J. Phys. Chem. 1989, 93, 6622-6625
6622
possibility. In fact, the related molecule quinacrine mustard shows multiexponential decay of fluorescence that has been attributed to multiproton transfer in the excited state.* However, proton transfer cannot explain the observed multiexponential fluorescence decay of quinacrine in pure alcohols where the dye molecule is in a neutral form, and one might expect the fluorescence decay to be single exponential. The results from ACMA suggest that excited-state cis to trans isomerization of the methoxy group combined with motions of the side chain at the 9 position could also contribute to the complex fluorescence decay of quinacrine, which requires at least three exponentials to describe.
Acknowledgment. This work was supported by a grant from the American Cancer Society (CH32 to D.R.K.) and a postdoctoral fellowship from the Swedish Natural Sciences Research Council (T.H.). We are greatly indebted to our colleagues Dr. Y. Ni and Professors J. D. Simon and J. S. Siege1 for numerous helpful discussions and many valuable suggestions. We are also grateful to Prof. D. Magde for the assistance with the time-resolved fluorescence studies. Registry No. ACMA, 3548-09-2; NaCI, 7647-14-5;NaC104, 760189-0; D,, 7782-39-0; sodium acetate, 127-09-3; ammonium acetate, 631-61-8;quinacrine, 83-89-6.
Origin of the Complex Fluorescence Emission of Q-Amlno-6-chloro-2-methoxyacrldine. 2. Theory Yong Ni and David R. Kearns* Department of Chemistry, University of California, San Diego, La Jolla. California 92093-0342 (Received: January 18, 1989; In Final Form: April 6, 1989)
A series of calculations, based on the A M I program developed by Dewar et aL,I have been carried out on 9-amino-6chloro-2-methoxyacridine (ACMA) in an attempt to understand some of the unusual fluorescence emission properties of this and related compounds. The AM1 calculations predict that in the ground state both the neutral and protonated forms of ACMA are planar and that the cis and trans isomers (see Figure 1) differ in energy by only 0.4 kcal/mol. In the excited state, however, ACMA-H+ is bent, the trans isomer lies lower in energy (by 2.3 kcal/mol) than the cis isomer, and the barrier for cis trans isomerization of the methoxy group is predicted to be 5.4 kcal/mol. These theoretical predictions nicely account for the experimental observations on ACMA-H+ of a slow (24s) conversion from one excited-state species to a second excited-state species (here identified as the cis and trans isomers, respectively) with a barrier of 4.3 kcal/mol. The calculations also account for many other experimental observations (effect of temperature on the emission spectrum, lack of solvent effects on the room-temperature emission behavior, lack of a deuterium isotope effect, effect of deprotonation, and lack of counterion effect). In neutral ACMA, the barrier for the cis trans isomerization is predicted to be only 0.8 kcal/mol in the excited state and the difference in energy between the two isomers is only 1.3 kcal/mol. This accounts for the observation that the emission properties of ACMA are normal (single exponential). Electronic charge distributions in the ground and excited states of ACMA-H+ and ACMA are also presented for use in examining their complexes with DNA.
