Pliotoclwmistry und Photohioloyy. 1975.
Vol. 21, pp. 141- 145
Pergamon Press
Printed
in
Great Britain
LUMINESCENCE PROPERTIES AND RELATED PHOTOCHEMISTRY OF 4-AMINO-2-METHOXY P Y RIMIDINE A. G. SZABOand K. 9. BERENS* Division of Biological Sciences, National Research Council of Canada, Ottawa, Ontario K1A OR6, Canada (Received 1 1 June 1974; accepted 23 September 1974)
Abstract-The luminescence properties of 4-amino-2-methoxypyrimidine. I, have been studied in several solvents. These studies indicate that the photochemical reaction of 1 with dihydrogen phosphate buffer proceeds via an intermediate derived from the fluorescent singlet excited state of I.
MATERIALS AND METHODS
INTRODUCTION
The title compound, 4-amino-2-methoxypyrimidine, I, The ultraviolet photochemistry of nucleic acids has been extensively studied and at least 2 reactions, the was purchased from.Eastman Organic Chemicals, RochesN.Y. It was sublimed prior to use, mp 171-172"C (lit. photodimerization and photohydration of pyrimi- ter, Moore, 1963 mp 170-171°C). The diene 2,4-hexadien-l-ol dines, are considered to be biologically effective (Smith was purchased from Aldrich Chemical Co. and distilled and Hanawalt, 1969; McLaren and Shugar, 1964). Ear- prior to use. bp (22 mm) 8344°C. The ethanol used was purified according to the method lier, Moore (1963) and later Pitha and Butler (1968) demonstrated that the photochemistry of 4-amino-2- described by Parker (1968).Other solvents were MCB Spectroquality Solvents and used without further purification. methoxypyrimidine I, a cytosine derivative, and cerUltraviolet spectra were obtained from either a Cary I 5 tain other 4-aminopyrimidines varied depending on or Cary 14 spectrophotometer. Fluorescence and phosphorthe presence or absence of dihydrogen phosphate escence measurements were determined with an NRC-built buffers in solution. Moore found that compound I, spectrofluorimeter. This consisted of a 500 W xenon arc for when irradiated in aqueous solution, decomposed with the excitation source and two Jarrell-Ash, 82-410, 0.25 m,fi 3.5 monochromators, one being used as an excitation monoa quantum yield 4 = 6.5 x In a dihydrogen- chromator, the other as an emission monochromator. The phosphate-buffered solution, pH 7.6, photolysis of I emission was monitored at right angles and detected by an resulted in the formation of a new absorption band at EMI-6256B photomultiplier. The signal was amplified by a 300nm with q5 = 1.7 x The absorption at Keithley 610C Electrometer and recorded on a Moseley 7101B strip chart recorder. Phosphorescence was detected 300nm has been tentatively assigned by Pitha and using a rotating can. No light leakage was detectable when Butler to a ring cleavage product, 11. Moore studied a quinine sulphate sample was placed in the sample holder. the effect ofphosphate concentration and of pH on the The fluorescence and phosphorescence spectra were formation of the 300 nm product and concluded that determined with a narrow-band Bausch & Lomb 3.67 gm- I dihydrogen phosphate acted as a 'catalyst' in the pho- interference filter in the excitation beam. A Pyrex@filter was in the monitoring beam when phosphorescence was tolysis of I. This effect was shown to be specific for placed measured. The excitation spectra were recorded with 1 cm phosphate buffers, since photolysis of I in other buffers of a NiS04/CoS04 solution filter (Kasha, 1948). The slits a t the same pH gave the same results as irradiation in were 1 mm on the exkitation monochromator and 2 mrn on unbuffered solutions. It was also shown that a ground the emission monochromator (dispersion 1.65 nmimm). Emission and excitation spectra were corrected according state complex between I and dihydrogen phosphate to the methods outlined by Melhuish (1972). A sample of was not involved in the reaction. In addition the for- Rhodawine B was used to determine the spectral distribumation of 11 was not a dark reaction of another photo- tion of the excitation source, and samples of 2-aminopyriproduct and phosphate. dine (lo-' M in 0.5 M H2S04)and quinine sulphate ( 2 x NH,
NH, I
I
II Scheme I.
In this paper we have studied the photophysics of I in solution and at 77 K and its relation to the photochemical 'phosphate' effect. *N.R.C.C. Postdoctoral Fellow 1971-1973. N.R.C.C. No. 14521.
M in 025 M H,SO,) were used to calibrate the detection monochromator and photomultiplier response. Quantum yields for emission at 300 K were calculated in comparison to that of an aqueous solution of tryptophan (Chen, 1967)and for emissionat 77 K in comparison to that of benzophenone (Gilmore et nl., 1952, 1955). The single-photon counting apparatus is similar to that described by Ware (1971) using a free running lamp. The flash emission apparatus has been described elsewhere (Porter et al., 1971). The flash absorption apparatus (120 J. 1/2 bandwidth 0 8 p ) is essentially the same as that described by Yip e t al. (1970) except that each lamp circuit used two 0.05 pF. 40 kV capacitors and the lamps were filled with 6 cm xenon and 1 cm hydrogen. Samples for flash excitation were degassed by 4 freezepump-thaw cycles.
