J. Phys. Chem. 1986, 90, 5058-5063 Tai for help in acquisition of the data and the use of some computer programs. The electron impact mass spectra were provided by Mr. Dilip Sen Sharma and Mr. John Wells at the UCLA Chemistry Department Mass Spectrometry Laboratory. J.D.S. thanks Dr. Jory Yarmoff for his assistance in the development
of the computer hardware and software used in these experiments. D.M.S. appreciates useful discussion on the thermochemistry of fragment ions with Prof. R. L. Whetten. Registry No. p-DCB, 106-46-7.
Photophysics of the Aniline Blue Fluorophore: A Fluorescent Probe Showing Specificity toward (1-+3)-~-~-Giucans P. Thistlethwaite,*1. Porter, Department of Physical Chemistry, University of Melbourne, Parkville. Victoria 3052, Australia
and N . Evans CSIRO Division of Protein Chemistry, Parkville, Victoria 3052, Australia (Received: November 12, 1985; I n Final Form: April 18, 1986)
The photophysics of a fluorophore, sodium carbonylbis(4-(phenyleneamino)benzenesulfonate), which occurs as an impurity in the dye Aniline Blue, have been studied. This fluorophore has found important application in histological studies of certain polysaccharides in plant tissues. The fluorescence lifetime and quantum yield of the fluorophore show a dramatic solvent dependence resulting from changes in both the radiative and nonradiative decay rates. The photophysical data suggest that emission is from a highly dipolar, intramolecular charge-transfer excited state. The marked enhancement of fluorescence that occurs on complexing of the aqueous fluorophore by a (1+3)-p-~-glucan has also been studied, and thermodynamic parameters for the interaction have been obtained. The results provide some insight into the ability of the fluorophore to act as a specific fluorescent probe for polysaccharides having the (1-.3)-@-~-glucan structure.
Introduction
For many years the sulfonated triarylmethane dye, Aniline Blue, has been used as a specific stain in histological studies of the distribution and physiology of polysaccharides known as callose.’ Arens2 and later other^^-^ showed that when commercial Aniline Blue is used to stain callose tissues, an impurity present in the dye emits bright yellow-green fluorescence when irradiated with ultraviolet light. The distribution of fluorescence in tissues treated with the Aniline Blue fluorophore (ABF) is the same as that of the Aniline Blue itself, but the fluorescence method gives greater sensitivity and there is better contrast between the callose deposits and other tissue Recently, ABF has been isolated from commercial Aniline Blue and its structure confirmed by spectroscopic methods and synthesis as sodium carbonylbis(4-(phenyleneamino)benzenesulfonate)* (Figure 1). A preliminary study of the fluorescence properties of the fluorophore has shown that while the fluorescence emission and excitation spectra are not greatly affected by the solvent, the fluorescence quantum yield is dramatically solvent dependent.* In aqueous solution the fluorophore is virtually nonfluorescent but is much more fluorescent in alcohols, with the intensity increasing with decline in the alcohol polarity. One of the most interesting aspects of the fluorophore’s behavior is its apparent specificity. It is induced to fluoresce most strongly in the presence of ( 1 4 3 ) - P - ~ - g I u c a n s . ~ The nature of the binding of the fluorophore to the polysaccharide and the reason for the enhanced fluorescence of the (1) Fincher, G. B.; Stone, B. A. In Encyclopaedia of Plant PhysioiogyNew Series; Tanner, W., Loewus, E. A., Eds.: Springer-Verlag: Heidelberg, 1981; Vol. 13B,p 68. ( 2 ) Arens, K. Lilloa 1949, 18, 71. (3) Fidalgo, 0. Ann. Bot. (Rome) 1954, 24, 431. (4) Currier, H. B.; Strugger, S . Proloplasma 1956, 45, 552. (5) Currier, H. B. Am. J . Bot. 1957, 44, 478. (6) Eschrich, W.; Currier, H. B. Stain Technol. 1964, 39, 303. (7) Smith, M. M.; McCully, M. E. Protoplasma 1978, 95, 229. (8) Evans, N . A.: Hoyne, P. A. Aust. J . Chem. 1982, 35, 2571. (9) Evans, N. A,; Hoyne, P. A,; Stone, B. A. Carbohydr. Polym. 1984,4, 215.
0022-3654/86/2090-5058.$01.50/0
TABLE I: Absorption and Emission Maxima and Stokes Shift for the Aniline Blue Fluorophore in Various Solvents Stokes Xabmax, Xemmax,
solvent H20 MeOH
EtOH PrOH
DMF CH,CN 96% glycerol-water complexo
ET( 30) >
nm
nm
cm-’
e
kJ mol-’
377 368 372 374 371 360 377 390
520 496 490 487 499 497 500 510
7294 7013 6474 6204 6914 7657 6525 6033
78.3 32.6 24.3 20.3 36.7 31.5 42.5
264 232 211 212 183 192 238
shift,
“See text.
