1760
J . Phys. Chem. 1988, 92, 1760-1765
Keto-Enol Tautomer of Uracil and Thymine Yuko Tsuchiya, Teruhiko Tamura, Masaaki Fujii, and Mitsuo Ito* Department of Chemistry, Faculty of Science, Tohoku University, Sendai 980, Japan (Received: July 21, 1987; In Final Form: October 16, 1987)
The fluorescence excitation and dispersed fluorescence spectra of jet-cooled uracil, thymine, and their derivatives have been observed. Two band systems having well-resolved vibrational structures were found for uracil and thymine in the frequency region from 31 000 to 38 000 cm-', which corresponds to the region of the long tail in the vapor absorption spectrum. The shorter wavelength band system (system I) was identified as the Sl(n,r*) So transition of the diketo tautomer, while the longer wavelength system (system 11) was assigned to the Sl(n,r*) So transition of one of the keto-enol tautomers. Successful detection of a very small amount of the keto-enol tautomer is due to a high fluorescence yield of the keto-enol tautomer. The nature of the S,(n,r*) states of the tautomers and the hydrogen-bonded complexes with water are also discussed.
- -
Introduction The tautomeric equilibria of the nucleic acid bases have attracted a great deal of interest with respect to the mutations occurring during DNA replication by the possible existence of the rare enol tautomeric forms of the bases.' In uracil, for example, the following six tautomers are possible:
A is the 2,4-diketo tautomer, B-E are keto-enol tautomers, and F is the 2,4-dienol tautomer. All the experimental results so far available are consistent with the diketo tautomer being the most stable tautomer in the condensed phase? and tautomers other than the diketo tautomer have never been found. Even in the gas phase at high temperature (-200 "C), no sign has been obtained for the presence of tautomers other than the diketo t a ~ t o m e r . ~ The situation is also the same for thymine where only the diketo tautomer is believed to exist. There is little doubt therefore that the diketo tautomer is the exclusively abundant species, and the amount of other tautomers is extremely small even if they are present. Under such conditions, detection of the species at extremely low concentration is very difficult with any absorption spectroscopy which reflects the concentration of ground-state molecules. In analytical chemistry, fluorescence spectroscopy (fluorometry) is often employed in the detection of a small amount of impurity in a sample. If the impurity is highly fluorescent and the sample is nonfluorescent, the former can be easily detected even in the presence of a large amount of the latter. In the present case, the diketo tautomer and the other tautomers correspond to sample and impurity, respectively. The diketo tautomer is characterized by two C=O bonds, and nonbonding electrons are localized on each 0 atom. On the other hand, the other tautomers have at least one conjugated ring nitrogen atom where nonbonding electrons are localized. We can expect therefore two kinds of n,r* states depending on the excitation of a nonbonding electron localized on the oxygen atom or the nitrogen atom. It is known that the n,r* state of ketone is usually very weakly fluorescent or nonfluorescent. However, the n,r* state of a nitrogen heterocyclic aromatic ( 1 ) Pullman, B.; Pullman, A. AdG. Heferocycl. Chem. 1971, 13, 7 7 . (2) Becker, R. S.; Kogan, G . Photochem. Photobiol. 1980, 31. 5 and
references therein. ( 3 ) Nowak, M. J.; Szczespaniak, K.; Barski, A,; Shugar, D. Z . .%'muforsh., C Biosci. 1978, 33C. 876.