141
142
and K. B. BERENS A. G. SZABO
The phosphorescence lifetimes at 77 K were determined using the flash emission apparatus with a combination of a Corning 7-54 filter and the previously described Ni/Co filter in the excitation beam. The emission was detected by a Jarrell-Ash monochromator and a 1P28, 9-stage photomultiplier. The signal was transmitted to a Hewlett-Packard 5754A signal averager operating in a summation mode. The accumulated signal was recorded on a Moseley recorder. Synchronization of the signal averager with the lamp discharge is the same as has been described by Yip (1969).The phosphorescence decay was then digitized and the phosphorescence lifetime calculated by a least-squares fit of the data points using a program developed for the Wang model 370 Calculator. Two irradiation apparatuses were used in this work. For experiments with oxygen or 2,4-hexadien-1-01, 6 germicidal lamps were symmetrically placed 0.5cm from a quartz water jacket (1 cm path) into which a quartz tube (1 cm dia) could be placed. The reaction was monitored by removing aliquots from the sample tube and, after appropriate dilution, UV absorption spectra were measured on the Cary 15 spectrophotometer. The effect of oxygen was studied by bubbling oxygen through a solution of I (I x M) and dihydrogen phosphate (pH 7, 0 0 5 M ) during the photolysis. The effect of 2,4-hexadien-l-ol was studied by irradiation of a similar solution, except that the solution contained 2,4hexadien-1-ol(O.1M)and nitrogen was bubbled through the solution during the course of the reaction. The second irradiation apparatus consisted of a Pyrexjacketed Hanovia medium-pressure 450 W mercury arc, which was water-cooled and inserted into a reaction vessel containing the solution to be irradiated. The path length was I cm. The effect of acetone sensitization was studied by irradiation of a phosphate-buffered (0.1 M , pH 7) solution of aceM) for 16 h in the second tone (0.05 M ) and I (1 x apparatus.
RESULTS AND DISCUSSION
WAVELENGTH (nmj
Figure 1. Absorption and corrected relative fluorescence spectra of 4-amino-2-methoxypyrimidine (I) (4 x 10- M ) in water, 300 K. Excitation wavelength for fluorescence was 3.67 pm- ',excitation bandwidth 2 nm, emission bandwidth 3 nm.
'
The singlet lifetime of I at 77 K was measured and found to be 7, = 4 0.2 ns. This measured lifetiinc is in reasonable agreement with the calculated va!uc of z, = 3 ns, obtained from the relationship
4 p o = 7,
(1)
where 4f was directly measured (Table 2) and the radiative lifetime T,, was calculated from the integrated intensity of the absorption band (Turro, 1967). It is then possible to estimate the singlet lifetime of I in aqueous solution at 300 K using relationship (1). This value is calculated to be 3 x 10- l 1 s. If the singlet excited state of I were reacting with dihydrogen phosphate to form I1 the following processes could describe the deactivation of the excited singlet state of I:
The nature of the excited state involved in the 'phosphate' effect is of fundamental importance. Using a variety of luminescence and absorption techniques we were able to characterize the excited state properties of I. The fluorescence spectra of I in several solvents were 'I-+ 0 1 recorded at 300K and the respective values of 4,. 'I--* 3 1 determined (Table 1). The fluorescence spectrum of 'I-+ Product I in aqueous solution, pH 7.6, has a maximum at 'I-+''I + hv k, 315nm, from which a (pf = 3 x can be determined (Fig. 1).The excitation spectrum of I in aqueous H,PO, 'I-tII k, solution is superimposable on the absorption spectrum Moore measured the rate of formation of I1 and was of I. The fluorescence spectrum of I at 77 K in ethanol, able to determine the ratio k , + kr/kR = 0.007 M . Fig. 2, is similar to that in aqueous solution, and the From the calculated value of 3 x s - ' for k,, + 4,. =: 035. k, from the fluorescence results (uide supra), thc value of k R i s calculated to be 5 x 10' M - I s- ', This Table I. Fluorescence properties of I at 300 K high value suggests that the proposed mechanism involving a reaction of dihydrogen phosphate anion with Solvent the fluorescent singlet state of I is unlikely. Further, an optimum value of & = 0.9 (0.05 M H,PO,) (quanWater 315 3 tum yield of formation of 11) can be calculated, which 0 1 M H,PO; 315 3 is considerably greater than the measured value of 1.7 pH 7.6 buffer Acetonitrile 330 12 x 10-3. t-Butanol 322 10 Additionally if dihydrogen phosphate anion were inEthanol 3 20 9 teracting with the fluorescent singlet state of I with k K EPA 320 7 = 5 x 1012M - ' s - ' to give the observed photoche-
p-
+
-
Luminescence properties and related photochemistry
143
