituted to the benzene ring. The two in-plane (24) orientations of the hydroxyl group give rise to two distinct tyrosine conformers for every conformer of phenylalanine. In the tyrosine spectra a striking similarity in relative intensity and position is observed among conformers A, B, C, D, and conformers E, H, I, J. These two sets of conformers result from a splitting of the electronic origins of tyrosine. This splitting is caused by the hydroxyl group attached to the aromatic ring and is measured as 123 cm-‘. Although this splitting is larger than the 12-cm-’ splitting observed in its simpler analogs ( 15, Z7), it is of comparable magnitude with those observed in me&substituted phenols (25). In measuring the electronic spectra of m-fluorophenol, m-chlorophenol, and m-cresol in a supersonic jet, Ito and co-workers (2.5) observed a hydroxyl splitting of all the vibronic transitions ranging from 100 to 200 cm-‘. A similar process is occurring in tyrosine, splitting the origins of the various conformers by 123 cm-‘. This measured splitting is not observed between conformers F and G. The magnitude of the splitting directly reflects the degree of interaction between the hydroxyl group and the amino acid side chain. The splitting is expected to be large when the hydroxyl group and the amino acid side chain are in close proximity and small when they are far apart. Evidently, the hydroxyl group and the amino acid side chain are spaced further apart in conformers F and G than in the other conformers. Rotational spectroscopy can be used to determine the conformer structures of tyrosine and phenylalanine. Information available from conformer structure determinations of their analogs combined with conformational information available from
TABLE II Origin Transitions of Phenylalanine Conformer
m(cme’ 37
A
537
B
37
558
C
37
570
0
37
600
37
613
E
a
ja
Typical
_ uncertainty
1s ?I
cm-‘.
SPECTROSCOPY
OF TYR
AND
427
PHE
‘H NMR studies of these amino acids in solution (26-39) should facilitate the analysis of their rotational spectra. (B) Dispersed Emission Data Figure 4 shows the dispersed emission spectra of tyrosine produced by exciting the origin transitions of each of its 10 conformers. The similarity of these spectra to each other and to the dispersed emission spectrum of p-cresol produced by exciting its electronic origin transition (16) indicates that most of the vibrational activity in the tyrosine dispersed spectra is in the ring modes of the p-cresol chromophore. Figure 5 shows the dispersed emission spectra of phenylalanine produced by exciting the origin transitions of each of its five conformers. The similarity of these spectra to each other and to the dispersed emission spectrum of toluene produced by exciting its electronic origin transition (40) signifies that most of the vibrational activity in the phenylalanine dispersed spectra is in the ring modes of the toluene chromophore. Intramolecular exciplex formation does not occur in tyrosine and phenylalanine. as indicated by the absence of any broad, red-shifted emission in our spectra. Molecular beam studies of tryptophan and its derivatives indicate that the existence of conformers with broad, red-shifted emission require the presence of an intramolecular hydrogen bond in the amino acid or peptide backbone ( 11, 12). An analogous conformation
(0)
(b)
cc)
(d)
(~9
L
h h_ L
L
(f)
L
(9)
L
(h)
k_
(1)
h
(J)
_
h U-l
-36000
36000 WAVENUMBER
KM-‘)
WAVENUMBER
(CM-‘)
FIG. 4. Dispersed emission spectra of tyrosine. The spectra of (a ) conformer A. (b) conformer B, (c ) conformer C, (d) conformer D. (e) conformer E, (f) conformer F, (g) conformer G, (h) conformer H. (i ) conformer I. and (j) conformer J were produced by exciting their respective electronic origins. The spectral resolution is 94 cm-‘. The oven temperature is 230°C. the carrier gas is helium at a backing pressure of 3.4 atm, and the nozzle diameter is 0.2 mm. Approximately 15% of the intensity of the resonance transition originates from scattered laser light, Each spectrum is an average of four scans measured with an exposure time of 60 sec.
428
MARTINEZ,
ALFANO.
AND
LEVY
e1
(a)
(b)
(c)
2
J_L--l-37000
WAVENUMBER
33000
(CM-‘)
FIG. 5. Dispersed emission spectra of phenylalanine. The spectra of (a) conformer A, (b) conformer B. C. (d) conformer D. and (e ) conformer E were produced by exciting their respective electronic origins. The spectral resolution is 105 cm-‘. The oven temperature is 190°C. the carrier gas is helium at a backing pressure of 3.4 atm, and the nozzle diameter is 0.2 mm. Approximately 15% of the intensity of the resonance transition originates from scattered laser light. Each spectrum is an average of four scans measured with an exposure time of 30 sec.
