- Email: [email protected]
The electronic spectroscopy of tyrosine and phenylalanine analogs in a supersonic jet: Acidic analogs
JOURNAL
OF MOLECULAR
SPECTROSCOPY
145,
1OO- 111 ( 199 1)
The Electronic Spectroscopy of Tyrosine and Phenylalanine Analogs in a Supersonic Jet: Acidic Analogs SELSOJ. MARTINEZ III, JOSEPH C. ALFANO, AND DONALD H. LEVY The Jame.y Franck Institute and The Department qf‘Chemistry, The University ofChicago, Chicago, Illinois 6063 7
Fluorescence excitation spectra of the origin regions of 3-( 4-hydroxyphenyl ) propionic acid, 3-(4-methoxyphenyl) propionic acid, and hydrocinnamic acid have been measured in a supersonic jet. Power dependence studies reveal the existence of one, one, and two conformers, respectively. The dispersed emission spectra of these molecules were measured and analysis of the region of the spectrum near the electronic origin confirms our conformer assignments. Molecular modeling calculations are used to propose the most reasonable structures for these conformers. The fluorescence lifetimes of the conformers of these molecules were measured. The fluorescence decays of hydrocinnamic acid were found to be single exponential and the lifetimes of its two conformers are 67 t 6 and 76 + 5 nsec. The fluorescence decay of 3-( 4-methoxyphenyl ) propionic acid was also found to be single exponential and the lifetime of its conformer is 15 f 3 nsec. In the case of 3-(4-hydroxyphenyl) propionic acid only an upper limit of 10 nsec can be given to its conformer lifetime because of our limited time resolution. This work serves as a basis for interpreting the more complicated spectroscopy of the amino acids tyrosine and phenylalanine. 1~’1991Academic Press. Inc.
INTRODUCTION
The amino acids tryptophan, tyrosine, and phenylalanine dominate the near ultraviolet absorption and fluorescence of many proteins ( 1, 2). Hence, their electronic spectroscopy and photophysics have long been subjects of intense investigation (I20). Studies have focused on the effect of conformation and solvent on the time- and wavelength-resolved fluorescence of these amino acid residues in order to characterize their use as optical probes of protein structure and dynamics (4-6). However, the lack of spectral resolution in solution makes it impossible to resolve spectral features of individual conformers and to study their properties. Investigators are now studying the spectroscopy and dynamics of these amino acids in supersonic jets in an effort to elucidate the properties of these molecules in solution. The cooling resulting from the supersonic expansion greatly reduces spectral congestion and facilitates the assignment of the remaining sharp structure, and 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. Rizzo el al. reported the observation of the electronic spectrum of isolated tryptophan which was cooled in a supersonic expansion and presented evidence for several spectroscopically distinguishable conformers in the molecular beam (21). To develop a detailed understanding of the effect of conformation on the electronic spectrum of tryptophan, Park et al. then measured the electronic spectra of four analogs of tryptophan in a supersonic molecular 0022-2852/9 I $3.00 Copyright
0 1991 by Academic
All nghts of reproduction
100 Press, Inc.
in any form reserved.
SPECTROSCOPY
101
OF ACIDIC ANALOGS
beam (22). Since then, tryptophan and its analogs have been studied in great detail in a supersonic molecular beam (23-30). Li and Lubman reported the observation of the electronic spectrum of isolated tyrosine which was cooled in a supersonic expansion (31). Recently, we have measured the jet-cooled electronic spectrum of phenylalanine (32). Like tryptophan’s spectrum, the spectra of tyrosine and phenylalanine exhibit the spectral complexity that results from a combination of vibrational structure and the existence of several conformers. To interpret these complicated spectra, investigations of the spectroscopy of simpler analogs have been initiated. The first study of tyrosine analogs cooled in a supersonic jet was that of Song and Hayes in which they measured fluorescence excitation spectra and dispersed fluorescence spectra for a series of seven 4-alkyl-substituted phenols (33). 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 (34). Comparison of 4-propylphenol spectra with that of n-propylbenzene (35) led to the determination of an 11S-cm-’ splitting arising from an interaction between the phenolic hydroxyl group and the 4-substituted propyl group. In an attempt to further develop a detailed understanding of the effect of conformation on the electronic spectra of tyrosine and phenylalanine, we have measured the fluorescence excitation spectra, the dispersed emission spectra, and the fluorescence lifetimes of hydrocinnamic acid, 3-( 4-hydroxyphenyl) propionic acid, and 3-( 4-methoxyphenyl) propionic acid (shown in Fig. 1). As Fig. 1 shows, these molecules are acidic analogs of tyrosine and phenylalanine: thus, their spectroscopies will serve as important models which can be used in interpreting the more complicated electronic spectra of tyrosine and phenylalanine. EXPERIMENTAL
DETAILS
The compounds 3-( 4-methoxyphenyl ) propionic acid, 3- ( 4-hydroxyphenyl ) propionic acid, and hydrocinnamic acid were purchased from Aldrich Chemical Company and used without further purification. A standard oven source was used to volatilize the samples, and a free jet was produced by passing 2.7 atm of helium over the heated sample reservoir and expanding the mixture through a 0. l-mm-diameter nozzle. OH
OCH3
OH
@cQ Cl&
CH*
Cli,
CA I2 COOH
CA I2 COOH
Cl! I2 COOH
(a)
(b)
(c)
Q
CH* I H-C-NH, I COOH
(d)
Q
CHZ I H-C-NH2 I COOH (el
FIG. 1. (a) 3-(4-Hydroxyphenyl) propionic acid. (b) 3-( 4-methoxyphenyl ) propionic acid, ( c) hydrocinnamic acid, (d) phenylalanine, and (e) tyrosine.
102
MARTINEZ,
ALFANO,
AND
LEVY
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 3-( 4-hydroxyphenyl) propionic acid and 3-( 4-methoxyphenyl) propionic acid samples. The frequency doubled output of the same dye laser operating on Coumarin 500 laser dye was used to excite the hydrocinnamic acid sample. Total fluorescence and dispersed emission were detected at right angles to the jet propagation direction and the laser light path and collected with fluorescence collection optics systems similar to those described in Ref. (36). Because of low signal intensity, the total fluorescence of hydrocinnamic acid and the dispersed emission signals of all three molecules were amplified ( LeCroy, Model 6 12A ) by a factor of 10. 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 these cold isolated molecules and not from vibrationally hot molecules or from impurities or decomposition products. In our power-dependence studies a variable attenuator (Newport Corp.) 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. The fluorescence lifetimes of the origins of the three compounds were measured using the pulsed laser system described above used in combination with a transient recorder system ( LeCroy TR88 18/ MM8 103 ) having a 10-nsec per bin time resolution. Our overall instrument response was -20 nsec FWHM. The fluorescence decays were fit to single-exponential functions by the method of iterative convolution (37). The quality of the fit was judged by the x-squared criterion and by visual inspection for systematic deviations in the weighted residuals. RESULTS AND
DISCUSSION
(A) Total Fluorescence Data Fluorescence excitation spectra of 3-( 4-hydroxyphenyl) propionic acid in the region 35 500 to 36 050 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 (21). Different transitions having a common initial level and different transition strength (e.g., vibronic bands with the zero-point vibrational level of the ground electronic state as the initial level and different vibrational levels of the excited electronic state as the final level) will power saturate at different rates, and their relative intensity will change as a function of laser power. Transitions having different initial levels and the same transition strength (e.g., different conformer origins) will power saturate at the same rate and will have a constant relative intensity that does not depend on laser power. In Fig. 2, all features grow in intensity relative to the feature at 35 552 cm-‘, and this feature has been assigned as the 0: band of the single conformer of 3-( 4-hydroxyphenyl) propionic acid. As was found for 4-ethylphenol(3#), the existence of only one conformer signifies that the propionic acid group must lie in an anti orientation with respect to the plane
SPECTROSCOPY
35500
35600
35700
OF ACIDIC
35800
WAVENUMBER b)
ANALOGS
35900
103
36000
(CM-‘)
1
35500
35600
35700
35800
WAVENUMBER
35900
36000
KM-‘)
FIG. 2. Fluorescence excitation spectra of 3-( 4-hydroxyphenyl) propionic acid in the region 35 500 to 36 050 cm -’ taken at different laser power densities. The spectrum in (b) is taken at - 100 times the power density of that in (a). The oven temperature is 14O”C, the carrier gas is helium at a backing pressure of 3.4 atm. and the nozzle diameter is 0. I mm.