-
-
Introduction Derivatives of 9-aminoacridine are often used as cytological stains2” and as fluorescent probes in biophysical studies of DNA,7-12and some have found use as therapeutic agents.I3-I5 It was in connection with their use as fluorescent probes of DNA that we became interested in the fluorescence properties of the DNA-binding drug quinacrine (I) and the related compound (1) Dewar, M. J. S.; Zoebisch, E. G.; Healy, E. F.; Stewart, J. J. P. J. Am. Chem. SOC.1985, 107, 3902-3909. (2) Lin, C. C.; van de Sande, H.; Smink, W.K.; Newton, D. R. Can. J . Genet. Cytol. 1974, 17, 81-92. (3) Andreoni, A,; Cova, S.; Bottiroli, G.; Prenna, G. Photochem. Photobiol. 1979, 29, 951-957. (4) Lurquin, P. F. Chem.-Biol.Interact. 1974, 8, 303-313. (5) Gatti, M.; Pimpinelli, S.; Santini, G. Chromosoma 1976, 57, 351-375. (6) Weisblum, B.; DeHaseth, P. L. Proc. Natl. Acad. Sci. U.S.A. 1972, 69. 629-632. (7) Natasi, M.; Morris, J. M.; Rayner, D. M.; Seligy, V. L.; Szabo, A. G.; Williams, D. F.;Williams, R. E.; Yip, R. W. J . Am. Chem. SOC.1976, 98, 3979-3986. (8) Michelson, A. M.; Monny, C.; Kovoor, A. Biochimie 1972, 54, 1 1 29-1 136. (9) Woodson, S . A.; Crothers, D. M. Biochemistry 1988,27, 8904-8914. (IO) Andreoni, A.; Cubeddu, R.; De Silvestri, S.; Laporta, P. Opt. Commun. 1980, 33, 277-280. ( 1 1) Andreoni, A.; Cubeddu, R.; De Silvestri, S.; Laporta, P. Chem. Phys. Lert. 1981, 80,323-326. ( 1 2) Marty, M.; Bourdeaux, M.; Dell’Amico,M.; Viallet, P. Eur. Biophys. J . 1986, 13, 251-257. (13) Baguley, B. C.; Ferguson, L. R.; Denny, W. A. Chem.-Bo/.Interacr. 1982, 42, 97-105. (14) Ferguson, L. R.; Baguley, B. C. Murat. Res. 1981, 82, 31-39. ( 1 5 ) Wilson, W. R.; Baguley, B. C.; Wakelin, L. P. G.; Waring, M. J. Mol. Pharmacol. 1981, 20, 404-414.
9-amino-6-chloro-2-methoxyacridine (ACMA, 11). Both compounds exhibit complex emission behavior, including nonexponential fluorescence decay, time-dependent shifts in their emission spectra, and other unusual features.10v11-16Examination of the emission behavior of ACMA under a wide variety of experimental conditions led us to conclude that much of the complexity of the emission behavior was due to an excited-state interconversion between two different conformational states of ACMA, but the precise nature of the two different states was ~ n c 1 e a r . l ~In an effort to provide a possible explanation for our observations, as well as those presented by earlier investigators, we initiated a series of quantum mechanical calculations based on the AM1 program recently developed by Dewar et a1.I This particular program has been shown to provide excellent predictions regarding a wide variety of ground-state properties of organic molecules. While the AM1 program does not appear to have been widely used to describe the excited-state properties of aromatic molecules, we decided to use it for several reasons. First, the program includes some limited configuration interaction (CI), and this should provide a better description of excited states than do similar programs that do not allow for CI. Secondly, more rigorous self-consistent field quantum mechanical calculations on molecules this large are extremely time consuming, and we have little assurance that the results are actually superior to those obtained more easily by the semiempirical molecular orbital methods. We specifically chose the AMI program because it has been shown to (16) Amdt-Jovin, D. J.; Latt, S. A,; Striker, G.; Jovin, T. M. J . Hisrochem. Cytochem. 1979, 27, 87-95. (17) Fan, P.; Hard, T.; Kearns, D. R. J . Phys. Chem., preceding paper in this
issue.
0022-3654/89/2093-6622$01.50/00 1989 American Chemical Society
The Journal of Physical Chemistry, Vol. 93, No. 18, 1989 6623
Theory of Complex Fluorescence Emission of ACMA NH2
& l a , u
CI
TABLE I: Equilibrium Torsional Angles, 0, Rotational Barriers," Central Ring Geometry, and C2-0 Bond Order in ACMA ACMA-H+ ACMA
CH3 I
I
r+
so
SI
so
SI
180
180
180
2.32 7.75b 0.00 69.07
0.37 2.68 (71.02)
1.25 0.00 2.06 1.50 0.00 0.35 79.57 (81.17)
bent 1.18
planar 1.06
planar planar 1.06 1.03
0
energy (kcal/mol) in other forms
e= e = 900 e = 1800 00
H
Figure 1. The structure and the numbering of ACMA-H+ (in cis form).
give results for ground-state molecules that are demonstrably superior to those obtained with the M N D O ' ~and other semiempirical programs. The A M I results were found to be in better agreement with experimental data on the conformational states of phenyl pyrrole^'^ and alkenylarenesZ0than were the results of MNDO calculations. As we shall demonstrate, the AM1 calculations show that the complex emission behavior of ACMA can be understood in terms of an excited-state cis-trans isomerization of the methoxy substituent. The calculations also provide information on the geometry and charge distribution in both the ground and electronically excited states of ACMA that may prove useful in understanding the spectroscopic properties of DNA complexes of ACMA.