(c) conformer
of the amino acid glycine was found to have a very high dipole moment ( I I. 41-43). The dipole-dipole interaction between the amino acid or peptide backbone and the indole A electron system lowers the energy of the IL,state, allowing it to mix effectively with the 'Lbstate. Mixing between the two states produces a double-well excited state potential characteristic of an exciplex. Broad and red-shifted emission occurs through the ’ L,-type state, which is stabilized by the large backbone dipole moment and is strongly coupled to the initially excited 'Lblevels through the backbone coordinates. The amount of energy lowering of the ’ L, state depends on the dipole moment of the amino acid or peptide backbone, the distance from the backbone dipole to the chromophore dipole, and the orientation of the two dipoles. In indole, the 'Lh state has a dipole moment that is almost the same as the ground electronic state (12, 44), whereas the 'L,state has a dipole moment 3 D larger than that of the ground state (22, 45). The magnitude of the dipole-dipole interaction is strongly dependent upon the distance and orientation between the two dipoles. Assuming the most favorable orientation and a minimum distance of 3.5 A between the dipoles, Tubergen et al. tabulated that an increase of 3 D in a chromophore interacting with a 6-D backbone would produce a decrease in energy of 4200 cm-’ (12). Room-temperature vaporphase studies of indole place the onset of the IL,state 1400 cm-l above the origin of the 'Lb state (46, 47). Consequently, the dipole-dipole interaction is capable of sufficiently lowering the energy of the IL,state, allowing it to mix effectively with the 'Lbstate. As a result, broad, red-shifted emission is observed in the emission spectra
SPECTROSCOPY
OF TYR
AND
PHE
429
of the conformers of tryptophan and its derivatives whose backbones form an intramolecular hydrogen bond (9, 11, 12). In contrast, vapor-phase studies of benzene place the onset of the ‘L, state 10 609 cm-’ above the origin of the ‘Lb state (48). Consequently, in tyrosine and phenylalanine the energy difference between the ’ L, and ‘Lb states is too large to allow effective mixing between these states, even though the energy of the ‘L, state is stabilized by an equivalent type of dipole-dipole interaction. As a result, broad, red-shifted emission is not observed in the emission spectra of the conformers of tyrosine and phenylalanine, even if their conformer backbones form an intramolecular hydrogen bond. Assuming the most favorable orientation and a minimum distance of 3.5 A between the dipoles, an increase of 7.5 D in a chromophore interacting with a 6-D backbone is needed to produce a decrease in energy of 10 609 cm-‘. SUMMARY
The electronic spectra of the amino acids tyrosine and phenylalanine have been measured in a supersonic jet using laser-induced fluorescence spectroscopy. Volatilizing tyrosine and phenylalanine with virtually no thermal decomposition was accomplished using the same type of continuous supersonic jet source that was developed to produce gas-phase samples of tryptophan. Through power-dependent studies we have shown in a manner similar to tryptophan that tyrosine and phenylalanine have multiple conformers, and we have assigned spectral features produced by the individual conformers. Dispersed emission spectra of the conformers following excitation of their respective electronic origins reveal that the major vibrational activity occurring in tyrosine and phenylalanine originates from the ring vibrational modes of their respective chromophores. In contrast with previous tryptophan studies, no evidence of intramolecular exciplex formation was observed in any of the tyrosine and phenylalanine dispersed emission spectra. ACKNOWLEDGMENTS This work was supported by the National Science Foundation under Grant CHE-881832 1. S.J.M. acfrom the Illinois Minority Graduate Incentive Program and from the Dorothy Danforth Compton Fellowship Program. J.C.A. acknowledges support from the National Science Foundation Graduate Fellowship Program. knowledges support
RECEIVED:
May 2, 1992 REFERENCES
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OF MOLECULAR
SPECTROSCOPY
156,421-430
( 1992)
The Electronic Spectroscopy of the Amino Acids Tyrosine and Phenylalanine in a Supersonic Jet SELSOJ.MARTINEZIII, JOSEPH C. ALFANO,ANDDONALD The James
Fran&
Institute and The Department of Chemistry. Chicago. Illinois 60637
H. LEVY
The University qf Chicago.