s, indicating that the lowest triplet of I was Lo - n in character (Kasha and Rawls, 1968). At 300K, as expected, no phosphorescence of I could be observed. Using a flash absorption apparatus (120 J, 0-8p) (Yip et al., 1970), we flash-excited degassed aqueous solutions of I ( M) and degassed dihydrogen-phosphate (0.1 M)-buffered solutions of I 10-4M). Both solutions were at pH 7.6. No transient absorption was detected from 300 to 600 nm, indicating that either &c was very low and/or T, was very short ( < 1 p).A degassed aqueous solution of I ( M ) containing acetone (0.05 M ) was flash-excited using a Pyrex filter. Under these conditions acetone was the sole absorber of light (Yip et al., 1970). Again no transient absorption except that of the acetone triplet excited state was observed. However, energy transfer from acetone ( E , = 78 WAVENUMBER ( p n - ' l kcal/mol) (Borkman and Kearns, 1966) to I ET = 84 Figure 2. Corrected fluorescence and phosphorescence M ) kcal/mol) is endothermic, and the rate constant for spectra of 4-amino-2-methoxypyrimidine (I) (4 x quenching, k,, of acetone triplet, by I should be less in ethanol. 77 K. than that for a diffusion-controlled process (Herkmica1 effect. fluorescence quenching of I by dihydrogen stroeter and Hammond, 1966). Using the flash-emisphosphate would be expected ([H,PO;] required for sion apparatus, the phosphorescence of degassed 50% quenching = OG07 M). However, the fluorescence aqueous solutions of acetone and varying amounts of and excitation spectra of 1 in 0.1 M dihydrogen phos- I was monitored. The value of k, was obtained from a phate buffer, pH 7.6, were superimposable on those plot of the rate of acetone phosphorescence decay vs obtained from aqueous solution, and the 4fwas equal concentration of I (Fig. 3) and found to be 1 x 10' to that measured in aqueous solution. M - s- a value consistent with endothermic energy Clearly, these results together with Moore's mea- transfer. Although these results do not allow one to sured ratio of k,, k , / k , indicate that dihydrogen attribute the quenching of acetone phosphorescence to phosphate anion does not react with the fluorescent energy transfer unambiguously, the alternatives are singlet state of 1. less attractive. Hydrogen abstraction by acetone can The foregoing results suggested the intermediacy of be eliminated, since the acetone ketyl radical is not the triplet excited state of I in the reaction with dihyd- observed in the flash-absorption experiments with I rogen phosphate. The phosphorescence spectrum, Fig. (vide supra). Back-transfer from the triplet state of I to 2, of I in ethanol glass a t 77 K was recorded and the acetone would have a negligible contribution to the quantum yield 4p = 1 x measured. The phos- observed acetone phosphorescence decay. Electron phorescence excitation spectrum of I under similar transfer is an alternative quenching process. However, conditions was the same as the absorption spectrum of based on the ionization potential of pyrimidines [cytoI. A value of 15, = 84 kcal/mol for I was estimated from sine, I.P. - 8.90eV (Bergman et al.. 1969) the rate I the phosphorescence spectrum. The phosphorescence lifetime was determined using a flash emission apparatus (4 J, 0.5 ps) (Porter et al., 197I ) and a computer-averaging transient (CAT) device. The technique involved repetitive flashing of the sample and summation of the weak emission signal in the CAT. The stored phosphorescence decay was then traced out on a recorder and the phosphorescence lifetime determined by a least-squares fit of the exponential decay. For 1 in an ethanol glass at 77 K the phosphorescence decay was measured at 370 nm. Sixteen repetitions were required, from which the phosphorescence lifetime was determined to be 0 1 4 0.01 WAVELENGTH (nml
',
+
Tahle 2. Luminescence properties of I in ethanol at 77 K
Property
'
111.1,
(b
Li fct i me
Fluorescence
Phosphorescence
314nm 0.35
372 nm
4 2 0211s
0.14 f 0.01 s
1
I
I
I
2
3
[I]
10-3
x
104 M
Figure 3. Plot of rate constant for decay (kobs)of acetone emission vs concentration of 4-amino-2-methoxypyrimidine (1) for determination of quenching rate constant (k,) of 1.
144
A. G. SZABO and K. B. BERENS
constant for quenching of acetone triplet by this mechanism can be estimated to be -106M-’s-’ (Yip et al., 1972). In addition Helene (1972) has measured the k, of the triplet state of benzophenone by thymine and adenine to be 1 2 x 10’ M-’ s - ’ , a process which he attributes to a charge-transfer mechanism. Our measured value of 108M-’ s - l for quenching of acetone triplet by I, while not rigorously excluding electron transfer as the quenching process, is more consistent with an endothermic energy transfer process. These experiments allow us to conclude that I quenches the acetone triplet state with sufficient efficiency (20%) that the T-Tabsorption of I should have been observable in the flash-absorption experiment with acetone. Obviously the 7T must be less than 1 ps, the lower limit of detectibility of our flash instrumentation. Steady-state experiments allow us to exclude the triplet state of I as an intermediate in the ‘phosphate’ M) reaction. A phosphate-buffered solution of I and acetone (0.1 M ) was irradiated for 16 h using a Pyrex filter, conditions in which acetone is the sole absorber of light. No reaction of I occurred under these conditions. Unfortunately, the choice of water-soluble triplet sensitizers with triplet energies comparable to that of I and with an appropriate absorption spectrum is limited, so that other sensitizers could not be used. The intermediacy of a triplet excited state in this photoreaction could be definitively excluded, since neither oxygen [ 1 x M ] nor 2,4-hexadienol [0.1 M ] [ET = 56 kcal M-’ (Yip et al., 1970)], both effective quenchers of triplet excited states, had no effect whatsoever on the rate of formation of I1 nor on the photoreaction of I. It can be shown that even if the triplet lifetime of I was as short as 1 ns, at least 10 per cent quenching by hexadienol would be expected. These results, taken together with the previous flash emission results, indicate that the triplet state of I is not an intermediate in the reaction of I with dihydrogen phosphate buffers. We conclude that neither the triplet nor the fluorescent singlet state of I react with dihydrogen phosphate. Rather, some other intermediate derived from the singlet state of I must be involved in the ‘phosphate’ effect. It is evident from the sum of the luminescence quantum yields at 77 K, #, + bp,for I that radiationless deactivation processes of the excited states of I are very efficient. At 77 K in an ethanol glass this sum is 0.35, considerably less than I . It appears that this is a general property of the excited states of aminopyrimidine derivatives. Berens et al. (1971) made similar