Methods All calculations were performed with the MOPAC program package21 using the A M I option.'-22 Figure 1 illustrates the structure and the numbering of atoms in the cis form of protonated ACMA (ACMA-H+). In the calculations, the two benzene rings and all directly bonded H, C1, and 0 atoms were individually constrained to remain planar, but no other geometrical constraints were imposed, unless otherwise specified. As a starting point in the calculations, we assumed a planar geometry with normal bond lengths (as in Appendix C of ref 23). As will be shown, the rotation of the methoxy group plays an important role in the interpretation of the unusual emission properties of ACMA. The torsional angle, e, between the methoxy group and the aromatic ring was taken as Oo in cis conformation. In our studies, the torsional potential was calculated in steps of 30° with optimization of all remaining geometrical parameters and fitted to a six-term Fourier e x p a n ~ i o n : l ~ * ~ ~ AE = E ( 0 ) - E(Oo) = !12/2cVn[l -COS (ne)] n
energy diff (kcal/mol) between trans (cis) forms in SI and So states central ring geom bond order'
0.00
'The precise maxima are within 0.1 kcal/mol of the values quoted. *In fact, this is the barrier at 0 = -goo, which is slightly lower than that at 0 = +90°. The conventionz1about dihedral angle is used here. cThe bond order between Cz-0 in the bond order matrixz1 is listed.
TABLE II: Fourier Coeficients (kcal/mol) of Methoxy Group Torsional Potential' ACMA-H+ ACMA S, S, SI S" -0.55 2.48
-1.86 6.48 -0.40 -0.14 -0.07 0.11
Vl
VZ v3 v4
v5 v6
-1.13 1.41 -0.08 0.14 -0.05 0.02
0.20 0.14 -0.02
0.01
0.06 1.32 0.3 1 0.09 -0.02 0.00
'See eq 1 in the text.
(1)
These calculations were performed for both the ground (So) and first excited singlet (SI) states of the neutral and protonated forms of ACMA. For comparison, some additional calculations were also carried out for protonated 9-aminoacridine. We also examined the effect of buckling of the central ring on the energetics of the excited-state molecules. The effect of the orientation of the ring nitrogen proton in ACMA-H+ was also examined for its possible role in contributing to the unusual ACMA emission properties. The computations were carried out on a S U N 3/140M-4 workstation with floating-point accelerator. Typically, one calculation required 2-3 h for the ground state and 2-6 days for the excited state, approximately the same as on a VAX 780/VMS computer.
Results and Discussion The principal results of our calculations on ACMA and ACMA-H+ are summarized in Tables I and I1 and in Figures 2-4. (18) Dewar, M. J. S.;Thiel, W. J. Am. Chem. Soc. 1977,99,4899-4907. (19) Fabian, W. Z . Naturforsch. 1987, 42A, 641-644. (20) Ni, Y.; Kearns, D. R. To be submitted for publication. (21) Stewart, J. J. P. QCPE 1983,No. 455. Version 4.00 released in 1987 was used. (22) Dewar, M. J. S.; Zoebisch, E. G. Submitted for publication. (23) Clark, T. A Handbook of Computarional Chemistry; Wiley: New York, 1985. (24) Barone, V.; Commisso, L.; Lelj, F.; Russo, N. Tetrahedron 1985, 41, 1915-1918.
Torsional Angle (Degrees)
Figure 2. Torsional potentials (kcal/mol) for rotation of the methoxy group of ACMA-H+ in the S, (top) and So (bottom) states. 3,
U
5 w 0
20
40
60
BO
1 0 0 120 1 4 0 1 6 0 I 8 0
Torsional Angle (Degrees)
Figure 3. Torsional potentials (kcal/mol) for rotation of the methoxy group of ACMA in the SI (top) and So (bottom) states.
