J. Phys. Chem. 1982,8 6 , 5223-5226
5223
Photophysical Properties of Benzimidazole and Thiabendazole and Their Homologues. Effect of Substituents and Solvent on the Nature of the Transition Patricia C. Tway’ and L. J. Cline Love’ Department of Chemistry, Seton Hall University, South Orange. New Jersey 07079 (Received: March 10, 1982)
The photophysical characteristics of benzimidazole and thiabendazole, and ten of their derivatives, are evaluated to determine the nature of the transitions occurring and how the excited states interact with the species microenvironment. On the basis of the fluorescence and phosphorescence energies, quantum yields, and lifetimes, these benzimidazole homologues appear to be excited to a manifold of singlet states. Which excited state predominates, either r a* or charge transfer, depends on the side-chain substituent, solvent system, pH, and temperature.
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Introduction Our interest in the luminescence properties of the benzimidazole analogues arose from earlier work utilizing the fluorescence properties of thiabendazole and its major metabolite, 5-hydro~ythiabendazole.~ Thiabendazole is a human and veterinary anthelmintic drug and is used as a fungicide on fruits and vegetables. The drug levels of thiabendazole and its metabolite are monitored in people and animals by measuring the fluorescence of these compounds. Thiabendazole and 5-hydroxythiabendazoleboth absorb in the 300-320-nm spectral region but have very different fluorescence characteristics, expecially in acid solutions. Although the fluorescence properties of thiabendazole are used to monitor drug residues, these properties have never been studied in any detail. In order to better understand the microenvironmental influences on the luminescence properties of these compounds and to determine the nature of the luminescence transition(s), we have undertaken a detailed excited-state study of the fluorescence and phosphorescence properties as a function of the side-chain substituent and solvent and the results are reported here. Benzimidazole and a series of substituted benzimidazole analogues were also studied since thiabendazole is a substituted benzimidazole and any proposed transition should explain the behavior of both series of compounds. The luminescence properties of benzimidazole have been extensively studied3* but little work has been reported on substituted species. In neutral solutions the fluorescence maximum of benzimidazole occurs at 290 nm with a quantum yield of -0.7, but in acid the maximum is at 360 nm with a quantum yield of 0.06.3 This energy shift has been ascribed to possible solvent exciplex formationB6In this paper, we report the photophysical characterization of all of these molecular species and propose a transition mechanism to explain their luminescence properties. Experimental Section The benzimidazole and thiabendazole analogues studied, given in Figure 1, were obtained from the sample collection (1)Merck and Co., Rahway, NJ 07065. (2)‘Food Additive Analytical Manual”; Food and Drug Adminstration: Washington, DC, 1973;Food Additive Reg. 121.260. (3) Longworth, J. W.; Rahn, R. 0.; Schulman, R. G. J. Chem. Phys. 1966,45,2930-9. (4)Undenfriend, S. “Fluorescence Assay in Biology and Medicine”; Academic Press: New York, 1969;pp 269-71. (5)Svejda, P.; Anderson, R. R.; Maki, A. H. J. A m . Chem. SOC.1978, 100, 7131-8. (6)Barresen, H. C. Acta Chem. Scand. 1963,17,921-9.
of Merck, Inc., Rahway, NJ, and were better than 98% pure. All organic solvents were Burdick & Jackson “distilled in glass”, and all aqueous solutions were made with doubly distilled water. Solutions were prepared to be M or less to avoid concentrational quenching. The fluorescence of these compounds was found to be insensitive to oxygen quenching and the solutions were not deaerated. All absorption measurements were made on a Beckman Acta 111. Fluorescence and phosphorescence spectra were measured on a laboratory-constructed fluorometer described previo~sly.’~~ Uncorrected spectra were used to measure energies since relative energies were sufficient for comparison among groups of compounds or solvents. Fluorescence lifetimes were measured by use of the time-correlated single-photon techniquegJOwith the system described previ~usly.~ The lifetimes were calculated by a reiterative convolution program written in Fortran and used on a Burroughs B6800 computer. The precision of the data run in triplicate was f0.04 ns. Phosphorescence lifetimes were measured as described previously,” and calculated by using the Guggenheim method,12 with a relative standard deviation of 5%. The fluorescence quantum yields were determined by the corrected spectra method13 or the photon-counting method14and were calculated by the comparative method relative to quinine sulfate (a = O.55).l3J5 The phosphorescence quantum efficiencies were estimated indirectly by comparison of corrected low-temperature phosphorescence spectra with low-temperature fluorescence spectra.
