49
J. phys. Chem. 1982,86,49-55
work suggests that comparison of IRMPD in other twochannel reactions with well-defined pulses will provide additional insight into the characteristics of IRMPA in the quasicontinuum above and below threshold. Acknowledgment. This work was carried out a t Brookhaven National Laboratory under contract with the U.S.Department of Energy and supported by its Office of Basic Energy Sciences.
Appendix RRKM Calculations. The theoretical basis of these RRKM calculations is described completely in Robinson and Holbro~k.'~These calculations are based on the computer program developed by Hase and Bunker%which computes k(E), A subroutine has been added which calculates kmi and k, for comparison with thermal data. The ground-state frequencies and assignments of EVE are estimated from the IR spectrum of pent-l-ene31with appropriate changes made for the C-O frequencies based on those known for vinyl ethers.32 A similar procedure is used for DHF in which cyclopentenea is the model compound. Ring puckerings" frequencies of DHF are taken from the literature. Tables I11 and IV list the frequency assignments of the ground states for EVE and DHF. The activated complex in the unimolecular decomposition of EVE via the lower-energy channel is expected to be six-centered with partial bond character between breaking and forming bonds. The frequencies of half-chair geometry (by analogy with the Cope rearrangement)%are (31)M. J. Pearson and B. S. Rabinovitch, J. Chem. Phys., 42,1624 (1965). (32)Y. Mikawa, Bull. Chem. SOC.Jpn., 29,110 (1956);J. R.During and D.A. C. Compton, J. Chem. Phys., 66,2028 (1978). (33)C. W. Beckett, N. K. Freeman, and K. S. Pitzer, J.Am. Chem. SOC.,70,4227 (1978). (34)T.Ueda and T. Shimanouchi, J. Chem. Phys., 47,4042 (1967). (35)W.von E.Doering and W. R. bth, Angew. Chem.,Int. Ed. Engl., 2, 115 (1963).
estimated from those of the similar complex in the sixcentered reaction of ethyl acetate.% Frequencies are optimized to reproduce the observed entropy of activation AS'* and are given in Table 111. The relatively small preexponential factor of this reaction suggests that there is a loss of internal degrees of freedom (internal rotors) in the course of reaction.22 Moments of inertia for internal rotors are calculated from the method of B e n ~ o nand ~~ Pitzer and Brewer.37 Overall moments of inertia for ground state and activated complex are calculated by diagonalizing the appropriate matrix for moments of inertia derived from an arbitrary set of Cartesian coordinates. The critical energy is calculated from the vibrational/rotational partition function. This energy was varied within a range of 2 kcal mol-' to give a best fit to the thermal rate constants. The activated complex for loss of H2 from DHF is assumed to involve a concerted 2 4 elimination and is modeled on the basis of the theory developed by Woodward and Hoffmann.ls The frequencies are assigned to reproduce AS* and to be consistent with a molecule changing in structure from DHF to furan. These frequencies are listed in Table I11 along with other relevant information. A comparison between calculated and observed thermal rate constants is given in Table V. It was found that optimization between calculated and observed kd required that overall rotation be inactive. Use of Marcus' adiabatic treatment%of overall rotation led to rate constants that were too large. Various models for the activated complex satisfying the constraints of Woodward-Hoffman rules were considered. These variations did not make noticeable differences in the results and required that overall rotation be inactive.
+
(36)H.E.ONeai and S. W. Benson, J.Phys. Chem., 71,2903(1967). (37)K.S.Pitzer and L. Brewer, 'Thermodynamics", revised ed., G. N. Lewis and M. Randall, E%., McGraw-Hill,New York, 1961,pp 439-46. (38)R.A. Marcus, J. Chem. Phys., 43,2658 (1965).
Fluorescence from Adenine Cations Waiter B. Knlghton, Gary 0. Gkkaas, and Patrlk R. Callls' Depemnent of C b d t r y , Montana State Univemtfy, Bozeman, Montana 59717 (Received Augwt 3, 1981; I n Final Form: September 11. 1981)
The unusual red-shifted fluorescence from several adenines in room temperature acidic solutions is totally depolarized. Studies of the polarization, excitation spectra, quantum yields, and quenching for 11closely related adenine derivatives lead to the conclusion that the fluorescence from adenine and adenosine is primarily from a minor tautomer protonated at the 7 position. This tautomer probably has a fluorescence lifetime of a few nanoseconds, a quantum yield 20.1,and is probably present at the level of 0.145%. CNDO/S calculations suggest that the large Stokes shift results from the loss of a large dipole moment upon excitation. Adenines having one or two methyls on the amino nitrogen do not have a highly fluorescent tautomer in most cases.
