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Static and dynamic fluorescence quenching of carbazole by tropanic alkaloids
1. Photochern. Photobiof. A:
Chem., 66 (1992)
171
171-184
Static and dynamic fluorescence quenching of carbazole by tropanic alkaloids E. M. Talavera,
B. Quintero
and J. M. Alvarez
Departamento de Quimica Fisica, Facultad de Farmacia, Campus Universitario de Cartuja, 18071 Granada (Spain) (Received
December
3, 1991; accepted January 30, 1992)
Abstract state fluorescence quenching of carbazole by tropanic derivatives in cyclohexane and acetonitrile was performed. The results obtained using cyclohexane as solvent support the existence of two species in equilibrium: free and hydrogen-bonded complexed carbazole in both the ground and excited states. The parameters involved in the static and dynamic quenching were obtained separately and the thermodynamic parameters corresponding to these associations were calculated. In acetonitriJe, the equilibrium in the excited state is established between carbazole and its proton phototransfer product. Steady
1. Introduction Fluorescence quenching can provide valuable information about the structure and dynamics of proteins and receptors [l-3]. TJ-ms the quenching of fluorescent probes by ions in the presence and absence of agonists and antagonists has been used in the characterization of the nicotinic receptor of acetylcholine (ACH) 141. However, a full identification of the subtypes of the muscarinic receptor of ACH as well as its nature and structure still remain to be characterized. Since tropanic alkaloids act by the specific blocking of the muscarinic receptor, we have investigated the fluorescence quenching of carbazole induced by tropanic alkaloids in order to improve the knowledge of the physical and chemical properties of these antagonists of ACH in the nanosecond domain. The fluorescence properties of carbazole and its derivatives have been widely studied because of the structural relationship with tryptophan. Due to the presence of an electron-deficient nitrogen atom and the high polarity of the amine group, fluorescence quenching of carbazole is mainly caused by the formation of a hydrogenbonded complex [5-71 and increases when the polarity of the solvent is such that it acts as a proton donor rather than a proton acceptor in the excited state [8]. However, it has also been reported that a charge-transfer interaction, other than hydrogen bonding, may contribute to the fluorescence quenching of carbazole. In a previous paper [9], we reported some aspects of the fluorescence quenching of carbazole by tropanic derivatives in polar and non-polar homogeneous media. In this paper, the dependence of the equilibrium on temperature has been studied and the essentially dynamic nature of the quenching is established. In addition, the existence of only two emitting species under the experimental conditions used has been checked.
lOlO-6030/92/$5.00
Q 1992 - Elsevier Sequoia.
AiJ rights reserved
172 2. Experimental
details
Carbazole and N-ethylcarbazole (99%, Aldrich) were recrystallized twice from absolute ethanol, dried and resublimated before use. Endo-S-methyl-S-azabicyclo[3.2.1 Joctan-3-01 (tropine) and e-(hydroxymethyl)benzeneacetic acid S-methyl-8-azabicyclo[3.2.l]oct-3-yl ester (atropine) (both of analytical grade) were donated by Boehringer Ingelheim; they were recrystallized twice from n-heptane, dried and stored in a desiccator. The desiccator containing the tropanic derivative was kept at 45 “C (tropine) or 90 “C (atropine) for 24 h. Cyclohexane (CH), ethanol, butanol and acetonitrile (AN) (for fluorescence spectroscopy, Merck) were used as solvents without further purification. All solvents were checked for purity by absorption and emission spectroscopy. The absorption spectra were recorded using a Perkin-Elmer Lambda 5 spectrophotometer. A solution containing the same concentration of tropanic derivative was used as a blank. Solutions were kept for at least 30 min in the thermostatically controlled sample compartment before the measurements were performed. Measurements of stead state fluorescence were made using a Shimadzu RF-5000 spectrofluorometer with 15 K and 30 A bandpass for excitation and emission respectively, Excitation was performed at an isosbestic point. All spectra were corrected for the photomultiplier response. The temperatures of the samples were controlled by a watercirculating external bath. In order to eliminate the inner filter effect due to weak absorption of tropanic alkaloids at the excitation wavelength, the ceI1 used in the fluorescence measurements had light paths of 2 mm in the excitation direction and 10 mm in the emission direction. A front-face arrangement was also used, but an increase in the amount of scattered light produced poorly resolved spectra. In some experiments molecular oxygen was removed by bubbling with argon for 3 min; this enabled fluorescence signals with higher intensity and accuracy to be obtained. However, since the flow of argon into the cell leads to a decrease in temperature, air enters the cell while the desired temperature is attained (cell not air tight)_ More reliable results were obtained when the sample was kept under conditions in which the desired temperatures (f 1 K) were reached after bubbling with argon. Lifetime measurements were obtained using an EEY nanosecond fluorometer operating in time-correlated single-photon counting mode. A hydrogen lamp (approximately 10 atm) was used with a frequency of 30 kHz, supplying very stable lamp pulses with a halfwidth of 2.2 ns. Sample excitation was performed at 300 nm using an interference filter FT-300 (bandwidth, 15 nm) and the emission was filtered by a Corning 7-60 filter. All the samples were aerated and controlled at 293 K. The decay curves were analysed by a least-squares reconvolution program. The goodness of the fit was estimated from the reduced ,$ value.
3. Results
and discussion
The spectral modifications caused by the addition of tropanic derivatives (atropine or tropine) to carbazole solutions in apolar solvents, together with the absence of such modifications in hydroxylated solvents and the inability of these alkaloids to quench the fluorescence of N-ethylcarbazole, have been interpreted to be due to the formation of a hydrogen-bonded complex in both the ground and excited states 191. The appearance of isosbestic points in the absorption spectrum [9] and an isoemissive point in the emission spectrum (Fig. l(a)) suggests that the equilibrium involves only
173
a
b
Fig. 1. Change in carbazole fluorescence with tropine concentration in CH solution (A,,=321 nm). Concentrations: (a) carbazole, 2.5 X lo-§ M; tropine, 0.0,0.25 X lo-*, 1.5 x lo-* and4.75 x lo-’ M; (b) carbazole, 2.5 x lo-’ M; tropine, 0.1 M. two species: free and complexed carbazole [lo, 111. Moreover, it is observed that the emission band (about 334 nm) decreases in intensity as the tropine concentration is increased and at a concentration above 0.1 M becomes almost negligible (Fig. l(b)). Considering that this emission is due to free carbazole, excitation spectra were recorded by fixing the emission wavelength at 334 nm. As shown in Fig. 2(a), the spectra obtained with the carbazole-tropine system vary in intensity, but no change is observed in the shape or the positions of the band maxima which are identical to those found in the absorption spectrum of carbazole. By fixing the emission wavelength at 350 nm, the spectra shown in Fig. 2(b) are obtained which match the absorption spectrum recorded with the carbazole-tropine system [9]. These results indicate that the emission at 334 nm is due to excited free carbazole, whereas the emission at 350 nm results from the superimposed emission of the two species involved in the excited state equilibrium. Under these conditions, it is possible to separate the spectrum of each species. Thus, the fluorescence of the carbazole-tropine system in Fig. 3 has been normalized by taking as reference the intensity measured at 334 nm for carbazole in CH. From the resulting spectrum, the reference spectrum of carbazole solution is subtracted.