Results Fluorescence Properties. Initial studies were done to obtain the absorption (A,) and the fluorescence (Af) maxima (7)Cline Love, L. J.; Upton, L. M.; Ritter, A. W. Anal. Chem. 1978, 50,2059-64. (8) Cline Love, L. J.; Skrilec, M.; Habarta, J. G. Anal. Chem. 1980,52, 754-9. (9)Schlag, E. W.in ”Spectroscopy of the Excited State”; Di Bartolo, B., Ed.; Plenum Press: New York, 1976;pp 225-48. (10)Isenberg, I. In “Biochemical Fluorescence”; Chen, R. F.; Edelhoch, H., Ed.; Marcel Dekker: New York, 1975;Vol. 1, pp 43-78. (11)Cline Love, L. J.; Habarta, J. G.; Skrilec, M. Anal. Chem. 1981, 53,437-44. (12)Margerison, D. In “Comprehensive Chemical Kinetics”, Bamford, C. H.; Tipper, C. F. H. Ed.; Elsevier: New York, 1969;pp 388-91. (13)Parker, C. A. ‘Photoluminescence of Solutions with Applications to Photochemistry and Analytical Chemistry”; Elsevier: Amsterdam, 1968;pp 261-8. (14)Upton, L. M.; Cline Love, L. J. Anal. Chem. 1979,51, 1941-5. (15)Demas, J.; Crosby, G. J. Phys. Chem. 1971,75,991-1024.
QQ22-3654/02/ 2Q06-5223$Q1.25/ 0 63 1982 American Chemical Society
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The Journal of Physical Chemistry, Vol. 86, No. 26, 1982
Tway and Cline Love
TABLE I : Fluorescence and Absorption Energy Maxima of Benzimidazole and Thiabendazole Analogues in Polar Solventsa wavelength maxima, nm EtOH benzimidazole 5-methylbenzimidazole 5-methoxybenzimidazole 5-aminobenzimidazole thiabendazole 1-hydroxythiabendazole 5-methylthiabendazole 5-methoxythiabendazole 5-hydroxy thiabendazole 5-aminothiabendazole cambendazole 1-aminocambendazole a
273, 277, 287 300 293, 302, 305, 318 319 330 320 319
279 283 299 308 318
0.1 N HCI
MeOH 29 1 297 314 358 350 357 3 53 373 388 457 385 390
271, 278 277, 283 288 298 298 300 305, 317 318 316 3 27 317 315
291, 299 297 314 358 346 34 7 353 377 400 464 398 397
267, 273, 286 268, 296, 301 307 317 317 300 319 320
274 281 274 302
360 362 340-360 444 3 54 3 50 369 4 14 540 520 440 445
All wavelengths listed in nm, t 2 nm
TABLE 11: Fluorescence Energy Maxima in Solvents Less Polar Than Ethanol wavelength maxima. nm
a
compd
hexane
chloroform
butanol
ethyl acetate
dioxane
tetrahydrofuran
acetonitrile
benzimidazole 5-aminobenzimidazole thiabendazole 5-methylthiabendazole 5-hy droxy thiabendazole 5-aminothiabendazole cam be n daz ol e
293
292 3 03 345 345 375 432 377
292 363 345 350 390 4 55 392
2 93 350 345 351 377 435 377
293 353 352 355 3 74 431 381
293 355 3 55 352 380 438 385
292 3 53 350 3 56 3 84 450 390
a 336 344 3 56 380 360
5-Aminobenzimidazole is insoluble in hexane TABLE 111: Fluorescence Lifetimes and Quantum Yields MeOH compd
I1
Flgure 1. Structures of the benzimidazole (I) and thiabendazole (11) analogues: (I) R, = H, benzimidazole: R, = CH,, 5-methylbenz-
imidazole: R, = CH30, 5-methoxybenzimidazole; R, = NH,, 5amine benzimidazole; (11) R, = R, = H, thiabendazole; R, = H, R2 = OH, l-hydroxythiibendazole;R, = CH,, R, = H, 5-methylthiibendazol: R, = CH,O, R, = H, 5-methoxythiabendazole;R1 = NH,, R, = H, 5aminothiibendazole,R, = OH, R2 = H, 5hydroxythiabendazole:R, = NHCOOCH(CH,),, R, = H, cambendazole; R, = NHCOOCH(CH,),, R, = NH,, 1-aminocambendazole.
