Investigation of hydrogen bonding and proton transfer of aromatic

Steven M. Bishop, J. B. Alexander Ross, and Ronald A. Kohanski ... Wyssbrod , Richard A. Porter , Gerald P. Schwartz , Chris A. Michaels , and William...
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J . Phys. Chem. 1991, 95, 2995-3005

2995

Investigation of Hydrogen Bondlng and Proton Transfer of Aromatic Alcohols in Nonaqueous Solvents by Steady-State and Time-Resolved Fluorescence+ C. A. Hasselbacber, Evan Waxman, Lisa T. Galati, Paul B. Contino, J. B. Alexander ROSS,* and William R. Laws* Department of Biochemistry, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, New York 10029 (Received: September 4, 1990)

Aromatic alcohols in aqueous solution become stronger acids upon excitation from the singlet ground state to the first excited singlet state. Hydrogen-bondingand excited-state proton-transfer reactions might also occur in the nonaqueous environment of a protein steroid-binding pocket or a tyrosine residue. Using absorption, steady-state fluorescence, and time-resolved fluorescencespectroscopies,we investigated these processes by titrating the aromatic alcohols 2-naphtho1, l'Ifl-dihydroequilenin, and phenol with the strong proton acceptor triethylamine in cyclohexane or toluene, organic solvents of different polarity and polarizability. Analysis of the absorption and steady-state fluorescencespectra as a linear combination of spectra (LINCS) shows that in the titration with triethylamine there are two separate ground-state and two separate excited-state species. Decay associated spectra (DAS) also show that there are two separate excited-statespecies. The ground-state and excited-state species that are formed from the interaction between the alcohol and triethylamine are hydrogen-bonded complexes. The LINCS analysis of the steady-state fluorescence emission and the fluorescence decay kinetics of the free alcohol indicate that formation of the hydrogen-bonded complex is diffusion-limited in the excited state. Excited donors (free alcohol) that subsequently form exciplexes in either nonpolar or polar solvents have the same emissive properties as those hydrogen-bonded complexes excited directly from the ground state. In addition, the degree of charge transfer in the hydrogen bond depends on solvent polarity and polarizability. On the basis of these observations, we can make predictions about the interactions of the tyrosine residue in a protein or a 2-naphthol-containing estrogen in the presence of a strong proton acceptor (Le., an unprotonated amino group). We conclude that emission in such a nonaqueous environment will be from the excited states of the free and hydrogen-bonded species; the fully ionized alcohol does not occur in the excited state.

Introduction As a result of the differences in the electronic configurations of the electronic ground and first excited singlet states of planar aromatic hydrocarbons, the two states often have dipole moments with different direction and/or magnitude. Consequently, these two states are chemically distinct species and can, therefore, exhibit different behavior in condensed media. For example, the differences in the electronic configurations of the ground and excited singlet states of molecules with ionizable groups may cause the ground- and excited-state proton offlon rates to differ by several others of magnitude. This is the case for 2-naphtho1, which has a hydroxyl group with a ground-state pK, of 9.5 and an excited-state pK,* of 2.8.' Thus, even if only the nonionic form is present in the ground state, fluorescent molecules with ionizable groups potentially can have emission from two excited-state species. The pH dependence of this type of two-state, excited-state reaction can be monitored spectrally by steady-state fluorescence methods, and in the general case the fluorescence intensity decay kinetics are well understod2 This two-state, excited-state proton-transfer reaction is sensitive to the proximity of proton donors or acceptors. Certain estrogens and tyrosine have the capability of undergoing excited-state proton transfer and, therefore, may be used to detect the presence of proton-accepting or -donating groups in their local environment. We have been investigating the steroid-binding site of the sex-steroid-binding protein from both human and rabbit sera using differences in both the ground-state and excited-state properties of the estrogens equilenin and 178-dihydroeq~ilenin.~>~ These steroids have a 2-naphthol moiety as their A and B rings, and their pK, and pK,* are similar to those of 2-naphth01.~ When bound to the protein, however, the fluorescence properties are quite different. Analysis of the excitation and emission spectra of the bound steroid suggest that it is neither fully protonated nor deprotonated. Instead, the spectra are consistent with the formation of a ground-state, hydrogen-bonded species which either does not ~~~~

~

Authors to whom correspondence should be addressed. 'Preliminary aspects of this research were presented at the 34th Annual Meeting of the Biophysical Society in Baltimore, MD, February 18-22, 1990, and at the 10th International Biophysics Congress in Vancouver, British Columbia, Canada, July 29-August 3, 1990.

ionize in the excited state or else forms a "dark", nonfluorescent

specie^.^,^ We have also been investigating the fluorescence of tyrosine in proteins and polypeptide hormones. The phenol group of tyrosine should be able to participate in hydrogen bonding as well as excited-state proton transfer. The pK,* of tyrosine (4.5) is higher than that of 2-naphthol (2.8),6-8 indicating that in the excited state tyrosine is a weaker acid than 2-naphthol. In contrast to 2-naphthol which shows emission from both its ionized and protonated forms at neutral pH, tyrosine shows only emission from the protonated form because its excited-state deprotonation rate is much slower than other depopulation rates. The local environment of a tyrosine in a protein, however, could include a strong proton acceptor that might help promote either hydrogen bonding or excited-state proton transfer. In fact, there have been several reports, based on fluorescence spectra, of tyrosine ionization in proteins at neutral pH; several of these have subsequently been attributed to tryptophan-containing contaminanh6 In the present studies, we examine the interaction of the aromatic alcohols phenol and 2-naphthol with the strong proton acceptor triethylamine in organic solvents of different polarity and different polarizability. Our purpose is to establish the requirements needed to promote either hydrogen bond formation, ion pair formation, or proton transfer in a nonaqueous environment, either in the ground state or in the excited state, so that we can use fluorescence methods to interpret the hydrogen-bonding or proton-transfer interactions occurring in a steroid-binding pocket (1) Weller, A. Prog. Reacr. Kiner. 1961, 1, 187-214. (2) Laws, W. R.; Brand, L. J . Phys. Chem. 1979.83, 795-802. (3) Orstan, A.; Lulka, M.; Eide, B.; Petra, P. H.; Ross, J. B. A. Biochemisrry 1986, 25, 2686-2692. (4) Casali, E.; Petra, P. H.; Ross, J. B. A. Biochemistry 1990, 29,

9334-9343. (5) Davenport, L.; Knutson, J. R.; Brand, L. Biochemisrry 1986, 25, 1186-1 195. ( 6 ) Ross, J. B. A.; Laws, W. R.; Rousslang, K. W.; Wyssbrod, H. R. Fluorescence Spectroscopy III. Biochemical Applicarions; Lakowicz, J. R., Ed.; Plenum Press: New York, in press. (7) Rayner, D. M.; Krajcarski, D. T.: Szabo, A. G. Can. J . Chem. 1978, 56, 1238-1245. ( 8 ) Feitelson, J. J . Phys. Chem. 1964, 68, 391-397.

0022-365419 112095-2995$02.50/0 0 1991 American Chemical Society

2996 The Journal of Physical Chemistry, Vol. 95, No. 8,1991 or at the site of a tyrosine residue. Of special importance is the present controversy concerning the nature of the excited-state donor/acceptor complex of an aromatic alcohol. Based on time-resolved fluorescence decay kinetics, Bisht et al.”’ have argued that excited molecules which form an excited-state, hydrogen-bonded complex (exciplex) in nonpolar solvents are nonemissive. They further state that an emissive hydrogen-bonded species is formed by direct excitation of the ground-state complex. This interpretation means that the exciplex and the directly excited complex are distinct species. Previously, however, Matsuzaki et aLi2made no distinction between the exciplex and the directly excited species. Our time-resolved fluorescence decay kinetics and steady-state absorption and emission spectra support the latter view. We find no evidence for a nonemissive exciplex; the complete kinetic analysis of the spectroscopic data requires that the hydrogen-bonded species formed in the excited state be indistinguishable from that directly excited from the ground state. Our data from the model systems suggest that a ground-state and excited-state interaction of a tyrosine residue or an estrogen with a strong proton acceptor in a nonaqueous environment results in a shift in the emission to lower energy, consistent with formation of a hydrogen bond. The magnitude of the shift reflects the nature of the hydrogen bond in environments of different polarity and polarizability. Because we do not see spectral and fluorescence lifetime changes consistent with excited-state ionization, we do not predict that complete excited-state proton dissociation will occur when an aromatic alcohol is buried in a protein site. Theory