0022-3654/88/2092-1760$01.50/0
molecule is usually highly fluorescent with few exceptions. Specifically, a large fluorescence quantum yield is known for pyrimidine. Therefore, it may be possible to detect a small amount of the keto-enol or dienol tautomer by observing fluorescence from n,r*. Figure 1 shows the electronic absorption spectrum of uracil vapor at 21 1 O C . The spectrum exhibits a broad and structureless band at 240 mn, which is assigned to the S(r,x*) state.4 There exists a long tail on the longer wavelength side of the peak extending until 330 nm. It may be suggested that the tail corresponds to the n,a* state of uracil. However, the n,r* assignment has never been proved. In any case, such a broad spectrum does not provide any detailed information on the structure of the tautomer. Under such a circumstance, we observed the electronic absorption spectra of uracil, thymine, and their derivatives under supersonic jet conditions by monitoring the fluorescence from the excited state; that is, the fluorescence excitation spectra of the jet-cooled molecules were observed. Two band systems having well-resolved vibrational structures were found for uracil in the frequency region from 3 1 000 to 38 000 cm-I, which corresponds to the region of the long tail in the vapor absorption spectrum. From the measurement of the dispersed fluorescence spectra, one of the systems was identified as the diketo tautomer and another as one of the keto-enol tautomers. It was also established that these systems are due to Sl(n,r*) Sotransitions of the respective species. A similar result was also obtained for thymine. The fluorescence excitation spectra of jet-cooled 1,3-dimethyluraciI and 4(3H)-pyrimidone were also observed and used for supporting the assignments of the species and the electronic state. This paper represents the first observation of well-resolved electronic spectra of uracil and thymine and of the tautomer other than the diketo form. A preliminary report on uracil has been made in a previous paper.5
-
Experimental Section The experimental apparatus for fluorescence excitation and dispersed fluorescence spectra of a jet-cooled molecule were described elsewhere.6 The pulsed supersonic free-jet apparatus for high-temperature usage was also described in a previous paper.5 Each sample was heated in a nozzle chamber to obtain sufficient vapor pressure: 1,3-dimethyIuracil at 130 O C ; uracil, thymine, and 4(3H)-pyrimidone at about 200 O C . The vapor was seeded in 4 atm of He gas, and then the gaseous mixture was expanded into a vacuum chamber at 10-Hz repetition through a 0.4-mmdiameter orifice. The exciting light was the second harmonic of a dye laser pumped by a N2 laser (Molectron). The fluorescence (4) Clark, L.B.; Peschel, G. G.; Tinoco, I., Jr. J. Phys. Chem. 1965, 69, 3615. ( 5 ) Fujii, M.; Tamura, T.; Mikami, N.; Ito, M. Chem. Phys. Lett. 1986, 126, 5 8 3 . ( 6 ) Mikami. N.; Hiraya, A.; Fujiwara, I.; Ito, M. Chem. Phys. Lett. 1980, 74. 531.
0 1988 American Chemical Society
The Journal of Physical Chemistry, Vol. 92, No. 7, 1988 1761
Keto-Enol Tautomer of Uracil and Thymine Vapor
Abs
,>,,:
I
\ 1500 1000 500 0 RELATIVEWAVENUMBER I cm-'
Figure 5. Dispersed fluorescence spectra of keto-enol tautomer of uracil in a supersonic jet obtained by exciting the (a) 0; (30917 cm-I), (b) 92 cm-I, (c) 147 cm-I, (d) 550 cm-', and (e) 803 cm-' bands in system 11.
Exciting positions are indicated by arrows. URACIL System I1
FREE
roo
+
DIOXANE 803
550 COMPLEX-59 ' -60 -50
n nn ' I '
-71
n
- 71
n
31 000 31 500 WAVENUMBER I cm-' Figure 6. Fluorescence excitation spectrum of the gaseous mixture of uracil and dioxane in a supersonic jet for system I1 region. Bands due to the uracil-dioxane complex are indicated by arrows. The pressure of dioxane was 10 Torr. because the ground-state frequencies of this species are entirely different from those of the diketo tautomer. Second, the species involves at least one C=O group, because of the appearance of the C=O stretching mode (181 1 cm-' in So) and the C=O bending mode (838 cm-' in So and 803 cm-' in SI).This automatically excludes the possibility of the dienol tautomer. The dienol tautomer can be also excluded by the nonplanarity of the species suggested by the anharmonic coupling between the in-plane and out-of-plane modes mentioned before. Third, the species contains a peptide group of ROC-NHR, indicated by the ground-state frequency of 1257 cm-I. When all the above facts are combined together, the species responsible for system I1 is concluded to be the keto-enol tautomer of B, C, or E. In order to obtain further evidence supporting the above conclusion, we observed the fluorescence excitation spectrum of a
31500 WAVENUM BER ( cm-' )
32000
Figure 8. Fluorescence excitation spectra of (a) diketo tautomer (system I) and (b) keto-enol tautomer (system 11) of thymine in a supersonic jet.