b
Fig. 2. Excitation spectra centrations of tropine: (a) (-) and 14.35 x 10e2 M 14.35 x 1O-2 M (- - a) (Aa
of carbazole (2.5X lo-’ M) (-) and carbazole-tropine system. Con0.75 X lo-’ (- - -), 2.27 X lo-’ (- - -), 4.75 X lo-* (. . -), 10.57 x IO-’ (a * *) (AEM= nm); (b) 2.27~ IO-’ (--- -), 10.57~ 10m2 (- - -) and -350 nm).
The spectra obtained in this way are shown in Fig. 4 together with the emission spectrum recorded on excitation at 337 nm of a carbazole-tropine solution (carbazole, 5 x lo-’ M; tropine, 0.1 M). This wavelength (337 nm) is characteristic of the new band which appears in the absorption spectrum of the carbazole-tropine system [9]. It can be seen that the shape of the spectrum obtained by excitation at 337 nm is similar to that derived from the normalization-subtraction procedure. Since free carbazole does not absorb at 337 nm, it seems reasonable to attribute the spectra in Fig. 4 to the complexed species. By measuring the absorbance at 340 nm of carbazole solutions in CH (5 x lo-’ M) containing different amounts of tropine, it is possible to cakulate Kg, the equilibrium constant for the ground state reaction, using
(1)
175
Fig. 3. Emission spectra of carbazole (5 X lo-’ M) and tropine (0.0, 1 X 10B3, 2 X lo-‘, 4 X 10e3, 5 X 10e3, 6 X 10e3, 8 X lop3 and 10X 10e3 M) in CH (A,, =321 nm).
3 X lo-‘,
An appropriate plot of the experimental data gives straight lines. The ordinate of the lines is equal to -Kg_ The values of Kg can, in turn, be plotted vs. l/T according to the van? Hoff equation to calculate Lw, AS and AG corresponding to the association reaction in the ground state. Table 1 summarizes the results obtained in this analysis. The thermodynamic data can be interpreted as a consequence of a spontaneous exothermic reaction where a molecular reorganization process takes place. The determination of I& could not be performed for the carbazole-atropine system because of the low solubility of atropine in CH. From Kg, we can calculate K,, the equilibrium constant for the association reaction in the excited state, using [9, lo] logK,=log&+
0.625 8y T a
where 6v, represents the spectral shift determined as either the difference between the absorption maxima positions expressed in wavenumbers (cm-‘) or one-half of the sum of the frequencies of the absorption and fluorescence peaks (cm- ‘). Some discrepancy can arise from the method used to determine 6u but, in any case, KB is much lower than K, [lo].
Fig. 4. Emission spectra of complexed species obtained 321 nm and spectra recorded for a carbazole-tropine [tropine] = 9~ lo-’ M) at hnx = 337 nm.
TABLE
from normalization
procedure
system ([carbazole]
=5
x
lo-’
at Anx= M and
1
Values of the equilibrium states, and thermodynamic
constants for the association reactions in the ground and excited parameters corresponding to the carbazole-tropine system in CH - AC,=
- AGeb
T WI
Kg
KG”
KGb
(1 mol-‘)
-AC, (kI mol-‘)
(kJ mol-‘)
(W mol-‘)
283 293 303 313
25.11 18.34 10.57 5.11
387.27 257.64 136.09 60.63
1111.25 715.64 365.47 157.77
7.58 7.09 5.95 4.24
14.02 13.52 12.38 10.68
16.51 16.01 14.87 13.17
(1 mol-‘)
(1 mol-‘)
-AHS=39.0 W mol-‘; -AHe”=45.61 kJ mol-‘; -AHeb=48.04 mol-‘; -AS,‘= 110.5 J K-r mol-‘; -ASeb= 110.2 J K-r mol-‘. ‘Values obtained using au,=538 cm-‘. bValues obtained using Sv,=746 cm-‘.
kJ mol-‘;
-AS,=llO.O
J K-’
The results obtained from the analysis performed according to eqn. (2) and by application of the van’t Hoff equation are also given in Table 1. In cases where combined static and dynamic quenching are present and the cause of the quenching is a hydrogen-bond interaction, Weller’s reaction scheme [12] can be accepted in principle. This provides a very complicated equation for the determination of the relative fluorescence yields [lo] if an appreciable overlap occurs between the
177
spectra of the free and complexed species. However, for the carbazole-tropine system a simplification can be made. Thus, on the basis of the assignment of the signal at 334 nm to free carbazole, the equation for the determination of the relative fluorescence yields can be written as (3) where F and F0 are the fluorescence intensities in the presence and absence of tropine respectively, 7 is the fluorescence lifetime of carbazole, k, is the bimolecular constant for the quenching process, [Q] is the quencher concentration and 6 is the light fraction absorbed by free carbazole, obtained from
6 = l/(1 + (E&E&[Q]}
(4)
This expression becomes more simple when excitation is carried out at- an isosbestic point (as has been performed in our experiments). The relative fluorescence intensity data measured at 334 nm for solutions of carbazole-tropine in CH (tropine concentration, not greater than 0.03 M) were used to plot eqn. (3). Ordinate values close to unity and determination coefficients within the range 0.99-1.00 were found in all cases. In Fig. 5 the fluorescence decay curve of a carbazole solution (5X 10e5 M) in CH is shown together with the value of the fluorescence lifetime at 293 K. From the fluorescence intensities obtained for carbazole solutions at different temperatures in
20,
1
0 0
5
10
15
20 TIME
25
30
35
40
45
50
(NSECI
Fig. 5. Fluorescence decay curve of carbazole the fluorescence lifetime.
solution (5 X10w5 M)
in CH and the value of
178
the range 283-313 K, no significant change in the value of 7 is expected. Taking the slope of the plot of eqn. (3) and 7, the value of the forward constant kr for the association reaction in the excited state can be calculated; in our case, this corresponds to the value of the bimolecular constant for the diffusion-controlled process of quenching, since the inverse rate constant for dissociation of the complex in the excited state (I+) is negligible in comparison with the rate of unimolecular deactivation of the complex [13]. All the above-mentioned values are included in Table 2. From these values and the CH viscosities (also included in Table 2), a plot of kI vs. T/v was made. A straight line is obtained (Fig. 6) supporting the dynamic nature of the quenching of the carbazole fluorescence by tropine in CH. According to the Smoluchowski-Einstein diffusion theory, the bimolecular constant (k,,) for a diffusion-controlled process is given by k,(r) = 477..