The electronic spectra of the amino acids tyrosine and phenylalanine have been measured in a supersonic jet using laser-induced fluorescence spectroscopy. Through power-dependent studies we have shown that tyrosine and phenylalanine have multiple conformers, and we have assigned spectral features produced by the individual conformers. Dispersed emission spectra of the conformers following excitation of their respective electronic origins reveal that the major vibrational activity occurring in tyrosine and phenylalanine originates from the ring vibrational modes of their respective chromophores. In contrast with previous tryptophan studies, no evidence of intramolecular exciplex formation was observed in any of the tyrosine and phenylalanine dispersed emission spectra. The lack of broad, red-shifted emission characteristic of intramolecular exciplex formation was attributed to the large energy difference between the ‘L, and ‘Lb states. E 1992 Academx
Press, Inc.
INTRODUCTION
The amino acid tryptophan (shown in Fig. 1) dominates the near-ultraviolet absorption and fluorescence of many proteins ( 1, 2). However, this amino acid is often not present in large amounts in common proteins. Although tyrosine (see Fig. 1 ) is a weaker emitter, it contributes significantly because there is often more of it present. However, tyrosine usually is quenched by any nearby tryptophans because of energy transfer (3, 4). Phenylalanine (see Fig. 1) is a much weaker emitter. Consequently. it is nearly impossible to observe any optical contribution from phenylalanine in a protein if the other aromatic amino acids are present. Until recently, molecules of biological interest have been studied almost exclusively in solution. Many of these studies have examined the effect of conformation and solvent on the time- and wavelength-resolved fluorescence of amino acid residues so that the spectra of the aromatic amino acids could be used as optical probes of protein structure and dynamics (5- 7). However, poor spectral resolution in solution makes it impossible to resolve spectral features of individual conformers and to study their properties. To more completely understand the role of the solvent on the spectroscopy and dynamics of amino acids in solution, investigations of these molecules in supersonic jets were initiated. The cooling resulting from the supersonic expansion greatly reduces spectral congestion and facilitates the assignment of the remaining sharp structure. The cold, isolated environment of a molecular beam allows the study of dynamical processes intrinsic to the molecule of interest, in the absence of solvent. The amino acid tryptophan has recently been studied in detail. These studies have revealed the presence of six noninterconverting conformers, one of which exhibits a nearly harmonic 26-cm-’ vibrational progression which did not appear in the spectra of other indole derivatives (8). Analysis of the dispersed emission spectrum of this 421
0022-2852192
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422
MARTINEZ. COOH I H-C-NH2 I
ALFANO,
AND
COOH I H-C-NH2 I C’H 2
LEVY COOH I H-C-NH2 I
3 0
OH
(4 FIG. 1. The molecular
structures
(b) of the amino acids (a) tryptophan,
(cl (b) tyrosine,
and (c) phenylalanine.
conformer revealed broad red-shifted fluorescence produced by the formation of an intramolecular exciplex (9). The fluorescence lifetime of this same conformer was found to be shorter than those of three other tryptophan conformers (10). To gain an understanding of the mechanism responsible for the formation of the intramolecular exciplex in tryptophan, Cable et al. (II) and Tubergen et al. ( 12) measured the electronic spectra of tryptophan peptides and tryptophan derivatives in a supersonic jet. They discovered that the existence of conformers with broad emission spectra appears to require the presence of an intramolecular hydrogen bond in the peptide or derivative backbone, which results in a large backbone dipole moment which strongly perturbs the electronic structure of the indole chromophore. Li and Lubman reported the observation of the electronic spectrum of isolated tyrosine cooled in a supersonic expansion (13). Like tryptophan’s spectrum, the spectrum of tyrosine exhibits the spectral complexity that results from a combination of vibrational structure and the existence of several conformers. In an effort to interpret this complicated spectrum, investigations of the spectroscopy of simpler analogs have been initiated. Song and Hayes measured fluorescence excitation spectra and dispersed fluorescence spectra for a series of seven 4-alkyl-substituted phenols ( 14). They assigned many vibrational bands and discussed the effect of multiple structural conformers on the spectra of these molecules. Through power dependent studies, Martinez et al. definitively identified the origin transitions of several molecular conformers (15). The study of 4-propylphenol revealed that two of its three origins resulted from a splitting of gauche structural conformers measured as 12 cm-’ and arising from the two different orientations of the hydroxyl group with respect to the gauche oriented propyl group. The third origin resulted from an anti structural conformer. Power-dependent fluorescence excitation spectra of 3-( 4-hydroxyphenyl) propionic acid, the acidic analog of tyrosine, revealed the existence of one conformer which had the propionic acid group in an anti orientation with respect to the plane perpendicular to the benzene ring and passing through carbon atoms 1 and 4 ( 16). Power dependent fluorescence excitation spectra of tyramine, the basic analog of tyrosine, revealed the existence of six conformers consisting of two distinct sets of gauche and anti conformers, each of which arises from a distinct orientation of the amino group ( 17).