gaseous mixture of uracil and dioxane in a supersonic jet. The addition of dioxane gives no change in the spectrum of system I, but it causes the Occurrence of new bands in system 11. As seen in Figure 6, several new bands appear in the spectrum of the mixture which are red-shifted by 60-70 cm-' from the main vibronic bands in system 11. These new bands are interpreted as due to a hydrogen-bonded complex formed between the ketc-enol tautomer and dioxane. The result clearly shows that the species responsible for system I1 has an acidic OH group, consistent with the conclusion derived above. We also observed the fluorescence excitation spectrum of 1,3-dimethyluracil which has only the diketo form. In the spectral region corresponding to system I, the spectrum having a wellresolved vibrational structure is found with the band origin at 35354 cm-', as shown in Figure 7. However, the spectrum corresponding to system I1 is completely absent. This is a natural consequence of the assignments that systems I and I1 are the diketo and ketc-enol tautomers. The very low frequency vibronic bands appearing near the band origin in the figure are obviously assigned to the internal rotation of the CH3 group. Figure 8 shows the fluorescence excitation spectrum of jetcooled thymine. Similar to uracil, there exist two band systems having well-resolvedvibrational structure, corresponding to systems I and I1 of uracil. Therefore, the spectrum having the band origin at 33 724 cm-I is called system I of thymine and that with the origin at 31 11.1 cm-I system 11. We tried to observe the dispersed fluorescence spectra. However, spectra with a good S/N ratio could not be obtained because of a lack of vapor pressure under our conditions. Although a detailed vibrational analysis is not possible without the data of the dispersed fluorescence spectra, it is apparent from the good correspondence with the uracil spectra that system I is due to the diketo tautomer and system I1 to one of the keto-enol tautomers. The latter is probably ascribed to a species corresponding to B, C, or E of uracil. The results obtained for uracil and thymine clearly show that both molecules have at least two tautomers; one is the diketo
I
~
I
Tsuchiya et al.
1764 The Journal of Physical Chemistry, Vol. 92, No. 7, 1988 (a) System
TABLE I V Comparison of Frequencies of Band Origins in Systems I and I1 among Uracil and Its Derivatives" system I system I1
uracil thymine 1,3-dimethyluracil 4(3H)-pyrimidone
35 288 33724 (-1564) 35 354 ( + 6 6 ) 35 337 (+49)
I U
30917 31 111 (+194) 30 528 (-389)
Frequency shifts from uracil are shown in parentheses. tautomer and the other the keto-enol tautomer. These two tautomers coexist in the equilibrium in the vapor at 200 "C. All the experimental results so far available show that only the diketo tautomer exists even in the vapor at a high temperat~re.~ Ab initio calculation also predicts that the keto-enol tautomer is higher in energy than the diketo tautomer by 72.1 kJ/mol.I2 Assuming this energy separation, the concentration of the keto-enol tautomer in the vapor equilibrium at 200 "C is negligibly small. The concentration of the keto-enol tautomer will be discussed later. The successful detection of such a small amount of the keto-enol tautomer will be ascribed to its very high fluorescence quantum yield, which will be described below. For both uracil and thymine, systems I and I1 appear with comparable intensities in the fluorescence excitation spectrum. Since the diketo tautomer associated with system I is in far greater abundance than that of the keto-enol tautomer, the comparable intensities imply a very high fluorescence quantum yield of the keto-enol tautomer over the diketo tautomer. The n,n* excited state of the diketo tautomer arises from the excitation of a nonbonding electron localized on the oxygen atom. In the keto-enol tautomer, we have two different types of n,n*: one arises from the excitation of a nonbonding electron localized on the oxygen atom of C=O (no,**) similar to that for the diketo tautomer, and another is n,n* resulting from the excitation of a nonbonding electron localized on the ring nitrogen atom (nN,n*). It is well-known that nO,n* states of ketones generally have a very small fluorescence quantum yield. On the other hand, it is not easy to predict whether the nN,n* state is highly fluorescent or not. For example, the nN,n* state of pyridine is almost nonfluorescent, but the nN,n* state of pyrimidine is highly fluorescent. As far as ring framework is