TABLE
oD{ 1 + a/( &r)“L)
(5)
2
CH viscosities and photophysical, kinetic and dynamic parameters process of the carbazole-tropine system in CH
K, (1 mol-‘) kIX1O-g (I mol-’ k,XlO_’ (s--l) 7 (-9 D,X106 (cm’ s-l) D,X lo6 (cm’ s-‘) k, x lo-’ (I mol-’ Y R:,=3.93 R,=3.26
s-‘)
s-l)
for the bimoIecular deactivation
283 K
293 K
303 Is
313 K
34.36 4.17 0.37
36.99 4.48 0.63
38.36 4.65 1.27
44.25 5.36 3.4O
A A.
k+O-'
6
4
31 200
I
I
400
300 T'rl
Fig. 6. Plot of k, vs. T/T for the carbazole-tropine
system in CH.
600
179
After electronic excitation, excited molecules with a quencher molecule inside their active sphere will be immediately deactivated (in less than lo-” s); only those molecules not deactivated in this way will suffer decay as a consequence of normal emission and difFusiona quenching. Several experimental and theoretical studies by Ware and coworkers [14-161 have been focused on the investigation of Stern-Volmer deviations due to static quenching in the active sphere. No upward deviations were observed in the system studied here and the viscosities of the solutions used were low; therefore the influence of static quenching in the active sphere has been ruled out. The straightforward application of eqn. (5) allows us to determine k. using the value of the diffusion coefficient D obtained from the Stokes-Einstein equation and the molecular radii calculated from the van der Waals’ volumes [17]. A comparison of k. with kl gives the value of the efficiency parameter y for diffusional encounters. The values of y shown in Table 2 indicate that tropine is an efficient quencher of carbazole fluorescence. The influence of oxygen on the kinetic parameters was tested since most of the experiments were carried out with aerated samples. Data from deaerated samples (argon bubbling at 303 K) were analysed by applying the same methodology as used with the aerated samples. The Stern-Volmer constant found for deaerated samples is 75.03 1 mol-‘. Using the carbazole lifetime in the absence of oxygen reported in the literature (16.1 ns [18]), a value of 4.66 X 10m9 mol-l s-l is obtained for kl in good agreement with the value found for aerated samples at the same temperature (see Table 2). Similar results were reported by Martin and Ware [6] for the carbazole-pyridine system. In Fig. 7 plots of Fe/F (fluorescence intensities measured at 350 nm) ~1s. tropine concentration at the four temperatures used in our study are shown. The shapes of these plots obey the general equation derived from Weller’s scheme [12]; admitting that the two species emit at 350 nm and free carbazole fluorescence is more intense
FJF 1.3
1,2
101
1
0
1
2
3
4
5
6
[QLlOe+2
Fig. 7. Plots of F,JF vs.tropine concentration: 0, 283 K, A, 293 K, *, 303 K; 0,
313 K.
180
than that of complexed carbazole. It can be seen that the asymptotic portion occurs at smaller F,/F values with increasing temperature which can be justified by the decrease in K, as the temperature is increased. Moreover, the asymptotic portion is reached at a smaller concentration of quencher as the temperature is increased which is in good agreement with the expected relationship between kl and T/q. The qualitative similarity between the carbazole-atropine and carbazole-tropine systems in CH must be emphasized. Nevertheless because of the low solubility of atropine, Kg cannot be calculated. This is the reason for restricting the analysis to only two temperatures, 303 K and 313 K. In general, it is expected that, as the temperature is increased, the amount of atropine dissolved will also increase; the choice of these temperatures is related to the range of biological interest and the limitations imposed by the solvent. Under our experimental conditions, it is assumed that there is no association in the ground state; in addition, the light fraction S is approximately unity due to the low concentration of the quencher. Under these circumstances, the K,, values shown in Table 3 were obtained by applying the Stern-Volmer equation to the fluorescence data measured at 334 nm. Other photophysical parameters are also included in Table 3. When AN is used as solvent, the free carbazole absorption spectrum shifts 270 cm-’ to the red with respect to that recorded in CH. Moreover, the three isosbestic points obtained on addition of tropanic derivatives are also red shifted with respect to the isosbestic points in CH (323,332 and 336 nm). However, the absorption spectral change induced by tropanic derivatives is very slight despite their high concentration due to good solubility in AN. Figure 8 shows the emission spectra obtained on addition of tropine (concentration range, 2.5 x 10V3-2.5 x 10-l M). Excitation was performed at the lower energy isosbestic point. Appreciable fluorescence quenching is observed as well as the appearance of a new well-structured band with maxima at 418 nm and 443 nm and a shoulder at 478 nm. An isoemissive point located at 397 nm is also present. Concentrations of tropine higher than 0.95 M produce changes in the spectral shape to give a structureless band centred at 420 nm. In addition, the emission spectra of carbazole in AN made alkaline with NaOH or NH40H were recorded; a new band appears which has similar features to that described above on addition of the tropanic derivatives. Emission spectra were also recorded using carbazole-tropinesolutions before and after acidification with acetic acid. Acetic acid inhibits the fluorescence quenching and, as a consequence, leads to the disappearance of the band at longer wavelengths [9]_
TABLE
3
Photophysical, kinetic and dynamic parameters for the carbazole-atropine
K, (1 mole’) /clX foe9 (1 mol-’ RQ DQX
/c,X
Y
s-l)
CA) 10”
(cd
s-l)
10m9 (I mol-’
s-l)
system in CH
303 K
313 K
47.57 5.77 4.07 6.57 8.19 0.70
55.07 6.68 4.07 8.16 10.10 0.66
181
.,.~.,._ . -... _.