as a function of solvent. These data are presented in Table I. The absorption transition energies show little shift with solvent, while the fluorescence transition energies shift to longer wavelengths as the solvent becomes more polar (ethanol < methanol < aqueous acid). To confirm the effects of solvent polarity on the fluorescence transition energies, we measured the absorption and fluorescence energies of several of these compounds in solvents less polar than ethanol and the data are presented in Table 11. In all cases the absorption energy is not solvent dependent. On the basis of Taft's T* functions which are a measure of solvent polarity,16the solvents studied, hexane, chloroform, butanol, ethyl acetate, dioxane, tetrahydrofuran, and acetonitrile, are listed in order of increasing polarity. The fluorescence energy maximum shifts to longer wavelengths in all cases as the solvent becomes more polar, except for benzimidazole itself which exhibits no solvent effects except in acid solutions. (16)Kamlet, M. J.;Abboud, J. L.; Taft, R. W. J. Am. Chem. SOC.1977, 99, 6027-38.
benz im id azo1e thiabendazole 1-hydroxythiabendazole 5-hydroxythiabendazole 5-aminothiabendazole cambendazole 1-aminocambendazole a
i0.04.
i6%.
Tf,ans
Qfb
0.11 0.18 0.48 0.24 1.07 1.25
0.6Ic 0.037 0.006 0.035 0.007 0.089 0.064
0.1 N HC1 Tf,a
ns
0.53 0.49 0.27 0.80 1.78 0.99
0.06c 0.041 0.003 0.006 0.003 0.28 0.092
Reference 5.
Although certain deviations from the trend are seen, solvent polarity appears to be a major factor affecting the fluorescence energy. These solvent effects are discussed in more detail in a previous paper." Anomalously large shifts of the fluorescence transition energy which cannot be explained by solvent polarity are seen for certain compounds in acid solution, and are the subject of the following paper.l8 Additional information about the luminescence transitions of these compounds were obtained by measuring the fluorescence lifetimes and quantum yields in methanol and aqueous acid (Table 111). The fluorescence lifetimes of all the thiabendazole analogues are on the order of 0.2-2 ns. Generally, the lifetimes are longer in aqueous acid solution than in methanol which suggests increased stabilization in the more polar solvent. The fluorescence lifetime of benzimidazole could not be measured because of instrumental limitations. The quantum yields of the thiabendazole analogues in methanol range from 0.006 to 0.09. These quantum yields are low relative to that of benzimidazole (0.67), probably (17) Tway, P. C.; Cline Love, L. J. Chem. Phys. Lett. 1982,87,204-7. (18) Tway, P. C.; Cline Love, L. J. J. Phys. Chem., following paper in this issue.
The Journal of Physical Chemistry, Vol. 86,No. 26, 1982 5225
Photophysical Properties of Benzimidazole Analogues
TABLE IV: Phosphorescence Energy Maxima and Lifetimes of Thiabendazole Analogues ~~
compd benzimidazole thiabendazole 5-hydroxythiabendazole 5-aminothiabendazole cam bendazole 1-aminocambendazole (I
~~~
TP
h p F nm
376,398, 453,478, 491 5 04 476,482, 454,477,
:
TABLE V : Summary of Fluorescence Transition Mechanism in Benzimidazole and Thiabendazole and Some Analogues transition
ms
421 500
6200 157 136
498, 510 504
152 185
Energy and lifetime measured in ethanol a t 77 K.
because of the increased modes of vibrational deactivation introduced by the thienyl sidechain. This supposition is supported by the quantum yield data at 77 K where internal conversion does not occur appreciably. Benzimidazole and thiabendazole have fluorescence quantum yield of 1 and -0.7, respectively, in methanol at 77 K, which strongly suggests that at room temperature radiationless internal conversion from the excited singlet, not intersystem crossing, is the predominant mode of deactivation. Phosphorescence Characteristics. I t was known that benzimidazole phosphoresces ~ e a k l y but , ~ the phosphorescence properties of thiabendazole and it analogues have never been reported. It was found that all of the thiabendazole analogues phosphoresce in ethanol at 77 K in the wavelength range 470-510 nm. The phosphorescence quantum efficiencies of benzimidazole and thiabendazole were determined by a comparative method and are low, 0.0003 and 0.07 (f15%), respectively. The data are presented in Table IV. The phosphorescence energies and lifetimes are less dependent on the nature of the side-chain substituent than the fluorescence data, as would be expected.