-
observations in their studies on the luminescence properties of 2-aminopyrimidine (111) and 4-aminopyrimidine (IV). They found values of q5f bP of 0.40 and 0.039 for 111 and IV, respectively. Inspection of Table 1 reveals that bf of I at 300 K increases with decreasing hydrogen-bonding ability of the solvent. This suggests that one deactivation process is influenced by hydrogen bonding with the solvent. One might well inquire whether the efficient radiationless deactivation pathway of I proceeds via an intermediate that is responsible for the ‘phosphate’ effect. The nature of this intermediate is unknown but may be either a lower, non-fluorescent singlet state or an isomer or tautomer derived from the single state. Alternatively the ‘phosphate’ effect may be the reaction of the dihydrogen phosphate anion with an upper vibrational level of the ground state reached after deactivation of the singlet excited state. Recently Summers et al. (1973) and Summers and Burr (1972) suggested that the nucleophilic photoaddition to uracil involved an excited singlet state of uracil lower in energy than the fluorescent state of uracil. Whitten and Lee (1971) presented evidence that an nn* singlet of acridine was responsible for the photoreduction of acridine and that this state was lower in energy than the n-n* fluorescent state. Li and Lim (1971) observed that fluorescence from pyrimidine originated from the second singlet state, which is very close in energy to the first singlet state. Thus evidence is accumulating that the wn* and A n* singlet states are of similar energy in aza aromatics such as 1, and the reactive state may be the lowest state while the fluorescent state is a n upper singlet state. The dihydrogen phosphate anion likely reacts through a nucleophilic addition to C-6 of the pyrimidine ring with subsequent ring cleavage and hydrolysis of the adduct, leading to 11. The work of Pitha and Butler (1968) indicates that C-6 substituents apparently prevent the ‘phosphate’ effect. In summary, we have demonstrated that the photoinduced reaction of I in dihydrogen phosphate buffer is a reaction with an intermediate derived from the fluorescent singlet excited state. This has important implications in the photobiology of nucleic acids. It suggests that intermediates other than the fluorescent singlet excited states of nucleic acids can be involved in photochemical modifications of nucleic acids. Obviously, the photochemistry of the nucleic acids would vary according to the nature of these states. Moreover, dihydrogen phosphate, a molecule ubiquitous in cellular systems, reacts with these intermediates.
+
REFERENCES Berens, K., J. Smagowicz and K. L. Wierzchowski (1971) I n t . Eur. Biophys. Congr, Abstract pp. 63- 67. Bergman, E. D., H. Weiler-Feiechenfeld and C . Lifshitz (1969) Jerusalem Symposium 1, 72-77. Borkman, R. F. and D. R. Kearns (1966) J. Clwm. Phys. 44,945-949. Chen, R. F. (1967) Aiiul. Letters 1, 35-42. Gilmore, E. H., G . E. Gibson and D. S. McClure (1952) J . Chcm. Phys. 20, 829-836.
Luminescence properties and related photochemistry Gilmore, E. H., G. E. Gibson and D. S. McClure (1955) J . Chem. Phys. 23,399. Helene, C. (1972) Photochem. Photobiol. 16, 519-522. Herkstroeter, W. G. and G. Hammond (1966) J . Am. Chem. Sac. 88,4769-4777. Li, Y. H. and E. C. Lim (1971) Chem. Phys. Letters 9, 514-516. Kasha, M. (1948) J . Opt. Soc. Am. 38,929-934. Kasha. M. and H. R. Rawls (1968) Photochem. Photobiol. 7, 561-569. McLaren, A. D. and D. Shugar (1964) In Photochemistry of Proteins and Nucleic Acids, Pergamon Press, New York. Melhuish, W. H. (1972) J . Res. N.B.S. 76A, 547-560. Moore, A . M . (1963) Can. J . Chem. 41, 1937-1950. Parker, C. A. (1968) In Photoluminescence ofSolutions, p. 420. Elsevier, New York. Pitha, P. M. and G. C. Butler (1968) Can. J . Biochem. 46, 893-897. Porter, G., R. W. Yip, J. M. Dunston, A. J. Cessna and S. E. Sugamori (1971) Trans. Faraday Soc. 67, 3149-3154. Smith, K. C. and P. C. Hanawalt (1969) In Molecular Photobiology, Inactivation and Recovery, Academic Press, New York. Summers, W. A,, Jr. and J. G. Burr (1972) J . Phys. Chem. 76,3137-3141. Summers, W. A,, Jr., C. Enwall, J. G. Burr and R. L. Letsinger (1973) Photochem. Photobiol. 17, 295301. Turro, N. J. (1967) In Molecular Photochemisfry, pp. 48-49. Benjamin, New York. Ware, W. R. (1971) In Creation and Detection qfthe Excited State, Vol. 1, Part A, p. 213. Marcel Dekker, New York. Whitten, D. G. and Y. J. Lee (1971) J . Am. Chem. Soc. 93, 961-966. Yip, R. W. (1969) Rev. Sci. Instr. 40, 1035-1040. Yip, R. W., R. 0. Loutfy, Y. L. Chow and L. K. Magdzinski (1972) Can. J . Chem. 50,34263431. Yip, R. W., W. D. Riddell and A. G . Szabo (1970) Can. J . Chem. 48,987-999.