perpendicular to the benzene ring and passing through carbon atoms 1 and 4. As this conformer is symmetric with respect to this plane, the two different orientations of the hydroxyl group give rise to indistinguishable geometries and therefore yield only a single origin transition. On the other hand, if the propionic acid group was not symmetric with respect to this plane, then two distinct conformers would have been observed, each corresponding to a different orientation of the hydroxyl group. Molecular modeling calculations (PCMODEL, Serena Software) proposed the optimized geometry shown in Fig. 3. PCMODEL is a full molecular modeling program which uses an MMX force field based on the MM2 ( 77 ) force field of Allinger with extensions and modifications to handle more functional groups. As Fig. 3 reveals, the calculations support the anti orientation of the propionic acid group. In the 3-( 4-hydroxyphenyl) propionic acid spectrum two intense vibrational features occur 15 1 and 170 cm -’ to higher wavenumbers from the origin. Two possible assignments exist for these features. Either they are torsions of the propionic acid group or ring vibrational modes. In the excitation spectra of seven different 4-alkylphenols the lowest wavenumber ring vibrational mode is 16a ’ with a wavenumber no lower than 360 cm-’ (33). In the 3-( 4-hydroxyphenyl) propionic acid spectrum the weak features detected 374 and 476 cm-’ to higher wavenumbers from the origin are tentatively assigned as the 16~ ’ and 6a ’ ring vibrational modes. In n-propylbenzene the anti conformation of the propyl group exhibited a propyl torsion 150 cm -’ to higher wavenumbers from the origin (35). Taken together, these observations strongly suggest that the two intense vibrational features occurring 15 1 and 170 cm-’ to higher wavenumbers from the origin are torsions of the propionic acid group. Fluorescence excitation spectra of 3-( 4-methoxyphenyl) propionic acid in the region 35 500 to 35 975 cm-’ taken at low and high power densities are shown in Fig. 4. All features grow in intensity relative to the feature at 35 604 cm-‘, and this feature has been assigned as the 0: band of the single conformer of 3-( 4-methoxyphenyl) propionic
MARTINEZ,
104
FIG. 3. The proposed modeling
optimized
geometry
ALFANO,
AND
of 3-(4-hydroxyphenyl)
LEVY
propionic
acid resulting from molecular
calculations.
acid. As Fig. 5 shows, the smaller feature to the red of the origin changes in intensity relative to the origin as a function of backing pressure: therefore, it is assigned as a hot band. The two other intense features in the spectrum occur 146 and 164 cm -’ to
WAVENUMBER
35600
35700
WAVENUMBER
(CM-‘)
35800
35900
(CM-‘)
FIG. 4. Fluorescence excitation spectra of 3-( 4-methoxyphenyl) propionic acid in the region 35 525 to 35 925 cm-’ taken at different laser power densities. The spectrum in (b) is taken at - 100 times the power density of that in (a). The oven temperature is 8O”C, the carrier gas is helium at a backing pressure of 2.7 atm, and the nozzle diameter is 0. I mm.
105
SPECTROSCOPY OF ACIDIC ANALOGS
I
I
35530
I
35580 WAVENUMBER
I
I
35630 (CM“)
FIG. 5.Fluorescence excitation spectra of the origin region of 3-( 4-methoxyphenyl) propionic acid taken at different backing pressures. The spectrum in (a) is taken at 5.4 atm, in (b) at 2.7 atm, and in (c) at I .4 atm. The oven temperature is 8O”C, and the nozzle diameter is 0.1 mm.
higher wavenumbers from the origin. Because of their similarity to the two intense features occurring 15 1 and 170 cm -’ to higher wavenumbers from the origin in the 3-(4-hydroxyphenyl) propionic acid excitation spectrum, these features are presumably torsions of the propionic acid group. Additional low wavenumber features in these spectra not seen in the spectra of 3-( 4-hydroxyphenyl ) propionic acid are presumably methyl torsions. As in the case of 3-(4-hydroxyphenyl) propionic acid, the existence of only one conformer for 3-( 4-methoxyphenyl) propionic acid signifies that the propionic acid group must lie in an anti orientation symmetric with respect to the plane perpendicular to the benzene ring and passing through carbon atoms 1 and 4. Molecular modeling calculations propose an optimized geometry similar to that shown in Fig. 3 for 3-( 4hydroxyphenyl) propionic acid, except that the hydroxyl group is replaced with a methoxy group. Fluorescence excitation spectra of hydrocinnamic acid in the region 37 54 1 to 38 459 cm-’ taken at low and high power densities are shown in Fig. 6. All features grow in intensity relative to the two features at 37 624 and 37 648 cm-‘, and these features have been assigned as the 0: bands of two conformers of hydrocinnamic acid. Added evidence supporting the assignment of the latter feature as a conformer and not as a Au = 1 vibration built on the former feature lies in the absence of Au = 2, 3, - - transitions which would form a 24-cm-’ vibrational progression. Based on the intensity of the latter feature it seems very unlikely that the Franck-Condon factors would suddenly go to zero for all Au > 2 transitions. One would instead expect to see a 24cm-’ progression with the Franck-Condon factors gradually falling to zero, which is not what we observe. Note now that, in contrast with the excitation spectra of the two other analogs, the two most intense vibrational features occur 533 and 558 cm-’ to higher wavenumbers from the redmost origin. The spacing between these two features is 25 cm-‘, matching the spacing between the two conformers. Comparison with the fluorescence excitation
106
MARTINEZ,
ALFANO,
‘)> 37600
38000
37800
WAVENUMBER
AND
LEVY
38200
38400
(CM-‘)
b)
--4UL
I
‘.
37600
37800
A
I 38000
WAVENUMBER
38200
38400
(CM-‘)
FIG. 6. Fluorescence excitation spectra of hydrocinnamic acid in the region 37 54 I to 38 459 cm -’ taken at different laser power densities. The spectrum in (b) is taken at - 100 times the power density of that in (a). The oven temperature is 13O”C, the carrier gas is helium at a backing pressure of 2.7 atm. and the nozzle diameter is 0. I mm.
spectrum
of toluene measured in a supersonic jet (38) suggests that these features are from each origin. The existence of 533 cm-’ to higher wavenumbers two conformers and the difference in vibrational structure indicate that the electronic structure of hydrocinnamic acid is significantly different from that of the two other analogs. Apparently, the presence or absence of a parusubstituted hydroxy or methoxy group has a significant effect on the electronic spectroscopy of these acidic analogs of tyrosine and phenylalanine. Although two conformers of hydrocinnamic acid were identified. the exact nature of these conformers is still in question. Molecular modeling calculations propose an optimized geometry very similar to those obtained for 3-( 4-hydroxyphenyl) propionic acid and 3-( 4-methoxyphenyl) propionic acid with no new added insights as to why more than one stable conformer exists.