Fig. 8. Fluorescence spectra of carbazole (5 X 10e5 M) and tropine (0.0, 1 X lo-‘, nm). 4X 10e2, 7X lo-‘, 0.1, 0.15, 0.2, 0.25 and 0.95 M) in AN solution (A,,=323
~X~CJ-~,
Interaction between heteroatomic compounds and bases in aprotic solvents produces the formation of hydrogen-bonded complexes and ionic pairs; these pairs may or may not be separated by solvent molecules depending on the strength of the base and the polarity of the solvent [19]_ Mataga and Kubota [lOI have emphasized that ionic pair formation and ionic separation by solvent molecules can occur due to a hydrogenbonded interaction in the fluorescent state. Therefore the long-wavelength band and the experimental behaviour shown by the carbazole-tropanic derivatives in AN can be attributed to a proton phototransfer product. The recently reported pK,* value of carbazole in the excited state [20], the polarity of AN and similar assignments found in the literature [21-231 indicate that the new band in these systems can be attributed to the anion derived from carbazole. The spectra of free carbazole and its anion can be separated using the normalization-subtraction procedure described earlier. In this case the fluorescence at 343 nm clearly originates from free carbazole since the shape of this band is unaffected by the addition of tropine (Fig, 8). In Fig. 9 the spectra of carbazole and its anion are shown separately. The anion emission is less structured and bathochromically shifted with respect to the emission of the hydrogen-bonded complexed carbazole (Fig. 4).
182
P
183 TABLE
4
Photophysical, T (K)
kinetic
and dynamic kr x 10-s (1 mol-’
&v (1 mol-‘)
parameters 77(cP)
s-‘)
for carbazole-tropanic DIX 16 (cm’ s-t)
derivatives
DoXld (cm2 s-*)
in AN
k,x lo-‘0 (1 mol-’ s-t)
’ l
283” 293a 303” 313a
12.15 12.42 12.54 12.68
1.63 1.67 1.69 1.70
0.394 0.358 0.333 0.315
1.34 1.53 1.70 1.85
1.61 1.84 2*04 2.23
1.61 1.83 2.03 2.22
0.10 0.09 0.08 0.08
283b 313b
6.08 6.14
0.82 0.83
0.394 0.315
1.34 1.85
1.29 1.79
1.59 2.21
0.05 0.04
“Carbazole-tropine bCarbazole-atropine
system. system.
The results and interpretation discussed in this paper suggest that the reaction between carbazole and the tropanic derivatives in polar aprotic solvents proceeds via the following mechanism
FH
+
Q
+ Kil hvjaji~t”
F*H
+
Q
+
kz
F*H...Q
k3,
F*-
+ HQ+
scheme is similar to that proposed for the reaction in apolar media, although some significant variations have been introduced, such as the absence of an equilibrium in the formation of ionic products due to proton transfer; this means that k3x=kl,k2 or, in other words, the proton transfer and ionic solvation take place in a time shorter than the complex lifetime in the excited state. Another differential characteristic is the very inefficient formation of products due to proton phototransfer in the ground state, Photophysical, kinetic and dynamic parameters for the carbazole-tropanic derivatives in AN are given in Table 4. Since the studies using tropine revealed only a slight difference in kr when the temperature was varied and since atropine is less soluble than tropine, the values of kl for atropine have been calculated from the data obtained at the extreme temperatures (283 and 313 K, Table 4). This
Acknowledgments This work was supported and the Junta de Andalucia.
by the I and D area of Boehringer
References 1 M. R. Eftink and C. A. Ghiron, Anal. Biochem., 2 G. P. Hess, D. J. Cash and H. Aoshima, Nature,
114
(1981) 199.
282
(1979)
329.
Ingelheim
(Spain)
184 3 J. W. Karpen, A. B. Sachs, D. J. Cash, E. B. Pasquale and G. P. Hess, AnaL Biochem., I35 (1983) 83. 4 M. Martinez-Carrion, J. Clarke, J. M. Gonzalez-Ros and J. C. Garcia-Borro, Subcell. Biochem., 13 (1988) 363. 5 R. Roy and S. Mukherjee, Chem. Phys. Left., 140 (1987) 210. 6 M. Martin and W. R. Ware, J. Phys. Chem., 82 (X978) 2770. 7 M. *Martin, H. Miyasaka, A. Karen and N. Mataga, J. Phys. Chem., 89 (1985) 182. 8 M. Krishnamurthy and S. K. Dogra, Spectrochim. Acta. Part. A, 42 (1986) 793. 9 E. M. Talavera, M. JimCnez, B. Quintero and J. M. Alvarez, J. Photochem. PhotobioZ. A: Chem., 54 (1990) 49. 10 N. Mataga and T. Kubota, Molecular lirteracfion and Electronic Spectra, Marcel Dekker, New York, 1970. 11 I. Yu. Martynov, A. B. Demyashkevich, B. M. Uzhinov and M. G. Kuz’min, mss. Chem. Rev., 46 (1971) 1. 12 A. Weller, Z. Phys. Chem. N. F., 13 (1957) 335. 13 A. Weller, Z. Elektrochem., 56 (1952) 662. 14 W. R. Ware and J. S. Novros, J. Phys. Chem., 70 (1966) 3246. 15 J. C. Andre, M. Niclause and W. R. Ware, Chem. Phys., 28 (1978) 371. 16 J. C. Andre, M. Bouchy and W. R. Ware, Chem. Phys., 37 (1979) 103. 17 A. Bondi, .l. Phys. Chem., 48 (1964) 441. 18 I. B. Berlman, Handbook of Fluorescence Spectra of Aromatic Mobcules, Academic Press, New York, 1971. 19 H. Baba, A. Matsujama and M. Kokubun, Spectrochim. Acta, 25 (1969) 709. 20 J. Catalan, R. M. Claramunt, J. Elguero, J. Laynez, M. Mentndez, F. Anvia, J. H. Quian, M. Taagepera and R. W. Taft, J. Am. Chem. Sot., 110 (1988) 4106. 21 T. Kunitake, M. Shimomura, Y. Hashiguchi and T. Kawanaka,.T. Chem. Sot., Chem. Commun., (1985) 833. 22 N. Chattopadhyay and M. Chowdhury, .l. Photochem., 28 (1987) 301. 23 N. Chattopadhyay, R. Dutta and M. Chowdhury, J. Photochem. Photobiol. A: Chem., 47 (1989) 249.