SPECTROSCOPY
OF TYR
AND PHE
423
The spectroscopy of the simpler analogs of phenylalanine has also been investigated. Breen et al. measured one-color two-photon ionization spectra of ethylbenzene and n-propylbenzene (18, 19). The spectrum of ethylbenzene contains a single origin transition at 37 587.8 cm-‘, with weaker features at 28.6 and 98.0 cm-’ to higher wavenumber of the origin, presumably due to torsions of the ethyl group. The spectrum of n-propylbenzene contains two origin transitions at 37 533.9 and 37 583.1 cm-‘. These two features arise from the existence of two stable and distinct conformations of the propyl group. The former was assigned as a gauche conformer origin, while the latter was assigned as an anti conformer origin. Power-dependent fluorescence excitation spectra of hydrocinnamic acid, the acidic analog of phenylalanine, revealed the existence of two conformers whose structures are presently undetermined ( 16). Power-dependent fluorescence excitation spectra of phenethylamine, the basic analog of phenylalanine, revealed the existence of four conformers consisting of two distinct sets of gauche and anti conformers, each of which arises from a distinct orientation of the amino group ( 17). The techniques developed in earlier tryptophan studies are used in this study to examine the properties of isolated tyrosine and phenylalanine molecules. The powerdependent fluorescence excitation spectra and the dispersed emission spectra of tyrosine and phenylalanine have been measured to determine the number of noninterconverting conformers and to determine whether any of these conformers exhibit intramolecular exciplex formation. The present study indicates that although the spectroscopy of tyrosine and phenylalanine is similar to that of tryptophan in some respects, significant differences exist which limit the generalizations which can be drawn from the tryptophan studies. EXPERIMENTAL
DETAILS
The compounds DL-tyrosine, DL-phenylalanine, and uracil were purchased from Sigma Chemical Company and used without further purification. Like tryptophan, the amino acids tyrosine and phenylalanine have low vapor pressures and are thermally unstable. Volatilizing tyrosine and phenylalanine with virtually no thermal decomposition was accomplished using the same type of continuous supersonic jet source that was developed to produce gas-phase samples of tryptophan ( 10). That is, a continuous supersonic jet source of tyrosine or phenylalanine was formed by passing helium over an equally weighted mixture of the amino acid and uracil heated in a standard oven. Specifically, 0.25 g of DL-tyrosine or DL-phenylalanine and 0.25 g of uracil were weighed and mixed before being loaded behind a 0.2-mm-diameter nozzle. The mixture was heated and a continuous jet was produced with 3.4 atm of helium. If the temperature was controlled to t15”C, suitable amounts of gas-phase sample could be maintained for up to 3 hr. Fluctuations in temperature ( f 1“C) were minimized by heating the sample reservoir with a I “-diameter by 1“-width mica band heater (Watlow Electric Mfg. Co.) regulated by a single setpoint proportional controller ( Model 400 1A, OMEGA Engineering, Inc.). The output of a Nd:YAG laser excited dye laser operating on Rhodamine 590 laser dye was frequency doubled in a KDP crystal, producing about 1 mJ of ultraviolet light. This was used to excite the tyrosine sample. The frequency doubled output of the same dye laser operating on Coumarin 500 laser dye was used to excite the phenylalanine sample. Total fluorescence and dispersed emission were detected at right angles to the jet propagation direction and the laser light path. Total fluorescence was