Ground-State Properties. In the ground electronic state, both ACMA and ACMA-H+ are predicted to be most stable in a planar geometry. The cis isomer is predicted to be slightly (0.4
6624
The Journal of Physical Chemistry, Vol. 93, No. 18, 1989 0 251 0 262
a)
0 247 0 2 9 3 0 266
H
H
0 1 0 4 0 086 0 086
H
H
H
0 169
0 262 0 275
0 169 0 166
0 175
0 231
0 231
H -0 106 0 1 4 0 147
!
q
1
~
I
H -0063
% ~ ~ 0 086
~
1
c % 0 2 0 184
I
c
-0,134
,
~
c
,
-0.080 Q
0,108
.0.077
4 l>c4,1&
.o
190
H
H
0 162
0 154 0 157
0 156
0 109 0.098 0.063
-0,207
c q ,‘U
Figure 4. Charge distribution for the equilibrium forms of (a) transACMA-H+ in the SI (top) and So (bottom) states and (b) trans-ACMA in the S , state (top) and cis-ACMA in the So state (bottom).
Figure 5. Three-dimensionalstructure of ACMA-H+ (trans form) in the SI state.
kcal/mol) more stable than the trans isomer in the neutral ACMA, but the reverse is true for the protonated ACMA. In both cases, the barrier to rotation of the methoxy group is predicted to be quite small, and therefore, we would expect to see rapid equilibration between the cis and trans isomers at room temperature. The ground-state electronic charge distributions in ACMA-H+ and ACMA are displayed in Figure 4. Excited-State Properties. ( i ) Ring Buckling. In the SI state, ACMA-H+ prefers a bent structure (see Figure 5) in which the central ring is in a boat conformation and the angle between the two flanking rings is 160O. A chair conformation was calculated to be energetically unstable. For the trans isomer of ACMA-H+, the planar geometry was found to be 1 kcal/mol higher in energy than the bent structure, after optimization of all other geometrical parameters. For neutral ACMA in the SI state, the planar structure is predicted to be more stable than is the bent conformation. Analogous calculations were carried out for the parent molecule, 9-aminoacridine (9AA), and as with ACMA-H+, we found that in the S, state of protonated 9AA a bent structure is favored with an angle of 162O between the two benzene rings. The difference between the bent and planar structures is, again, only about 1 kcal/mol. (ii) Orientation of the Ring Nitrogen Proton. We explored the effect of the orientation of the ring nitrogen proton on the energy of the trans isomer of ACMA-H+ in the S, state. In the most stable conformation, the N-H proton is equatorial. When the N-H proton was oriented in an “axial” position, the molecule was calculated to be unstable relative to reversion back to an equatorial orientation. (iii) Orientation of the Methoxy Group and Barrier to Rotation. In the SIstate of ACMA-H+, theory predicts that the trans isomer is more stable than the cis isomer by 2.3 kcal/mol (see Figure
Ni and Kearns
2) and that there is a relatively large barrier (5.4kcal/mol) for the cis-trans conversion (7.8 kcal/mol for the reverse transition). This is to be. contrasted with the behavior of the neutral ACMA molecule where the difference in the stabilities of the cis and trans isomers differ by only 1.3 kcal/mol and the barrier is reduced to only 0.8 kcal/mol (see Figure 3). It is therefore clear that protonation of ACMA has a pronounced effect on the barrier to rotation of the methoxy group. As may be seen in Table I, this is due to enhancement of the double bond character in the Cz-0 bond between the methoxy group and the ring. (iv) Excited-State Charge Distributions. The distributions of charge in the SIstates of ACMA-H+ and ACMA are also shown in Figure 4. The changes in the charge distribution that occur upon excitation will lead to altered interactions of the ACMA-H+ with the solvent and therefore give rise to a red shift of the emission relative to the absorption. Since recent theoretical studies support the notion that electrostatic interactions between the bases are a major factor determining the sequence-dependent helical properties of DNA,2Sit is not unreasonable to expect that electrostatic interactions will also play an important role in determining the geometry of drug complexes with DNA. Furthermore, changes that occur in the charge distribution of a drug upon excitation might also lead to alterations in the geometry of the ACMA complex with DNA. With these possibilities in mind, we have summarized the calculated charge distributions for ACMA-H+ and for neutral ACMA in both the ground and excited states (see Figure 4). Cis-Trans Isomerization of the Methoxy Group Accounts for the Unusual Emission Properties of ACMA-H+. In the preceding paper” in this issue, we summarized a set of experimental observations that demonstrate that the complex emission behavior observed for ACMA-H+ is due to a slow (2-11s) conversion from an initially formed excited-state species to a second form. By a series of experiments, we were able to eliminate all of the previously suggested explanations such as interconversion between two differently