6bA vibrations
(B) Dispersed Emission Duta Figure 7a shows the dispersed emission spectrum of 3-( 4-hydroxyphenyl) propionic acid produced by exciting its origin at 35 552 cm-‘. For comparison, the dispersed emission spectrum of the 0: region ofp-cresol(33) is shown in Fig. 7b. The similarity of the two spectra signifies that most of the vibrational activity in the 3-( 4-hydroxyphenyl) propionic acid spectrum is in the ring modes of the p-cresol chromophore. Figure 8a shows the dispersed emission spectrum of 3-( 4-methoxyphenyl ) propionic acid produced by exciting its origin at 35 604 cm-‘. The strong similarity to the dispersed emission spectrum of the 08 region of I-methoxy-4-methylbenzene (39), shown in Fig. 8b, indicates that most of the vibrational activity in the 3-( 4-methoxyphenyl) propionic acid spectrum is in the ring modes of the l-methoxy-4-methylbenzene chromophore. Figures 9a and 9b show the dispersed emission spectra of hydrocinnamic acid produced by exciting its origins at 37 624 and 37 648 cm -‘, respectively. The spectra are
SPECTROSCOPY
OF ACIDIC
107
ANALOGS
-4
35000
34000
33000
WAVENUMBER b)
32000
31000
(CM-])
I
35000
33000
34000
32000
WAVENUMBER
31000
(CM-‘)
FIG. 7. Dispersed emission spectra of (a) 3-( 4-hydroxyphenyl) propionic acid and (b) p-cresol produced by exciting their respective origins. In (a) the monochromator slits are 0.5 mm producing a resolution of -25 cm -‘. The oven temperature is 143°C. the carrier gas is helium at a backing pressure of 2.7 atm. and the nozzle diameter is 0. I mm. In (b) the monochromator slits are 0.3 mm producing a resolution of - 15 cm-‘. The oven temperature is 25°C. the carrier gas is helium at a backing pressure of 1.4 atm. and the nozzle diameter is 0.1 mm. In each case. the spectrum was corrected for laser scattering at the resonant transition.
similar to the emission spectrum of the 0: region of toluene (40), demonstrating that most of the vibrational activity is in the ring modes of the toluene chromophore. Although most of the ground state vibrational frequencies and intensity patterns of
35000
33000
34000
WAVENUMBER
b, * 35000
34000
33000
WAVENUMBER
32000
KM-‘)
32000
31000
(CM-‘)
FIG. 8. Dispersed emission spectra of (a) 3-(4-methoxyphenyl) propionic acid and (b) 1-methoxy-4methylbenzene produced by exciting their respective origins. In (a) the monochromator slits are 0.5 mm producing a resolution of -25 cm-‘. The oven temperature is 95°C. the carrier gas is helium at a backing pressure of 2.7 atm, and the nozzle diameter is 0.1 mm. In (b) the monochromator slits are 0.25 mm the carrier gas is helium producing a resolution of - 12 cm _’ The sample was kept at room temperature. at a backing pressure of 2.7 atm, and the nozzle diameter is 0.1 mm. In each case. the spectrum was corrected for laser scattering at the resonant transition.
108
MARTINEZ, ALFANO, AND LEVY
A
0)
Jl
I
+
37000
36000
WAVENUMBER
b,
35000
34000
(CM-‘)
D 37000
36000
WAVENUMBER
35000
34000
(CM-‘)
FIG. 9. Dispersed emission spectra of hydrocinnamic acid (a) conformer A and ( b) conformer B produced by exciting their respective origins. The monochromator slits are 1.O mm producing a resolution of --57 cm-‘. The oven temperature is 166”C, the carrier gas is helium at a backing pressure of 2.7 atm, and the nozzle diameter is 0.1 mm. The spectrum was corrected for laser scattering at the resonant transition.
conformers A and B coincide, some notable differences do exist. Consider, for example, the peak marked with an asterisk in both spectra. In conformer A this feature is 8 12 cm-’ from the resonant peak, whereas in conformer B it is 778 cm -‘. Another example is the peak marked with a plus sign. In conformer A this feature is 1440 cm-’ from the resonant peak, whereas in conformer B it is 1394 cm-‘. Note also the difference in intensity of the peak centered at 37 000 cm-‘. Its intensity in conformer B is about one and a half times that in conformer A. Since the emission of different conformers is likely to have different ground state vibrational frequencies and different emission intensity patterns, these emission spectra support our assignment of these features in the excitation spectrum as conformers. The observation of different emission spectra from excitation of the origins of different conformers indicates that the conformers do not interconvert in the excited state during the fluorescence lifetime. (C) Fluorescence
Decay Data
The measured conformer lifetimes of the three molecules studied are presented in Table I. The decays of the two hydrocinnamic acid conformers were fit to single exponentials and their lifetimes were measured as 76 & 5 and 67 -+ 6 nsec for conformers A and B, respectively. The decay of the one conformer of 3-( 4-methoxyphenyl) propionic acid was fit to a single exponential and its lifetime was measured as 15 f 3 nsec. Attempts to fit the decay of 3-( 4-hydroxyphenyl) propionic acid’s one conformer yielded a lifetime lower than our time resolution of 10 nsec. Therefore, only an upper limit of 10 nsec can be assigned as the lifetime of the origin of 3-(4-hydroxyphenyl) propionic acid. An example of fits to the data for conformers A and B of hydrocinnamic acid is shown in Fig. 10. The different conformer peaks in hydrocinnamic acid presumably correspond to different molecular geometries; thus, hydrocinnamic acid may be an example of a
109
SPECTROSCOPY OF ACIDIC ANALOGS TABLE I
Fluorescence Lifetimes of the Origin Transitions of Hydrocinnamic Acid (HA). 3-( 4-Hydroxyphenyl ) Propionic Acid (34HPPA). and 3-( 4-Methoxyphenyl) Propionic Acid (34MPPA )
Molecule
Origin(s)
34HPPA
Lifetime
35 552
34MPPA
5
(us)
10’
35 604
15f3
HA Conformer
A
37 624
76f5
HA Conformer
B
37 648
67k6
’ Typical
’
(cm-‘)”
The
uncertainty
measured
is ztl
lifetime
.
cm-’
is shorter
than
OUT time
resolution
of 10 ns
molecule where conformation affects the fluorescence decay rate of the molecule in the gas phase. To definitively demonstrate this, better time resolution is needed. The single exponential decay of both conformers demonstrates the absence of conformer interconversion on the time scale of emission. This same type of behavior was found to occur in 3-indolepropionic acid, the acidic analog of the amino acid tryptophan. The lifetimes for the A and B conformers of 3indolepropionic acid are, respectively, 16.2 and 13.5 nsec (41). In each instance the decay was well fitted with a single exponential component and the lifetimes typically
3. Be+0
_
-3.9e+o
-
.
.
.
.
.
.
.
.
-~~~~-.
-
.--:
100.
200.
TIME
.
*
_.
300.
(ns)
3.90+0 -3.
ge+
b)
-
.
-
* -
* _ .
.
-
JJj
100.
200.
TIME
300.
hs)
FOG. 10. Time-resolved fluorescence decays of (a) conformer A and (b) conformer B of hydrocinnamic acid that have been fit to single exponential decay rates of 76 + 5 and 67 + 5 nsec, respectively. The normalized residuals from the fits to each trace are given at the top of the figure.
MARTINEZ,
110
ALFANO.