I45
Vol. 21, pp. 141- 145
Pergamon Press
Printed
in
Great Britain
LUMINESCENCE PROPERTIES AND RELATED PHOTOCHEMISTRY OF 4-AMINO-2-METHOXY P Y RIMIDINE A. G. SZABOand K. 9. BERENS* Division of Biological Sciences, National Research Council of Canada, Ottawa, Ontario K1A OR6, Canada (Received 1 1 June 1974; accepted 23 September 1974)
Abstract-The luminescence properties of 4-amino-2-methoxypyrimidine. I, have been studied in several solvents. These studies indicate that the photochemical reaction of 1 with dihydrogen phosphate buffer proceeds via an intermediate derived from the fluorescent singlet excited state of I.
MATERIALS AND METHODS
INTRODUCTION
The title compound, 4-amino-2-methoxypyrimidine, I, The ultraviolet photochemistry of nucleic acids has been extensively studied and at least 2 reactions, the was purchased from.Eastman Organic Chemicals, RochesN.Y. It was sublimed prior to use, mp 171-172"C (lit. photodimerization and photohydration of pyrimi- ter, Moore, 1963 mp 170-171°C). The diene 2,4-hexadien-l-ol dines, are considered to be biologically effective (Smith was purchased from Aldrich Chemical Co. and distilled and Hanawalt, 1969; McLaren and Shugar, 1964). Ear- prior to use. bp (22 mm) 8344°C. The ethanol used was purified according to the method lier, Moore (1963) and later Pitha and Butler (1968) demonstrated that the photochemistry of 4-amino-2- described by Parker (1968).Other solvents were MCB Spectroquality Solvents and used without further purification. methoxypyrimidine I, a cytosine derivative, and cerUltraviolet spectra were obtained from either a Cary I 5 tain other 4-aminopyrimidines varied depending on or Cary 14 spectrophotometer. Fluorescence and phosphorthe presence or absence of dihydrogen phosphate escence measurements were determined with an NRC-built buffers in solution. Moore found that compound I, spectrofluorimeter. This consisted of a 500 W xenon arc for when irradiated in aqueous solution, decomposed with the excitation source and two Jarrell-Ash, 82-410, 0.25 m,fi 3.5 monochromators, one being used as an excitation monoa quantum yield 4 = 6.5 x In a dihydrogen- chromator, the other as an emission monochromator. The phosphate-buffered solution, pH 7.6, photolysis of I emission was monitored at right angles and detected by an resulted in the formation of a new absorption band at EMI-6256B photomultiplier. The signal was amplified by a 300nm with q5 = 1.7 x The absorption at Keithley 610C Electrometer and recorded on a Moseley 7101B strip chart recorder. Phosphorescence was detected 300nm has been tentatively assigned by Pitha and using a rotating can. No light leakage was detectable when Butler to a ring cleavage product, 11. Moore studied a quinine sulphate sample was placed in the sample holder. the effect ofphosphate concentration and of pH on the The fluorescence and phosphorescence spectra were formation of the 300 nm product and concluded that determined with a narrow-band Bausch & Lomb 3.67 gm- I dihydrogen phosphate acted as a 'catalyst' in the pho- interference filter in the excitation beam. A Pyrex@filter was in the monitoring beam when phosphorescence was tolysis of I. This effect was shown to be specific for placed measured. The excitation spectra were recorded with 1 cm phosphate buffers, since photolysis of I in other buffers of a NiS04/CoS04 solution filter (Kasha, 1948). The slits a t the same pH gave the same results as irradiation in were 1 mm on the exkitation monochromator and 2 mrn on unbuffered solutions. It was also shown that a ground the emission monochromator (dispersion 1.65 nmimm). Emission and excitation spectra were corrected according state complex between I and dihydrogen phosphate to the methods outlined by Melhuish (1972). A sample of was not involved in the reaction. In addition the for- Rhodawine B was used to determine the spectral distribumation of 11 was not a dark reaction of another photo- tion of the excitation source, and samples of 2-aminopyriproduct and phosphate. dine (lo-' M in 0.5 M H2S04)and quinine sulphate ( 2 x NH,
NH, I
I
II Scheme I.
In this paper we have studied the photophysics of I in solution and at 77 K and its relation to the photochemical 'phosphate' effect. *N.R.C.C. Postdoctoral Fellow 1971-1973. N.R.C.C. No. 14521.
M in 025 M H,SO,) were used to calibrate the detection monochromator and photomultiplier response. Quantum yields for emission at 300 K were calculated in comparison to that of an aqueous solution of tryptophan (Chen, 1967)and for emissionat 77 K in comparison to that of benzophenone (Gilmore et nl., 1952, 1955). The single-photon counting apparatus is similar to that described by Ware (1971) using a free running lamp. The flash emission apparatus has been described elsewhere (Porter et al., 1971). The flash absorption apparatus (120 J. 1/2 bandwidth 0 8 p ) is essentially the same as that described by Yip e t al. (1970) except that each lamp circuit used two 0.05 pF. 40 kV capacitors and the lamps were filled with 6 cm xenon and 1 cm hydrogen. Samples for flash excitation were degassed by 4 freezepump-thaw cycles.