Chem., 66 (1992)
171
171-184
Static and dynamic fluorescence quenching of carbazole by tropanic alkaloids E. M. Talavera,
B. Quintero
and J. M. Alvarez
Departamento de Quimica Fisica, Facultad de Farmacia, Campus Universitario de Cartuja, 18071 Granada (Spain) (Received
December
3, 1991; accepted January 30, 1992)
Abstract state fluorescence quenching of carbazole by tropanic derivatives in cyclohexane and acetonitrile was performed. The results obtained using cyclohexane as solvent support the existence of two species in equilibrium: free and hydrogen-bonded complexed carbazole in both the ground and excited states. The parameters involved in the static and dynamic quenching were obtained separately and the thermodynamic parameters corresponding to these associations were calculated. In acetonitriJe, the equilibrium in the excited state is established between carbazole and its proton phototransfer product. Steady
1. Introduction Fluorescence quenching can provide valuable information about the structure and dynamics of proteins and receptors [l-3]. TJ-ms the quenching of fluorescent probes by ions in the presence and absence of agonists and antagonists has been used in the characterization of the nicotinic receptor of acetylcholine (ACH) 141. However, a full identification of the subtypes of the muscarinic receptor of ACH as well as its nature and structure still remain to be characterized. Since tropanic alkaloids act by the specific blocking of the muscarinic receptor, we have investigated the fluorescence quenching of carbazole induced by tropanic alkaloids in order to improve the knowledge of the physical and chemical properties of these antagonists of ACH in the nanosecond domain. The fluorescence properties of carbazole and its derivatives have been widely studied because of the structural relationship with tryptophan. Due to the presence of an electron-deficient nitrogen atom and the high polarity of the amine group, fluorescence quenching of carbazole is mainly caused by the formation of a hydrogenbonded complex [5-71 and increases when the polarity of the solvent is such that it acts as a proton donor rather than a proton acceptor in the excited state [8]. However, it has also been reported that a charge-transfer interaction, other than hydrogen bonding, may contribute to the fluorescence quenching of carbazole. In a previous paper [9], we reported some aspects of the fluorescence quenching of carbazole by tropanic derivatives in polar and non-polar homogeneous media. In this paper, the dependence of the equilibrium on temperature has been studied and the essentially dynamic nature of the quenching is established. In addition, the existence of only two emitting species under the experimental conditions used has been checked.
lOlO-6030/92/$5.00
Q 1992 - Elsevier Sequoia.
AiJ rights reserved
172 2. Experimental
details
Carbazole and N-ethylcarbazole (99%, Aldrich) were recrystallized twice from absolute ethanol, dried and resublimated before use. Endo-S-methyl-S-azabicyclo[3.2.1 Joctan-3-01 (tropine) and e-(hydroxymethyl)benzeneacetic acid S-methyl-8-azabicyclo[3.2.l]oct-3-yl ester (atropine) (both of analytical grade) were donated by Boehringer Ingelheim; they were recrystallized twice from n-heptane, dried and stored in a desiccator. The desiccator containing the tropanic derivative was kept at 45 “C (tropine) or 90 “C (atropine) for 24 h. Cyclohexane (CH), ethanol, butanol and acetonitrile (AN) (for fluorescence spectroscopy, Merck) were used as solvents without further purification. All solvents were checked for purity by absorption and emission spectroscopy. The absorption spectra were recorded using a Perkin-Elmer Lambda 5 spectrophotometer. A solution containing the same concentration of tropanic derivative was used as a blank. Solutions were kept for at least 30 min in the thermostatically controlled sample compartment before the measurements were performed. Measurements of stead state fluorescence were made using a Shimadzu RF-5000 spectrofluorometer with 15 K and 30 A bandpass for excitation and emission respectively, Excitation was performed at an isosbestic point. All spectra were corrected for the photomultiplier response. The temperatures of the samples were controlled by a watercirculating external bath. In order to eliminate the inner filter effect due to weak absorption of tropanic alkaloids at the excitation wavelength, the ceI1 used in the fluorescence measurements had light paths of 2 mm in the excitation direction and 10 mm in the emission direction. A front-face arrangement was also used, but an increase in the amount of scattered light produced poorly resolved spectra. In some experiments molecular oxygen was removed by bubbling with argon for 3 min; this enabled fluorescence signals with higher intensity and accuracy to be obtained. However, since the flow of argon into the cell leads to a decrease in temperature, air enters the cell while the desired temperature is attained (cell not air tight)_ More reliable results were obtained when the sample was kept under conditions in which the desired temperatures (f 1 K) were reached after bubbling with argon. Lifetime measurements were obtained using an EEY nanosecond fluorometer operating in time-correlated single-photon counting mode. A hydrogen lamp (approximately 10 atm) was used with a frequency of 30 kHz, supplying very stable lamp pulses with a halfwidth of 2.2 ns. Sample excitation was performed at 300 nm using an interference filter FT-300 (bandwidth, 15 nm) and the emission was filtered by a Corning 7-60 filter. All the samples were aerated and controlled at 293 K. The decay curves were analysed by a least-squares reconvolution program. The goodness of the fit was estimated from the reduced ,$ value.
3. Results
and discussion
The spectral modifications caused by the addition of tropanic derivatives (atropine or tropine) to carbazole solutions in apolar solvents, together with the absence of such modifications in hydroxylated solvents and the inability of these alkaloids to quench the fluorescence of N-ethylcarbazole, have been interpreted to be due to the formation of a hydrogen-bonded complex in both the ground and excited states 191. The appearance of isosbestic points in the absorption spectrum [9] and an isoemissive point in the emission spectrum (Fig. l(a)) suggests that the equilibrium involves only
173
a
b
Fig. 1. Change in carbazole fluorescence with tropine concentration in CH solution (A,,=321 nm). Concentrations: (a) carbazole, 2.5 X lo-§ M; tropine, 0.0,0.25 X lo-*, 1.5 x lo-* and4.75 x lo-’ M; (b) carbazole, 2.5 x lo-’ M; tropine, 0.1 M. two species: free and complexed carbazole [lo, 111. Moreover, it is observed that the emission band (about 334 nm) decreases in intensity as the tropine concentration is increased and at a concentration above 0.1 M becomes almost negligible (Fig. l(b)). Considering that this emission is due to free carbazole, excitation spectra were recorded by fixing the emission wavelength at 334 nm. As shown in Fig. 2(a), the spectra obtained with the carbazole-tropine system vary in intensity, but no change is observed in the shape or the positions of the band maxima which are identical to those found in the absorption spectrum of carbazole. By fixing the emission wavelength at 350 nm, the spectra shown in Fig. 2(b) are obtained which match the absorption spectrum recorded with the carbazole-tropine system [9]. These results indicate that the emission at 334 nm is due to excited free carbazole, whereas the emission at 350 nm results from the superimposed emission of the two species involved in the excited state equilibrium. Under these conditions, it is possible to separate the spectrum of each species. Thus, the fluorescence of the carbazole-tropine system in Fig. 3 has been normalized by taking as reference the intensity measured at 334 nm for carbazole in CH. From the resulting spectrum, the reference spectrum of carbazole solution is subtracted.