AND
LEVY
were determined with a precision of ~0.1 nsec. The lifetimes of these isolated conformers were roughly an order of magnitude longer than those in solution, indicating the importance of solvent interaction in explaining solution emission. The relative lifetimes exhibited by the acidic analogs in this study are consistent with the gas phase lifetime measurements of their respective chromophores. In the gas phase, toluene’s origin has a lifetime of 82 nsec (42), anisole’s origin has a lifetime of -20 nsec (43). and p-cresol’s origin has a lifetime of -4.1 nsec (43). This relative behavior is attributed to the extent to which the benzene De,, symmetry is perturbed: the greater the perturbation, the shorter the radiative lifetime becomes. SUMMARY
The spectroscopy of the acidic analogs of phenylalanine and tyrosine has been studied in the gas phase in a supersonic jet using laser-induced fluorescence. Powerdependence studies revealed the existence of two conformers for hydrocinnamic acid and one conformer for 3-(4-hydroxyphenyl) propionic acid and 3-(Cmethoxyphenyl) propionic acid. The conformer structures of the latter two compounds are proposed to have the propionic acid group in an unti orientation with respect to the plane perpendicular to the benzene ring and passing through carbon atoms 1 and 4. Molecular modeling calculations support this orientation. The conformer structures of hydrocinnamic acid are still undetermined. Dispersed emission spectra of hydrocinnamic acid. 3-( 4-hydroxyphenyl) propionic acid, and 3-( 4-methoxyphenyl) propionic acid confirm our conformer assignments. These spectra reveal that the major vibrational activity occurring in these molecules originates from the ring vibrational modes of their respective chromophores. The fluorescent lifetimes of the conformers of these molecules were measured and found to exhibit single exponential behavior. The conformer lifetimes were found to differ among the different molecules. The relative lifetimes of these molecules are consistent with those of their respective chromophores. ACKNOWLEDGMENTS
This work was supported by the National Science Foundation under Grant CHE-88 1832 I. SJM acknowlsupportfrom the Illinois Minority Graduate Incentive Program. JCA acknowledges support from the National Science Foundation Graduate Fellowship Program. We thank Dr. Dan Eads for assisting us in fitting lifetimes using programs developed by Dr. Graham Fleming’s group. We also thank Basil Paulson for assisting us in using Dr. Gerhard Gloss’ molecular modeling program. edges
RECEIVED:
August 6, 1990 REFERENCES
I. I.
WEINRYB AND R. F. STEINER, in “Excited
States of Proteins and
Nucleic Acids” (R. F. Steiner and
I. Weimyb. Eds.), Plenum. New York. 1971. 2. D. CREED, Photochem. Photobiol. 39, 537-575 ( 1984). 3. C. TANFORD, J. D. HAUENSTEIN. AND D. G. RANDS, 1. ,4mer. c’henz. Sm. 77,6409-6413 4. I. MUNRO, I. PECHT, AND L. STRYER, Proc. Natl. Acad. Sri. US.4 76, 56-60 ( 1979). 5. M. R. EFTINK AND C. A. GHIRON. Blochemistr~~ 15, 672-680 ( 1976). 6. J. B. A. Ross, K. W. ROUSSLANG, AND L. BRAND, Biochemistry 20,436 l-4369 ( 198 I )_ 7. G. WEBER, Biochem. J. 75,335-345 (1960).
( 1955
).
SPECTROSCOPY 8 M. MARTINAUD AND A. KADIRI, Chem. Phjx
OF -ACIDIC
28,473-485
ANALOGS
111
(1978).
9. Y. YAMAMOTO AND J. TANAKA, Bull. Chem. SOL,. Japan 45, 1362-1366 ( 1972). 10. S. R. MEECH, D. PHILLIPS, AND A. G. LEE, Chem. Php. 80,317-328 (1983). Il. II. 13 14. 15. 16. 17. 18. 19. 20. -71. 22. 23. 24. 25. 26. 27. 28. ?V 30. 31. 3-7. 33. 34. 3.5. 36. 37. 38. 39. 40. 41. 42. 43.
G. R. FLEMING. J. M. MORRIS, R. J. ROBLUNS,G. J. WOOLFE, P. J. THISTLETHWAITE,AND G. W. ROBINSON.Proc. Natl. Acad. Sci. USA. 75.4652-4656 ( 1978). M. C. CHANG, J. W. PETRICH, D. B. MCDONALD, AND G. R. FLEMING, J. .4mrr. Chem Sot. 105, 3819-3831 (1983). A. G. SZABO AND D. M. RAYNER, J. Amer. Chem. Sot. 102,554-563 ( 1980). E. F. GUDCIN-TEMPLETON AND W. R. WARE, J. Phys. Chem. 88,4626-4631 ( 1984). D. V. BENT AND E. HAYON, J. ‘4mer. Chem. Sot. 97,2599-2619 ( 1975). C. PIGAULT. C. HASSELMANN,AND G. LAUSTRIAT, J. Phys. Chem. 86. 1755-1757 ( 1982). H. EDELHOCH,L. BRAND. AND M. WILCHECK. Iv. J. C’hem. 1,216-2 17 ( 1963 ). R. W. COWGILL, Biochim. Biophys. .4cta 75, 272-273 ( 1963). A. WHITE, Biochem. J. 71,217-220 (1960). J. FEITELSON.J. Phy.s. Chem. 68,391-397 ( 1964). T. R. RIZZO, Y. D. PARK, L. A. PETEANU,ANDD. H. LEVY. J. Chem. Phys. 84(5),2534-2541 ( 1986). Y. D. PARK, T. R. RIZZO, L. A. PETEANU. AND D. H. LEVY. J. Chem. Phw. 84( 12), 6539-6549 (1986). T. R. RIZZO. Y. D. PARK, AND D. H. LEVY. J. Chetn. PhJx 85( 12), 6945-695 1 ( 1986). L. A. PHILIPS. S. P. WEBB, S. J. MARTINEZ III, G. R. FLEMING. AND D. H. LEVY, J. .4mer. Chem. SOC,. 110, 1352-1355 (1988). L. A. PETEANU AND D. H. LEVY, J. Php. Chem. 92,6554-6561 ( 1988). J. R. CABLE, M. J. TUBERGEN.AND D. H. LEVY, J. ,4mer. Chem. SOC. 109, 6198-6199 ( 1987 ). J. R. CABLE, M. J. TUBERGEN,AND D. H. LEVY, J. .4mer. Chem. SM. 110, 7349-7355 (1987). J. R. CABLE, M. J. TUBERGEN.AND D. H. LEVY, J. Amer. Chem. SOL.. 111,9032-9039 ( 1987). J. R. CABLE. M. J. TUBERGEN.AND D. H. LEVY, Faraday Discu.s.s. Chem. Sot. 86, 143-l 52 ( 1988). M. J. TUBERGEN,J. R. CABLE, AND D. H. LEVY, J. Chem. Phs. 92( I ), 51-60 (1990). L. LI AND D. M. LUBMAN Appl. Spectros. 42(3), 418-424 ( 1988). S. J. MARTINEZ III, J. C. ALFANO. AND D. H. LEVY, in preparation. K. SONG AND J. M. HAYES, J. Mol. Speczrosc. 134, 82-97 ( 1989). S. J. MARTINEZ III, J. C. ALFANO, AND D. H. LEVY, J. Mol. Spec‘frasc. 137, 420-426 ( 1989). P. J. BREEN. J. A. WARREN, E. R. BERNSTEIN,AND J. I. SEEMAN. J. Chem. Phys. 87(4), 1927-1935 (1987). W. SHARFIN, K. E. JOHNSON, L. WHARTON, AND D. H. LEVY, J. Chem. Phys. 71, 1292-1299 ( 1979). M. C. CHANG, S. H. COURTNEY, A. J. CROSS,R. J. GULOTTY. J. W. PETRICH, AND G. R. FLEMING, Anal. Instrum. 14, 433-464 ( 1985). J. B. HOPKINS, D. E. POWERS,AND R. E. SMALLEY, J. Chem. Phy. 72(9), 5039-5048 ( 1980). P. J. BREEN, E. R. BERNSTEIN,H. V. SECOR,AND J. I. SEEMAN, J. .4mer. Chem. Sot. 111(6), 19581968 (1989). J. I. SELCOAND P. G. CARRICK, J. Mol. Spectrosc. 137. 13-23 ( 1989). J. SIPIOR, M. SULKES,R. AUERBACH,AND M. BOIVINEAU, J. Phys. Chem 91.2016-2018 (1987 ). M. JACON. C. LARDEUX, R. LOPEZ-DELGADO.AND A. TRAMER, Chem. Php. 24, 145-157 ( 1977). R. J. LIPERT. S. D. COLSON, AND A. SUR J. Phy.s. Gem. 92, 183-l 85 ( 1988 ).