Many aromatic alcohols exhibit fluorescence from two species in aqueous systems. When only the protonated form of the alcohol exists in the ground state, the dual emission results from the formation of the ionized alcohol by excited-state proton transfer. This reaction occurs because the alcohol is a much stronger acid in the excited state. Both steady-state and time-resolved fluorescence studies indicate that this process is a simple two-state mechanism in water.’Y2 Depending upon the rate constants for the loss of the proton and the bimolecular rate constants for the reprotonation in both the ground and excited states, the radiative and nonradiative decay rates for both the protonated and unprotonated species, and the pH of the solution, the excited-state reaction may or may not be reversible. The observed fluorescence decay kinetics will therefore be dependent upon all of these conditions. For example, when the pH is near the pKa*, both excited-state species decay as the sum of two exponentials, and the time constants and their associated amplitudes are kinetically derived. A kinetic signature of this process is that when only the protonated species is excited, the decay of the ionized species will have the same two time constants as the protonated species, but the respective amplitudes will be equal in magnitude and opposite in sign since this excited-state species is created by the protontransfer reaction.* As we have shown for para-substituted phenols, which have a higher pK,* than 2-naphtho1, the constant fluorescence yield and the absence of multiexponential kinetics as a function of pH in the range of the pKa* suggest that they do not undergo significant excited-state proton transfer in water.I3 In addition, Willis and Szabo do not find emission from tyrosinate, even in the presence of high concentrations of a proton a ~ c e p t 0 r . l ~Instead, their fluorescence decay studies indicate the presence of a ground-state, hydrogen-bonded complex that persists in the excited state. Thus, the fluorescence decay kinetics of aromatic alcohols in water depend strongly upon the difference between pKa and pKa*. (9) Bisht, P. B.; Tripathi, H. B.; Pant, D. D. Chem. Phys. Lett. 1987, 142, 291-297. (10) Bisht, P. B.; Tripathi. H. B.; Pant, D. D. J . Lumin. 1989,43, 301-308. (1 I ) Bisht. P. 8.; Tripathi, H. B.; Pant, D. D. J. Lumin. 1990, 46, 25-31. (12) Matsuzaki, A.; Nagakura, S.; Yoshihara, K.Bull. Chem. Soc. Jpn. 1974, 47, 1152-1157. (13) Laws, W. R.;Ross, J. B. A.; Wyssbrod, H. R.;Beechem, J. M.; Brand, L.; Sutherland, J. C. Biochemistry 1986, 25, 599407. (14) Willis, K. J.; Szabo, A. G. J. Phys. Chem. 1991, 95, 1585-1589.

Hasselbacher et al. SCHEME I %e*

kM*

A*+X

A+X

r

B?X

C*+HX

B*X

C+HX

A more complete spectroscopic description of aromatic alcohols interacting with a proton donor/acceptor involves at least three different species in both the ground state and the excited state: the free protonated alcohol, the hydrogen-bonded complex, and the fully ionized alcohol formed by proton dissociation. More species might be possible if, for example, the proton donor/acoeptor is also involved in solvation of the alcohol. The equilibria and kinetics will be solvent, alcohol, and proton acceptor dependent. In Scheme I, A, B-X, and C represent the protonated, hydrogen-bonded, and deprotonated species, respectively, and A*, B*.X, and C* denote the excited-state forms. The rates kf,A,kf$, and k , , represent the sum of the rates for radiative and nonradiative decay processes of A*, B*.X, and C*, respectively. Depending on the solvent, the alcohol, and the proton acceptor, the mechanism in Scheme I can be simplified by the exclusion of one or more species. For example, we expect that in water, which is a hydrogen-bonding solvent, the pH-dependent, ground-state equilibrium will involve the hydrogen-bonded and ionized species, B.X and C. In non-hydrogen-bonding solvents, however, the ground-state equilibrium will involve the free alcohol and the hydrogen-bonded species, A and B-X, provided a proton acceptor is present. In water, the excited-state equilibrium will favor the formation of C* from B*.X, due to the difference in pK, and pKa*. Since kBC and kBc* are diffusion-controlled bimolecular reprotonation rates, they are likely to have similar values. This means that the greater acidity of aromatic alcohols in the excited state is due to kcB* being orders of magnitude greater than kcB. Observation of C*, formed by excited-state proton transfer during the lifetime of B*.X, will depend on the relative magnitude of kcB* versus k[,B. In a non-hydrogen-bonding solvent, both A and B.X will exist in the presence of a proton acceptor. A change in the subsequent excited-state concentrations of A* and B*.X will depend on three factors. First, kAB*, the rate of excited-state complex dissociation, must be compared to kf,B;if it is less than k,,, the back reaction to re-form A* from B*-X will be less likely. Second, keA*, the rate of the diffusion-controlled bimolecular formation of B*.X from A*, must be compared to k , A , the rate of depopulation of A*. Finally, in the limit of slow decay from the excited state, KO*,the ratio of kBA* to k A B * , must be considered. Ionization to form C* from B*-X in non-hydrogen-bonding solvents will be unlikely due to poor solvation of free ions. Materials and Methods

Chemicals. 2-Naphthol (Kodak) and 2-methoxynaphthalene (Aldrich) were recrystallized twice from spectral grade cyclohexane, dried under nitrogen, and stored as concentrated solutions in cyclohexane. Spectrophotometric grade cyclohexane and toluene were purchased from Aldrich. Sequanal grade triethylamine (TEA) was obtained from Pierce. Newly opened bottles of the solvents and TEA were used to avoid problems due to deterioration of “older”, opened solvents. 17B-Dihydroequilenin (Steraloids) was purified by thin layer silica gel chromatography using a cyc1ohexane:ethyl acetate (1:l) solvent system and stored in absolute ethanol. Phenol (Baker) was purified by distillation and stored as a concentrated ethanolic solution. All solutions were stored in the dark in the presence of desiccant. Samples were prepared by drying an aliquot of the stock solution on the walls of a test tube with nitrogen and adding solvent to give an optical density of about 0.3 at the excitation wavelength.

The Journal of Physical Chemistry, Vol. 95, No. 8, 1991 2997

Interaction of Aromatic Alcohols with Triethylamine

5

3

n\

7

NdYAQ b u r

FDC

I

bur Electmnlu

Pambollc Minor

Y ultlctmnn6i AnalyHr CPU

1START STOP TAC

(Hamamatsu R2809U-06) operated at 3.4 KV (Bertan 365). The generated electronic pulse was amplified (Phillips 6954), constant fraction discriminated (Phillips 6915), and used as one timing signal (start) for a time-to-amplitude converter (Tennelec 863). The other timing signal (stop) was derived from the square-wave output of the cavity-dumper electronics by conversion to a NIM pulse using our own shaping circuit. The start signal was delayed relative to the stop signal to use the linear region of the timeto-amplitude converter. The voltage corresponding to the time difference between events was then processed by a multichannel analyzer (LeCroy 35 12 analog-to-digital converter and LeCroy 3588 histogramming memory). Decay curves were typically collected into 2000 channels at a resolution of 22 ps/channel. The instrument response function, obtained from light scattered off of a silica gel suspension (Ludox, Dupont), had a full width at half-maximum of 225 ps. Data acquisition and analysis were performed on an H P 1000 A700 minicomputer using software developed in this laboratory. Data Analysis. The steady-state spectral titration data were initially analyzed at single wavelengths by the change in absorbance (or fluorescence) as a function of the logarithm of the TEA concentration. To determine whether the titration involved a two-state equilibrium or involved additional intermediates, the data were also analyzed as a linear combination of spectra (LINCS). Individual spectra were fit to the equation

+ biAn(X)

Ai@) = aiAo(X)

(1)