bound fluorophore are not at present understood. The aim of this work was the elucidation of the fundamental photophysics of ABF with a view to understanding the reason for the fluorescence enhancement in the presence of (1+3)-@-D-glucans. Experimental Section
ABF was synthesized as described earlier.* All solvents were checked for fluorescing impurities and redistilled as required. Absorption spectra were recorded on a Cary 17 spectrophotometer. Uncorrected emission spectra were recorded on a Perkin-Elmer MPF-44A spectrofluorimeter and then corrected by using a previously determined correction curve.1° Fluorescence quantum yield measurements were made on solutions of absorbance less than 0.05 with recrystallized quinine bisulfate in 0.5 M H2S04 as reference.” The sample and reference solutions had approximately equal absorbances at the excitation wavelength in order to minimize errors due to differences in optical geometry. No evidence of sample deterioration with the time was found. Steady-state fluorescence measurements below room temperature were made using an Oxford Instruments DN 704 cryostat. Above (10) Ghiggino, K. P.; Skilton, P. F.; Thistlethwaite, P. J. J . Phorochem. 1985, 31, 111.
(11) Demas, J. N.; Crosby, G. A. J . Phys. Chem. 1971, 75, 991.
0 1986 American Chemical Society
Photophysics of Aniline Blue Fluorophore
The Journal of Physical Chemistry, Vol. 90, No. 21, 1986 5059 TABLE II: Fluorescence Quantum Yields, Lifetimes, and Radiative and Nonradiative Rates for the Aniline Blue Fluorophore in a Series of Solvents
Figure 1. Structure of the Aniline Blue fluorophore.
room temperature a water-heated cell holder supplied by a thermostated water bath was used to give temperature control to within 0.2 "C. Fluorescence decay times were measured by using a mode-locked Nd:glass laser/streak camera system. Single pulses of the third harmonic (A = 351 nm, T~ = 8 ps, E = 0.1 mJ) were used to excite the sample, and the emission was observed at right angles through a polarizer set at 54.7O in order to prevent distortion of decay curves by rotational relaxation. Decay curve analysis, including streak camera response function deconvolution, was accomplished using a standard least-squares iterative convolution technique.
Results Absorption Spectrum. The absorption spectrum of the aqueous fluorophore consists of a strong band at 371 nm and a weaker band at 275 nm. The position of the longest wavelength absorption band varies considerably with solvent (Table I). The molar absorptivity (e, N 27000 M-'cm-I in water and within experimental error the same in other solvents) indicates a *T,T transition. However, as in the related molecules 1-(phenylamin0)naphthalene (1 - A N ) and 8-(pheny1amino)-1naphthalenesulfonate (1,8-ANS),I2 it is likely that excitation leads to some transfer of electronic charge from the N lone pairs into the aromatic rings, and possibly to the CO or SO< groups. The solvent-induced spectral shifts are rather irregular. The absorption lies furthest to the red in the most polar solvent, water. However, in methanol it is 4 nm to the blue of its position in the less polar ethanol. The observed shifts in the protic solvents can be explained in terms of an interplay of polarity and H-bonding effects. If the excited state has some charge-transfer character, polar solvents would be expected to give a red shift of the absorption because of the greater dipole-dipole stabilization of the polar excited state relative to the ground state. On the other hand, interaction of H-bond donor solvents with the N lone pairs will tend to stabilize the ground state more than the excited state, leading to a blue shift in more H-bonding solvents. This argument implies a blue shift going from an aprotic solvent such as DMF to a protic solvent such as MeOH, of comparable dielectric constant. It cannot however explain the large blue shift observed in acetonitrile. Emission Spectrum. The corrected emission maxima and Stokes shift (taken as the difference in frequency between absorption and emission maxima) are also given in Table I, together with some solvent properties. While among the protic solvents there is a trend to a more red-shifted emission with an increase in polarity, over the whole range of solvents there is no clear correlation between either the emission maximum or Stokes shift and either the dielectric constant or ET(30) parameter.I3 The Stokes shift is large and approximately equal in all solvents, suggesting that the excited state is highly polar and interacts strongly with polar solvents. Radiative and Nonradiative Rates. Table I1 gives the fluorescence decay times and quantum yields, at room temperature, together with the derived radiative and nonradiative rates. In all cases the fluorescence decays were well fitted to singleexponential functions. The results show that the dramatic solvent dependence of quantum yield is the result of changes in both the radiative and nonradiative rates. Unlike 1,8-ANS,I2the quantum yield is low in all the polar solvents studied. The radiative rates of Table I1 may be compared with the predictions of the Strickler-Berg relationI4 k , = 2.88
X
10-9n2(~;3)a;'Je d In B
(12) Sadkoski, P. J.; Fleming, G. R. Chem. Phys. 1980, 54, 79. (13) Reichardt, C . Angew. Chem., In?. Ed. Engl. 1979, 18, 98.
solvent
H2O MeOH EtOH PrOH DMF
CH,CN 96% glycerol-water
complex
of 0.0003 0.0030 0.0088 0.032 0.041 0.026 0.041 0.043
10-7kr, Tf,
ns
0.027 0.048 0.068 0.127 0.379 0.122 0.136 0.370
s-I 1 6.3 13 25 11 21 31 12
io-9kn,, S-'
37 21 15 7.66 2.53 7.98 6.95 2.67
A
Figure 2. Temperature dependence of the fluorescence spectrum of the Aniline Blue fluorophore in 95% ethanol-water: 1, 110 K 2, 120 K; 3, 130 K, 4, 140 K; 5, 150 K; 6, 160 K; 7, 180 K.