OF MOLECULAR
SPECTROSCOPY
145,
1OO- 111 ( 199 1)
The Electronic Spectroscopy of Tyrosine and Phenylalanine Analogs in a Supersonic Jet: Acidic Analogs SELSOJ. MARTINEZ III, JOSEPH C. ALFANO, AND DONALD H. LEVY The Jame.y Franck Institute and The Department qf‘Chemistry, The University ofChicago, Chicago, Illinois 6063 7
Fluorescence excitation spectra of the origin regions of 3-( 4-hydroxyphenyl ) propionic acid, 3-(4-methoxyphenyl) propionic acid, and hydrocinnamic acid have been measured in a supersonic jet. Power dependence studies reveal the existence of one, one, and two conformers, respectively. The dispersed emission spectra of these molecules were measured and analysis of the region of the spectrum near the electronic origin confirms our conformer assignments. Molecular modeling calculations are used to propose the most reasonable structures for these conformers. The fluorescence lifetimes of the conformers of these molecules were measured. The fluorescence decays of hydrocinnamic acid were found to be single exponential and the lifetimes of its two conformers are 67 t 6 and 76 + 5 nsec. The fluorescence decay of 3-( 4-methoxyphenyl ) propionic acid was also found to be single exponential and the lifetime of its conformer is 15 f 3 nsec. In the case of 3-(4-hydroxyphenyl) propionic acid only an upper limit of 10 nsec can be given to its conformer lifetime because of our limited time resolution. This work serves as a basis for interpreting the more complicated spectroscopy of the amino acids tyrosine and phenylalanine. 1~’1991Academic Press. Inc.
INTRODUCTION
The amino acids tryptophan, tyrosine, and phenylalanine dominate the near ultraviolet absorption and fluorescence of many proteins ( 1, 2). Hence, their electronic spectroscopy and photophysics have long been subjects of intense investigation (I20). Studies have focused on the effect of conformation and solvent on the time- and wavelength-resolved fluorescence of these amino acid residues in order to characterize their use as optical probes of protein structure and dynamics (4-6). However, the lack of spectral resolution in solution makes it impossible to resolve spectral features of individual conformers and to study their properties. Investigators are now studying the spectroscopy and dynamics of these amino acids in supersonic jets in an effort to elucidate the properties of these molecules in solution. The cooling resulting from the supersonic expansion greatly reduces spectral congestion and facilitates the assignment of the remaining sharp structure, and 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. Rizzo el al. reported the observation of the electronic spectrum of isolated tryptophan which was cooled in a supersonic expansion and presented evidence for several spectroscopically distinguishable conformers in the molecular beam (21). To develop a detailed understanding of the effect of conformation on the electronic spectrum of tryptophan, Park et al. then measured the electronic spectra of four analogs of tryptophan in a supersonic molecular 0022-2852/9 I $3.00 Copyright
0 1991 by Academic
All nghts of reproduction
100 Press, Inc.
in any form reserved.
SPECTROSCOPY
101
OF ACIDIC ANALOGS
beam (22). Since then, tryptophan and its analogs have been studied in great detail in a supersonic molecular beam (23-30). Li and Lubman reported the observation of the electronic spectrum of isolated tyrosine which was cooled in a supersonic expansion (31). Recently, we have measured the jet-cooled electronic spectrum of phenylalanine (32). Like tryptophan’s spectrum, the spectra of tyrosine and phenylalanine exhibit the spectral complexity that results from a combination of vibrational structure and the existence of several conformers. To interpret these complicated spectra, investigations of the spectroscopy of simpler analogs have been initiated. The first study of tyrosine analogs cooled in a supersonic jet was that of Song and Hayes in which they measured fluorescence excitation spectra and dispersed fluorescence spectra for a series of seven 4-alkyl-substituted phenols (33). 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 (34). Comparison of 4-propylphenol spectra with that of n-propylbenzene (35) led to the determination of an 11S-cm-’ splitting arising from an interaction between the phenolic hydroxyl group and the 4-substituted propyl group. In an attempt to further develop a detailed understanding of the effect of conformation on the electronic spectra of tyrosine and phenylalanine, we have measured the fluorescence excitation spectra, the dispersed emission spectra, and the fluorescence lifetimes of hydrocinnamic acid, 3-( 4-hydroxyphenyl) propionic acid, and 3-( 4-methoxyphenyl) propionic acid (shown in Fig. 1). As Fig. 1 shows, these molecules are acidic analogs of tyrosine and phenylalanine: thus, their spectroscopies will serve as important models which can be used in interpreting the more complicated electronic spectra of tyrosine and phenylalanine. EXPERIMENTAL
DETAILS
The compounds 3-( 4-methoxyphenyl ) propionic acid, 3- ( 4-hydroxyphenyl ) propionic acid, and hydrocinnamic acid were purchased from Aldrich Chemical Company and used without further purification. A standard oven source was used to volatilize the samples, and a free jet was produced by passing 2.7 atm of helium over the heated sample reservoir and expanding the mixture through a 0. l-mm-diameter nozzle. OH
OCH3
OH
@cQ Cl&
CH*
Cli,
CA I2 COOH
CA I2 COOH
Cl! I2 COOH
(a)
(b)
(c)
Q
CH* I H-C-NH, I COOH
(d)
Q
CHZ I H-C-NH2 I COOH (el
FIG. 1. (a) 3-(4-Hydroxyphenyl) propionic acid. (b) 3-( 4-methoxyphenyl ) propionic acid, ( c) hydrocinnamic acid, (d) phenylalanine, and (e) tyrosine.
102
MARTINEZ,
ALFANO,
AND
LEVY
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 3-( 4-hydroxyphenyl) propionic acid and 3-( 4-methoxyphenyl) propionic acid samples. The frequency doubled output of the same dye laser operating on Coumarin 500 laser dye was used to excite the hydrocinnamic acid sample. Total fluorescence and dispersed emission were detected at right angles to the jet propagation direction and the laser light path and collected with fluorescence collection optics systems similar to those described in Ref. (36). Because of low signal intensity, the total fluorescence of hydrocinnamic acid and the dispersed emission signals of all three molecules were amplified ( LeCroy, Model 6 12A ) by a factor of 10. 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 these cold isolated molecules and not from vibrationally hot molecules or from impurities or decomposition products. In our power-dependence studies a variable attenuator (Newport Corp.) 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. The fluorescence lifetimes of the origins of the three compounds were measured using the pulsed laser system described above used in combination with a transient recorder system ( LeCroy TR88 18/ MM8 103 ) having a 10-nsec per bin time resolution. Our overall instrument response was -20 nsec FWHM. The fluorescence decays were fit to single-exponential functions by the method of iterative convolution (37). The quality of the fit was judged by the x-squared criterion and by visual inspection for systematic deviations in the weighted residuals. RESULTS AND
DISCUSSION
(A) Total Fluorescence Data Fluorescence excitation spectra of 3-( 4-hydroxyphenyl) propionic acid in the region 35 500 to 36 050 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 (21). Different transitions having a common initial level and different transition strength (e.g., vibronic bands with the zero-point vibrational level of the ground electronic state as the initial level and different vibrational levels of the excited electronic state as the final level) will power saturate at different rates, and their relative intensity will change as a function of laser power. Transitions having different initial levels and the same transition strength (e.g., different conformer origins) will power saturate at the same rate and will have a constant relative intensity that does not depend on laser power. In Fig. 2, all features grow in intensity relative to the feature at 35 552 cm-‘, and this feature has been assigned as the 0: band of the single conformer of 3-( 4-hydroxyphenyl) propionic acid. As was found for 4-ethylphenol(3#), the existence of only one conformer signifies that the propionic acid group must lie in an anti orientation with respect to the plane
SPECTROSCOPY
35500
35600
35700
OF ACIDIC
35800
WAVENUMBER b)
ANALOGS
35900
103
36000
(CM-‘)
1
35500
35600
35700
35800
WAVENUMBER
35900
36000
KM-‘)
FIG. 2. Fluorescence excitation spectra of 3-( 4-hydroxyphenyl) propionic acid in the region 35 500 to 36 050 cm -’ taken at different laser power densities. The spectrum in (b) is taken at - 100 times the power density of that in (a). The oven temperature is 14O”C, the carrier gas is helium at a backing pressure of 3.4 atm. and the nozzle diameter is 0. I mm.