b
Fig. 2. Excitation spectra centrations of tropine: (a) (-) and 14.35 x 10e2 M 14.35 x 1O-2 M (- - a) (Aa
of carbazole (2.5X lo-’ M) (-) and carbazole-tropine system. Con0.75 X lo-’ (- - -), 2.27 X lo-’ (- - -), 4.75 X lo-* (. . -), 10.57 x IO-’ (a * *) (AEM= nm); (b) 2.27~ IO-’ (--- -), 10.57~ 10m2 (- - -) and -350 nm).
The spectra obtained in this way are shown in Fig. 4 together with the emission spectrum recorded on excitation at 337 nm of a carbazole-tropine solution (carbazole, 5 x lo-’ M; tropine, 0.1 M). This wavelength (337 nm) is characteristic of the new band which appears in the absorption spectrum of the carbazole-tropine system [9]. It can be seen that the shape of the spectrum obtained by excitation at 337 nm is similar to that derived from the normalization-subtraction procedure. Since free carbazole does not absorb at 337 nm, it seems reasonable to attribute the spectra in Fig. 4 to the complexed species. By measuring the absorbance at 340 nm of carbazole solutions in CH (5 x lo-’ M) containing different amounts of tropine, it is possible to cakulate Kg, the equilibrium constant for the ground state reaction, using
(1)
175
Fig. 3. Emission spectra of carbazole (5 X lo-’ M) and tropine (0.0, 1 X 10B3, 2 X lo-‘, 4 X 10e3, 5 X 10e3, 6 X 10e3, 8 X lop3 and 10X 10e3 M) in CH (A,, =321 nm).
3 X lo-‘,
An appropriate plot of the experimental data gives straight lines. The ordinate of the lines is equal to -Kg_ The values of Kg can, in turn, be plotted vs. l/T according to the van? Hoff equation to calculate Lw, AS and AG corresponding to the association reaction in the ground state. Table 1 summarizes the results obtained in this analysis. The thermodynamic data can be interpreted as a consequence of a spontaneous exothermic reaction where a molecular reorganization process takes place. The determination of I& could not be performed for the carbazole-atropine system because of the low solubility of atropine in CH. From Kg, we can calculate K,, the equilibrium constant for the association reaction in the excited state, using [9, lo] logK,=log&+
0.625 8y T a
where 6v, represents the spectral shift determined as either the difference between the absorption maxima positions expressed in wavenumbers (cm-‘) or one-half of the sum of the frequencies of the absorption and fluorescence peaks (cm- ‘). Some discrepancy can arise from the method used to determine 6u but, in any case, KB is much lower than K, [lo].
Fig. 4. Emission spectra of complexed species obtained 321 nm and spectra recorded for a carbazole-tropine [tropine] = 9~ lo-’ M) at hnx = 337 nm.
TABLE
from normalization
procedure
system ([carbazole]
=5
x
lo-’
at Anx= M and
1
Values of the equilibrium states, and thermodynamic
constants for the association reactions in the ground and excited parameters corresponding to the carbazole-tropine system in CH - AC,=
- AGeb
T WI
Kg
KG”
KGb
(1 mol-‘)
-AC, (kI mol-‘)
(kJ mol-‘)
(W mol-‘)
283 293 303 313
25.11 18.34 10.57 5.11
387.27 257.64 136.09 60.63
1111.25 715.64 365.47 157.77
7.58 7.09 5.95 4.24
14.02 13.52 12.38 10.68
16.51 16.01 14.87 13.17
(1 mol-‘)
(1 mol-‘)
-AHS=39.0 W mol-‘; -AHe”=45.61 kJ mol-‘; -AHeb=48.04 mol-‘; -AS,‘= 110.5 J K-r mol-‘; -ASeb= 110.2 J K-r mol-‘. ‘Values obtained using au,=538 cm-‘. bValues obtained using Sv,=746 cm-‘.
kJ mol-‘;
-AS,=llO.O
J K-’
The results obtained from the analysis performed according to eqn. (2) and by application of the van’t Hoff equation are also given in Table 1. In cases where combined static and dynamic quenching are present and the cause of the quenching is a hydrogen-bond interaction, Weller’s reaction scheme [12] can be accepted in principle. This provides a very complicated equation for the determination of the relative fluorescence yields [lo] if an appreciable overlap occurs between the
177
spectra of the free and complexed species. However, for the carbazole-tropine system a simplification can be made. Thus, on the basis of the assignment of the signal at 334 nm to free carbazole, the equation for the determination of the relative fluorescence yields can be written as (3) where F and F0 are the fluorescence intensities in the presence and absence of tropine respectively, 7 is the fluorescence lifetime of carbazole, k, is the bimolecular constant for the quenching process, [Q] is the quencher concentration and 6 is the light fraction absorbed by free carbazole, obtained from
6 = l/(1 + (E&E&[Q]}
(4)
This expression becomes more simple when excitation is carried out at- an isosbestic point (as has been performed in our experiments). The relative fluorescence intensity data measured at 334 nm for solutions of carbazole-tropine in CH (tropine concentration, not greater than 0.03 M) were used to plot eqn. (3). Ordinate values close to unity and determination coefficients within the range 0.99-1.00 were found in all cases. In Fig. 5 the fluorescence decay curve of a carbazole solution (5X 10e5 M) in CH is shown together with the value of the fluorescence lifetime at 293 K. From the fluorescence intensities obtained for carbazole solutions at different temperatures in
20,
1
0 0
5
10
15
20 TIME
25
30
35
40
45
50
(NSECI
Fig. 5. Fluorescence decay curve of carbazole the fluorescence lifetime.
solution (5 X10w5 M)
in CH and the value of
178
the range 283-313 K, no significant change in the value of 7 is expected. Taking the slope of the plot of eqn. (3) and 7, the value of the forward constant kr for the association reaction in the excited state can be calculated; in our case, this corresponds to the value of the bimolecular constant for the diffusion-controlled process of quenching, since the inverse rate constant for dissociation of the complex in the excited state (I+) is negligible in comparison with the rate of unimolecular deactivation of the complex [13]. All the above-mentioned values are included in Table 2. From these values and the CH viscosities (also included in Table 2), a plot of kI vs. T/v was made. A straight line is obtained (Fig. 6) supporting the dynamic nature of the quenching of the carbazole fluorescence by tropine in CH. According to the Smoluchowski-Einstein diffusion theory, the bimolecular constant (k,,) for a diffusion-controlled process is given by k,(r) = 477..