concerned, the keto-enol tautomer is similar to pyrimidine. Then, it might be reasonable that the nN,n* state of the keto-enol form is more fluorescent than the no,** state. We interpret therefore that the appearance of a small amount of the keto-enol tautomer is due to a high fluorescence quantum yield of the nN,n* state and this nN,n* state is responsible for system I1 of uracil and thymine. A large amount of the diketo tautomer with its very small fluorescence quantum yield is to be compared to a very small amount of the keto-enol tautomer and its very high fluorescence yield, explaining the comparable intensities of systems I and I1 in the fluorescence excitation spectrum. In the diketo tautomer, we have two no,n* states arising from the excitation of a nonbonding electron of the oxygen atom (0,) connected to ring carbon atom C2 and of the oxygen atom (0,) connected to C4. It is of particular interest which no,r* state is responsible for system I. In Table IV are summarized the frequencies of the band origins in systems I and I1 of uracil, thymine, 1,3-dimethyluracil, and 4(3H)-pyrimidone. The last molecule has only one carbonyl oxygen atom at C4as shown in Figure 9. The fluorescence excitation spectrum of this molecule in a supersonic jet is shown in this figure. Again, two band systems corresponding to systems I and I1 appear with the origin bands at 35 337 and 30 528cm-I. As seen from Table IV, the origin band of the system I (diketo or keto tautomer) has nearly the same frequency for all the molecules except for thymine. Thymine exhibits a red shift as large as 1564 cm-I. This large red shift in thymine is induced by the methyl group at the Cscarbon atom which is adjacent to the O4atom. This seems to support the assignment of no,,r* for system I; that is, the nonbonding orbital of the O4atom is greatly perturbed by the nearby CH, group and also the Cs carbon atom (12) Scanlan, M. J.; Hiller, I. H. Chem. Phys. Lett. 1983, 98, 545.
35500
30000 WAVEN UMBER I c m-1
( b ) S y s t e m 11
(+361)
0" 30528cm-'
I
I
I
I
1
I
I
I
I
I
30500
31 000 WAVENUMBER cm-' Figure 9. Fluorescence excitation spectra of (a) keto tautomer (system I) and (b) enol tautomer (system 11) of 4(3H)-pyrimidone in a supersonic jet.
to which the CH, group is attached is in n-conjugation involving C=O. The nonbonding orbital is not completely localized on the O4atom, but it actually spreads over the whole molecule including the O2atom. The nonbonding orbital is approximately expressed by n+ = ano4 + bnO2and n- = bnOn- ano2. Recent CNDO CI calculation shows a >> b for ~ r a c i 1 . I ~It also predicts that the n-,a* state is much higher in energy than the nt,a* state. Therefore, the nonbonding electron in n+ is practically localized on the O4 atom and its excitation to r* gives the low-energy transition of system I. Water is essential in any biological system. The state of the nucleic acids in the presence of water is therefore attracting a great deal of interest. With this in mind, we studied the hydrogenbonded complex of uracil and water. Figure 10a shows the fluorescence excitation spectrum of a gaseous mixture of uracil and water in a supersonic jet in the region of system I (diketo tautomer). In addition to the vibronic bands of the bare molecule, new bands appear at 34 872,35 077,35 201, and 35 499 cm-'. The first three bands are situated at -416, -211, and -87 cm-' from the origin of the diketo tautomer, and the last band is situated at +211 cm-'. Since all the bands except for the weak band at -87 cm-' are fairly strong and have comparable intensities, it is likely that these three bands represent different hydrogen-bonded complexes. In hydrogen bonding, water acts as a proton acceptor as well as a proton donor. Therefore, we can assume various kinds of the hydrogen-bonded complexes. The diketo tautomer has two proton-accepting sites at O2and O4and two proton-donating sites at the hydrogen atoms (HI and H,) attached to the two nitrogen atoms N l and N,. The hydrogen-bonded complexes appearing in the spectrum will be ones in which the no,,n* state is weakly perturbed because the frequency shifts of the complex bands from the bare molecule are rather small. A hydrogen-bonded complex with a water molecule at O4will greatly stabilize the nonbonding orbital of this atom and result in a great blue shift of system I. It is also expected that the fluorescence quantum yield of this complex becomes much smaller than that of the bare molecule. (13)
Hug,W.; Tinoco, I., Jr. J . Am. Chem. SOC.1974, 96, 665.