protonated forms of ACMA, unusually slow (2-11s) relaxation of solvent water molecules following excitation of the ACMA, excimer formation, and other more unusual possibilities. The results of the theoretical calculations that we have presented here appear to provide a straightforward explanation of all our experimental observations with the protonated and neutral forms of ACMA in terms of an excited-state cis-trans isomerization of the methoxy group. The AM1 calculations show that in the ground state of ACMA-H+ the barrier to rotation of the methoxy group is quite small (2.3 kcal/mol) and the cis and trans isomers, which are nearly isoenergetic, are predicted to be about equally populated. At room temperature, interconversion between the two isomers is expected to be rapid. In the excited state, the trans isomer is more stable than the cis isomer by 2.3 kcal/mol and the barrier to cis-trans conversion increases to 5.4kcal/mol. These results, which are shown schematically in Figure 2, provide the following explanation for our experimental observations. In the ground state, both the cis and trans isomers are present and excitation in the main So-S1 absorption band would generate comparable populations of both cis and trans excited-state species. However, because the trans isomer lies lower in energy in the excited state, conversion of the cis isomer to the trans isomer is predicted to occur with an activation energy of 5.4 kcal/mol. Experimentally, we observe an activation energy of 4.3 kcal/mol for the depletion of the higher energy emitting species. Because the trans isomer lies lower than the cis isomer, we further predict a red shift of about 200 A in the emission of the trans isomer relative to the cis. Experimentally, we find time-dependent red shift of the ACMA-H+ emission corresponding to about 250 A. Another consequence of the relative energies of the cis and trans isomers in the excited state is that the trans to cis conversion is predicted to be slower by about a factor of 46 at room temperature. Assuming that the experimental value of 2 ns reflects the cis to trans ( 2 5 ) Sarai, A,; Mazur, J.; Nussinov, R.; Jernigan, R. L. Biochemistry 1988, 27, 8498-8502.
J. Phys. Chem. 1989, 93, 6625-6628
excited-state conversion, we would predict a time constant of over 90 ns for the reverse process. This is considerably longer than the normal fluorescence lifetime of ACMA-H+ (18 ns) and explains why the excited-state trans to cis process can be neglected at room temperature. The observed dependence of the fluorescence emission spectrum on the excitation wavelength a t low temperatures can also be accounted for in terms of the theoretical results depicted in Figure 2. At low temperatures, cis-trans isomerization is "frozen out", and consequently, excitation at the red edge of the ACMA-H+ absorption spectrum will lead preferentially to excitation and emission from the trans isomer. Excitation within the main absorption band will, on the other hand, lead to emission from both the cis and trans isomers. In this way, we can account for the observed wavelength dependence of the low-temperature spectra. It also follows from the proposed role of cis-trans isomerization that the nature of the solvent and the solvent viscosity are not expected to have much effect on the ACMA-H+ emission behavior. The intrinsic barrier to cis-trans isomerization in the excited state arises from the generation of partial double bond character in the Cz-0 bond between the ring and the methoxy group and does not depend upon some special solvent effect. Moreover, rotation of the methoxy group is expected to have only a small solvent viscosity dependence, judging from studies of the excited-state cis-trans isomerization of analogous vinyl-substituted anthracene.% We should note, however, that since the calculations are for ACMA in a vacuum, we have not taken into account the interactions with solvent molecules. Since the changes in electronic charge distribution are not large, this may not be too serious. According to our interpretation, we expect no effect of salt, no counterion effect, no deuterium isotope effect, and no effect of pH in the range where the ring nitrogen is protonated. All of these predictions are in agreement with experimental observations. Finally, our theoretical calculations provide a clear prediction regarding the effects of deprotonation on cis-trans isomerization. As shown in Table I, the excited-state cis-trans barrier is reduced from 5.4 to 0.8 kcal/mol when the ring nitrogen proton is removed. Because of the very low barrier, cis-trans isomerization is predicted to be approximately 2000 times faster in ACMA relative to ACMA-H+ at room temperature. This is clearly too fast to be followed with nanosecond resolution and explains why neutral (26) Flom, R.;Nagarajan, V.; Barbara, P. F. J . Phys. Chem. 1986, 90, 2085-2092.