424
MARTINEZ,
ALFANO,
AND
LEVY
collected with a fluorescence collection optics system similar to that described elsewhere (20). Dispersed emission was collected by f/1.O optics, imaged onto the entrance slit of a 0.275-m monochromator (SpectraPro 275, Acton Research Corporation) and detected by an optical multichannel analyzer system (OMA III, EG & G Princeton Applied Research). The mass spectra and resonantly enhanced two-photon ionization spectra of these molecules were measured prior to collecting their fluorescence, and the usual diagnostics were performed to ensure that all spectral features arose from cold isolated molecules and not from vibrationally hot molecules or from impurities or decomposition products. In our power-dependence studies a variable attenuator (Newport Corporation ) was used to reduce the laser power before the laser beam was directed into the vacuum chamber. When saturation of the spectral transitions was desired, the attenuator was replaced by a 50-cm focal length lens which focused the excitation beam into the vacuum chamber. RESULTS
AND
DISCUSSION
(A) Total Fluorescence Data Fluorescence excitation spectra of tyrosine in the region 35 455 to 35 800 cm-’ taken at low and high power densities are shown in Fig. 2. As in previous studies. the power saturation behavior of these spectra is used to identify different molecular conformers (8). The features labeled A through J are assigned as origin transitions of ten distinct conformers of tyrosine, and their positions are listed in Table I. Our spectrum of tyrosine exhibits significant differences from that obtained by Lubman et al. using a pulsed laser desorption method for sample introduction into the jet expansion (13). The high-wavenumber region of 35 600 to 35 700 cm-’ is similar
(a)
I
I
35500
I
35600
WAVENUMBER
35700
(CM-‘)
(b)
I
35500
-’
, 35600
WAVENUMBER
I 35700
(CM-‘>
FIG. 2. Fluorescence excitation spectra of tyrosine in the region 35 455 to 35 800 cm-’ taken at different laser power densities. The spectrum in (b) is taken at about 100 times the power density of that in (a), The oven temperature is 23O”C, the carrier gas is helium at a backing pressure of 3.4 atm, and the nozzle diameter is 0.2 mm.
SPECTROSCOPY
OF TYR
AND
425
PHE
TABLE I Origin Transitions
of Tyrosine
A
35
492
B
35
519
C
35
524
D
35
540
E
35
615
F
35
624
G
35
637
H
35
644
35
646
35
660
J
a
Typical
uncertainty
1s 21
cm-’
in both spectra, but the low-wavenumber region of 35 455 to 35 600 cm-’ is quite different. This low-wavenumber region has no intense features in Lubman’s spectrum; however, our spectrum has features of comparable intensity to those observed in the high-wavenumber region. These were observed whether we used the thermospraypulsed jet technique (8), or the tyrosine/uracil continuous jet technique as the sample source. We attribute these differences to both different source preparation techniques. Fluorescence excitation spectra of phenylalanine in the region 37 520 to 37 625 cm-’ taken at low and high power densities are shown in Fig. 3. The features labeled
d’
(O) 37550
WAVENUMBER
(b)
37600
37650
KM-‘)
A(J_
I 37600
37550
WAVENUMBER
37650
(CM“)
FIG. 3. Fluorescence excitation spectra of phenylalanine in the region 37 520 to 37 625 cm-’ taken at different laser power densities. The spectrum in (b) is taken at about 100 times the power density of that in (a). The oven temperature is 183°C. the carrier gas is helium at a backing pressure of 3.4 atm. and the nozzle diameter is 0.2 mm.