TABLE
oD{ 1 + a/( &r)“L)
(5)
2
CH viscosities and photophysical, kinetic and dynamic parameters process of the carbazole-tropine system in CH
K, (1 mol-‘) kIX1O-g (I mol-’ k,XlO_’ (s--l) 7 (-9 D,X106 (cm’ s-l) D,X lo6 (cm’ s-‘) k, x lo-’ (I mol-’ Y R:,=3.93 R,=3.26
s-‘)
s-l)
for the bimoIecular deactivation
283 K
293 K
303 Is
313 K
34.36 4.17 0.37
36.99 4.48 0.63
38.36 4.65 1.27
44.25 5.36 3.4O
A A.
k+O-'
6
4
31 200
I
I
400
300 T'rl
Fig. 6. Plot of k, vs. T/T for the carbazole-tropine
system in CH.
600
179
After electronic excitation, excited molecules with a quencher molecule inside their active sphere will be immediately deactivated (in less than lo-” s); only those molecules not deactivated in this way will suffer decay as a consequence of normal emission and difFusiona quenching. Several experimental and theoretical studies by Ware and coworkers [14-161 have been focused on the investigation of Stern-Volmer deviations due to static quenching in the active sphere. No upward deviations were observed in the system studied here and the viscosities of the solutions used were low; therefore the influence of static quenching in the active sphere has been ruled out. The straightforward application of eqn. (5) allows us to determine k. using the value of the diffusion coefficient D obtained from the Stokes-Einstein equation and the molecular radii calculated from the van der Waals’ volumes [17]. A comparison of k. with kl gives the value of the efficiency parameter y for diffusional encounters. The values of y shown in Table 2 indicate that tropine is an efficient quencher of carbazole fluorescence. The influence of oxygen on the kinetic parameters was tested since most of the experiments were carried out with aerated samples. Data from deaerated samples (argon bubbling at 303 K) were analysed by applying the same methodology as used with the aerated samples. The Stern-Volmer constant found for deaerated samples is 75.03 1 mol-‘. Using the carbazole lifetime in the absence of oxygen reported in the literature (16.1 ns [18]), a value of 4.66 X 10m9 mol-l s-l is obtained for kl in good agreement with the value found for aerated samples at the same temperature (see Table 2). Similar results were reported by Martin and Ware [6] for the carbazole-pyridine system. In Fig. 7 plots of Fe/F (fluorescence intensities measured at 350 nm) ~1s. tropine concentration at the four temperatures used in our study are shown. The shapes of these plots obey the general equation derived from Weller’s scheme [12]; admitting that the two species emit at 350 nm and free carbazole fluorescence is more intense
FJF 1.3
1,2
101
1
0
1
2
3
4
5
6
[QLlOe+2
Fig. 7. Plots of F,JF vs.tropine concentration: 0, 283 K, A, 293 K, *, 303 K; 0,
313 K.
180
than that of complexed carbazole. It can be seen that the asymptotic portion occurs at smaller F,/F values with increasing temperature which can be justified by the decrease in K, as the temperature is increased. Moreover, the asymptotic portion is reached at a smaller concentration of quencher as the temperature is increased which is in good agreement with the expected relationship between kl and T/q. The qualitative similarity between the carbazole-atropine and carbazole-tropine systems in CH must be emphasized. Nevertheless because of the low solubility of atropine, Kg cannot be calculated. This is the reason for restricting the analysis to only two temperatures, 303 K and 313 K. In general, it is expected that, as the temperature is increased, the amount of atropine dissolved will also increase; the choice of these temperatures is related to the range of biological interest and the limitations imposed by the solvent. Under our experimental conditions, it is assumed that there is no association in the ground state; in addition, the light fraction S is approximately unity due to the low concentration of the quencher. Under these circumstances, the K,, values shown in Table 3 were obtained by applying the Stern-Volmer equation to the fluorescence data measured at 334 nm. Other photophysical parameters are also included in Table 3. When AN is used as solvent, the free carbazole absorption spectrum shifts 270 cm-’ to the red with respect to that recorded in CH. Moreover, the three isosbestic points obtained on addition of tropanic derivatives are also red shifted with respect to the isosbestic points in CH (323,332 and 336 nm). However, the absorption spectral change induced by tropanic derivatives is very slight despite their high concentration due to good solubility in AN. Figure 8 shows the emission spectra obtained on addition of tropine (concentration range, 2.5 x 10V3-2.5 x 10-l M). Excitation was performed at the lower energy isosbestic point. Appreciable fluorescence quenching is observed as well as the appearance of a new well-structured band with maxima at 418 nm and 443 nm and a shoulder at 478 nm. An isoemissive point located at 397 nm is also present. Concentrations of tropine higher than 0.95 M produce changes in the spectral shape to give a structureless band centred at 420 nm. In addition, the emission spectra of carbazole in AN made alkaline with NaOH or NH40H were recorded; a new band appears which has similar features to that described above on addition of the tropanic derivatives. Emission spectra were also recorded using carbazole-tropinesolutions before and after acidification with acetic acid. Acetic acid inhibits the fluorescence quenching and, as a consequence, leads to the disappearance of the band at longer wavelengths [9]_
TABLE
3
Photophysical, kinetic and dynamic parameters for the carbazole-atropine
K, (1 mole’) /clX foe9 (1 mol-’ RQ DQX
/c,X
Y
s-l)
CA) 10”
(cd
s-l)
10m9 (I mol-’
s-l)
system in CH
303 K
313 K
47.57 5.77 4.07 6.57 8.19 0.70
55.07 6.68 4.07 8.16 10.10 0.66
181
.,.~.,._ . -... _.