Introduction Early studiesL2 of the fluorescence from adenine (6aminopurine) and its derivatives were limited to low pH solutions where the fluorescence quantum efficiency (&) was large enough for detection, though certainly weak by (1)S. Udenfriend and P. Zaltman, Analy. Biochem., 3, 49 (1962). (2) H.C. Borreeen, Acta Chem. Scand., 21,11 (1967).
In neutral aqueous solutions most standards (& z at room temperature the fluorescence of d l nucleic acid components, including the adenines, is generally an order of "tude weaker (hzz lo4) but can be reliably Studied due to the fact that Preparations are remarkably free of fluorescent impurities.3~~A striking general characteristic (3)M.Daniels and W. Hauswirth, Science, 171,675 (1971).
0022-3654l82/2086-0049$01.2510 0 1982 American Chemical Society
50
The Journal of Physical Chemistry, Vol. 86, No. 1, 1982
of the weak emission from neutral species is its large anisotropy (p~larization),"~ which approaches that seen in rigid media. The meaning is clearly that nonradiative processes leading to low 4f are more rapid than the process of rotational diffusion. The excited state lifetimes are estimated to be a few picose~onds.~J This paper stems from our discovery that the fluorescence of several protonated adenine derivatives is completely depolarized, a decided curiosity because we had found many other systems with 4f whose fluorescence was only partially depolarized as expected.8 Furthermore, Borresen's2 study of the protonated adenines has revealed two other aspects which are unusual. (1)The fluorescence excitation spectrum in every case fails to coincide with the absorption spectrum and, (2) protonation has only a small effect on the position and width of the absorption spectrum whereas the fluorescence is greatly broadened and red-shifted over that of the corresponding neutral species. The red-shifted fluorescence is characteristic of molecules in polar solvents in which a significant solvent reorientation takes place following excitation because of accompanying changes in charge distrib~tion.~ Indeed, EisingerlO has shown that the adenine fluorescence in acidic glycol-water shifts back to a typical, "mirror image" shape and position as the temperature is lowered and the solvent becomes too viscous to allow solvent reorientation during the excited state lifetime. Regarding the excitation spectra, Borresen2 postulated the existence of a minor but highly fluorescent tautomer whose presence does not influence the absorption but does dictate the fluorescence excitation spectrum. The excitation spectrum is the same as the absorption spectrum of the fluorescent tautomer. Until recently it was difficult to accept the mismatch of excitation and absorption as definite proof of a fluorescent tautomer; virtually all reported excitation spectra €or nucleic acid components showed similar mismatches, a fact which prompted a proposal that the behavior was physically linked to the rapid nonradiative decay process." However, the situation is now quite different; as part of the present study (to be presented separatelf) we have found the excitation and absorption spectra to agree precisely for 13 purine and pyrimidine free bases, nucleosides, and their ions in room temperature aqueous solution. Our findings agree with those of Vigny and D~quesne.~ Clearly, the above findings imply that virtually all the fluorescence from the protonated adenines comes from a highly fluorescent tautomer as postulated by Borresen.2 The absence of polarization and the large red-shift both imply a long lifetime, perhaps several nanoseconds. In the first part of this paper we describe fluorescence quenching and fluorescence polarization experiments in viscous solvents which establish that the fluorescence lifetime of the emitting species is indeed -1 ns.
=
(4)P. Vigny and M. Duquesne, 'Excited State of Biological Molecules", J. B. Birks, Ed.,Wiley-Interscience, New York, 1976 pp 166-177. (5) J. P. Morgan and M. Daniels, Photochem. Photobiol., 27,73 (1978). (6)P.R. Callis, Chem. Phys. Lett., 61,563 (1979). (7)B. E. Anderson and P. R. Callis, Photochem. Photobiol., 32, 1 (1980). (8) W. B. Knighton and P. R. Callis, in preparation.
(9) (a) D. M. Hercules and L. G. Rogers, J. Phys. Chem., 64, 397 (1960); (b) J. Eisinger and G. Navon, J. Chem. Phys., 60, 2069 (1969); (c) K.A. Al-Hasean and M. A. El-Bayoumi, Chem. Phys. Lett., 76,121 (1980). (10)J. Eieinger, Photochem. Photobiol., 9, 247 (1969). (11)R.W. Wilson, J. P. Morgan, and P. R. Callis, Chem. Phys. Lett., 36,618 (1975).
Knighton et al.