perpendicular to the benzene ring and passing through carbon atoms 1 and 4. As this conformer is symmetric with respect to this plane, the two different orientations of the hydroxyl group give rise to indistinguishable geometries and therefore yield only a single origin transition. On the other hand, if the propionic acid group was not symmetric with respect to this plane, then two distinct conformers would have been observed, each corresponding to a different orientation of the hydroxyl group. Molecular modeling calculations (PCMODEL, Serena Software) proposed the optimized geometry shown in Fig. 3. PCMODEL is a full molecular modeling program which uses an MMX force field based on the MM2 ( 77 ) force field of Allinger with extensions and modifications to handle more functional groups. As Fig. 3 reveals, the calculations support the anti orientation of the propionic acid group. In the 3-( 4-hydroxyphenyl) propionic acid spectrum two intense vibrational features occur 15 1 and 170 cm -’ to higher wavenumbers from the origin. Two possible assignments exist for these features. Either they are torsions of the propionic acid group or ring vibrational modes. In the excitation spectra of seven different 4-alkylphenols the lowest wavenumber ring vibrational mode is 16a ’ with a wavenumber no lower than 360 cm-’ (33). In the 3-( 4-hydroxyphenyl) propionic acid spectrum the weak features detected 374 and 476 cm-’ to higher wavenumbers from the origin are tentatively assigned as the 16~ ’ and 6a ’ ring vibrational modes. In n-propylbenzene the anti conformation of the propyl group exhibited a propyl torsion 150 cm -’ to higher wavenumbers from the origin (35). Taken together, these observations strongly suggest that the two intense vibrational features occurring 15 1 and 170 cm-’ to higher wavenumbers from the origin are torsions of the propionic acid group. Fluorescence excitation spectra of 3-( 4-methoxyphenyl) propionic acid in the region 35 500 to 35 975 cm-’ taken at low and high power densities are shown in Fig. 4. All features grow in intensity relative to the feature at 35 604 cm-‘, and this feature has been assigned as the 0: band of the single conformer of 3-( 4-methoxyphenyl) propionic
MARTINEZ,
104
FIG. 3. The proposed modeling
optimized
geometry
ALFANO,
AND
of 3-(4-hydroxyphenyl)
LEVY
propionic
acid resulting from molecular
calculations.
acid. As Fig. 5 shows, the smaller feature to the red of the origin changes in intensity relative to the origin as a function of backing pressure: therefore, it is assigned as a hot band. The two other intense features in the spectrum occur 146 and 164 cm -’ to
WAVENUMBER
35600
35700
WAVENUMBER
(CM-‘)
35800
35900
(CM-‘)
FIG. 4. Fluorescence excitation spectra of 3-( 4-methoxyphenyl) propionic acid in the region 35 525 to 35 925 cm-’ taken at different laser power densities. The spectrum in (b) is taken at - 100 times the power density of that in (a). The oven temperature is 8O”C, the carrier gas is helium at a backing pressure of 2.7 atm, and the nozzle diameter is 0. I mm.
105
SPECTROSCOPY OF ACIDIC ANALOGS
I
I
35530
I
35580 WAVENUMBER
I
I
35630 (CM“)
FIG. 5.Fluorescence excitation spectra of the origin region of 3-( 4-methoxyphenyl) propionic acid taken at different backing pressures. The spectrum in (a) is taken at 5.4 atm, in (b) at 2.7 atm, and in (c) at I .4 atm. The oven temperature is 8O”C, and the nozzle diameter is 0.1 mm.
higher wavenumbers from the origin. Because of their similarity to the two intense features occurring 15 1 and 170 cm -’ to higher wavenumbers from the origin in the 3-(4-hydroxyphenyl) propionic acid excitation spectrum, these features are presumably torsions of the propionic acid group. Additional low wavenumber features in these spectra not seen in the spectra of 3-( 4-hydroxyphenyl ) propionic acid are presumably methyl torsions. As in the case of 3-(4-hydroxyphenyl) propionic acid, the existence of only one conformer for 3-( 4-methoxyphenyl) propionic acid signifies that the propionic acid group must lie in an anti orientation symmetric with respect to the plane perpendicular to the benzene ring and passing through carbon atoms 1 and 4. Molecular modeling calculations propose an optimized geometry similar to that shown in Fig. 3 for 3-( 4hydroxyphenyl) propionic acid, except that the hydroxyl group is replaced with a methoxy group. Fluorescence excitation spectra of hydrocinnamic acid in the region 37 54 1 to 38 459 cm-’ taken at low and high power densities are shown in Fig. 6. All features grow in intensity relative to the two features at 37 624 and 37 648 cm-‘, and these features have been assigned as the 0: bands of two conformers of hydrocinnamic acid. Added evidence supporting the assignment of the latter feature as a conformer and not as a Au = 1 vibration built on the former feature lies in the absence of Au = 2, 3, - - transitions which would form a 24-cm-’ vibrational progression. Based on the intensity of the latter feature it seems very unlikely that the Franck-Condon factors would suddenly go to zero for all Au > 2 transitions. One would instead expect to see a 24cm-’ progression with the Franck-Condon factors gradually falling to zero, which is not what we observe. Note now that, in contrast with the excitation spectra of the two other analogs, the two most intense vibrational features occur 533 and 558 cm-’ to higher wavenumbers from the redmost origin. The spacing between these two features is 25 cm-‘, matching the spacing between the two conformers. Comparison with the fluorescence excitation
106
MARTINEZ,
ALFANO,
‘)> 37600
38000
37800
WAVENUMBER
AND
LEVY
38200
38400
(CM-‘)
b)
--4UL
I
‘.
37600
37800
A
I 38000
WAVENUMBER
38200
38400
(CM-‘)
FIG. 6. Fluorescence excitation spectra of hydrocinnamic acid in the region 37 54 I to 38 459 cm -’ taken at different laser power densities. The spectrum in (b) is taken at - 100 times the power density of that in (a). The oven temperature is 13O”C, the carrier gas is helium at a backing pressure of 2.7 atm. and the nozzle diameter is 0. I mm.
spectrum
of toluene measured in a supersonic jet (38) suggests that these features are from each origin. The existence of 533 cm-’ to higher wavenumbers two conformers and the difference in vibrational structure indicate that the electronic structure of hydrocinnamic acid is significantly different from that of the two other analogs. Apparently, the presence or absence of a parusubstituted hydroxy or methoxy group has a significant effect on the electronic spectroscopy of these acidic analogs of tyrosine and phenylalanine. Although two conformers of hydrocinnamic acid were identified. the exact nature of these conformers is still in question. Molecular modeling calculations propose an optimized geometry very similar to those obtained for 3-( 4-hydroxyphenyl) propionic acid and 3-( 4-methoxyphenyl) propionic acid with no new added insights as to why more than one stable conformer exists.