In this equation, Ai@) is the absorbance (or fluorescence) at each wavelength X at the ith titration point. Ao(X) is the spectrum in neat solvent and An(X)is the spectrum at any arbitrarily chosen TEA concentration. Since the entire set of spectra defined by eq 1 can be treated as a vector space of rank 2, any two spectra obtained at arbitrary TEA concentrations can be used as basis spectra. In particular, spectra obtained in neat solvents are not required, and in fact can be extrapolated from this analysis. The coefficients ai and bi were determined by nonlinear least-squares regression.21 The quality of the fit was evaluated by visual inspection and the residuals, and checked by varying the basis set spectra. Fluorescence intensity decay data analysis was by nonlinear least-squares regression,21 fitting the individual intensity decay curves, I ( t ) , to a sum of exponentiaIs22s23

where ai is the amplitude and ri is the lifetime of the ith component. The reduced chi square (x2)and weighted residuals with their autocorrelation were used as best fit criteria. Fluorescence intensity decay curves, obtained as a function of emission wavelength, were also analyzed by a global nonlinear least-squares fitting p r o c e d ~ r e . ~In ~ ,this ~ ~ analysis, the decay constants were assumed to be emission wavelength independent and were iterated as common parameters for all the decay curves in a particular data set. Decay-associated spectra (DAS)26J7 were generated, using fluorescence decay parameters obtained from either global or single-curve analyses, by the equation

(21)Bevington, P.R.Data Reduction and Error Analysis for the Physical Sciences; McGraw-Hill: New York, 1969. (22) Knight, A. E.W.; Selinger, B. K. Chem. Phys. Leir. 1971, IO, 43-48. (23)Grinvald, A.; Steinberg, I. Z . Anal. Biochem. 1974, 59, 583-598. (24)Knutson, J. R.;Becchem, J. M.; Brand, L. Chem. Phys. Lett. 1983,

(IS) Badea, M. G.; Brand, L. Methods Enzymol. 1979, 61, 378-425. (16)Holtom. G. Proc. SPIE 1990, 1204, 2-12. (17)Paoletti, J.; LePecq, J.-B. Anal. Eiochem. 1969, 31, 33-41. (18) Azumi, 7.: McGlynn, S. P. J . Chem. Phys. 1962, 37, 2413-2420. (19) Kalantar, A. H. J . Chem. Phys. 1968, 48, 4992-4996. (20)Shinitzky, M.J . Chem. Phys. 1972, 56, 5979-5981.

102, 501-507.

(25)Beechem, J. M.;Knutson, J. R.; Ross,J. B. A.; Turner, B. W.; Brand, L. Biochemistry 1983, 22, 6054-6058. (26)Donzel, B.; Gauduchon, P.; Wahl, P. J . Am. Chem. Soc. 1974, 96, 801-808. (27)Knutson, J. R.;Walbridge, D. G.; Brand, L. Biochemistry 1982, 21, 4671-4679.

2998 The Journal of Physical Chemistry, Vol. 95, No. 8, 1991

I

A

t

Hasselbacher et al. B O A

0

E

300

320

340 360 Wavelength (nmJ

380

4

Wavelength (nm)

Figure 2. Absorption spectra (panels A and C) and fluorescence spectra (panels B and D) for the TEA titration of 2-naphthol (100 pM)in cyclohexane (panels A and B) and toluene (panels C and D). Panels E and F are the absorbance and fluorescence, respectively, of 2-naphthol in water as a function i f pH.

where Fi(A)is the decay-associated fluorescence emission spectrum for component i (intensity as a function of emission wavelength A), F,(A) is the overall steady-state fluorescence emission spectrum of the sample, andJ.(A) is the fractional intensity of component i. The fractional intensity is defined as

(4) i= I

Results Absorbance Spectra in Nonaqueous Solvents. The absorbance spectra of 2-naphthol in cyclohexane and toluene, shown in panels A and C of Figure 2, respectively, are shifted to lower energies and exhibit loss of fine structure after titration with TEA. At the titration end point (0.1-0.2 M TEA), the spectra are intermediate between those of the aromatic alcohols in either neat cyclohexane or toluene and in basic aqueous solution, shown in panel E of Figure 2, where the hydroxyl group is completely ionized. Similar spectral shifts have been previously observed for the titration of 2-naphthol by TEA in heptane, and they have been ascribed to hydrogen bonding between the oxygen of 2-naphthol and the amine nitrogen of TEA.** The spectra of 178-dihydroequilenin in cyclohexane and toluene, as well as those resulting from the TEA titrations, are similar to those observed for 2-naphtho1, but shifted 8-10 nm to lower energy. We have verified that phenol has its absorption maximum near 275 nm, and its spectrum displays absorbance spectral shifts similar to those described above on titration by TEA in cyclohexane.I0 Corresponding titrations in toluene were not done because toluene has strong absorption in the same spectral region as phenol. TEA titrations of 2methoxynaphthalene in cyclohexane and toluene and of anisole in cyclohexane result in no spectral shifts. Thus, replacement of the hydroxyl hydrogen with a methyl group blocks perturbation of the absorption spectrum by TEA. (28) Nagakura, S.; Gouterman,

M.J . Cfiem. Phys.

1957, 26, 881-886.

Steady-State Fluorescence Spectra in Nonaqueous Solvents. Using excitation at an isosbestic point, the emission band observed in neat solvent decreases in intensity and a new emission band appears at lower energy upon TEA titration of 2-naphthol in cyclohexane or toluene. As indicated in panels B and D of Figure 2, the fluorescence spectra have an isoemissive point at relatively low concentrations of TEA (2-24 mM). Fluorescence difference spectra show that the new band has a maximum near 370 nm in cyclohexane and 410 nm in toluene (see Figure 5 ) . In addition, the toluene difference spectrum has the same emission maximum and spectral envelope as the emission spectrum of 2-naphthol in neat TEA. TEA titration of 17@-dihydroequileninin cyclohexane and toluene results in fluorescence spectra similar to those observed for 2-naphtho1, but shifted to lower energies by about 10 nm. The TEA titration of phenol, which was only carried out in cyclohexane (see above), results in the appearance of a lower energy emission band near 333 nm at low TEA concentrations. TEA titration has no effect upon the position or shape of the fluorescence emission band of 2methoxynaphthalene in cyclohexane or toluene, or upon the emission band of anisole in cyclohexane. There is weak quenching in all cases, however. The quenching was analyzed according to the Stern-Volmer relationshipZ9 FO

-= 1

F

+ &[Q1

(5)

where Fo is the fluorescence intensity in absence of quencher, F is the fluorescence intensity in presence of quencher, and [Q] is the quencher concentration. The slope is the Stern-Volmer constant, Ksv, which is the ratio of the bimolecular collisional rate constant, k,, to the sum of the rates for radiative and nonradiative decay of the fluorophore in the absence of added quencher. The values for k were difficult to determine accurately but are in the range of lo9 M-' s-I. A large inner filter correction due to the absorbance of TEA is the source of the uncertainty. These values should be contrasted with the k, values, discussed below, that are in the range of (2-6) X lo9 M-' s-' for the quenching of the "neat (29) Stern, 0.; Volmer, M. Pfiys. Z. 1919, 20, 183-188.