The major influence on the radiative rate comes from the solvent dependence of the emission frequency. A further smaller effect arises from small variations in molar absorptivity and from the variation in absorption frequency with solvent. The radiative rate is predicted to be lowest in the aqueous solution which has the most red-shifted emission. ( P{~),, was determined by taking the ratio of areas under plots of I#{3 and If vs. B ~ ,where If is the fluoresceRce intensity. For aqueous solution the calculated radiative rate is 13 X IO7 s-l. It is clear that the observed variation is too large to be explained by the variation in emission frequency. The latter can only account for a variation in k , by a factor of less than 2. This suggests that, in water and the more polar alcohols, relaxation from the Franck-Condon state is to a fluorescent state of much lower oscillator strength and that the oscillator strength varies with the solvent. Temperature Effects. The temperature dependence of the emission spectrum of the fluorophore in 95% ethanol-water is shown in Figure 2. (95% ethanol-water was chosen because of its good glass-forming properties.) As the temperature is raised from 110 to 180 K, the uncorrected emission maximum shifts from 428 to 457 nm. At the same time the width at half-maximum of the spectrum increases from ca. 2400 to 4300 cm-I, suggestive of incomplete solvent relaxation round an increasing solvent dipole, No isoemissive point is seen. The temperature dependence of the fluorescence quantum yield in pure ethanol was studied over the temperature range 200-250 K. Assuming the radiative rate is approximately constant over this small temperature range, and given that k,, >> k,, the quantum yield is inversely proportional to kn,. A plot of In Ifvs. 103/T was found to be linear, confirming that k,, follows an Arrhenius relation and supporting the assumption of constant radiative rate. The activation energy was found to be 11.2 kJ mol-'. For comparison, a value of 6.4 kJ mol-' has been reported for the activation energy of the nonradiative rate of 1,8-ANS in e t h a n ~ l . ' ~As the temperature is raised from 200 to 250 K, the (14) Strickler, S . J.; Berg, R. A. J . Chem. Phys. 1962, 37, 814.
5060 The Journal of Physical Chemistry, Vol. 90, No. 21, 1986
/,’
\
t-
2
\
Iio
Thistlethwaite et al.
4 1
II 1000
10YT
(K-’)
Figure 3. Temperature dependence of fluorescence intensity for the Aniline Blue fluorophore: (a) in water; (b) in 96% glycerol-H,O; (c) complexed by polysaccharide;(d) fit of experimental data for the complexed fluorophore using W = -51 kJ mol-’ and E A = 7.6 kJ mol-’ (see text). 0,experimental points; solid line, fitted curve.
uncorrected emission maximum shifts from 466 to 472 nm and the width at half-maximum rises to 4700 cm-I. The reducing rate of red shift with increase in temperature at these higher temperatures suggests that solvent relaxation is likely to be complete by room temperature, in line with the observation of single-exponential decay. The temperature dependence for the fluorophore in water, in 96 wt % glycerol-water, and complexed by aqueous polysaccharide (see later), was studied in the temperature range 25-50 “C. The results are shown in Figure 3. In water the activation energy is 5.2 kJ mol-I. In this regard the fluorophore’s behavior parallels that of 1,8-ANS where a near-zero activation energy is reported in water and a somewhat higher value in ethan01.l~The data for 96 wt % glycerol-water show a slight deviation from Arrhenius behavior. Fitting to an Arrhenius relation gives an activation energy of approximately 34.9 kJ mol-]. It will be suggested elsewhere that the radiative rate in ABF is dependent on relaxation to an intramolecular charge-transfer state. If this is so, then the radiative rate might be expected to be temperature dependent, and this would invalidate the assumption of the foregoing section. However, over a small temperature range and in temperature regions where the solvent is quite fluid and solvent relaxation is fast, the radiative rate variation is likely to be small. The observation of approximate Arrhenius behavior of the nonradiative rate derived as above supports this view. The polysaccharidecomplexed fluorophore exhibits a large temperature dependence which deviates markedly from Arrhenius behavior (see later). Isotope Effect. The Aniline Blue fluorophore shows an appreciable solvent deuterium isotope effect. The values for 4(D20)/4(H@), d(MeOD)/4(MeOH), and d(EtOD)/d(EtOH) are 1.38, 1.60, and 1.52, respectively. These values are somewhat larger than those reported for 1,8-ANS.12 Fluorescence Enhancement by Binding to a (1-3)-p-Oligoglucoside. The effect on the fluorescence quantum yield of complexing of the fluorophore by a (1-+3)-~-oligoglucoside was studied. The sample of polysaccharide was supplied by Professor B. Stone of LaTrobe University, Melbourne, and had been prepared by Dr. K. Ogawa, Laboratory of Biophysical Chemistry, College of Agriculture, University of Osaka. It was prepared by hydrolysis of Curdlan, and a narrow molecular weight range sample was isolated by chromatography. The degree of polymerization was ca. 14. Binding of the fluorophore to the oligoglucoside affects the absorption spectrum which now exhibits a maximum at 390 nm and a shoulder at 368 nm. The total integrated absorption intensity, however, remains constant. The equilibrium constant for complexing can be determined by absorption measurements provided the free and complexed fluorophore differ appreciably in absorption spectrum. This has been done for example in the case of complexing of 2-naphthol by (15) Nakamura, H.; Tanaka, J. Chem. Phys. Lett. 1981, 78, 5 7
2000
[PIo-’ (M-’)
Figure 4. Plot of the reciprocal of the fluorescence intensity vs. the reciprocal of the initial polysaccharide concentration for the Aniline Blue fluorophore in aqueous solutions of a (1-.3)-~-~-0ligoglucoside(degree of polymerization, 14).