6bA vibrations
(B) Dispersed Emission Duta Figure 7a shows the dispersed emission spectrum of 3-( 4-hydroxyphenyl) propionic acid produced by exciting its origin at 35 552 cm-‘. For comparison, the dispersed emission spectrum of the 0: region ofp-cresol(33) is shown in Fig. 7b. The similarity of the two spectra signifies that most of the vibrational activity in the 3-( 4-hydroxyphenyl) propionic acid spectrum is in the ring modes of the p-cresol chromophore. Figure 8a shows the dispersed emission spectrum of 3-( 4-methoxyphenyl ) propionic acid produced by exciting its origin at 35 604 cm-‘. The strong similarity to the dispersed emission spectrum of the 08 region of I-methoxy-4-methylbenzene (39), shown in Fig. 8b, indicates that most of the vibrational activity in the 3-( 4-methoxyphenyl) propionic acid spectrum is in the ring modes of the l-methoxy-4-methylbenzene chromophore. Figures 9a and 9b show the dispersed emission spectra of hydrocinnamic acid produced by exciting its origins at 37 624 and 37 648 cm -‘, respectively. The spectra are
SPECTROSCOPY
OF ACIDIC
107
ANALOGS
-4
35000
34000
33000
WAVENUMBER b)
32000
31000
(CM-])
I
35000
33000
34000
32000
WAVENUMBER
31000
(CM-‘)
FIG. 7. Dispersed emission spectra of (a) 3-( 4-hydroxyphenyl) propionic acid and (b) p-cresol produced by exciting their respective origins. In (a) the monochromator slits are 0.5 mm producing a resolution of -25 cm -‘. The oven temperature is 143°C. the carrier gas is helium at a backing pressure of 2.7 atm. and the nozzle diameter is 0. I mm. In (b) the monochromator slits are 0.3 mm producing a resolution of - 15 cm-‘. The oven temperature is 25°C. the carrier gas is helium at a backing pressure of 1.4 atm. and the nozzle diameter is 0.1 mm. In each case. the spectrum was corrected for laser scattering at the resonant transition.
similar to the emission spectrum of the 0: region of toluene (40), demonstrating that most of the vibrational activity is in the ring modes of the toluene chromophore. Although most of the ground state vibrational frequencies and intensity patterns of
35000
33000
34000
WAVENUMBER
b, * 35000
34000
33000
WAVENUMBER
32000
KM-‘)
32000
31000
(CM-‘)
FIG. 8. Dispersed emission spectra of (a) 3-(4-methoxyphenyl) propionic acid and (b) 1-methoxy-4methylbenzene produced by exciting their respective origins. In (a) the monochromator slits are 0.5 mm producing a resolution of -25 cm-‘. The oven temperature is 95°C. the carrier gas is helium at a backing pressure of 2.7 atm, and the nozzle diameter is 0.1 mm. In (b) the monochromator slits are 0.25 mm the carrier gas is helium producing a resolution of - 12 cm _’ The sample was kept at room temperature. at a backing pressure of 2.7 atm, and the nozzle diameter is 0.1 mm. In each case. the spectrum was corrected for laser scattering at the resonant transition.
108
MARTINEZ, ALFANO, AND LEVY
A
0)
Jl
I
+
37000
36000
WAVENUMBER
b,
35000
34000
(CM-‘)
D 37000
36000
WAVENUMBER
35000
34000
(CM-‘)
FIG. 9. Dispersed emission spectra of hydrocinnamic acid (a) conformer A and ( b) conformer B produced by exciting their respective origins. The monochromator slits are 1.O mm producing a resolution of --57 cm-‘. The oven temperature is 166”C, the carrier gas is helium at a backing pressure of 2.7 atm, and the nozzle diameter is 0.1 mm. The spectrum was corrected for laser scattering at the resonant transition.
conformers A and B coincide, some notable differences do exist. Consider, for example, the peak marked with an asterisk in both spectra. In conformer A this feature is 8 12 cm-’ from the resonant peak, whereas in conformer B it is 778 cm -‘. Another example is the peak marked with a plus sign. In conformer A this feature is 1440 cm-’ from the resonant peak, whereas in conformer B it is 1394 cm-‘. Note also the difference in intensity of the peak centered at 37 000 cm-‘. Its intensity in conformer B is about one and a half times that in conformer A. Since the emission of different conformers is likely to have different ground state vibrational frequencies and different emission intensity patterns, these emission spectra support our assignment of these features in the excitation spectrum as conformers. The observation of different emission spectra from excitation of the origins of different conformers indicates that the conformers do not interconvert in the excited state during the fluorescence lifetime. (C) Fluorescence
Decay Data
The measured conformer lifetimes of the three molecules studied are presented in Table I. The decays of the two hydrocinnamic acid conformers were fit to single exponentials and their lifetimes were measured as 76 & 5 and 67 -+ 6 nsec for conformers A and B, respectively. The decay of the one conformer of 3-( 4-methoxyphenyl) propionic acid was fit to a single exponential and its lifetime was measured as 15 f 3 nsec. Attempts to fit the decay of 3-( 4-hydroxyphenyl) propionic acid’s one conformer yielded a lifetime lower than our time resolution of 10 nsec. Therefore, only an upper limit of 10 nsec can be assigned as the lifetime of the origin of 3-(4-hydroxyphenyl) propionic acid. An example of fits to the data for conformers A and B of hydrocinnamic acid is shown in Fig. 10. The different conformer peaks in hydrocinnamic acid presumably correspond to different molecular geometries; thus, hydrocinnamic acid may be an example of a
109
SPECTROSCOPY OF ACIDIC ANALOGS TABLE I
Fluorescence Lifetimes of the Origin Transitions of Hydrocinnamic Acid (HA). 3-( 4-Hydroxyphenyl ) Propionic Acid (34HPPA). and 3-( 4-Methoxyphenyl) Propionic Acid (34MPPA )
Molecule
Origin(s)
34HPPA
Lifetime
35 552
34MPPA
5
(us)
10’
35 604
15f3
HA Conformer
A
37 624
76f5
HA Conformer
B
37 648
67k6
’ Typical
’
(cm-‘)”
The
uncertainty
measured
is ztl
lifetime
.
cm-’
is shorter
than
OUT time
resolution
of 10 ns
molecule where conformation affects the fluorescence decay rate of the molecule in the gas phase. To definitively demonstrate this, better time resolution is needed. The single exponential decay of both conformers demonstrates the absence of conformer interconversion on the time scale of emission. This same type of behavior was found to occur in 3-indolepropionic acid, the acidic analog of the amino acid tryptophan. The lifetimes for the A and B conformers of 3indolepropionic acid are, respectively, 16.2 and 13.5 nsec (41). In each instance the decay was well fitted with a single exponential component and the lifetimes typically
3. Be+0
_
-3.9e+o
-
.
.
.
.
.
.
.
.
-~~~~-.
-
.--:
100.
200.
TIME
.
*
_.
300.
(ns)
3.90+0 -3.
ge+
b)
-
.
-
* -
* _ .
.
-
JJj
100.
200.
TIME
300.
hs)
FOG. 10. Time-resolved fluorescence decays of (a) conformer A and (b) conformer B of hydrocinnamic acid that have been fit to single exponential decay rates of 76 + 5 and 67 + 5 nsec, respectively. The normalized residuals from the fits to each trace are given at the top of the figure.
MARTINEZ,
110
ALFANO.
