1
Laser-Induced Recombination Delayed Fluorescence of Tryptophan and Indole Solution at Room Temperature. V.M. Mazhul 1, D.G Scharbin, V.A. Shashilov, 2A.A. Sukhodola 2, E.M. Zaitseva, G.B. Tolstorozhev 2 1
Institute of Photobiology, National Academy of Sciences of Belarus, Minsk, Belarus. Institute of Molecular & Atomic Physics, National Academy of Sciences of Belarus, Minsk, Belarus, e-mail:
[email protected]. Fax:375-172-84-00-30. 2
Received May
, 2002.
The specific delayed fluorescence properties for biological molecules tryptophan and its model analog indole has been discovered. Three different mechanisms of second order processes were analyzed. The formation and subsequent recombination of ion - radicals is proposed.
KEY WORDS: Tryptophan, Indole, Solution, Temperature, Recombination Delayed Fluorescence
Room
INTRODUCTION Delayed luminescence of complex compounds is usually associated with a longleaved triplet states [1]. In the 60-70s three mechanisms of delayed luminescence in a gas phase, liquid solution, and in solid media: direct phosphorescence, thermally activated delayed fluorescence (E-type), and delayed annihilation fluorescence (P-type) were established and studied in detail. The E type and P-type fluorescence spectrum coincides with a fluorescence spectrum and a phosphorescence spectrum is shifted to a more long wavelength region. At present phosphorescence in frozen solutions [2] is intensively studied for biologically important molecules of tryptophan and its model compound of indole. Lifetime of triplet states under such conditions lies in the range of 100 ms to 10s. A maximum of a phosphorescence spectrum for both compounds is near 450 nm. When passing to liquid solutions, it is difficult to observe long-leaved luminescence of these compounds at room temperature because of a sharp decrease in the lifetime of triplet states by several orders of magnitude. Long-leaved luminescence of indole derivatives in liquid solutions at room temperature was fist observed in 1995 [3]. Phosphorescence and P-type fluorescence were recorded at an exciting radiation of λ exc = 292 nm and low solution concentration of 5.10 -6 M. The lifetime of triplet state was 1.2 ms. In this article, it was also noted that the obtained value of the lifetime of triplet state measured by phosphorescence technique differs noticeable from the one measured by the induced triplet-triplet triplet absorption (17 – 29 s) [4–7] at a concentration of 10-4 M. This difference might be associated with forming photoproducts (solvated electrons, cations, anions and neutral radicals) at higher concentrations of solution that effectively quench triplet states.
2 Recombination luminescence of tryptophan [8] was also seen in frozen solutions in a mixture with water and ethylene glycol. It was suggested that cation-radicals and electrons are formed under the two - quantum step-by-step excitation of molecules through triplet states. Diffusion of electrons and their subsequent recombination with cation-radicals results in populating the excited singlet and triplet states.
RESULTS The recombination delayed fluorescence of indole and tryptophan in liquid solutions at room temperature was observed and examined in detail in our experiments. Measurements were made on an automated laser spectrometer that permitted the luminescence kinetics measurements for different luminescence recording wavelengths as well as instantaneous luminescence spectra to be recorded at different luminescence decay stages in the micro- and millisecond time ranges. The third - harmonic pulses of a titanium sapphire laser with a time duration of 20 ns that were tuned in the spectral region 230 - 320 nm with energy up to 1 mJ served as exciting radiation. The titanium - sapphire laser was pumped by the second - harmonic pulses of an Nd3+ : YAG laser. Luminescence in a narrow spectrum range, selected by a monochromator MDR-23, was recorded by the photoelectric method using an analog -to-digital converter and computer data output. Water, ethanol, acetonitrile and hexane were used as solvents. When indole and tryptophan molecules were excited by laser the radiation of exc = 265 nm in the first band of electron absorption, delayed fluorescence was observed, decaying in the microsecond time range. Figure 1 plots the spectra of this luminescence measured in 100 s after being excited by pulses as well as the steady fluorescence spectra at exc = 265 nm excitation for tryptophan solutions in hexane and water (pH=7.4). The specific feature of delayed fluorescence is that a long-leaved fluorescence spectrum for both compounds is somewhat shifted in short wavelength region relative to a steady fluorescence one. The value of this shift for indole and tryptophan in different solvents is listed in Table 1. A small decrease in shift is seen when passing from the polar to non-polar solvents. The delayed fluorescence spectrum for tryptophan in hexane (Fig. 2) has also a structure. Its maxima at 290 nm and 300 nm coincide with two short - wavelength maxima of the fluorescence spectrum.