426
MARTINEZ, ALFANO, AND LEVY
A through E are assigned as origin transitions of five conformers of phenylalanine, and their positions are listed in Table II. In both amino acids the redmost origin transition occurs fairly close to the origin transition of their respective chromophores. The redmost origin transition of tyrosine occurs at 35 492 cm-’ while the origin transition of its chromophore, p-cresol, occurs at 35 329 cm-’ ( 14. 21). The redmost origin transition of phenylalanine occurs at 37 537 cm-’ while the origin transition of its chromophore, toluene, occurs at 37 477 cm-’ (22, 23). Evidently, the amino acid side chain exerts only a minor perturbation on the electronic states of tyrosine and phenylalanine. The spectra of tyrosine and phenylalanine are too complex to assign conformer structures to the observed origin transitions using our modular approach. However. the determination of IO tyrosine conformers is consistent with the determination of five phenylalanine conformers. Tyrosine is simply phenylalanine with a hydroxyl group aura-substituted to the benzene ring. The two in-plane (24) orientations of the hydroxyl group give rise to two distinct tyrosine conformers for every conformer of phenylalanine. In the tyrosine spectra a striking similarity in relative intensity and position is observed among conformers A, B, C, D, and conformers E, H, I, J. These two sets of conformers result from a splitting of the electronic origins of tyrosine. This splitting is caused by the hydroxyl group attached to the aromatic ring and is measured as 123 cm-‘. Although this splitting is larger than the 12-cm-’ splitting observed in its simpler analogs ( 15, Z7), it is of comparable magnitude with those observed in me&substituted phenols (25). In measuring the electronic spectra of m-fluorophenol, m-chlorophenol, and m-cresol in a supersonic jet, Ito and co-workers (2.5) observed a hydroxyl splitting of all the vibronic transitions ranging from 100 to 200 cm-‘. A similar process is occurring in tyrosine, splitting the origins of the various conformers by 123 cm-‘. This measured splitting is not observed between conformers F and G. The magnitude of the splitting directly reflects the degree of interaction between the hydroxyl group and the amino acid side chain. The splitting is expected to be large when the hydroxyl group and the amino acid side chain are in close proximity and small when they are far apart. Evidently, the hydroxyl group and the amino acid side chain are spaced further apart in conformers F and G than in the other conformers. Rotational spectroscopy can be used to determine the conformer structures of tyrosine and phenylalanine. Information available from conformer structure determinations of their analogs combined with conformational information available from
TABLE II Origin Transitions of Phenylalanine Conformer
m(cme’ 37
A
537
B
37
558
C
37
570
0
37
600
37
613
E
a
ja
Typical
_ uncertainty
1s ?I
cm-‘.
SPECTROSCOPY
OF TYR
AND
427
PHE
‘H NMR studies of these amino acids in solution (26-39) should facilitate the analysis of their rotational spectra. (B) Dispersed Emission Data Figure 4 shows the dispersed emission spectra of tyrosine produced by exciting the origin transitions of each of its 10 conformers. The similarity of these spectra to each other and to the dispersed emission spectrum of p-cresol produced by exciting its electronic origin transition (16) indicates that most of the vibrational activity in the tyrosine dispersed spectra is in the ring modes of the p-cresol chromophore. Figure 5 shows the dispersed emission spectra of phenylalanine produced by exciting the origin transitions of each of its five conformers. The similarity of these spectra to each other and to the dispersed emission spectrum of toluene produced by exciting its electronic origin transition (40) signifies that most of the vibrational activity in the phenylalanine dispersed spectra is in the ring modes of the toluene chromophore. Intramolecular exciplex formation does not occur in tyrosine and phenylalanine. as indicated by the absence of any broad, red-shifted emission in our spectra. Molecular beam studies of tryptophan and its derivatives indicate that the existence of conformers with broad, red-shifted emission require the presence of an intramolecular hydrogen bond in the amino acid or peptide backbone ( 11, 12). An analogous conformation
(0)
(b)
cc)
(d)
(~9
L
h h_ L
L
(f)
L
(9)
L
(h)
k_
(1)
h
(J)
_
h U-l
-36000
36000 WAVENUMBER
KM-‘)
WAVENUMBER
(CM-‘)
FIG. 4. Dispersed emission spectra of tyrosine. The spectra of (a ) conformer A. (b) conformer B, (c ) conformer C, (d) conformer D. (e) conformer E, (f) conformer F, (g) conformer G, (h) conformer H. (i ) conformer I. and (j) conformer J were produced by exciting their respective electronic origins. The spectral resolution is 94 cm-‘. The oven temperature is 230°C. the carrier gas is helium at a backing pressure of 3.4 atm, and the nozzle diameter is 0.2 mm. Approximately 15% of the intensity of the resonance transition originates from scattered laser light, Each spectrum is an average of four scans measured with an exposure time of 60 sec.
428
MARTINEZ,
ALFANO.