141
142
and K. B. BERENS A. G. SZABO
The phosphorescence lifetimes at 77 K were determined using the flash emission apparatus with a combination of a Corning 7-54 filter and the previously described Ni/Co filter in the excitation beam. The emission was detected by a Jarrell-Ash monochromator and a 1P28, 9-stage photomultiplier. The signal was transmitted to a Hewlett-Packard 5754A signal averager operating in a summation mode. The accumulated signal was recorded on a Moseley recorder. Synchronization of the signal averager with the lamp discharge is the same as has been described by Yip (1969).The phosphorescence decay was then digitized and the phosphorescence lifetime calculated by a least-squares fit of the data points using a program developed for the Wang model 370 Calculator. Two irradiation apparatuses were used in this work. For experiments with oxygen or 2,4-hexadien-1-01, 6 germicidal lamps were symmetrically placed 0.5cm from a quartz water jacket (1 cm path) into which a quartz tube (1 cm dia) could be placed. The reaction was monitored by removing aliquots from the sample tube and, after appropriate dilution, UV absorption spectra were measured on the Cary 15 spectrophotometer. The effect of oxygen was studied by bubbling oxygen through a solution of I (I x M) and dihydrogen phosphate (pH 7, 0 0 5 M ) during the photolysis. The effect of 2,4-hexadien-l-ol was studied by irradiation of a similar solution, except that the solution contained 2,4hexadien-1-ol(O.1M)and nitrogen was bubbled through the solution during the course of the reaction. The second irradiation apparatus consisted of a Pyrexjacketed Hanovia medium-pressure 450 W mercury arc, which was water-cooled and inserted into a reaction vessel containing the solution to be irradiated. The path length was I cm. The effect of acetone sensitization was studied by irradiation of a phosphate-buffered (0.1 M , pH 7) solution of aceM) for 16 h in the second tone (0.05 M ) and I (1 x apparatus.
RESULTS AND DISCUSSION
WAVELENGTH (nmj
Figure 1. Absorption and corrected relative fluorescence spectra of 4-amino-2-methoxypyrimidine (I) (4 x 10- M ) in water, 300 K. Excitation wavelength for fluorescence was 3.67 pm- ',excitation bandwidth 2 nm, emission bandwidth 3 nm.
'
The singlet lifetime of I at 77 K was measured and found to be 7, = 4 0.2 ns. This measured lifetiinc is in reasonable agreement with the calculated va!uc of z, = 3 ns, obtained from the relationship
4 p o = 7,
(1)
where 4f was directly measured (Table 2) and the radiative lifetime T,, was calculated from the integrated intensity of the absorption band (Turro, 1967). It is then possible to estimate the singlet lifetime of I in aqueous solution at 300 K using relationship (1). This value is calculated to be 3 x 10- l 1 s. If the singlet excited state of I were reacting with dihydrogen phosphate to form I1 the following processes could describe the deactivation of the excited singlet state of I:
The nature of the excited state involved in the 'phosphate' effect is of fundamental importance. Using a variety of luminescence and absorption techniques we were able to characterize the excited state properties of I. The fluorescence spectra of I in several solvents were 'I-+ 0 1 recorded at 300K and the respective values of 4,. 'I--* 3 1 determined (Table 1). The fluorescence spectrum of 'I-+ Product I in aqueous solution, pH 7.6, has a maximum at 'I-+''I + hv k, 315nm, from which a (pf = 3 x can be determined (Fig. 1).The excitation spectrum of I in aqueous H,PO, 'I-tII k, solution is superimposable on the absorption spectrum Moore measured the rate of formation of I1 and was of I. The fluorescence spectrum of I at 77 K in ethanol, able to determine the ratio k , + kr/kR = 0.007 M . Fig. 2, is similar to that in aqueous solution, and the From the calculated value of 3 x s - ' for k,, + 4,. =: 035. k, from the fluorescence results (uide supra), thc value of k R i s calculated to be 5 x 10' M - I s- ', This Table I. Fluorescence properties of I at 300 K high value suggests that the proposed mechanism involving a reaction of dihydrogen phosphate anion with Solvent the fluorescent singlet state of I is unlikely. Further, an optimum value of & = 0.9 (0.05 M H,PO,) (quanWater 315 3 tum yield of formation of 11) can be calculated, which 0 1 M H,PO; 315 3 is considerably greater than the measured value of 1.7 pH 7.6 buffer Acetonitrile 330 12 x 10-3. t-Butanol 322 10 Additionally if dihydrogen phosphate anion were inEthanol 3 20 9 teracting with the fluorescent singlet state of I with k K EPA 320 7 = 5 x 1012M - ' s - ' to give the observed photoche-
p-
+
-
Luminescence properties and related photochemistry
143