cyclodextrins.I6 Where the absorption spectra differ only slightly, this method will be subject to large error. Moreover, the absorption method requires that the spectrum of the completely complexed fluorophore be known. This is a disadvantage in the present case where a strictly limited amount of the purified oligoglucoside was available. For this reason the equilibrium constant for complexing was determined from fluorescence measurements. In solutions in which the initial polysaccharide concentration is much greater than the initial fluorophore concentration, the equilibrium constant for complex formation is given by
where [C] is the concentration of complex, [PI, is the initial concentration of polysaccharide, and [F], is the initial concentration of fluorophore. Thus
But [C] is proportional to the fluorescence intensity, If,provided (1) the fluorescence of uncomplexed fluorophore may be neglected and (2) the concentration of complex is sufficiently low that concentration is proportional to absorbed, and hence emitted, light intensity. In this case - =- k
If
[FIO
+
k ~e,~Plo[Flo
where k is a constant. Thus, a plot of l / I f vs. 1/[P], at fixed [F], will give a slope of k/(Keq[FIo)and an intercept of k/[F],, thus enabling determination of Kq. Figure 4 shows such a plot (for [F], = 7.8 X M) and yields a value of Kq of 570 f 50 M-I. This value is comparable to those found for the complexing of 2-naphthol16 and 2-p-toluidino-6-naphthalenesulfonate(2,6-TNS)” by P-cyclodextrin. For the complexing of 2,6-TNS by amylose of degree of polymerization 17, an association constant of 17.6 M-’ has been reported.” The temperature dependence of the nonradiative rate for the aqueous fluorophore has been found to be small. If the polysaccharide-complexed fluorophore still experiences an essentially aqueous environment, then the large temperature dependence seen for the complexed fluorophore (Figure 3) must be mainly the result of shifts in the ground-state equilibrium constant for complex formation, and this gives rise to the curvature in the plot of Figure 3. If the nonradiative rate for the complexed fluorophore follows an Arrhenius relation, and given that the fluorescence quantum (16) Yorozu, T.; Hoshino, M.; Imamura, M.; Shizuka, H. J . Phys. Chem. 1981, 85, 1820. ( 1 7 ) Nakatani, H . ; Shibata, K. I.; Kondo, H.; Hiromi, K. Biopolymers 1971, 16, 2363.
Photophysics of Aniline Blue Fluorophore
The Journal of Physical Chemistry, Vol. 90, No. 21, 1986 5061
These changes were attributed to a relaxation from the FranckCondon state to an intramolecular charge-transfer (ICT) state, this relaxation being facilitated by suitable polar solvents and possibly also by a change in molecular g e ~ m e t r y . ’ ~Intramo-~’ IT - (1 + c) exp[(A + _ lecular charge transfer in the related arylaminonaphthaleneI298 1 Cexp(Ax) sulfonates has been extensively studied by Kosower and cow o r k e r ~ . ~The ~ - ~above ~ observations suggest that the emission Here, C = K2ss[P]o, A = A H o / R , where AHo is the enthalpy of ABF might also be from a highly dipolar excited state in which change in complex formation, B = -EA/R, where EA is the acelectronic charge has been transferred away from the N atoms. tivation energy for the nonradiative rate, and x = (298-’ - T’). In the cases cited above, a progressive decline in radiative rate C is a known constant with a value of 1.247. The experimental (increasing charge-transfer character) correlated with an indata of Figure 3 are well fitted with E A = 7.6 kJ mol-’ and AH creasing Stokes shift, as the solvent polarity (measured by the = -51 kJ mol-’. As expected, the value of 7.6 kJ mol-’ is close ET(30) parameter) increased. The absence of this correlation in to that found for the fluorophore in water, supporting the view the present case may possibly be explained by the argument that the complexed fluorophore experiences an essentially aqueous already advanced in relation to the absorption shifts. environment. Increased charge-transfer character in the relaxed excited state The quantum yield for the second solution in Figure 4 (2.2 X will result in increased dipole-dipole stabilization compared to M in oligoglucoside) was measured and found to be 0.0239. the ground state, implying an increased Stokes shift. At the same The fraction of fluorophore complexed is calculated from time depletion of N lone-pair charge leads to lower stabilization of the excited state by H-bond donor solvents than occurs in the KPIo CY= ground state, thus tending to decrease the Stokes shift. The Stokes 1 + KPIO shift will also be influenced by how the N lone-pair charge is redistributed. In the case of 1,8-ANSit was suggested that charge to be 0.56. The quantum yield for the complex-uncomplexed transferred from the N atom migrated to the SO< group, making fluorophore mixture can be calculated from it a much better H-bond acceptor in the excited state.I2 This view ~5 = 0.56& 0.44& = 0.0239 was