The Journal of Physical Chemistry, Vol. 95, No. 8, 1991 2999

Interaction of Aromatic Alcohols with Triethylamine 1.o

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350

400

460

500

300

400

350

Wavelength (nm)

450

500

Wavelength (nm)

Figure 3. Fluorescence decay parameters for the TEA titration of 2-naphthol as a function of emission wavelength. Panels A and B, in cyclohexane; panels C and D, in toluene. Panels A and C: fractional intensity (eq 4) of each kinetic component; the lines have been drawn for clarity. Panels B and D: lifetimes; the lines are the average values at a given TEA concentration. Open symbols: short-lifetime component; solid symbols: long-lifetime component.

solvent” fluorescence emission of 2-naphthol or 170-dihydroequilenin in either cyclohexane or toluene, or that of phenol in cyclohexane. As the titration of either 2-naphthol or 176-dihydroequilenin in cyclohexane proceeds to higher concentrations of TEA (above 0.1 M), the isoemissive point is lost; the spectra gradually shift further to lower energy and finally approach that of either 2naphthol or 176-dihydroequilenin in neat TEA (data shown for 2-naphthol in panel B of Figure 2). At high concentrations of TEA, there is also shifting of the phenol emission band (333 to 345 nm). By contrast, the TEA titration of 2-naphthol and 176-dihydroequileninin toluene results in a single isoemissivepoint, even at high concentrations of TEA (250-500 mM; data shown for 2-naphthol in panel D of Figure 2). Steady-State Absorbance and Fluorescence Spectra in Water. In neutral and acidic water, the So SI transition of 2-naphthol has resolved vibrational bands. The maximum is 328 nm (panel E of Figure 2). 17a-Dihydroequilenin has an essentially identical spectrum with a maximum at 338 nm. At pH 12, the absorptions of 2-naphthol and 176-dihydroequilenin shift to lower energy (about 345 and 355 nm, respectively) with a concomitant loss of vibrational structure (shown for 2-naphthol in panel E of Figure 2). The fluorescence emission spectra of 2-naphtho1, 176-dihydroequilenin, and phenol in water also shift to lower energies at alkaline pH (see panel F of Figure 2). Time-Resolved Fluorescence. The fluorescence lifetimes at 20 OC of the aromatic alcohols in cyclohexane, toluene, or TEA are listed in Table I. In neat cyclohexane, toluene, and TEA, the fluorescence decays of 2-naphthol and 176-dihydroequilenin are essentially single e x p ~ n e n t i a l . ~Whereas ~ in cyclohexane and toluene the lifetimes are 7.7 and 6.8 ns, respectively, in TEA the lifetime is shorter with a value of 1.3 ns. When TEA is added to 2-naphthol or 176-dihydroequilenin in cyclohexane or toluene,

TABLE I: Time-Resolved Fluorescence Decay Parameter9

(30) In neat cyclohexane or toluene a small but significant improvement could be obtained in the data analysis when the decay curves were fit for a double exponential. The second lifetime component, which is poorly determined, represents about 0.5% of the total intensity decay (eq 4) and has a time constant between 0.5 and 1.E ns. This second component cannot be resolved after addition of TEA probably because the hydrogen-bonded species formed with TEA has a similar lifetime (1-2 ns). We are not certain whether the second component in neat solvent is from soluttsolvent interaction or whether it is a trace impurity. Efforts to purity solvents and sample failed to eliminate this component. Its fluorescence decay did show one interesting, reproducible pattern: the amplitude term is positive on the high-energy side of the alcohol emission and negative on the low-energy side. While this could be rationalized as an excited-state interaction, we do not believe that this component alters the results presented here. We are currently trying to understand its origin.

the fluorescence lifetime decreases. In addition, a second exponential with a decay constant in the range observed for neat TEA is required to adequately fit the data. At TEA concentrations less than 0.1 M, the time constant for the second component of 2-naphthol is 2.3 ns in cyclohexane and 1.O ns in toluene. In both cases, these shorter lifetimes appear to be essentially independent of TEA concentration up to 0.1 M. At TEA concentrations between 0.1 and 1.O M, there is a small decrease in the lifetime of the 2.3-11s component in cyclohexane with a k, value of 5 X 107 M-l s-1 . N o significant change is observed, however, for the 1 .O-ns component in toluene.

comDound 2-naphthol

-

170-dihydroequilenin

2-methoxynaphthalene

1TEAl. mM n.C ns 0 7.70 6.71 2.3 6.31 4.7 5.67 8.2 12.9 4.84 200 400 600 800 1000 toluene 0 6.60 6.26 2.5 6.09 5 5.50 IO 4.92 20 triethylamine ( 100%)

solventb cyclohexane

cyclohexane toluene triethylamine

0 0 ( 100%)

7.90 7.30

cyclohexane

0 4 20 200 0 4 20 200 ( 100%)

7.68 7.66 7.64 7.54 7.28 7.48 7.39 7.49 4.18

toluene

triethylamine

T-.C ns

2.36 2.28 2.34 2.34 2.32 2.25 2.20 2.16 2.11 1.02 1.04 1.04 1.02 1.32

2.96

“Measured at 20 OC;standard deviations are less than 100 ps for repeated measurements. bThe fluorescence decays in neat cyclohexane and toluene could also be fit by a sum of two exponential^.'^ ‘The lifetimes associated with the ‘free” and hydrogen-bonded species are and T,, respectively.

3OOO The Jotrrnal of Physical Chemistry, Vol. 95, No. 8, 1991 1.8

1

0

5

10

15

20

25

[TEA], mM

Figure 4. Stern-Volmer plots (eq 6 ) for the TEA dependence of the longer lifetime of 2-naphthol. The lines represent the best least-squares linear regression. The slope, which equals KSV. is 46 and 17 M-l in cyclohexane and toluene, respectively.

Fluorescence decay data were collected as a function of emission wavelength for 2-naphthol in cyclohexane and in toluene, each with TEA concentrations up to 20 mM. Both lifetimes are invariant as a function of emission wavelength a t each TEA concentration (panels B and D, Figure 3). At wavelengths less than 370 nm, the intensity contribution of the short-lifetime component to the total ffuoreScence emission is small (panels A and C, Figure 3), and in an analysis with unrestricted parameters, the precision of the values obtained a t these wavelengths for the short lifetime was poor. When the value of the short lifetime was held constant a t the average value obtained a t wavelengths greater than 370 nm, fits of equal quality were obtained, consistent with the conclusion that the value of the short-lifetime component is constant across the entire emission spectrum. Moreover, a global analysis as a function of emission wavelength of the data for each TEA concentration, iterating for two common, emission-wavelengthindependent lifetimes, yielded similar results. The relative amplitudes of both decay components resemble a typical titration plot for a two-state system: the amplitude of the shorter lifetime increases with increasing wavelength as the amplitude for the longer lifetime decreases, consistent with a two-state, ground-state model.13 This amplitude behavior is represented by the fractional intensity plots for each kinetic component as given in panels A and C of Figure 3. A two-state, ground-state model, however, does not explain the TEA-concentration dependence of the longer lifetime of 2-naphthol in cyclohexane and toluene. The decrease can be described by the Stern-Volmer relationship3' where r0 and T are lifetimes in the absence and presence of quencher (TEA), respectively. An analysis of the effect of TEA on the longer lifetime is shown in Figure 4. The kqvalues obtained for the TEA-dependent quenching of the longer lifetime in cyclohexane and toluene were 6.0 X lo9 and 2.6 X lo9 M-' s-', respectively. Furthermore, a two-state, ground-state model does not account for the smaller k, value of 5 X IO7 M-' s-I, described above for the quenching observed at higher concentrations of TEA for the shorter lifetime component in cyclohexane. As indicated in Table I, the fluorescence lifetime of 2-methoxynaphthalene in cyclohexane or toluene is also constant as a function of emission wavelength. In cyclohexane, the lifetime appears to decrease slightly with increasing TEA concentration with an approximate k, value of IO7 M-' s-', which is the same as that obtained from the decrease in steady-state quenching. The fluorescence lifetime of anisole in cyclohexane also appears to decrease slightly with increasing TEA concentration, yielding a similar value for k,. By contrast, as indicated in Table I, the fluorescence lifetime of 2-methoxynaphthalene in toluene appears to increase slightly at low concentrations of TEA. This is at variance with the steady-state results. It should be noted, however, as explained in the Discussion, that these are small perturbations (3 1) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Plenum Press: New York, 1983.