AND
LEVY
e1
(a)
(b)
(c)
2
J_L--l-37000
WAVENUMBER
33000
(CM-‘)
FIG. 5. Dispersed emission spectra of phenylalanine. The spectra of (a) conformer A, (b) conformer B. C. (d) conformer D. and (e ) conformer E were produced by exciting their respective electronic origins. The spectral resolution is 105 cm-‘. The oven temperature is 190°C. the carrier gas is helium at a backing pressure of 3.4 atm, and the nozzle diameter is 0.2 mm. Approximately 15% of the intensity of the resonance transition originates from scattered laser light. Each spectrum is an average of four scans measured with an exposure time of 30 sec.
(c) conformer
of the amino acid glycine was found to have a very high dipole moment ( I I. 41-43). The dipole-dipole interaction between the amino acid or peptide backbone and the indole A electron system lowers the energy of the IL,state, allowing it to mix effectively with the 'Lbstate. Mixing between the two states produces a double-well excited state potential characteristic of an exciplex. Broad and red-shifted emission occurs through the ’ L,-type state, which is stabilized by the large backbone dipole moment and is strongly coupled to the initially excited 'Lblevels through the backbone coordinates. The amount of energy lowering of the ’ L, state depends on the dipole moment of the amino acid or peptide backbone, the distance from the backbone dipole to the chromophore dipole, and the orientation of the two dipoles. In indole, the 'Lh state has a dipole moment that is almost the same as the ground electronic state (12, 44), whereas the 'L,state has a dipole moment 3 D larger than that of the ground state (22, 45). The magnitude of the dipole-dipole interaction is strongly dependent upon the distance and orientation between the two dipoles. Assuming the most favorable orientation and a minimum distance of 3.5 A between the dipoles, Tubergen et al. tabulated that an increase of 3 D in a chromophore interacting with a 6-D backbone would produce a decrease in energy of 4200 cm-’ (12). Room-temperature vaporphase studies of indole place the onset of the IL,state 1400 cm-l above the origin of the 'Lb state (46, 47). Consequently, the dipole-dipole interaction is capable of sufficiently lowering the energy of the IL,state, allowing it to mix effectively with the 'Lbstate. As a result, broad, red-shifted emission is observed in the emission spectra
SPECTROSCOPY
OF TYR
AND
PHE
429
of the conformers of tryptophan and its derivatives whose backbones form an intramolecular hydrogen bond (9, 11, 12). In contrast, vapor-phase studies of benzene place the onset of the ‘L, state 10 609 cm-’ above the origin of the ‘Lb state (48). Consequently, in tyrosine and phenylalanine the energy difference between the ’ L, and ‘Lb states is too large to allow effective mixing between these states, even though the energy of the ‘L, state is stabilized by an equivalent type of dipole-dipole interaction. As a result, broad, red-shifted emission is not observed in the emission spectra of the conformers of tyrosine and phenylalanine, even if their conformer backbones form an intramolecular hydrogen bond. Assuming the most favorable orientation and a minimum distance of 3.5 A between the dipoles, an increase of 7.5 D in a chromophore interacting with a 6-D backbone is needed to produce a decrease in energy of 10 609 cm-‘. SUMMARY
The electronic spectra of the amino acids tyrosine and phenylalanine have been measured in a supersonic jet using laser-induced fluorescence spectroscopy. Volatilizing tyrosine and phenylalanine with virtually no thermal decomposition was accomplished using the same type of continuous supersonic jet source that was developed to produce gas-phase samples of tryptophan. Through power-dependent studies we have shown in a manner similar to tryptophan that tyrosine and phenylalanine have multiple conformers, and we have assigned spectral features produced by the individual conformers. Dispersed emission spectra of the conformers following excitation of their respective electronic origins reveal that the major vibrational activity occurring in tyrosine and phenylalanine originates from the ring vibrational modes of their respective chromophores. In contrast with previous tryptophan studies, no evidence of intramolecular exciplex formation was observed in any of the tyrosine and phenylalanine dispersed emission spectra. ACKNOWLEDGMENTS This work was supported by the National Science Foundation under Grant CHE-881832 1. S.J.M. acfrom the Illinois Minority Graduate Incentive Program and from the Dorothy Danforth Compton Fellowship Program. J.C.A. acknowledges support from the National Science Foundation Graduate Fellowship Program. knowledges support
RECEIVED:
May 2, 1992 REFERENCES
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