s, indicating that the lowest triplet of I was Lo - n in character (Kasha and Rawls, 1968). At 300K, as expected, no phosphorescence of I could be observed. Using a flash absorption apparatus (120 J, 0-8p) (Yip et al., 1970), we flash-excited degassed aqueous solutions of I ( M) and degassed dihydrogen-phosphate (0.1 M)-buffered solutions of I 10-4M). Both solutions were at pH 7.6. No transient absorption was detected from 300 to 600 nm, indicating that either &c was very low and/or T, was very short ( < 1 p).A degassed aqueous solution of I ( M ) containing acetone (0.05 M ) was flash-excited using a Pyrex filter. Under these conditions acetone was the sole absorber of light (Yip et al., 1970). Again no transient absorption except that of the acetone triplet excited state was observed. However, energy transfer from acetone ( E , = 78 WAVENUMBER ( p n - ' l kcal/mol) (Borkman and Kearns, 1966) to I ET = 84 Figure 2. Corrected fluorescence and phosphorescence M ) kcal/mol) is endothermic, and the rate constant for spectra of 4-amino-2-methoxypyrimidine (I) (4 x quenching, k,, of acetone triplet, by I should be less in ethanol. 77 K. than that for a diffusion-controlled process (Herkmica1 effect. fluorescence quenching of I by dihydrogen stroeter and Hammond, 1966). Using the flash-emisphosphate would be expected ([H,PO;] required for sion apparatus, the phosphorescence of degassed 50% quenching = OG07 M). However, the fluorescence aqueous solutions of acetone and varying amounts of and excitation spectra of 1 in 0.1 M dihydrogen phos- I was monitored. The value of k, was obtained from a phate buffer, pH 7.6, were superimposable on those plot of the rate of acetone phosphorescence decay vs obtained from aqueous solution, and the 4fwas equal concentration of I (Fig. 3) and found to be 1 x 10' to that measured in aqueous solution. M - s- a value consistent with endothermic energy Clearly, these results together with Moore's mea- transfer. Although these results do not allow one to sured ratio of k,, k , / k , indicate that dihydrogen attribute the quenching of acetone phosphorescence to phosphate anion does not react with the fluorescent energy transfer unambiguously, the alternatives are singlet state of 1. less attractive. Hydrogen abstraction by acetone can The foregoing results suggested the intermediacy of be eliminated, since the acetone ketyl radical is not the triplet excited state of I in the reaction with dihyd- observed in the flash-absorption experiments with I rogen phosphate. The phosphorescence spectrum, Fig. (vide supra). Back-transfer from the triplet state of I to 2, of I in ethanol glass a t 77 K was recorded and the acetone would have a negligible contribution to the quantum yield 4p = 1 x measured. The phos- observed acetone phosphorescence decay. Electron phorescence excitation spectrum of I under similar transfer is an alternative quenching process. However, conditions was the same as the absorption spectrum of based on the ionization potential of pyrimidines [cytoI. A value of 15, = 84 kcal/mol for I was estimated from sine, I.P. - 8.90eV (Bergman et al.. 1969) the rate I the phosphorescence spectrum. The phosphorescence lifetime was determined using a flash emission apparatus (4 J, 0.5 ps) (Porter et al., 197I ) and a computer-averaging transient (CAT) device. The technique involved repetitive flashing of the sample and summation of the weak emission signal in the CAT. The stored phosphorescence decay was then traced out on a recorder and the phosphorescence lifetime determined by a least-squares fit of the exponential decay. For 1 in an ethanol glass at 77 K the phosphorescence decay was measured at 370 nm. Sixteen repetitions were required, from which the phosphorescence lifetime was determined to be 0 1 4 0.01 WAVELENGTH (nml
',
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Tahle 2. Luminescence properties of I in ethanol at 77 K
Property
'
111.1,
(b
Li fct i me
Fluorescence
Phosphorescence
314nm 0.35
372 nm
4 2 0211s
0.14 f 0.01 s
1
I
I
I
2
3
[I]
10-3
x
104 M
Figure 3. Plot of rate constant for decay (kobs)of acetone emission vs concentration of 4-amino-2-methoxypyrimidine (1) for determination of quenching rate constant (k,) of 1.
144
A. G. SZABO and K. B. BERENS
constant for quenching of acetone triplet by this mechanism can be estimated to be -106M-’s-’ (Yip et al., 1972). In addition Helene (1972) has measured the k, of the triplet state of benzophenone by thymine and adenine to be 1 2 x 10’ M-’ s - ’ , a process which he attributes to a charge-transfer mechanism. Our measured value of 108M-’ s - l for quenching of acetone triplet by I, while not rigorously excluding electron transfer as the quenching process, is more consistent with an endothermic energy transfer process. These experiments allow us to conclude that I quenches the acetone triplet state with sufficient efficiency (20%) that the T-Tabsorption of I should have been observable in the flash-absorption experiment with acetone. Obviously the 7T must be less than 1 ps, the lower limit of detectibility of our flash instrumentation. Steady-state experiments allow us to exclude the triplet state of I as an intermediate in the ‘phosphate’ M) reaction. A phosphate-buffered solution of I and acetone (0.1 M ) was irradiated for 16 h using a Pyrex filter, conditions in which acetone is the sole absorber of light. No reaction of I occurred under these conditions. Unfortunately, the choice of water-soluble triplet sensitizers with triplet energies comparable to that of I and with an appropriate absorption spectrum is limited, so that other sensitizers could not be used. The intermediacy of a triplet excited state in this photoreaction could be definitively excluded, since neither oxygen [ 1 x M ] nor 2,4-hexadienol [0.1 M ] [ET = 56 kcal M-’ (Yip et al., 1970)], both effective quenchers of triplet excited states, had no effect whatsoever on the rate of formation of I1 nor on the photoreaction of I. It can be shown that even if the triplet lifetime of I was as short as 1 ns, at least 10 per cent quenching by hexadienol would be expected. These results, taken together with the previous flash emission results, indicate that the triplet state of I is not an intermediate in the reaction of I with dihydrogen phosphate buffers. We conclude that neither the triplet nor the fluorescent singlet state of I react with dihydrogen phosphate. Rather, some other intermediate derived from the singlet state of I must be involved in the ‘phosphate’ effect. It is evident from the sum of the luminescence quantum yields at 77 K, #, + bp,for I that radiationless deactivation processes of the excited states of I are very efficient. At 77 K in an ethanol glass this sum is 0.35, considerably less than I . It appears that this is a general property of the excited states of aminopyrimidine derivatives. Berens et al. (1971) made similar