Hasselbacher et al. compared to those produced by TEA titration of the alcohols. Comparison of Decay-Associated Spectra (DAS) and Linear Combination of Spectra (LINCS).The DAS generated for 2naphthol in cyclohexane and toluene as a function of TEA concentration are shown in Figure 5. At low TEA concentrations ( IO9 M-' s-I), resemble the steady-state spectra of 2-naphthol in the respective solvents. As the concentration of TEA increases, the individual DAS curves begin to shift to lower energy; this is more evident in cyclohexane than in toluene. The DAS as a function of TEA concentration for the shorter lifetime resemble the difference of the steady-state spectrum for 2-naphthol in the presence of TEA less its spectrum in neat solvent and are independent of TEA concentration. The fluorescence lifetime data and the DAS suggest that the TEA titration of the aromatic alcohols in both cyclohexane and toluene results in two distinct species. The absorption and steady-state fluorescence emission spectra at low TEA concentrations (50.02 M) likewise suggest two distinct species. Based on the absorption and fluorescence changes at single wavelengths, apparent equilibrium constants were estimated and found to be in the range observed by others for hydrogen For example, we observe from absorption changes values of about 100 and 35 M-I, respectively, for 2-naphthol in cyclohexane and in toluene. According to these estimates, the strengths of the complexes are solvent dependent. In addition, the estimates based on fluorescencechanges suggest tighter complexes than those based on absorbance changes. To understand the reasons for the different estimates of the equilibrium constants from absorption and fluorescence data, we carried out a more complete, detailed spectral analysis. Titration of a simple, two-state system should produce spectra with clear, well-defined isosbestic or isoemissive point(s), provided that appropriate corrections have been made for sample dilution as well as for primary and secondary inner filter errors.31 Although isosbestic points are seen in both solvents and an isoemissive point is apparently obtained for the TEA fluorescence titrations in toluene, the isoemissive point is lost at high TEA concentrations in the cyclohexane fluorescence titrations. If, on one hand, we assume that our corrections are adequate, the loss of the isoemissive point suggests that additional fluorescent species are being formed by the TEA titration. If, on the other hand, additional species are not being formed, both the absorption and steady-state fluorescence spectra should be linear combinations of two basis spectra, one for the free and the other for the TEA-complexed alcohol; there should be no need for additional spectral components. We analyzed the TEA titration data for 2-naphthol in cyclohexane and in toluene as linear combination of spectra (LINCS; see Figure 6) in terms of the two-state model. Both the absorbance and fluorescence data can be fit by a linear combination of two basis set spectra. As shown in panels A and C of Figure 7, the variation in the coefficients for absorbance basis spectra as a function of the logarithm of TEA concentration corresponds to a two-state titration. The equilibrium constants were determined by fitting the basis set coefficients, and they agree well with those obtained by absorbance changes at a single wavelength, described above. The fluorescence titration data appear to be more complex. As shown in panels B and D of Figure 7,rather than the smooth sigmoidal titration of both coefficients as seen for the absorption basis spectra, the relative intensity of the fluorescence emission spectrum of the TEA-complexed alcohol reaches a maximum near saturating TEA concentration and then decreases at higher TEA concentrations. As explained in the Discussion, this fluorescence titration behavior is a consequence of the ground-state and excited-state kinetics of the entire system.

Discussion In their pioneering studies on the effects of hydrogen-bond formation upon the absorption spectra of 2-naphthol in n-heptane, Nagakura and Gouterman showed that the magnitude of the shifts toward longer wavelengths of the near-ultraviolet absorption bands,

The Journal of Physical Chemistry, Vol. 95, No. 8, 1991 3001

Interaction of Aromatic Alcohols with Triethylamine

41.

300

350

400

450

500 300

Wavelength (nm)

I

350

.

.

.

I

.

.

.

400

.

I

450

.

.

.

500

Wavelength (nm)

Figure 5. Decay associated spectra (DAS) as a function of TEA concentration for 2-naphthol in cyclohexane (panels A and B) and toluene (panels C and D). Panels A and C are the DAS for the longer lifetime component while panels B and D are the DAS for the shorter lifetime component. The lines in panels A and C are the steady-state emission spectra in neat solvent. The lines in panels B and D are steady-state emission difference

spectra.

A

0

ITEAl

2.4 mM (fit1 2.4

300

350

mM (data)

400

450

500

Wavelength (nm)

Figure 6. An example of the linear combination of spectra (LINCS) of

2-naphthol steady-state fluorescence emission in cyclohexane.

appearing in the region of 280-340 nm, depends directly upon the strength of the proton acceptor.28 For formation of a single hydrogen bond with triethylamine (TEA), they obtained an equilibrium constant of about 100 M-' at 25 O C , which is comparable to the values we obtained in cyclohexane. They also suggested that the hydrogen bond is not purely electrostatic but appears to have its origin in a charge-transfer mechanism as described earlier in the quantum mechanical analysis by MulMataga and Kaifu examined the steady-state fluorescence of the 2-naphthol complex with TEA in cyclohexane and benze r ~ e . ~On~ the basis of spectral shifts in these solvents, they concluded that the complex has considerable charge-transfer character in the excited state. The first time-resolved fluorescence studies on the singlet excited-state interaction of 2-naphthol and TEA were made by Matsuzaki et a1.I2 They found that the rate constant for formation of the exciplex in toluene was 2.2 X IO9 M-l s-I at 30 "C. This can be compared to the bimolecular rate constant of 2.6 X lo9 M-' s-I that we obtained for the quenching of 2-naphthol by TEA in the same solvent at 20 OC. Bisht et al. have also studied the time-resolved fluorescence of 2-naphthol in polar and nonpolar solvent^.^^^^ They argue that in nonpolar solvents the exciplex differs from the excited-state species obtained by direct excitation of the ground-state hydrogen-bonded complex in that the exciplex is nonfluorescent. The basis of their argument is that in nonpolar solvents the fluorescence decay kinetics fail to display characteristic signatures for a species formed in the excited state, specifically a double-exponential decay with one negative preexponential ~

~

~~

(32) Mulliken, R. S. J . Am. Chem. Soc. 1952, 74, 811-824. (33) Mataga, N.; Kaifu, Y . J. Chem. Phys. 1962, 36, 2804-2805.

amplitude at the low-energy side of the emission. By contrast, they observe this kinetic trait for the fluorescence decay in polar solvents. Bisht et al. also make the same argument for the phenol/TEA complex in polar solvents and find no evidence of an excited-state reaction in nonpolar solvents.IO As mentioned earlier, aromatic alcohols in aqueous solution can be assigned to species B-X and C in Scheme I. That these alcohols are hydrogen bonded in water can be demonstrated by the shifts to lower energies of both the absorption and fluorescence spectra compared to the spectra obtained in either neat cyclohexane or neat toluene. The relative concentrations of B.X and C will depend on the pK, of the alcohol as well as the pH; the pK, of many of the aromatic alcohols is near 10. In the case where only B-X exists in the ground state, the only way C*emission can be observed is through excited-state proton transfer. A necessary requirement for formation of C* is that kcB* has to be a significant fraction of kf,&for example, kCB*is 2 x IO7 s-I and kf,B is 1.3 X lo8 s-I for 2-naphthol in water.2 If a constituent group added to the ring system makes the alcohol a stronger acid in the excited state, as for example 2,6-naphtholsulfonateP4 then the pK,* decreases due to an increase in kCB*s3' Those aromatic alcohols that do not exhibit the formation of C* from B*.X, for example, phenol and tyrosine, are relatively weaker acids in the excited state with a concomitant decrease in kCB*.7'8 To observe C* emission from tyrosinate, it has been shown that the pH must be high enough to have C in the ground state for direct ex~itati0n.l~ According to our absorbance data, the titration of 2-naphthol, 17&dihydroequilenin, and phenol by TEA in cyclohexane all result in the formation of a weak hydrogen-bonded complex in the ground state. The fluorescence maximum of the corresponding B*.X species at low TEA concentration in cyclohexane also indicates hydrogen bonding since the emission maximum is intermediate between that of the "free" alcohol in neat solvent and that of the ionized alcohol in water at high pH. The maximum for B*.X emission of 2-naphthol is at lower energy than for the alcohol in water (375 versus 360 nm, respectively), which reflects the correlation between the position of the hydrogen atom with respect to the donor and the acceptor due to the strength of the proton acceptor (TEA > water).28 The steady-state fluorescence intensity decrease and the corresponding decrease in the lifetime of the free alcohol as a function (34) Loken, M. R.; Hayes, J. W.; Gohlke, J. R.;Brand, L.Biochemisrry 1912, 11,4779-4186. ( 3 5 ) That this effect is due to kce* alone is a consequence of the reprotonation being a diffusion-controlled rate, kw*.

3002 The Journal of Physical Chemistry, Vol. 95, No. 8,1991

Hasselbacher et al.