supported by the observed correlation of the Stokes shift with solvent ET(30)parameter. As the ET(30) scale assigns a higher provided the molar absorptivity at the excitation wavelength is “polarity” value to a protic solvent, compared to an aprotic solvent the same in both cases. Thus, & = 0.0427. This result confirms of the same dielectric constant,13 it was concluded that a major the earlier assumption that even for the solution of lowest olifraction of the observed Stokes shift was due to stabilization of goglucoside concentration, where CY is calculated to be 0.167, the the excited state by H bonding at the SO< group. If, in the present fluorescence is virtually all from complexed fluorophore. Comcase, formation of the ICT state is not accompanied by increased plexing increases the quantum yield by a factor of approximately H-bond accepting character of the SO3-groups, a major source 140. This figure agrees well with an earlier relative quantum yield of excited-state stabilization is removed. Smaller Stokes shifts of 124 obtained by Evans et aL8g9 might then be expected, and the correlation between Stokes shift The fluorescence decay time of the fluorophore in the most and ET(30) would be less likely. The importance of such specific concentrated of the foregoing oligoglucoside solutions was meainteractions has previously been noted in other cases where highly sured and found to be 370 f 50 ps. In this solution the fluorophore dipolar excited states are i r n p l i ~ a t e d . de ~ ~Haas and Warman,26 is ca. 80% complexed. On the streak camera sweep speed used, using the time-resolved microwave conductivity technique, have no fast component due to the uncomplexed fluorophore was deobserved large variations in photophysical behavior of 4-(ditected. The radiative and nonradiative rates for the complex are methy1amino)nitrostilbene in a range of solvents having a very given in Table 11. similar e and ET(30) values and have attributed these variations Because of the possibility that the fluorophore is loosely held to specific short-range interactions that influence the relative in some sort of inclusion complex, it is of interest to use positions of * X , B and * r , n states having markedly different pofluorescence polarization measurements to assess the degree to larities. These observations suggest that a solute-solvent exciplex which the complexed fluorophore is free to move independently could provide an alternative explanation to that of a solventof the polysaccharide. Polarized emission spectra were obtained stabilized large dipole. Because of the systematic change in by the use of polarizes placed in the emission and excitation beams radiative rate going from water to propanol and the increase in of the fluorimeter, and the emission anisotropy was calculated fluorescence bandwidth with temperature, the ICT hypothesis must by using the method of Price et a1.18 The emission anisotropy be favored. However, the likelihood that specific, as well as found for the aqueous complexed fluorophore was equal within dipole-dipole, interactions are important suggests that it is unexperimental error to that found for the fluorophore in a highly reasonable to expect a smooth variation in photophysical properties viscous medium (96 wt % glycerol-water). From this lack of with ET(30). Unfortunately, studies of ABF in other less polar rotational depolarization it can be concluded, via the Perrin solvents are very difficult because of the extremely low solubility. equation, that in an aqueous solution of the complex the fluoroStudies of the N,N-dimethylamide derivative, which would be more phore and oligoglucoside rotate as a single unit with a rotational soluble in low-polarity solvents, would be of interest. correlation time (as would be expected from the oligoglucoside The formation of ICT states has previously been linked to a molecular weight, ca 2300) much greater than the fluorescence change in the configuration of amino groups.19-21,27~28 For 1decay time of 370 ps. yield is inversely proportional to the nonradiative rate, it can be shown that the ratio of the fluorescence intensity at T to that a t 298 K is given by
+
+
Discussion Photophysics of the Aniline Blue Fluorophore. ( a ) Nature of the Excited State. The observed variation in radiative rate is much too large to be explained by the shift in emission frequency and indicates that in the more polar solvents the oscillator strength of the fluorescent state is much lower than that of the FranckCondon state. Changes in radiative rate with solvent that exceed those predicted from the Strickler-Berg relation have previously been reported for 1-aminonaphthalene and its derivative^.'^-^^ (18) Price, T.M.; Kaihara, M.; Howerton, H. K. Appl. Opt. 1962, I , 521. (19) Meech, S. R.; O’Connor, D. V.; Phillips, D. J. Chem. SOC.,Faraday Trans. 2 1983, 79, 1563.
(20) Drew, J.; Thistlethwaite, P. J.; Woolfe, G. Chem. Phys. Lett. 1983, 96, 296.