Table 1
Tryptophan Indole
Water, pH=7.4 26 20
Ethanol
Acetonitrile
Hexane
22 20
23
10 —
—
Figure 3a shows the long-leaved fluorescence decay measured at the wavelength of the fluorescence spectrum maximum for tryptophan molecules in hexane. Fluorescence decay in the microsecond time range, and the fluorescence decay law is nonexponential. The time dependence of the luminescence intensity 1/I is linear in the measured time range and points to the diffusional nature of fluorescence. The fluorescence intensity measured at the initial decaying moment depends linearly on the exciting radiation intensity, pointing to its one-photon character. Only a slight, approximately 1.5-fold increase in the delayed fluorescence intensity is seen in deoxygenized tryptophan solutions.
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DISCUSSION The observed laser induced long luminescence of indole and tryptophan at room temperature cannot be thermally activated delayed fluorescence (E-type), since an energy interval between the lower triplet and first excited singlet levels is relatively large ( e.g. , for indole its value is equal to 7000 cm-1 ) [1]. The delayed fluorescence of organic compound solutions under large singlet – triplet splitting is usually due to triplet-triplet annihilation when two molecules colliding in the triplet state form molecules in excited singlet and ground states. In this case the P-type spectrum coincides with the fluorescence one. It is also known that the P-type intensity at the initial time instant is proportional to the squared intensity of an exiting pulse [1]. The observed regularities for long luminescence in nondeoxygenized of indole and tryptophan eliminates the annihilation mechanism of this luminescence. First, it is known [3] that the oxygen concentration in the air - saturated solution constitutes = 1.2·10-3 M while the rate constant of the oxygen quenching of indole triplet states in aqueous solutions is 5.3·10 9 M-1s-1. The lifetime of indole triplet states under these conditions must be about 160 ns and the measured long luminescence time falls in the microsecond time range. Second, as already noted, the delayed fluorescence intensity depends in a linear manner on the exciting radiation one, which is not characteristic of P-type. This is also supported by the experiments on the heavy atom influence. So, adding KI molecules to the solution of indole and tryptophan that augment a molecule yield into a triplet state did not increase the long luminescence intensity but decreased it. Thus, proceeding from the results discussed it may be concluded that the triplet states do not contribute to the observed delayed luminescence. We proposed that the recorded delay fluorescence of indole and tryptophan can be connected with recombination of long - living cation-,anion-, and neutral radicals that have been already recorded for these compounds [4, 9 - 11]. Such an assumption is also supported by our experiments when addition of inhibitors of free radicals (reduced glutadione) has resulted in a considerable decrease in the delayed luminescence intensity of the investigated compounds. Radicals can be formed as a result photochemical processes such as photoionization and photodissociation. The ionization of indole and tryptophan has been earlier seen in aqueous and alcohol solutions under one-photon excitation of molecules in the first band of electron absorption. In this situation, it was established by the methods of laser flashphotolysis that solvated electrons as well as cations–,anions, and neutral radicals [12] are the photoionization products. Solvated electrons are efficiently quenched with oxygen with a rate constant of 1.9·1010 M-1s-1, and, hence, the lifetime of solvated electrons in nondeoxygenized aqueous solutions is about 100 ns [9]. On the other hand, in deoxygenized solutions approximately 80% of the solvated electrons formed decay exponentially due to geminate recombination with cation –radicals already with the time of the order of 800 ns. The solvated electrons, dissociated from the geminate pairs then disappear at diffusional collisions with other particles [10, 11]. Also, note that the lifetime of cation-radicals in aqueous solutions depends on pH of a medium. The lifetimes of cation-radicals in neutral solutions is about 1 s and is less than 1 s in alkali ones. They damp due to the separation of a proton, followed by the formation of neutral radicals in acid solutions is the hundreds of microseconds [4, 9]. Decaying of neutral radicals occurs for the hundreds of microseconds and is governed be the second-order curve [13, 14]. The formation of neutral radicals due to photoionization of an N-H bond is seen for indole derivatives in cyclohexane [15].
4 A detailed discussion of the main properties of the observed long luminescence of indole and tryptophan, with invoking the above-mentioned literature data, enables one to consider possible mechanisms for laser-induced fluorescence. I. Photoionization in aqueous and alcohol solutions and recombination of cation, and anion radicals. As a result of photoionization of molecules that absorbed a quantum of exciting radiation, the geminate ion pairs are formed and then dissociated onto the separated cationradicals and solvated electrons. While colliding with unexcited molecules the solvated electrons form anion radicals. Subsequent diffusion of cation- and anion- radicals gives rise to their pair recombinations and causes molecules to form a fluorescence state. However, this mechanism cannot explain the experimental results for indole and tryptophan solutions in acetonitrile and hexane, for which no photoionization under radiation excitation ( exc = 265 nm) has been earlier observed [16]. Furthermore, the decay kinetics of delayed luminescence is inconsistent with the lifetime of cation-radicals in aqueous solutions at pH=7.4. II. Photodissociation of N-H bond in molecules and recombination of neutral radicals. The dissociation of the N-H bond in an excited state and formation of neutral radicals for indole solutions in cyclohexane were discussed in [14]. The decay kinetics of neutral radicals is the second-order curve in the microsecond time range [13, 14]. The delayed luminescence decay observed in our experiments is consistent with the decay kinetics of neutral radicals. In addition, it was noted in [14] that neutral radicals are not quenched with oxygen.