.

rn

1

1.40 I

1.00

0.80 0.60 0.40 (D

.-e

5

O.*O

f

0.00

e

1.00

1.oo

0.80

0.60

0.60

0.60

0.40

0.40

0.20

0.20

.-e2 U

0.00 0.0001

0.00 0.001

0.0 1

0.1

1

0.0001

0.001

0.0 1

0.1

1

[TEA],M

[TEA], M

Figure 7. Basis set coefficients for the LINCS of 2-naphthol for its absorbance (panels A and C) and fluorescence (panels B and D) in cyclohexane (panels A and B) and toluene (panels C and D). Closed symbols are for the basis spectra in neat solvent, and open symbols are for the arbitrarily chosen second basis spectra. The lines through the data represent the best fits.

of increasing concentration of TEA suggests that TEA is a collisional quencher of the A* species. The rate of the quenching (> IO9 M-I s-') being in the range of diffusion-controlled reactions suggests that this is in fact a collisional process. We will now show that this quenching of A* is actually the formation of an emitting B**X species, as suggested by Matsuzaki et al.,lz and not the formation of a dark, nonemissive complex as suggested by Bisht et al."' This means that the k, value determined from the steady-state and time-resolved data is kBA* of Scheme I. If B*-X is formed from A* by its reaction with X, and is no different than B*.X formed by direct excitation of B-X, then several kinetic signatures in the time-resolved data should be observed; these are discussed below. Another consequence of a two-state reaction occurring simultaneously in the ground state and in the excited state is that the spectral contours of both the absorption and steady-state fluorescence, as a function of TEA concentration, should be a linear combination of two basis spectra. This can be demonstrated in our data (Figures 6 and 7). The resulting coefficients for the contributions of the two basis absorbance spectra show typical sigmoid behavior as expected for a titration. The coefficients in the fluorescence spectra for the increase of the B*.X species as a function of TEA concentration show a distinct maximum and then begin to decrease. In our model, the spectral coefficient, b, for the hydrogenbonded species, B*.X, will be proportional to the concentration of B**Xformed by direct excitation of B.X plus the concentration of B*-X formed from A*, taking into account the back reaction to reform A*. This is represented by b

a

tB[B.X]

+ €A[A]PBA- [B*.X]PA,

(7)

where cA and cB are the extinction coefficients for A and B-X, respectively. During the lifetimes of the excited-state species, PEA is the probability of forming B*.X from A* and PABis the probability of the dissociation of the hydrogen-bonded complex to A* and X. We expect that PABis relatively small since the dissociation rate is slow compared to the other processes, as discussed below. Since the equilibrium association constant K, = [B.X]/[A][X], and since excitation is at an isosbestic point = €6, and by ignoring PAB

b

a

[Al([XlKa + PEA)

of A* with X is the ratio of the rate of that process to all of the rates of the kinetic pathways for decay of A*: (9)

With this substitution

The extent of the deviation from a typical sigmoid curve will depend on the ground-state equilibrium, K,, which is a function of the strength of the proton acceptor and the properties of the solvent, as well as the ratio of rates, Kdc for the aromatic alcohol. The Ka dependence accounts for tfe different magnitude of this effect in cyclohexane compared to toluene. The lines through the coefficients in panels B and D of Figure I are based on the K, and kBA*values obtained by nonlinear least-squares regression. The fit for the B*.X coefficients in cyclohexane is improved by the addition of the weak (2 X lo' M-l s-I) quenching process observed by directly measuring the decrease of the B*.X lifetime at high TEA concentration^.^^ In fact, iterating for this kq term in the LINCS analysis of the fluorescence spectra yields the same value as that obtained from the SternVolmer plots based on the fluorescence decay kinetics of the shorter lifetime component. The K, values calculated from the LINCS analysis of the fluorescence spectra agree with those obtained by LINCS analysis of the absorption spectra. The higher K, values initially estimated from the fluorescence change at a single emission wavelength as a function of TEA concentration are a consequence of k,, being greater than the back reaction, kAB*. Since kAB*has not been determined, it is difficult to know whether K,* is different than

(8)

The probability of forming B*.X by the excited-state interaction

(36)It only becomes significant (>I%) above 0.1 M TEA. This depends upon the relative magnitude of the product of k,[TEA] versus k,,p

Interaction of Aromatic Alcohols with Triethylamine

-

KO;the only way K,* can be determined is to measure kAB*and calculate the kBA*/kAB*ratio. We expect that kBA kBA* since both are rates of diffusion-controlled reactions. Consequently, we can estimate kAB to be 5 X 1O7 s-' based on a K, of 100 M-' for 2-naphthol in cyclohexane. Since the excited-state alcohol is a stronger acid, kAB is an upper limit for kAB*. This justifies the assumption, stated above, that the excited-state, hydrogenbonded dissociation rate is slow, resulting in net conversion of A* to B*.X; there is no excited-state equilibrium. Solvent Effects. Chignell and Gratzer have analyzed the effects of hydrogen-bond formation and the concomitant change of solvent composition upon the absorption spectra of aromatic chromop h o r e ~ . ' ~The shift produced by hydrogen-bond formation is considered a specific solvent effect, and the shift produced by the change in solvent composition is considered a general solvent effect. As pointed out by Pimer~tel,~* the magnitude of the spectral shift produced by hydrogen-bond formation for both absorption and fluorescence depends upon the strength of the acceptor. When the hydrogen bond is stronger in the excited state, the direction of the spectral shift is to lower energy (red shift) for both absorption and fluorescence. Any additional shift observed after saturation of the hydrogen-bonded alcohol/acceptor complex is a function of the solvent composition and will be, to the first approximation, a linear function of the volume percent of the hydrogen-bonding ~olvent.~' In the case of aromatic alcohols, as the concentration of TEA increases in cyclohexane, the spectrum of the species formed in the presence of TEA begins to noticeably shift toward that observed in toluene or neat TEA. The loss of the isoemissive point in cyclohexane can be explained by this shift in the spectrum at higher TEA concentrations. In addition, the weak quenching in cyclohexane of B*.X contributes to the loss of the isoemissivepoint. Loss of the isoemissive point is not observed in toluene largely because the spectrum of the complex in toluene and neat TEA are similar. In fact, we would predict the loss of the isoemissive point in toluene because instead of a decrease in the fluorescence lifetime of the hydrogen-bonded species, as observed during the titration in cyclohexane, there is an increase in the lifetime of this species during the titration in toluene. However, this effect is too small to affect the spectra in toluene. There is also weak quenching observed for TEA titration of the steady-state emission of 2-methoxynaphthalene in both toluene and cyclohexane. In cyclohexane, the slight decrease in the steady-state emission intensity is similar in magnitude to the decrease in the fluorescence lifetime. But in toluene opposite effects are observed: up to TEA concentrations of 200 mM, while the steady-state emission intensity decreases, the fluorescence lifetime appears to increase slightly. As indicated in Table I, the changes in the fluorescence lifetime are relatively small compared with the effect of TEA upon the alcohol. In addition, the effects of TEA on the fluorescence lifetime of 2-methoxynaphthalene in the two solvents are comparable in magnitude with the effects observed on the fluorescence lifetime of the hydrogen-bonded alcohol, B*.X. We attribute the differences of the lifetime of the excited-state 2-naphthol/TEA complex (B*.X) in cyclohexane (2.3 ns), toluene (1.0 ns), and TEA (1.3 ns) to a general solvent effect. The weak quenching observed in the TEA titrations in cyclohexane is thus due to the change in solvent composition and not Stern-Volmer quenching. We also attribute the emission maximum of the hydrogen-bonded species to a general solvent effect on the character of the hydrogen bond. The general solvent effects arise from the properties of each solvent. While cyclohexane, toluene, and TEA all have similar, low dielectric constant^,'^ toluene and TEA each have permanent dipole moments. By contrast, cyclohexane is inert due to its symmetry.40 Each aromatic alcohol

-

(37) Chignell, D. A.; Gratzer, W. B. J . Phys. Chem. 1968,72,2934-2941. (38) Pimentel, G. C. J . Am. Chem. Soc. 1957, 79, 3323-3326. (39) The dielectric constants of neat cyclohexane, toluene, and triethylamine are 2.02 (20 "C), 2.38 (25 "C), and 2.42 (25 "C), respectively;National Bureau of Standards Circular 514, abstracted in the CRC Handbook of Chemistry ond Physics, 52nd ed.;The Chemical Rubber Co.: Cleveland, OH.