(21) Li, Y. H.; Chan, L. M.; Tyer, L.; Moody, R. T.; Himel, C. M.; Hercules, D. M. J . Am. Chem. SOC.1975, 97, 3 1 18. (22) Huppert, D.; Kanety, H.; Kosower, E. M. Faraday Discuss. Chem. SOC. 1982, 72,161. (23) Kosower, E. M.; Huppert, D. Chem. Phys. Lett. 1983, 96, 433. (24) Kosower, E. M.: Kanetv. H.; Dcdiuk. H.; Striker. G.; Jovin. T.;Boni, H.;‘Huppert, D. J. Phys. Chem. 1983, 87, 2479. (25) Visser, R. J.; Varma, C. A. G . 0.;Konijnenberg, J.; Bergwerf, P. J. Chem. SOC.,Faraday Trans. 2 1983, 79, 347. (26) de Haas, M. P.; Warman, J. M. Chem. Phys. 1982, 73, 35. (27) Kirkor-Kaminska, E.; Rotkiewicz, K.; Grabowska, A. Chem. Phys. Lett. 1978. 58. 379. (28) Rotklewicz, K.; Grellmann, K. H.; Grabowski, Z. R. Chem. Phys. Lett. 1973, 19, 315. ~
5062 The Journal of Physical Chemistry, Vol. 90, No. 21, 1986
amin~naphthalene,'~ a red shift of emission occurred with increase of temperature in a temperature range where the solvent was essentially rigid and solvent relaxation could be ruled out. This was taken as evidence for excited-state stabilization as a result of a minor conformational change. Figure 2 shows that in the present case, although most of the observed Stokes shift arises at temperatures above the point where the solvent becomes fluid, there is an approximately 8-nm red shift between 110 and 140 K. This may indicate that the formation of the fluorescent state is associated with a change in configuration of the nitrogen atoms from tetrahedral to planar. ( b ) Decay Mechanism. The large temperature dependence of k,, and its marked variation with solvent suggest that solvent reorganization is involved in the decay. At the same time this evidence of solvent participation in the decay is a further argument for the existence of an ICT excited state. It would be expected that the reverse charge transfer involved in the decay of such a highly dipolar state would depend on the ability of the solvent to adjust to the altered polarity. A significant activation energy associated with solvent rearrangement and large solvent dependence could be anticipated. Although decay by intersystem crossing to the triplet state could also be highly solvent dependent, the large activation energies observed here would not be expected. Similarities of structure suggest that, as in 1,8-ANS,29photoionization of ABF might occur in aqueous solution. However, whereas in 1,8-ANS there is a 30-fold increase in nonradiative rate going from methanol to water,I2 there is a less than 2-fold increase in the present case, suggesting that the nonradiative pathways are substantially the same in both solvents. Photoionization of ABF is quite unlikely in the alcohols, where the rate of the necessary solvent reorganization is likely to be too slow to compete with other processes that deactivate the dipolar precursor state.30 If photoionization were a major decay pathway in aqueous solution, the decay rate would be expected to drop more dramatically going from water to ethanol. Decay by reverse charge transfer is expected to exhibit an appreciable solvent isotope effect as observed. Changes in equilibrium geometry, and hence vibrational energy, of solventsolute H bonds in going from the initial polar excited state to the much less polar ground state will introduce into the decay rate expression Franck-Condon factors which will be more favorable for OH than OD vibrations. A similar explanation has been given for the isotope effect in 1 ,8-ANS31 Decay by reverse charge transfer is expected to depend on both the polarity and the viscosity of the solvent.22 High solvent viscosity might also be expected to inhibit ICT state formation.22 This, and the higher radiative rate observed, might suggest that, in glycerol, the excited state lacks charge-transfer character and decays by some other means. The high activation energy for the decay is, however, most consistent with the appreciable solvent reorganization associated with reverse intramolecular charge transfer. Around room temperature the temperature variation of the decay rate of the excited state of ABF is less rapid than would be suggested by an inverse dependence of this rate on solvent viscosity. Kosower and ~ o - w o r k e r shave ~ ~ , suggested ~~ that the formation and decay of ICT states are governed by the solvent dielectric relaxation rates. The decay of the ICT state of four 6-(phenylamino)-N,N-dimethyl-2-naphthalenesulfonamidederivatives was studied in methanol-dioxane and water-dioxane mixed solvents.24 An increase in nonradiative decay rate with ET(30) was observed and was interpreted in terms of an increasing dielectric relaxation rate with increasing solvent polarity. The Debye relaxation rate of the polar component was implicated. The longest Debye relaxation time for the alkanols is associated with the breaking of H bonds followed by molecular reorientation. The longest Debye relaxation times (at 293 K) for the alcohols32and (29) Fleming, G. R.; Porter, G.; Robbins, R. J.; Synowiec. J . A . Chem. Phys. Lett. 19778, 52, 228. (30) Moore, R. A,; Lee. J ; Robinson, G. W. J . Phys. Chem. 1985, 89. 3648. (31) Lee, J.; Robinson, G. W. J. Phys. Chem. 1985, 89, 1872. (32) Chase, W. J.; Hunt, J. W. J. Phys. Chem. 1975, 79, 2835.