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Discussion Three different types of electronic transitions are possible in the benzimidazole analogues based on their molecular structure. The nonbonding electrons on the imidazole ring could be involved in 7 a* transitions, the aromatic system could undergo a a* transitions, or charge transfer could occur upon excitation. Mechanism of the Transition. The excited-state species in a a* and charge transfer transitions is generally more polar than the gound-state species because of the large dipole moment changes accompanying these electronic reorganizations. Consequently,a a* and charge transfer transitions are most affected by solvent polarity changes.lg In 17 T* transitions the fluorescence spectra are not markedly dependent on solvent polarity, but the absorption spectra depend strongly on the hydrogen-bonding ability of the solvent. The data from Tables 1-111 show that the fluorescence wavelength maxima shift to lower energies and the fluorescence lifetimes are longer in more polar solvents for these compounds. These results suggest that a a a* or charge transfer transition is taking place and that the excited-state species is more polar than the ground state species and is stabilized relative to the ground state in the more polar solvents. The absorption spectra are only slightly solvent dependent which strongly suggests that the nonbonding electrons on the imidazole ring are not involved in a 7 a* transition. The short fluorescence and phosphorescence lifetimes of these molecules also suggest
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+
+
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+
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(19) Schulman, S. G. “Fluorescenceand Phosphorescence Spectroscopy: Physicochemical Principals and Practice”; Pergamon Press: Oxford, 1977; p 25.
nonaqueous s olven tc
compd benzimidazole 5-methy1benzimi dazole 5-methoxybenzimidazole 5-ami nobe nz im idaz 01e thiabendazole 1-hydroxythiabendazole 5-methylthiabendazole 5-methoxy thiabendazole 5-hydroxythiabendazole 5-aminothiabendazole cambendazole 1-aminocambendazole
R
+
TI TI
+
aqueous acid (0.1 N ) CT (I
R* R*
CT CT
n*/CTb
CT
CT
71 +
n*
TI-
TI*
n+
R*
TI+-
TI*
77
+-
n*
TIc
TI*
TI
+
n*ICT
n
n*ICT
CT CT CT CT
+-
CT CT CT CT
CT represents charge transfer. Borderline between This c ategory includes solvents the t w o transitions. more polar than hexane and less polar than water.
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a a a* rather than a 7 IT* transition, since the latter generally have longer fluorescence and phosphorescence lifetimes. The phosphorescence quantum efficiencies are also low which contraindicates a 7 a* transition. All of the luminescence data for the benzimidazole and the thiabendazole analogues support a a a* or a charge transfer transition. It is proposed that the benzimidazoles are excited by absorption of light to a manifold of excited singlet states; one excited state has predominantly a ?r* character while the other has charge transfer characteristics. Which excited singlet state predominates depends on the side-chain substituents on the benzimidazole and the solvent system. Generally, in more polar solvents and in solutes with strong electron-donating substituents the charge transfer transition is stabilized relative to the a a* transition. The transition(s) believed to be occurring in each of the compounds studied are summarized in Table V and the results for selected species will be discussed briefly. Benzimidazole in neutral organic solvents exists as a neutral molecule, undergoes a relatively high-energy K a* transition and has a Stokes shift of 20-30 nm. The absorption and fluorescence bands are sharp, indicating little broadening from vibrational contributions, and the fluorescence energy is only weakly affected by solvent polarity. Similar behavior is seen for 5-methylbenzimidazole, 5-methoxybenzimidazole, thiabendazole, 5methylthiabendazole, and 1-hydroxythiabendazole in organic solvents. Benzimidazole in acid solutions, however, undergoes a charge transfer transition as indicated by a larger Stokes shift of 65-75 nm, a very broad fluorescence band, and a much lower quantum yield compared to that in methanol. This transition has lower energy than the a a* transition but a low probability of occurrence, Le., low quantum yield. The broadness of the band is a result of loss of vibrational quantization in the ground states because of the rapid nuclear adjustments accompanying the charge transfer.lg It is postulated that the charge transfer transition involves electron donation from the homocyclic to the heterocyclic ring, with protonation of the imidazole ring being the driving force. A second weak fluorescence band in the benzimidazole spectra at 291 nm (a a* transition) is seen in 0.1 M HC1 which demonstrates that both the a a* and the charge transfer transitions occur in acid media, although the latter is the predominant mode of radiative deactivation. On the other hand, the a a* transition
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Tway and Cline Love
The Journal of Physical Chemistry, Vol. 86,No. 26, 1982 I
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I