AND
LEVY
were determined with a precision of ~0.1 nsec. The lifetimes of these isolated conformers were roughly an order of magnitude longer than those in solution, indicating the importance of solvent interaction in explaining solution emission. The relative lifetimes exhibited by the acidic analogs in this study are consistent with the gas phase lifetime measurements of their respective chromophores. In the gas phase, toluene’s origin has a lifetime of 82 nsec (42), anisole’s origin has a lifetime of -20 nsec (43). and p-cresol’s origin has a lifetime of -4.1 nsec (43). This relative behavior is attributed to the extent to which the benzene De,, symmetry is perturbed: the greater the perturbation, the shorter the radiative lifetime becomes. SUMMARY
The spectroscopy of the acidic analogs of phenylalanine and tyrosine has been studied in the gas phase in a supersonic jet using laser-induced fluorescence. Powerdependence studies revealed the existence of two conformers for hydrocinnamic acid and one conformer for 3-(4-hydroxyphenyl) propionic acid and 3-(Cmethoxyphenyl) propionic acid. The conformer structures of the latter two compounds are proposed to have the propionic acid group in an unti orientation with respect to the plane perpendicular to the benzene ring and passing through carbon atoms 1 and 4. Molecular modeling calculations support this orientation. The conformer structures of hydrocinnamic acid are still undetermined. Dispersed emission spectra of hydrocinnamic acid. 3-( 4-hydroxyphenyl) propionic acid, and 3-( 4-methoxyphenyl) propionic acid confirm our conformer assignments. These spectra reveal that the major vibrational activity occurring in these molecules originates from the ring vibrational modes of their respective chromophores. The fluorescent lifetimes of the conformers of these molecules were measured and found to exhibit single exponential behavior. The conformer lifetimes were found to differ among the different molecules. The relative lifetimes of these molecules are consistent with those of their respective chromophores. ACKNOWLEDGMENTS
This work was supported by the National Science Foundation under Grant CHE-88 1832 I. SJM acknowlsupportfrom the Illinois Minority Graduate Incentive Program. JCA acknowledges support from the National Science Foundation Graduate Fellowship Program. We thank Dr. Dan Eads for assisting us in fitting lifetimes using programs developed by Dr. Graham Fleming’s group. We also thank Basil Paulson for assisting us in using Dr. Gerhard Gloss’ molecular modeling program. edges
RECEIVED:
August 6, 1990 REFERENCES
I. I.
WEINRYB AND R. F. STEINER, in “Excited
States of Proteins and
Nucleic Acids” (R. F. Steiner and
I. Weimyb. Eds.), Plenum. New York. 1971. 2. D. CREED, Photochem. Photobiol. 39, 537-575 ( 1984). 3. C. TANFORD, J. D. HAUENSTEIN. AND D. G. RANDS, 1. ,4mer. c’henz. Sm. 77,6409-6413 4. I. MUNRO, I. PECHT, AND L. STRYER, Proc. Natl. Acad. Sri. US.4 76, 56-60 ( 1979). 5. M. R. EFTINK AND C. A. GHIRON. Blochemistr~~ 15, 672-680 ( 1976). 6. J. B. A. Ross, K. W. ROUSSLANG, AND L. BRAND, Biochemistry 20,436 l-4369 ( 198 I )_ 7. G. WEBER, Biochem. J. 75,335-345 (1960).
( 1955
).
SPECTROSCOPY 8 M. MARTINAUD AND A. KADIRI, Chem. Phjx
OF -ACIDIC
28,473-485
ANALOGS
111
(1978).
9. Y. YAMAMOTO AND J. TANAKA, Bull. Chem. SOL,. Japan 45, 1362-1366 ( 1972). 10. S. R. MEECH, D. PHILLIPS, AND A. G. LEE, Chem. Php. 80,317-328 (1983). Il. II. 13 14. 15. 16. 17. 18. 19. 20. -71. 22. 23. 24. 25. 26. 27. 28. ?V 30. 31. 3-7. 33. 34. 3.5. 36. 37. 38. 39. 40. 41. 42. 43.
G. R. FLEMING. J. M. MORRIS, R. J. ROBLUNS,G. J. WOOLFE, P. J. THISTLETHWAITE,AND G. W. ROBINSON.Proc. Natl. Acad. Sci. USA. 75.4652-4656 ( 1978). M. C. CHANG, J. W. PETRICH, D. B. MCDONALD, AND G. R. FLEMING, J. .4mrr. Chem Sot. 105, 3819-3831 (1983). A. G. SZABO AND D. M. RAYNER, J. Amer. Chem. Sot. 102,554-563 ( 1980). E. F. GUDCIN-TEMPLETON AND W. R. WARE, J. Phys. Chem. 88,4626-4631 ( 1984). D. V. BENT AND E. HAYON, J. ‘4mer. Chem. Sot. 97,2599-2619 ( 1975). C. PIGAULT. C. HASSELMANN,AND G. LAUSTRIAT, J. Phys. Chem. 86. 1755-1757 ( 1982). H. EDELHOCH,L. BRAND. AND M. WILCHECK. Iv. J. C’hem. 1,216-2 17 ( 1963 ). R. W. COWGILL, Biochim. Biophys. .4cta 75, 272-273 ( 1963). A. WHITE, Biochem. J. 71,217-220 (1960). J. FEITELSON.J. Phy.s. Chem. 68,391-397 ( 1964). T. R. RIZZO, Y. D. PARK, L. A. PETEANU,ANDD. H. LEVY. J. Chem. Phys. 84(5),2534-2541 ( 1986). Y. D. PARK, T. R. RIZZO, L. A. PETEANU. AND D. H. LEVY. J. Chem. Phw. 84( 12), 6539-6549 (1986). T. R. RIZZO. Y. D. PARK, AND D. H. LEVY. J. Chetn. PhJx 85( 12), 6945-695 1 ( 1986). L. A. PHILIPS. S. P. WEBB, S. J. MARTINEZ III, G. R. FLEMING. AND D. H. LEVY, J. .4mer. Chem. SOC,. 110, 1352-1355 (1988). L. A. PETEANU AND D. H. LEVY, J. Php. Chem. 92,6554-6561 ( 1988). J. R. CABLE, M. J. TUBERGEN.AND D. H. LEVY, J. ,4mer. Chem. SOC. 109, 6198-6199 ( 1987 ). J. R. CABLE, M. J. TUBERGEN,AND D. H. LEVY, J. .4mer. Chem. SM. 110, 7349-7355 (1987). J. R. CABLE, M. J. TUBERGEN.AND D. H. LEVY, J. Amer. Chem. SOL.. 111,9032-9039 ( 1987). J. R. CABLE. M. J. TUBERGEN.AND D. H. LEVY, Faraday Discu.s.s. Chem. Sot. 86, 143-l 52 ( 1988). M. J. TUBERGEN,J. R. CABLE, AND D. H. LEVY, J. Chem. Phs. 92( I ), 51-60 (1990). L. LI AND D. M. LUBMAN Appl. Spectros. 42(3), 418-424 ( 1988). S. J. MARTINEZ III, J. C. ALFANO. AND D. H. LEVY, in preparation. K. SONG AND J. M. HAYES, J. Mol. Speczrosc. 134, 82-97 ( 1989). S. J. MARTINEZ III, J. C. ALFANO, AND D. H. LEVY, J. Mol. Spec‘frasc. 137, 420-426 ( 1989). P. J. BREEN. J. A. WARREN, E. R. BERNSTEIN,AND J. I. SEEMAN. J. Chem. Phys. 87(4), 1927-1935 (1987). W. SHARFIN, K. E. JOHNSON, L. WHARTON, AND D. H. LEVY, J. Chem. Phys. 71, 1292-1299 ( 1979). M. C. CHANG, S. H. COURTNEY, A. J. CROSS,R. J. GULOTTY. J. W. PETRICH, AND G. R. FLEMING, Anal. Instrum. 14, 433-464 ( 1985). J. B. HOPKINS, D. E. POWERS,AND R. E. SMALLEY, J. Chem. Phy. 72(9), 5039-5048 ( 1980). P. J. BREEN, E. R. BERNSTEIN,H. V. SECOR,AND J. I. SEEMAN, J. .4mer. Chem. Sot. 111(6), 19581968 (1989). J. I. SELCOAND P. G. CARRICK, J. Mol. Spectrosc. 137. 13-23 ( 1989). J. SIPIOR, M. SULKES,R. AUERBACH,AND M. BOIVINEAU, J. Phys. Chem 91.2016-2018 (1987 ). M. JACON. C. LARDEUX, R. LOPEZ-DELGADO.AND A. TRAMER, Chem. Php. 24, 145-157 ( 1977). R. J. LIPERT. S. D. COLSON, AND A. SUR J. Phy.s. Gem. 92, 183-l 85 ( 1988 ).