Thistlethwaite et al. TABLE III: Debve Relaxation Times at 293 K
solvent H2O MeOH EtOH
T, PS
9.2 52 191
solvent PrOH CH,CN
T, PS
430 3.9
the single relaxation time for a ~ e t o n i t r i l are e ~ ~listed in Table 111. Although the trend of nonradiative decay rates in the alcohols approximates very roughly to that of the dielectric relaxation rates, for acetonitrile there is no correlation at all. It is noteworthy that in the earlier studies carried out in mixed solvents the dielectric relaxation time in all cases decreased steadily with increase in solvent ET(30) parameter. For the solvents used in the present work this is not so. While it is reasonable that the rate of intramolecular charge transfer should be related to the ability of the solvent dipoles to accommodate the new charge distribution, there are likely to be other factors involved. The change in solvent-solute H bonding in passing from the dipolar ICT state to the ground state leads to the involvement of Franck-Condon factors in the radiationless transition rate. This would be expected to introduce differences in decay rate for the different solvents. Influence of Polysaccharide Complexing. For the complexed fluorophore, the increased quantum yield compared to that for the free, aqueous fluorophore is due to changes in both the nonradiative and radiative rates. The much higher radiative rate suggests an inhibition of ICT state formation in the complexed fluorophore. The Stokes shift is lower than for any other solvent. The nonradiative decay rate is reduced by a factor of 14 compared to that in water. Both these changes could be explained by the polysaccharide providing a partially hydrophobic, less polar environment for the fluorophore, as occurs in an inclusion complex. Alternatively, H bonding between the fluorophore and the polysaccharide, possibly via the N atoms, could inhibit ICT state formation. Our data show that the 14-fold decrease in k,, is due mainly to a reduction in the preexponential factor. The preexponential factor reflects the extent of solvent structure breaking associated with intramolecular charge transfer and rises from water, to ethanol, to 96 wt % glycerol. For the complexed fluorophore it must be concluded that the polysaccharide influences the solvent in the vicinity so as to make the entropy terms less favorable. No simple picture of this process is apparent at present, although thermodynamic studies confirm that the polysaccharide will have a major influence on the neighboring water s t r u c t ~ r e . ~ ~ ~ ~ ~ While modification of the interaction with water in the presence of the polysaccharide is plausible, there is the further problem of the particular ability of (1-+3)-P-~-glucansto diminish the nonradiative rate. Earlier observations implied the importance of polysaccharide conformation, both in relation to the superior fluorescence enhancement found for the (1-+3)-P-D-glUCanS compared to other polysaccharides and in relation to the differences between the (1+3)-P-glucans themselves. For the (1 -3)-P-~-oligoglucosides the fluorescence enhancement was found to increase markedly as the degree of polymerization (DP) rose to 20, the point at which the conformation changes from random coil to open Moreover, the fluorescence is reduced at pH greater than 12, when the (1-+3)-P-~-glucansadopt a random-coil c ~ n f o r m a t i o n . ~ These ~ observations do not allow a distinction to be drawn between differing degrees of complexing and differences in the extent of suppression of the complexed fluorophore's nonradiative rate. The determination of association constants for a wide range of complexes and the detection of any nonfluorescent complexes are beyond the scope of this paper. The idea of strong complex formation only with (1-+3)-@-~-glucans seems the more plausible, and our thermodynamic data allow some conclusions. (33) Colonomos, P.; Wolynes, P. G. J . Chem. Phys. 1979, 71, 2644. (34) Taylor, J. B.; Rowlinson, J. S. Trans. Faraday SOC.1955, 51, 1183. (35) Suggett, A. In Water-A Comprehensive Treatise; Franks, F., Ed.; Plenum: New York, 1973; Vol. 4, p 519. (36) Saito, H.; Ohki, T.; Sasaki, T. Biochemistry 1977, 16, 908. (37) Ogawa, K.: Tsurugi, J.; Watanabe, T. Carbohydr. Res. 1973, 29,397.