at the 5 position and the a-electronic system. The stronger the electron-donating side chain, the more stabilized the charge transfer transition is relative to the P a* transition. The electron-withdrawingnitrogen at the 8 position also strengthens this transition. All of the data are consistent with the presence of two types of transitions in this series. Both polar solvents and electron-donating substituents at the 5 position stabilize the excited state of the charge transfer transition relative P* transition. For example, 5-hydroxyto the A thiabendazole undergoes a charge transfer transition in dioxane, chloroform, acetonitrile, and methanol, but in hexane the fluorescence transition is very similar to the A a* fluorescence of thiabendazole. Apparently the nonpolar hexane solvent system does not stabilize the charge transfer transition sufficiently, and the a P* transition predominates. In hexane all of the thiabendazole analogues appear to undergo primarily a a* transitions with the exception of 5-aminothiabendazole in which both a a a* (A, = 367 nm) and a charge transfer (X, = 380 nm) transition is seen. This is not unreasonable since the amino group was the strongest electron-donating substituent studied. Hammett Constants Correlation. Additional evidence for the existence of a manifold of excited singlet states is provided by a study of the fluorescence transition energy as a function of the Hammett substituent constants. Processes with a small electron demand (a P* transitions) generally respond less to changes in substituents than processes with a high electron demand (charge transfer transitions) and consequently have smaller Hammett p values. Figure 2 shows a plot of fluorescence energy as a function of the Hammett constants, up0 and u+. The Hammett constant, u+, is used for the proposed charge transfer transitions, and up0 is transitions a A* transitions. The p value for thiabendazole and 5-methylthiabendazole in methanol is -13.7 kcal/mol, which is consistent with an inductive effect from the side chain to the aromatic a system in a a A* transition. The plots for the compounds with more polar substituents have p values of -26.9 and -27.4 kcal/mol in methanol and ethanol, respectively. These p values are large enough in magnitude to support a charge transfer mechanism. The plot of the fluorescence energy of the benzimidazole analogues vs. up0 gave a p value of -16.5 kcal/mol, indicating a P A* mechanism. These Hammett plots also support the proposal that both K a* and charge transfer transfer transitions occur depending on the side-chain substituent on the compound.
-
901
-
1-H
-
-
-
-‘rpo’
-b+)
Figure 2. Correlation of fluorescence emission energy with Hammett substituent constants for a series of thiibendarole derlvathres (-) and benzlmidazole derivatives (- -) In methanol (0)and ethanol (0).
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at 291 nm is the main mode of radiative deactivation in neutral solvents in which benzimidazole is not protonated. The existence of a manifold of excited singlet states in a molecule has been proposed previously to explain the fluorescence properties of indole-type molecules.20-22 Thiabendazole and 5-methylthiabendazole, in contrast to benzimidazole, undergo a a K* transition both in acid and neutral solutions. The fluorescence spectra, quantum yields, and lifetimes change only slightly as a function of solvent which suggests that there is no change of mechanism. Although a a* is the dominant transition, a weak charge transfer transition can be seen in acid solutions of thiabendazole and 5-methylthiabendazole at 375 and 398 nm, respectively. The driving force for the charge transfer transition is less in thiabendazole than in benzimidazole because in thiabendazole the positive charge on the protonated species is diffused over a larger area. From NMR data, a resonance structure which places some of the positive charge on the thienyl ring is known to exist.l8 Substituent Effects. Thiabendazole and benzimidazole analogues with electron-donating groups at the 5 position generally have lower fluorescence energies, larger Stokes shifts (Ax = 70-140 nm), and broadened fluorescence and absorption bands relative to thiabendazole. These data (Tables I and 11) suggest that the addition of a relatively strong electron-donating group at the 5 position changes the mechanism of the fluorescence transition from P a * to a charge transfer transition. 5-Hydroxythiabendazole, 5-aminothiabendazole, and cambendazole undergo a charge transfer transition from the homocyclic to the heterocyclic ring (similar to that proposed for benzimidazole in acid) in most solvent systems. The charge transfer transition is stabilized by resonance structures which show interactions between the nonbonding electrons
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(20)Szabo, A. G.;Rayner, D. M. J.Am. Chem. Soc. 1980,102,554-63. (21)Sun,M.;Song, P. S. Photochem. Photobiol. 1977,25,3. (22)Weber, G.Bzochem. J. 1960,75,335-45.
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Summary Through a complete study of the absorption and luminescence properties, namely, fluorescence and phosphorescence energies, quantum yields, and lifetimes, we have determined that benzimidazole, thiabendazole, and several of their derivatives are excited to a manifold of singlet states; one excited state has a a* character while the other has charge transfer characteristics. Which excited state(s) are appreciably populated depend on both molecular and environmental factors. Generally, in more polar solvents and in solutes with strong electron-donating substituents at the 5 position the charge transfer transition is stabilized relative to the a A* transition. +-
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Acknowledgment. We thank Jerry A. Hirsch for helpful comments on the data. P.C.T. also thanks Merck, Inc. for partial financial support.