III. Electron phototransfer. Colliding singlet-excited and unexcited molecules can give rise either to eximers or to colliding complexes which then decompose into cation- and anion-radicals. Such a mechanism of forming radicals was discussed in [17] by analyzing the luminescence of a perylene solution in acetonitrile and was suggested in [18] to elucidate the multiexponential character of the tryptophan fluorescence decay in proteins. Diffusion and pair recombination of cation- and anion-radicals appearing result in populating the excited singlet state. In this case, proton transfer from a cation-radical to an anion-radical being in a geminate pair is possible and is followed by the formation of a neutral radical and a protoned molecule. Within the framework of this recombination mechanism, the particles formed in the radiation state can differ from the singlet-excited particles directly of indole and tryptophan. So, the observed short-wavelength shift of the recombination fluorescence spectrum relative to the stationary fluorescence of indole and tryptophan can be explained using the above data. CONCLUDING REMARKS In our experiments the delayed fluorescence of tryptophan and indole in liquid solutions at room temperature not connected with the participation of long-living triplet states of these compounds was observed. By the proposed mechanisms for long luminescence are meant the bimolecular processes associated with the formation and subsequent recombination of ionradicals. REFERENCES 1.C.A. Parker, In: “Photoluminescence of Solutions”, Elsevier Publishing, Co., Amsterdam, 1968
5 2. A.P. Demchenko, In; “Luminescence and Dynamics of the Protein Structure ”, Kiev: Navukova Dumka, 1988. 3. G.B. Strambini, M. Gonnelli (1995) J. Am. Chem. Soc. 117, 7646-7651. 4. D.V. Bent, E. Hayon (1975) J. Am. Chem. Soc. 97,2612-2619. 5. L.I. Grossweiner, A.G. Kaliskar, J.F. Baugher (1976) Int. J. Radiant. Biol. 29, 1-16. 6. W.A. Volkert, R.R. Kuntz, C.A. Chiron, R. F. Evans, R. Santus (1977) Photochemi. Photobiol. 26, 3-9. 7. C. Pepmiller, E. Bedwell, R.R. Kuntz, C.A. Chiron (1983) Photochemi. Photobiol. 38, 273-280. 8. J. Moan, (1974) J. Chem. Phys. 60, 3859-3865. 9. W.G. McGimpsey, H. Corner (1996) Photochemi. Photobiol. 64, 501-509. 10. F.D. Bryant, R. Santus,. L.I. Grossweiner (1975) J. Phys. Chem. 79, 2711-2716. 11. J.F. Baugher, L.I. Grossweiner (1977) J. Phys. Chem. 81,1349-1354. 12. D. Greed (1984) ) Photochemi. Photobiol. 39, 537-562. 13. H. Templer, P.J. Thistlethwaite (1976) ) Photochemi. Photobiol. 23, 79-85. 14. D. Burdi et al. (1977)J. Am. Chem. Soc. 119, 6457-6460. 15. M.T. Pailthorpe, C.N. Nicolls (1971) ) Photochemi. Photobiol. 14, 135-145. 16. F. Saito, S. Tobita, H. Shizuka (1997) J. Photochemi. Photobiol. A. 106, 119-126. 17. K.H. Grellmann, A.R. Watkins (1971) Chem. Phys. Lett.9, 439. 18. B.S. Hudson, J.M. Huston, G. Soto-Campos (1999) J. Phys. Chem. A. 103, 2227-2234.
FIGURE CAPTIONS Fig. 1. Spectra of fluorescence (2, 4) and delayed fluorescence measured in 100 s after excitation for tryptophan (1, 2) and indole (3, 4) in water (pH=7.4) at room temperature. Fig. 2 Fluorescence spectra of (1) and delayed fluorescence for tryptophan in hexane at room temperature. Fig. 3. Damping kinetics I (a.u) of tryptophan delayed fluorescence in hexane (a), time dependence of the reciprocal of the delayed fluorescence intensity 1/I (a. u.) of tryptophan in hexane (b).
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I, отн.ед. 1
1.0
2
0.5
300
Fig 1
350
400 , нм
7
I, отн.ед. 1.0
12
3
4
0.5
300
350
FIG 2
400 , нм
8
Ln I/I 0
а
0
-1
-2
200
400
t, мкс
1/I 4
б
2
0
200
Fig 3
400
t, мкс