Photophysics of Aniline Blue Fluorophore The equilibrium constant of 570 M-’ corresponds to a AGO of -15.8 kJ mol-I. This, together with the AHo of -51 kJ mol-’, gives a ASoof -1 18 J K-’ mol-’. This large negative entropy change indicates a highly specific interaction in which the polysaccharide is induced to take up a much more ordered (probably helical) conformation. It has previously been reported that (1+3)-pglucans of D P < 20 take up an ordered conformation in the presence of Congo Red.38 The above hypothesis would be consistent with the higher fluorescence enhancement observed with oligoglucosides of higher degree of polymeri~ation.~ Where the polysaccharide tends to adopt the helical conformation before interaction with the fluorophore, a higher equilibrium constant for complexation would be anticipated. Moreover, it is known that the nature and extent of hydration of a sugar (and hence possibly its ability to inhibit interaction between the fluorophore and water) depend upon the s t e r e o ~ h e m i s t r y . ’ ~The ~ ~ ~AHo of -5 1 kJ mol-’ is equivalent to only one strong H bond. However, this AHD represents the difference in enthalpy between H bonds broken and those formed in complex formation. As the polysaccharide will be, initially, very strongly H bonded to solvent water, the AHo of -51 kJ mol-I indicates quite strong H bonding between the fluorophore and polysaccharide. Thus, it seems likely that the fluorescence enhancement in the presence of (1+3)-pglucans is the result of specific, strong H-bond interactions, rather than the formation of an inclusion complex, as is thought to occur in the well-known complex between iodine and amylosem and as has been suggested for the complexing of 2,6-TNS by amylose of degree of polymerization 17.I’ The stability of inclusion complexes is usually attributed to “hydrophobic bonding”, with an appreciable, if not major, contribution arising from the positive entropy change. For example, the AHo and ASofor the formation of an inclusion complex between 2,6-TNS and P-cyclodextrin are reported to be -8.8 kJ mol-’ and 15.5 J K-’ mol-’, respectively, while for the inclusion complex between 2,6-TNS and amylose (DP 17) the values are -2.1 kJ mol-’ and 17.6 J K-’ mol-’.” In the latter case the helical amylose chain was thought to provide a partly hydrophobic environment for the 2,6-TNS. However, molecular models of a single (1-3)-~-oligoglucoside chain show that it is unrealistic to envisage the helical structure enclosing the fluorophore in a substantially hydrophobic environment. Moreover, if the effect on the ABF was due to its enclosure in a hydrophobic environment provided by the helical (1+3)-P-glucan structure, it is not clear why amylose could not also provide a similar hydrophobic environment. The alternative hypothesis of strong H bonding to the polysaccharide inhibiting ICT state formation, and thus raising the radiative rate, together with the water structuring properties of the polysaccharide reducing the nonradiative decay rate seems more plausible. The idea of H bonding as the source of complexing is also supported by infrared data. The fluorophore shows two strong, sharp IR bands at 1593 (38) Ogawa, K.;Hatano, M. Carbohydr. Res. 1978, 67, 527. (39) Barone, G.;Cacace, P.; Castronuovo, G.; Elia, V. Carbohydr. Res. 1981, 91, 101.
(40) Foster, J. F. In Starch: Chemistry and Technology;Whistler, R. L., Parschall, E. F., Eds.; Academic: New York, 1965;Vol. 1, p 349.
The Journal of Physical Chemistry, Vol. 90, No. 21, 1986 5063 and 1640 cm-’ which are attributable to the C=O stretch and N-H bend, respectively. The rather low value for the C=O stretch is consistent with the presence of a para amino group. A sample of the fluorophore, together with a large excess of the (1+3)-p-D-glUCan, Sclerotan, was dissolved in water and subsequently freeze-dried. The 1640-cm-’ band is no longer seen, and the C=O stretch now occurs at 1580 cm-’. This drop in frequency by 13 cm-’ is consistent with H bonding of the C=O group of the fluorophore. The preceding discussion applies to complexing by low molecular weight oligomers which exist in solution as single polysaccharide chains. At much higher molecular weights in solution4143and in the solid the (1+3)-P-~-glucans will adopt a triple-helix structure, In this case molecular models suggest that provision of a partly hydrophobic environment by grooves in the triple-helix structure might further reduce the nonradiative decay.
Conclusion The dramatic solvent dependence of the fluorescence quantum yield of ABF results from changes in both the radiative and nonradiative rates. The marked reduction in radiative rate in polar protic solvents indicates that in these solvents emission is from a state of different electronic character to that attained on excitation. The radiative rate variation with solvent, together with other observations, suggests that emission occurs from an intramolecular charge-transfer state. The large Stokes shift at room temperature and the red shift in emission that accompanies the transition from rigid to fluid polar solvent point to a highly dipolar excited state. The large solvent-dependent activation energy of the nonradiative rate and the significant solvent isotope effect are compatible with a role for solvent reorganization in the decay, as would be expected for an ICT state. ABF forms a complex with a (1+3)-@-~-glucan(DP 14) which is ca. 140 times more fluorescent than the free aqueous fluorophore. The driving force for complexing is the large negative enthalpy change. The large negative entropy change on complexing suggests that H bonding, rather than hydrophobic bonding as occurs in inclusion complexes, is the source of binding.
Acknowledgment. We thank Professor B. Stone of La Trobe University for many helpful discussions on this topic and Professor G. W. Robinson of Texas Tech University for clarifying a number of points. Financial support by the Australian Research Grants Scheme is also gratefully acknowledged. Registry No. DMF, 68-12-2;HzO, 7732-18-5;MeOH, 67-56-1; EtOH, 64-17-5;PrOH, 71-23-8;CH,CN, 75-05-8;glycerol, 56-81-5; sodium carbonylbis(4-phenyleneamino)benzenesulfonate,103693-84-1; (1+3)-(3-~-glucan, 9051-97-2. (41) Yanaki, T.;Norisuye, T.; Fujita, H. Macromolecules 1980, 13, 1466. (42) Norisuye, T.;Yanaki, T.; Fujita, H. J . Polym. Sci., Polym. Phys. Ed. 1980, 18, 547. (43) Kashiwagi, Y.; Norisuye, T.; Fujita, H. Macromolecules 1981, 14, 1220. (44) Deslandres, Y.; Marchessault, R. H.; Sarko, A. Macromolecules 1980, 13, 1466.