Keto-Enol Tautomer of Uracil and Thymine (a) U R A C I L System I
+
H,O
The Journal of Physical Chemistry, Vol. 92, No. 7, I988
will lead a great blue shift of system 11, and its fluorescence will be greatly quenched for the same reason mentioned before. Therefore, the hydrogen-bonded complex with a water molecule at the conjugated nitrogen atom will escape from our detection. A complex having water at the oxygen atom of C=O will cause a slight stabilization of nN through a mixing of two nonbonding orbitals of nN and no, resulting in a small blue shift of system 11. The band at +426 cm-' may be assigned to this complex. The red-shifted bands at -1 14 and -73 cm-I are probably ascribed to the complexes in which the keto-enol tautomer is acting as a proton donor. The donating proton will be the hydrogen atoms of NH and O H groups of the molecule.
Frrr
-416
-211
n
H
__
H
I
I
35000
2 9'
'
I WAVENUMBER
I / cm-'
I
I
35500
( b ) U R A C I L System11 + Ii20
-114
1765
1
30800
31 000 ' 31200 WAVENUMBER I cm-' Figure 10. Fluorescence excitation spectra of (a) diketo tautomer of uracil-H20 complex and (b) keto-enol tautomer of uracil-H20 complex in a supersonic jet. Complex bands are indicated by arrows.
because the fluorescence from a n,x* is often quenched by hydrogen-bond formation. Therefore, there is little possibility that we can detect the hydrogen-bonded complex having a water molecule at O4by the fluorescence excitation method. A complex with H,O at 0, will slightly stabilize the O4 nonbonding orbital by mixing of no, and no4, resulting in a slight blue shift of system I. The band at +210 cm-I is probably assigned to this complex. The other two bands at -200 and -396 cm-' may be ascribed to the complexes in which the water molecule is acting as a proton acceptor. We have two such complexes having the water at HI and H3. It is difficult however to predict which band is assigned to which complex. In Figure 10b is shown the fluorescence excitation spectrum of the uracil-water mixture in the region of system I1 (the keto-enol tautomer). New bands appear at 30 803, 30 843, and 31 343 cm-I, which are shifted by -1 14, -73, and +426 cm-I from the origin of the bare molecule. Again, these three bands are strong with comparable intensities. Therefore, they probably represent hydrogen-bonded complexes. System I1 is associated with the nonbonding orbital nN of the keto-enol tautomer. A complex in which this nN directly participates in hydrogen bonding
Conclusion The fluorescence excitation spectra of jet-cooled uracil and thymine revealed the coexistence of the diketo and keto-enol tautomers. The first observation of the keto-enol tautomer has a profound implication in possible relation with the mutations in DNA replication. The successful detection of the keto-enol form by the fluorescence excitation method is due to a high fluorescence quantum yield of this tautomer. Although the structure of the keto-enol tautomer was not definitely determined, it was concluded to be B, C, or E. Recent ab initio calculation predicts the most stable tautomer next to the diketo tautomer to be E.', Therefore, E is tentatively assigned to the tautomer responsible for system 11. According to the calculation, the energy difference between the diketo and keto-enol (E) tautomers is 72.1 kJ/mol (6030 cm-'). However, this value is too large to explain our observation of comparable intensities of systems I and I1 in the fluorescence excitation spectrum. The energy difference of 72.1 kJ/mol means that the population of the keto-enol tautomer is of that of the diketo tautomer under Boltzmann distribution at 500 K. The fluorescence quantum yield afof the diketo tautomer has been measured to be 7 X to 1 X in the low-temperature matrix.' To obtain the comparable fluorescence intensities of both systems, the absorption coefficient of the keto-enol tautomer must be at least lo4 times larger than that of the diketo tautomer. Since both systems I and I1 are assigned to the n,x* transitions, such a large difference in the absorption coefficient is hardly acceptable. Therefore, the energy difference will be smaller than 72.1 kJ/mol. Assuming that the absorption coefficients of the two tautomers are comparable and af of the keto-enol tautomer is unity, the energy difference is estimated to be 40 kJ/mol, which will be the upper limit. Very recently, the energy difference between the tautomer E and the diketo tautomer is reported to be 39.0 kJ/mol by semiempirical calculation,14 which is in agreement with our estimation. Acknowledgment. We thank Dr. A. Y . Hirakawa for informing us of her data prior to publication. Registry No. Uracil, 66-22-8; thymine, 65-7 1-4; 1,3-dimethyluracil, 874- 14-6; 4(3H)-pyrimidone, 5 1953-17-4. (14) Norinder, U. J. J . Mol. Srruct. 1987, 151, 259