In the second part of the paper we introduce new spectral, quantum yield, and polarization data for methylated adenine cations which lead to a firm assignment of a single fluorescent tautomer having substitution at both the 7 and 9 positions. This differs from Borresen's assignment.2
Experimental Section Materials. Compounds utilized in this study were obtained from commercial sources as follows: adenosine and N6f16-dimethyhdeninefrom Calbiochem, 3-methyladenine from Vega Chemical Corp., and the remainder from Sigma Chemical Corp. No further purification was carried out. Except for N6-methyladenosinethe fluorescence spectrum of these compounds is independent of excitation wavelength (Aex). All measurements were for 0.1-4 mM pH 1.5 aqueous solutions which were 0.15 N in H3P04. The temperature was 20 f 2 "C. The buffer was prepared with singly distilled water with the criterion of purity being that its fluorescence amplitude be at least 20 times less than that of the prominent 3400 cm-' H 2 0 Raman line. The high absorbance of most of the solutions studied precluded the necessity for making corrections due to the solvent fluorescence in most cases. When this was done the solvent contribution was scaled according to the attenuation of the H20 Raman line due to absorption by the sample under conditions in which the Raman line suffered no reabsorption. Methods. The fluorimeter employed in this study has been described previously12and has been used in several similar s t ~ d i e s . ~ ~The J l excitation and viewing monochromator slits were set for a 13-nm band pass. The supracil fluorescence cuvettes were positioned on a translatable stage such that the detecting monochromator, viewing at 90" to the excitation beam, viewed the front 2 mm of the 10-mm cells. Excitation spectra were carried out on the optically dense solutions (up to 3.5/cm) with the aid of an empirically determined fluorescence intensity vs. absorbance function using tryptophan solutions in the same geometry. After normalizing the excitation intensity using a 3 g/L Rhodamine B-ethylene glycol solution as a quantum counter,13 the excitation spectrum was constructed by converting the observed fluorescence intensity (at the fluorescence maximum) vs. A,, into apparent absorbance vs. Lxusing the empirical conversion curve. Thii process gave excitation spectra for pH 6.7 solutions of tryptophan with absorbance = 1.5 to 2.8/cm which fit the absorption spectrum within 10% from 250-300 nm in agreement with previous findings.I4 Fluorescence quantum yields, reported here for excitation at the first absorption maximum, were determined by integrating the fluorescence spectrum (corrected for instrument response) and comparing with that expected for tryptophan (4f = 0.1515) at the same absorbance. Fluorescence polarization ratios (N) are measured for excitation and viewing at the absorption and emission maxima, respectively. To avoid excessive signal reduction we have used a single polacoat (PL-40) polarizing sheet on the excitation monochromator only. N is then defined as IvlIH where V and H mean electric field vector vertical and horizontal (the excitation and viewing axes form a horizontal plane). Both monochromators are equipped with quartz wedge depolarizers so that corrections due to instrument anisotropy were very (12)R.W. Wilaon and P. R. Calliis, J,Phys. Chem., 80,2280 (1976). (13)W. H. Melhuish, J. Opt. SOC.Am., 52, 1256 (1962). (14)G.Weber and F. W. J. Teale, am. Faraday SOC.,53,646 (1957). (15)R.J. Robbins, G. R. Fleming, G. S. Beddand, G. W. Robinson, P. J. Thistlethwaite, and G. J. Woolfe, J.Am. Chem. SOC.,102,6271(1980).
The Journal of hyslcal Chemistry, Vol. 86, No. 1, 1982 51
Fluorescence from Adenine Cations
TABLE I: Fluorescence Anisotropies, Quantum Yield, and Spectral Maxima for Adenine Cations in Aqueous Solution nm
h,
anisotropies, ra adenine adenosine 7-methyladenine' 2-methyladenine' 3-methyladenine' 1-methyladenine+ 1-methyladenosine N,-methyladenine+ N6-methyladenosine+
0 0 0 0 0.272 * 0.003 -0.21 t 0.04
* 0.03
-0.06
+
0.246
-f 0.015
@f
x
lo4
20.7 c 0.4 7.7 c 0.1 11.9 i 0.1 9.9 0.1 1.17 * 0.03 and the actual +f of the tautomer will be proportionately less, leading to larger values for the fraction of fluorescent tautomer. (26)J. T.Edward, J. Chem. Educ., 47,261 (1970). (27)S. J. Strickler and R. A. Berg, J. Chem. Phys., 37, 814 (1962). (28)M. Gueron, J. Eisinger, and R. G. Shulman, J. Chem. Phys., 47, 4077 (1967).