-
observations in their studies on the luminescence properties of 2-aminopyrimidine (111) and 4-aminopyrimidine (IV). They found values of q5f bP of 0.40 and 0.039 for 111 and IV, respectively. Inspection of Table 1 reveals that bf of I at 300 K increases with decreasing hydrogen-bonding ability of the solvent. This suggests that one deactivation process is influenced by hydrogen bonding with the solvent. One might well inquire whether the efficient radiationless deactivation pathway of I proceeds via an intermediate that is responsible for the ‘phosphate’ effect. The nature of this intermediate is unknown but may be either a lower, non-fluorescent singlet state or an isomer or tautomer derived from the single state. Alternatively the ‘phosphate’ effect may be the reaction of the dihydrogen phosphate anion with an upper vibrational level of the ground state reached after deactivation of the singlet excited state. Recently Summers et al. (1973) and Summers and Burr (1972) suggested that the nucleophilic photoaddition to uracil involved an excited singlet state of uracil lower in energy than the fluorescent state of uracil. Whitten and Lee (1971) presented evidence that an nn* singlet of acridine was responsible for the photoreduction of acridine and that this state was lower in energy than the n-n* fluorescent state. Li and Lim (1971) observed that fluorescence from pyrimidine originated from the second singlet state, which is very close in energy to the first singlet state. Thus evidence is accumulating that the wn* and A n* singlet states are of similar energy in aza aromatics such as 1, and the reactive state may be the lowest state while the fluorescent state is a n upper singlet state. The dihydrogen phosphate anion likely reacts through a nucleophilic addition to C-6 of the pyrimidine ring with subsequent ring cleavage and hydrolysis of the adduct, leading to 11. The work of Pitha and Butler (1968) indicates that C-6 substituents apparently prevent the ‘phosphate’ effect. In summary, we have demonstrated that the photoinduced reaction of I in dihydrogen phosphate buffer is a reaction with an intermediate derived from the fluorescent singlet excited state. This has important implications in the photobiology of nucleic acids. It suggests that intermediates other than the fluorescent singlet excited states of nucleic acids can be involved in photochemical modifications of nucleic acids. Obviously, the photochemistry of the nucleic acids would vary according to the nature of these states. Moreover, dihydrogen phosphate, a molecule ubiquitous in cellular systems, reacts with these intermediates.
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REFERENCES Berens, K., J. Smagowicz and K. L. Wierzchowski (1971) I n t . Eur. Biophys. Congr, Abstract pp. 63- 67. Bergman, E. D., H. Weiler-Feiechenfeld and C . Lifshitz (1969) Jerusalem Symposium 1, 72-77. Borkman, R. F. and D. R. Kearns (1966) J. Clwm. Phys. 44,945-949. Chen, R. F. (1967) Aiiul. Letters 1, 35-42. Gilmore, E. H., G . E. Gibson and D. S. McClure (1952) J . Chcm. Phys. 20, 829-836.
Luminescence properties and related photochemistry Gilmore, E. H., G. E. Gibson and D. S. McClure (1955) J . Chem. Phys. 23,399. Helene, C. (1972) Photochem. Photobiol. 16, 519-522. Herkstroeter, W. G. and G. Hammond (1966) J . Am. Chem. Sac. 88,4769-4777. Li, Y. H. and E. C. Lim (1971) Chem. Phys. Letters 9, 514-516. Kasha, M. (1948) J . Opt. Soc. Am. 38,929-934. Kasha. M. and H. R. Rawls (1968) Photochem. Photobiol. 7, 561-569. McLaren, A. D. and D. Shugar (1964) In Photochemistry of Proteins and Nucleic Acids, Pergamon Press, New York. Melhuish, W. H. (1972) J . Res. N.B.S. 76A, 547-560. Moore, A . M . (1963) Can. J . Chem. 41, 1937-1950. Parker, C. A. (1968) In Photoluminescence ofSolutions, p. 420. Elsevier, New York. Pitha, P. M. and G. C. Butler (1968) Can. J . Biochem. 46, 893-897. Porter, G., R. W. Yip, J. M. Dunston, A. J. Cessna and S. E. Sugamori (1971) Trans. Faraday Soc. 67, 3149-3154. Smith, K. C. and P. C. Hanawalt (1969) In Molecular Photobiology, Inactivation and Recovery, Academic Press, New York. Summers, W. A,, Jr. and J. G. Burr (1972) J . Phys. Chem. 76,3137-3141. Summers, W. A,, Jr., C. Enwall, J. G. Burr and R. L. Letsinger (1973) Photochem. Photobiol. 17, 295301. Turro, N. J. (1967) In Molecular Photochemisfry, pp. 48-49. Benjamin, New York. Ware, W. R. (1971) In Creation and Detection qfthe Excited State, Vol. 1, Part A, p. 213. Marcel Dekker, New York. Whitten, D. G. and Y. J. Lee (1971) J . Am. Chem. Soc. 93, 961-966. Yip, R. W. (1969) Rev. Sci. Instr. 40, 1035-1040. Yip, R. W., R. 0. Loutfy, Y. L. Chow and L. K. Magdzinski (1972) Can. J . Chem. 50,34263431. Yip, R. W., W. D. Riddell and A. G . Szabo (1970) Can. J . Chem. 48,987-999.
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