6625
ACMA exhibits single-exponential decay in our experiments. The key role of protonation in the complex emission behavior is thus accounted for. In some of our other theoretical studies, we looked for other possible explanations for the unusual fluorescence emission behavior of ACMA-H+. In the ground state, the molecule is predicted to be planar, whereas in the excited state it is predicted to be bent by about 20'. The possibility that the conformational rearrangement from a planar to bent geometry might be responsible for the slow 2-11s process is eliminated since no barrier is predicted to exist. We also explored the possibility that the orientation of the ring nitrogen proton might generate a metastable state and that interconversion between excited states with different N-H orientations might be involved. Again, this possibility was eliminated by the calculations, which show that the conformation with axial hydrogen is energetically unstable. Moreover, if reorientations of the N-H bond from axial to equatorial were involved, a deuterium isotope effect might have been expected, but none was observed. Calculations on the protonated 9AA show that in the ground state the molecule is planar, but bent by 18' in the SI state. Because of the absence of a methoxy group, however, no unusual emission behavior is expected or observed." Thus 9AA-H+ exhibits single-exponential decay.
Summary The theoretical calculations that we have presented here account for all of our experimental spectroscopic observations on ACMA-H+, including (i) emission from two different excited-state species, (ii) the barrier to cis-trans interconversion, (iii) the effect of deprotonation in removal of the excited-state interconversion barrier, (iv) the lack of solvent, salt, and buffer effects, and (v) the effect of temperature on the emission properties. Since similar experimental results are also obtained with quinacrine, we may presume that cis-trans isomerization contributes to the complex emission behavior of this molecule as well. It is known that the AM1 program gives a good account of ground-state conformational properties of organic molecules. Our results suggest that it is successful in accounting for the conformational properties of the excited-state molecules as well. Acknowledgment. This work was supported by grants from the National Science Foundation and the American Cancer Society (to D.R.K.). We especially thank Professors Jay Siege1 and John Simon for many helpful discussions.
Laser Ionization Studies of Organophosphonates and PO Radicals S. Randolph Long* and Steven D. Christesen US.Army Chemical Research, Development and Engineering Center, Aberdeen Proving Ground, Maryland 21 01 0-5423 (Received: January 23, 1989; In Final Form: April 26, 1989) Mass spectra of dimethyl methylphosphonate (DMMP) and diisopropyl methylphosphonate (DIMP) have been generated in a quadrupole mass spectrometer by ionization with the KrF (248 nm) and ArF (193 nm) excimer lasers. High mass ions, including the molecular ion, are prominent. PO radicals, produced by multiphoton dissociation of parent DMMP, are ionized in resonant two-photon ionization via the AZZ+-XZntransition with (0-0) bandheads near 247 nm.
Introduction Laser ionization of molecules via multiphoton processes holds considerable potential advantage for mass spectrometric analysis of chemical materials. Certainly this is true for molecules such as aromatic hvdrocarbons. whose ionization Dotentials are relativelv low and which have excited electronic statks which couple to thk ground state via strong transitions' The ionization Of such I'ilol@Xle permits the sensitive and Selectivedetection Of these species, while also making possible soft ionization (low degree of
fragmentation) by application of low laser intensities.' In order to ascertain the breadth of potential application of laser ~onizationas a of generating mass spectra, it is important to apply the technique to t n 0 h ~ l e not s apparently easily i o n i z d k (1) The following reviews provide ample background and references on multiphoton ionization mass spectrometry: Grotemeyer, J.; Schlag, E,W,
Anpew. Chem.. Int. Ed. E n d . 1988. 27. 447. Lubman, D. M. Anal. Chem. 19g7, 59, 31A:
This article not subject to US. Copyright. Published 1989 by the American Chemical Society