Fig. 8. Fluorescence spectra of carbazole (5 X 10e5 M) and tropine (0.0, 1 X lo-‘, nm). 4X 10e2, 7X lo-‘, 0.1, 0.15, 0.2, 0.25 and 0.95 M) in AN solution (A,,=323
~X~CJ-~,
Interaction between heteroatomic compounds and bases in aprotic solvents produces the formation of hydrogen-bonded complexes and ionic pairs; these pairs may or may not be separated by solvent molecules depending on the strength of the base and the polarity of the solvent [19]_ Mataga and Kubota [lOI have emphasized that ionic pair formation and ionic separation by solvent molecules can occur due to a hydrogenbonded interaction in the fluorescent state. Therefore the long-wavelength band and the experimental behaviour shown by the carbazole-tropanic derivatives in AN can be attributed to a proton phototransfer product. The recently reported pK,* value of carbazole in the excited state [20], the polarity of AN and similar assignments found in the literature [21-231 indicate that the new band in these systems can be attributed to the anion derived from carbazole. The spectra of free carbazole and its anion can be separated using the normalization-subtraction procedure described earlier. In this case the fluorescence at 343 nm clearly originates from free carbazole since the shape of this band is unaffected by the addition of tropine (Fig, 8). In Fig. 9 the spectra of carbazole and its anion are shown separately. The anion emission is less structured and bathochromically shifted with respect to the emission of the hydrogen-bonded complexed carbazole (Fig. 4).
182
P
183 TABLE
4
Photophysical, T (K)
kinetic
and dynamic kr x 10-s (1 mol-’
&v (1 mol-‘)
parameters 77(cP)
s-‘)
for carbazole-tropanic DIX 16 (cm’ s-t)
derivatives
DoXld (cm2 s-*)
in AN
k,x lo-‘0 (1 mol-’ s-t)
’ l
283” 293a 303” 313a
12.15 12.42 12.54 12.68
1.63 1.67 1.69 1.70
0.394 0.358 0.333 0.315
1.34 1.53 1.70 1.85
1.61 1.84 2*04 2.23
1.61 1.83 2.03 2.22
0.10 0.09 0.08 0.08
283b 313b
6.08 6.14
0.82 0.83
0.394 0.315
1.34 1.85
1.29 1.79
1.59 2.21
0.05 0.04
“Carbazole-tropine bCarbazole-atropine
system. system.
The results and interpretation discussed in this paper suggest that the reaction between carbazole and the tropanic derivatives in polar aprotic solvents proceeds via the following mechanism
FH
+
Q
+ Kil hvjaji~t”
F*H
+
Q
+
kz
F*H...Q
k3,
F*-
+ HQ+
scheme is similar to that proposed for the reaction in apolar media, although some significant variations have been introduced, such as the absence of an equilibrium in the formation of ionic products due to proton transfer; this means that k3x=kl,k2 or, in other words, the proton transfer and ionic solvation take place in a time shorter than the complex lifetime in the excited state. Another differential characteristic is the very inefficient formation of products due to proton phototransfer in the ground state, Photophysical, kinetic and dynamic parameters for the carbazole-tropanic derivatives in AN are given in Table 4. Since the studies using tropine revealed only a slight difference in kr when the temperature was varied and since atropine is less soluble than tropine, the values of kl for atropine have been calculated from the data obtained at the extreme temperatures (283 and 313 K, Table 4). This
Acknowledgments This work was supported and the Junta de Andalucia.
by the I and D area of Boehringer
References 1 M. R. Eftink and C. A. Ghiron, Anal. Biochem., 2 G. P. Hess, D. J. Cash and H. Aoshima, Nature,
114
(1981) 199.
282
(1979)
329.
Ingelheim
(Spain)
184 3 J. W. Karpen, A. B. Sachs, D. J. Cash, E. B. Pasquale and G. P. Hess, AnaL Biochem., I35 (1983) 83. 4 M. Martinez-Carrion, J. Clarke, J. M. Gonzalez-Ros and J. C. Garcia-Borro, Subcell. Biochem., 13 (1988) 363. 5 R. Roy and S. Mukherjee, Chem. Phys. Left., 140 (1987) 210. 6 M. Martin and W. R. Ware, J. Phys. Chem., 82 (X978) 2770. 7 M. *Martin, H. Miyasaka, A. Karen and N. Mataga, J. Phys. Chem., 89 (1985) 182. 8 M. Krishnamurthy and S. K. Dogra, Spectrochim. Acta. Part. A, 42 (1986) 793. 9 E. M. Talavera, M. JimCnez, B. Quintero and J. M. Alvarez, J. Photochem. PhotobioZ. A: Chem., 54 (1990) 49. 10 N. Mataga and T. Kubota, Molecular lirteracfion and Electronic Spectra, Marcel Dekker, New York, 1970. 11 I. Yu. Martynov, A. B. Demyashkevich, B. M. Uzhinov and M. G. Kuz’min, mss. Chem. Rev., 46 (1971) 1. 12 A. Weller, Z. Phys. Chem. N. F., 13 (1957) 335. 13 A. Weller, Z. Elektrochem., 56 (1952) 662. 14 W. R. Ware and J. S. Novros, J. Phys. Chem., 70 (1966) 3246. 15 J. C. Andre, M. Niclause and W. R. Ware, Chem. Phys., 28 (1978) 371. 16 J. C. Andre, M. Bouchy and W. R. Ware, Chem. Phys., 37 (1979) 103. 17 A. Bondi, .l. Phys. Chem., 48 (1964) 441. 18 I. B. Berlman, Handbook of Fluorescence Spectra of Aromatic Mobcules, Academic Press, New York, 1971. 19 H. Baba, A. Matsujama and M. Kokubun, Spectrochim. Acta, 25 (1969) 709. 20 J. Catalan, R. M. Claramunt, J. Elguero, J. Laynez, M. Mentndez, F. Anvia, J. H. Quian, M. Taagepera and R. W. Taft, J. Am. Chem. Sot., 110 (1988) 4106. 21 T. Kunitake, M. Shimomura, Y. Hashiguchi and T. Kawanaka,.T. Chem. Sot., Chem. Commun., (1985) 833. 22 N. Chattopadhyay and M. Chowdhury, .l. Photochem., 28 (1987) 301. 23 N. Chattopadhyay, R. Dutta and M. Chowdhury, J. Photochem. Photobiol. A: Chem., 47 (1989) 249.