The Journal of Physical Chemistry, Vol. 95, No. 8, 1991 3003 has a permanent dipole moment. It can be reasoned, on the basis of dipole-dipole interaction, that toluene and TEA will be better solvents for aromatic alcohols than cyclohexane. In addition, toluene and TEA will mutually compete for "solvation" of the aromatic alcohol. Thus, as the mole fraction of TEA increases in a cyclohexane solution, the free alcohol and the hydrogenbonded species are preferentially solvated by TEA. The net effect is that complex formation is more favorable in cyclohexane than toluene. In toluene, in addition to dipole-dipole interaction, there is dipole-induced dipole interaction due to the polarizability of the A electrons of toluene. The possibility for dipolar interactions results in a greater charge separation in the alcohol/TEA complex. Consequently, the hydrogen-bonded aromatic alcohol in toluene or neat TEA becomes more ion-like. Thus, the fluorescence lifetime of B*.X is more similar in toluene and neat TEA, and the emission band is at lower energies than in cyclohexane. The similarity of the emission energies of B*.X in toluene or in neat TEA to the emission of the fully ionized species, C*, in basic aqueous solution could suggest complete ionization of the complex in toluene or neat TEA. These solvents, however, as a result of their low dielectric constants and lack of a hydrogen-bonding network, will not support complete ionization. B*.X, therefore, must be essentially an ion pair in these polar solvents, and a hydrogen-bonded species in cyclohexane. Excited-State Kinetics. As noted earlier, if A* converts to B*-X at a diffusion-controlled rate in these organic solvents, then an excited-state reaction is occurring and the decay of B*.X should be the sum of two emission-wavelength-independent exponentials with one negative amplitude term. Our data, however, show that both A* and B*.X appear to decay as single exponentials. We also do not observe a negative amplitude term, as predicted by the excited-state reaction, even on the extreme low energy side of the B*-X emission where it should be easier to observe due to the decrease of spectral overlap from A* emission. This lack of the expected kinetic signatures for an excited-state reaction caused Bisht et al."' to infer that the collision of TEA with A* results in a dark complex that is different from the B*.X species formed by direct excitation of B.X. While the existence of a negative amplitude term in a multiexponential decay is proof of an excited-state reaction, the absence of the negative amplitude does not immediately eliminate the existence of an excited-state reaction. Furthermore, the fact that both A* and B*.X apparently decay as single exponentials does not rule out an excited-state reaction. The decay of the B*.X species can be evaluated from its differential rate expression:

Integration with an initial boundary condition that B-X is in the ground state, yields

[B**X]$-kf,~f(1 3) Thus, we expect the decay of B*.X to be the sum of two exponentials. There is a finite initial concentration of B*.X, formed by direct excitation of ground-state B-X, which will have the same lifetime, (kf,B)-', with a positive amplitude as the kinetic component for the decay of B*.X, formed in the excited state from A* by collision with X, which has a negative amplitude term. Consequently, depending on the relative ground-state concentrations of A and B.X, the initial excited-state concentrations of A* and B*.X, and the rate of formation of B*-X from A*, the resulting amplitude term for this kinetic component could be positive, negative, or even zero. This situation is demonstrated in Table (40) The dipole moments of toluene and triethylamine are 0.36 and 0.66, respectively; National Reference Data Series No. IO, National Bureau of Standards 10, from: CRC Handbook of Chemistry and Physics, 52nd ed.; The Chemical Rubber Co.: Cleveland, OH.

3004 The Journal of Physical Chemisrry, Vol. 95, No. 8, 1991 TABLE II: Estimates of B*.X Formation by Two Plthwr~s'

I

0.01 0.02 0.10 0.50 1.00

0.32 0.48 0.82 0.96 0.98

0.50 0.33 0.09 0.02 0.01

0.50 0.67 0.91 0.98 0.99

0.16 0.16 0.07 0.02 0.01

0.01 0.05 0.10 0.25

0.32 0.70 0.82 0.92 0.97 0.98 0.99

0.99 0.95 0.91 0.80 0.57

0.01 0.05 0.09 0.20 0.43 0.50 0.67

0.31 0.66 0.75 0.74

0.75 1.00

2.00

0.50 0.33

0.55 0.49 0.33

'Assumes equal extinctions for A and B-X, and [XI>> (A] + [B-X]. Based on values for 2-naphthol in cyclohexane: kBA* = 6 X IO9 M-' s-', and kt,a = 1.3 X IO8 s-'. "he probability of forming B*.X from A* (eq 9). CMolefractions of A and B-X; MA+ MB.x = I . 11, which summarizes the effects of K, on the sign and magnitude of this amplitude. When the value of K, is similar to that observed for the hydrogen-bonded complex of 2-naphthol and TEA in cyclohexane, the negative amplitude is masked because the concentration of the ground-state complex is greater than that which can be formed in the excited state. Even if the value of K, is a factor of 100 smaller, the negative amplitude will be observed up to only 1 M acceptor. The decay of A* is expected to be a TEA concentration dependent single exponential since the excited-state reaction is essentially irreversible on these time scales. Consequently, any kAB* terms can be neglected. The rate expression under these conditions is

which integrates to a single exponential with a decay time that depends on the concentration of X. At low TEA concentration (up to 20 mM), B*-X is actually decaying as a double exponential. The lifetime initially associated with the decay of A* has amplitude components from both the A* and the B*-X species. Thus, as the concentration of X increases, the fraction of the amplitude for this kinetic component that comes from B*-X kinetics will increase (Table 11). This explains the shift in the DAS to lower energy for this longer lifetime component as a function of TEA Concentration. This DAS, therefore, consists of spectral contributions from both the free alcohol and the hydrogen-bonded complex. If there were sufficient points in the DAS, it could be analyzed by LINCS. The shift in the DAS of the longer lifetime component as a function of TEA concentration is proof that the exciplex is emi~sive.~' The shorter lifetime associated with the decay of the B*.X species, which could have a negative amplitude based on the excited-state reaction, has a positive amplitude because of the initial boundary conditions having a finite initial concentration of B*-X due to direct excitation of B.X. Bisht et al."' do observe a negative amplitude in solvents which are more polar than toluene. Their results are consistent with our mechanism. As noted above, more polar solvents will tend to solvate the donor and the acceptor individually and thus inhibit complex formation. For example, they report ground-state equilibrium constants less than 10 M-' for TEA complexes with 2-naphthol in ethanol, acetonitrile, or dimethyl sulfoxide9 Consequently, at each concentration of TEA there is less B.X available for direct excitation to B*.X. Therefore, the negative amplitude associated with exciplex formation can be observed. At the higher TEA concentrations (20.2 M), where we examined the solvent composition effect, the decay of B*.X becomes a single exponential. This is because [BeX] >> [A] and, conse(41) An exception would bc the situation in which B*.X has complex decay kinetics leading to the sum of two exponentials with one lifetime the same as that for A*, including its TEA concentration dependence. This is not likely.

Hasselbacher et al. quently, there is effectively no A* to form the B*.X complex (Table 11). Elimination of the excited-state reaction results in a rate expression for the decay of B*.X that leads to a single exponential. Between these two concentration ranges, it might be possible to demonstrate the excited-state reaction by the decay kinetics. It will be complicated, however, since the lifetime of the A* species will first approach and then become shorter than the "lifetime" of the B*.X species. Since in this situation the decay rates will be similar, resolution of individual lifetimes will be difficult.

Summary Two views might be given to describe the ground-state species in which there is a "strong" hydrogen bond with an acceptor.42 At one extreme, there could be a resonance form, ArOH-X ArO--.HX+, which is characterized by one potential energy minimum, determined by the electron density properties of the donor and acceptor. Alternatively, there could be a tautomeric equilibrium, ArOH-X ArO--HX+, which possesses two energy minima. Both situations could result in a B*.X species with a single, unique lifetime (other than from an excited-state reaction). In the first situation, only the electron density needs to change following excitation. In the second situation, ArOH*-X, the form on the left, represents the initial Franck-Condon state constrained by the Born-Oppenheimer approximation. ArO*--HX+, the form on the right, would represent the final excited-state configuration in which the electrons have moved and the proton has shifted closer to the acceptor. In either view, the form on the right is the ion pair. If there is a tautomeric equilibrium rather than resonance, we expect that the rate for ion-pair formation is fast compared to that for fluorescence decay. Ion dissociation represents an additional step. Even though there might be only a small difference between the excited-state energies of the ion pair and the completely dissociated ions, for example, 2-naphthol in toluene versus basic water, the decay kinetics of these two forms are significantly different. Our results for the aromatic alcohols in low-dielectric polar and nonpolar solvents show that the degree of charge transfer from the alcohol to TEA in the hydrogen-bonded complex depends on solvent polarity and polarizability. In cyclohexane, which is nonpolar and has low polarizability, the complex is a hydrogenbonded species and formation of the ion pair is inhibited. In toluene, which is polar and more polarizable, the proton moves closer to'the acceptor and formation of the ion pair is essentially complete. The differences between cyclohexane and toluene are reflected by the fluorescence lifetimes and emission energies of B*-X in the two solvents. In either solvent, there is no observable difference between the excited-state properties of the exciplex or the complex directly excited from the ground state. On the basis of these observations, we can make predictions about the interactions of the tyrosine residue in a protein or a 2-naphthol-containing estrogen in the presence of a strong proton acceptor such as a primary amine. Examples of other acceptor groups in a protein include carboxyl and imidazole side chains; the acceptor strength of these groups is reflected by their pK,. We conclude that emission in these circumstances will be from the excited-state free and hydrogen-bonded species; the fully ionized alcohol does not occur in the excited state. Based on the lifetime and emission energy of the alcohol/acceptor complex in the protein, its local environment can be characterized in terms of polarity and polarizability, as well as the strength of the acceptor.

-

f

Acknowledgment. We thank Dr. John Spudich and his group for the use of their absorption spectrophotometer. We also thank Drs. Ludwig Brand, Robert DeToma, Jay Knutson, and Paolo Neyroz for helpful discussions, and Dr. Y. Nemerson for his generous support and continued interest. In particular, we thank Drs. Arthur Szabo and Kevin Willis for their detailed reading of this manuscript and their helpful comments, and for sharing (42) Ratajczak, H. J . Phys. G e m . 1972, 76, 3000-3004.

J . Phys. Chem. 1991,95, 3005-301 1

3005

A.R.). E.W. was supported by National Institutes of Health Grant HL-29019 (Y.Nemerson).

their data on the fluorescence decay kinetics of tyrosinate and tyrosine hydrogen-bonded complexes in water. This work was supported by National Institutes of Health Grants GM-12231 (C.A.H.), GM-39750 (J.B.A.R.), and DK-39548 (W.R.L.); and by National Science Foundation Grant DMB-85-16318 (J.B.

Registry No. TEA, 121-44-8; 2-naphtho1, 135-19-3; 17&dihydroequilenin, 1423-97-8; phenol, 108-95-2.

Reactions of Ground-State and Electronically Excited Sodium Atoms with Methyl Bromide and Molecular Chlorine P. S. Weiss,f J. M. Mestdagh,t H. Schmidt,* M. H. Covinsky, and Y. T. Lee* Department of Chemistry, University of California, and Chemical Sciences Division, Lawrence Berkeley Laboratory, Berkeley, California 94720 (Received: October 17, 1990)

The reactions of ground- and excited-state Na atoms with methyl bromide (CH3Br) and chlorine (CI,)have been studied by using the crossed molecular beams method. For both reactions, the cross sections increase with increasing electronic energy. The product recoil energies change little with increasing Na electronic energy, implying that the product internal energies increase substantially. For Na + CH,Br, the steric angle of acceptance opens with increasing electronic energy.

Introduction The reactions of ground-state alkali-metal atoms (M) with halogen-containing molecules (RX) have been extensively studied for more than 50 years.’-27 The present work is a continuation of the systematic study we have undertaken of the reactivity of excited Na The aim is to determine the changes in the reaction dynamics when electronic energy is deposited in a reactant, here the Na atom. In this paper, we study reactions which for ground-state N a atoms are representative of rebound and stripping mechanisms; these are the reactions of Na with methyl bromide and chlorine molecules, respectively. Na(3S,3P,4D) CH3Br NaBr CH, (1) Na(3S,3P) CI2 NaCl + CI (2) For the reactions of ground-state alkali-metal atoms with halogen and methyl halide molecules the mechanism has an electron transferring from the alkali-metal atom to the halogencontaining molecule. This electron goes into the lowest unoccupied molecular orbital which is an antibonding orbital of the R-X bond. For the methyl halide molecules this causes the rupture of this bond, as the negative ion is not bound. For the halogen molecules, the negative ion is formed on a repulsive part of the potential near the dissociation limit (to halide ion and halogen atom), and the field of the alkali-metal ion is strong enough to dissociate the halogen negative ion.*v9 Of the methyl monohalides, only the reactions of alkali-metal atoms with CH31and CH3Br have been studied in crossed molecular beams including the reactions of all the stable alkali-metal metals with CH31.’O

+ +

-

TABLE I: Measured Atomic a d Molecular Beam Velocities a d Speed Ratios beam seed gas beam velocity, cm/s s p e d ratiog Na helium 3.00 x 105 6 Na neon 1.60 x 105 5 Na argon 1.08 x 105 5 6.1 x 104 5 C12 5.8 x 104 5 CH,Br CH3Br helium 1.26 X IO5 4

“ v l b v fwhm.

+

pumped the Na atoms to the Na(32P3,2,42D5,2) levels in the interaction regiona2” The atomic and molecular beam conditions are given in Table I. The N a atomic velocity distributions were measured by the laser technique described in refs 4 and 5. The molecular beam velocity distributions were measured by the time-of-flight technique using a mass spectrometer.

Other Possible Processes A . Na + CH3Br. A number of processes other than (1) are possible in the collisions of electronically excited N a with CH3Br as discussed below. ~

(1) Polanyi, 1932.

Experimental Section As described previously, these experiments were conducted in a crossed molecular beams apparatus in which supersonic atomic sodium and molecular chlorine or methyl bromide beams crossed at 90°, and scattered products were detected with an ultrahigh-vacuum mass spectrometer equipped with an electron bombardment ionizer which rotates about the interaction r e g i ~ n . ~ - ~ In addition, one or two single-frequency CW,dye lasers optically ‘Permanent address: Department of Chemistry, The Pennsylvania State

University, University Park, PA 16802.

‘Permanent address: CEN Saclay, SPAS Bt. 62.91 191 Gif sur Yvette,

France.

‘Permanent address: Braun A. G. Forschung, Frankfurter Str. 145, D6242 Kronberg, Federal Republic of Germany.

0022-3654/91/2095-3005%02.50/0

~~

M. Atomic Reactions; Williams and Northgate: London,

(2) Vernon, M. F.; Schmidt, H.; Weiss, P. S.;Covinsky, M. H.; Lee, Y. T.J . Chem. Phys. 1986,84, 5580. (3) Weiss, P. S.;Mestdagh, 3. M.; Covinsky, M.H.; Balko, B. A.; Lee, Y. T.Chem. Phys. 1988, 126,93. (4) Weiss. P. S.Ph.D. Dissertation. Universitv of California. Berkelev. CA..

1986,‘ ( 5 ) Vernon, M.F. Ph.D. Dissertation, University of California, Berkeley, CA, 1983. (6) Weiss, P. S.;Covinsky, M. H.;Schmidt, H.; Balko, B. A.; Lee,Y. T.; Mestdagh, J. M. 2.Phys. D. 1988, I O , 227. (7) Mestdagh, J. M.; Balko, B. A.; Covinsky, M. H.; Weiss, P. S.;Vemon, M. F.; Schmidt, H.; Lee, Y. T. Furaduy Discuss. Chem. Soc. 1987,84, 145. (8) Birely, J. H.; Herschbach, D. R. J . Chem. Phys. 1966, 44, 1690. (9) Birely, J. H.; Herm, R. R.; Wilson, K.R.; Herschbach, D. R. J . Chem. Phys. 1967, 47, 993. (10) Herm, R. R. In Alkali Halide Vapors: Structure, Spectra, and Reuction Dynumics; Academic Press: New York, 1979; Chapter 6 and references therein.

0 1991 American Chemical Society