Photochemistr) and Photubioloyy. 1976, Vol. 24, pp. 83-86.
Pergamon Press. Printed in Great Britain
RESEARCH NOTE
TRIPLET ELECTRONIC STRUCTURE AND PHOTOREACTIVITY OF 8-METHOXYPSORALEN* THOMAS A. MOORE,ALANB. MONTGOMERY and ALVINL. KWIRAMt Department of Chemistry, University of Washington, Seattle, WA 98195, U.S.A. (Received 17 November 1975; accepted 23 February 1976)
troscopy, our measurements strongly suggest that there is a unique feature of the triplet electronic structure of the very active derivatives which distinguishes them from the less active derivatives and from the parent coumarin.
INTRODUCTION
The photochemical reactions of furocoumarin derivatives (see Fig. 1) with D N A have received widespread attention recently. These derivatives, especially 8-methoxypsoralen (8-MOP) are implicated in skin
photochemotherapy of psoriasis (Parrish et al., 1974). However, the derivatives differ markedly in their photosensitivity. Musajo and Rodighiero (1970) established a correlation between the photoreactivity of a variety of psoralen derivatives with DNA and their phototoxic potency in uiuo. The photochemical reactions of interest are most likely the cycloaddition to the DNA pyrimidine bases by the psoralen (Musajo and Rodighiero, 1970), a reaction which occurs via the psoralen excited triplet state (Mantulin and Song, 1973). Utilizing luminescence spectroscopy and theoretical techniques Song and coworkers (Mantulin and Song, 1973) have investigated the excited states of coumarin and the psoralen derivatives. It is somewhat surprising to find, based on these data, that the common photophysical parameters which characterize the triplet state (i.e. energy, quantum yield, and lifetime) are similar for most of the derivatives and do not show any correlation with the wide range of phototoxicities represented among the psoralen derivatives. Consequently, we have undertaken a detailed characterization of the excited triplet state of 8-MOP and several other derivatives utilizing optical detection of magnetic resonance (ODMR) and we report herein the zero-field splitting (ZFS)parameters for these compounds. In contrast to the results of optical spec-
6:0.142
8-methoxypsoralen
state
sac.
k sac:'
I
I
T
cm-'
relative kinetic const.
'k 0.5
Niol
A
I
I
u psoralen
I
1
I
I
I
0.3
0.1
1.46 GHz 4.0
*Presented at the First Chemical Congress of the North American Continent, Mexico City, Mexico, 4 December 1975, paper no. 102, BMPC session. Preliminary results presented at the 29th Northwest Regional Meeting, American Chemical Society, Cheney, Washington, 14 June 1974, abstract no. 234. t A u thor to whom correspondence should be addressed. 83
0.25
0.1
Figure 1. Lifetimes, relative kinetic constants and zerofield splittings for the lowest triplet state of S-methoxypsoralen. The labels x , y and z are arbitrary. k' is the relative radiative rate constant, N(0) is the relative steady state population and A is the relative populating rate constant. 7 and k are the state lifetime and rate constant, respectively.
84
T. A. MOORE,A. B. MONTGOMERY and A. L. KWIRAM
ing the populations of x and y, thus creating a population difference between x and z, and then sweeping through the 3.35 GHz transition and observing the concomitant change in phosphorescence intensity. Note that sublevel x plays only a minor role in determining the photophysical parameters of the triplet state. At temperature greater than approximately 4 K where spin-lattice relaxation will assure equilibrium populations of the three states, the observed phosphorescence is approximately 6% from x, 31% from z and 63% from y. In Table 1 we present for comparison the general ZFS parameter? D* for a selection of aromatic hydrocarbons and heterocyclic compounds together with the psoralens and coumarin. All of these comRESULTS AND DISCUSSION pounds have lowest triplet states of n. n* symmetry. Figure 1 presents the kinetic parameters as well One expects D* to decrease as the size of the delocaas the magnetic resonance parameters for the lowest lized n-system increases since the zero-field splitting excited triplet state of 8-MOP. The kinetic parameters is dominated in these systems by the dipole-dipole were obtained using the techniques described preinteraction of the two unpaired electrons.$ This trend viously (Moore and Kwiram, 1974). Spin lattice relaxis clearly evident in comparing D* for benzene, naphation processes were assumed to be slow compared thalene, and anthracene. The value, D* = 0.124cm-', with the lifetime of the state and were neglected. The for coumarin is typical for a molecule of its size. In emission was monitored at the origin of the phoscontrast to the parent coumarin which is not photophorescence spectrum and thus these kinetic partoxic, psoralen and 8-MOP have unusually high D* ameters characterize the relatively pure electronic values, 0.141 and 0.142 cm-I, respectively. transitions. The labels for the three spin states are 4,4'-Dimethylisopsoralen which is somewhat less phoarbitrary since the principal axes and state assigntotoxic than psoralen has D* = 0.135 cm-'. ments are not determined in the zero-field experiment. The measured D* values are consistent with the Although three transitions were observed, the transisuggestion (Mantulin and Song, 1973) that the lowest tion at 3.35 GHz is relatively weak due to the similartriplet state of psoralen has a rather localized distriity in radiative and kinetic properties of sublevels z bution of the unpaired electrons, and that this in turn and y. The 3.35 GHz transition was observed in a could facilitate triplet state reactivity. Whether in fact transient double resonance experiment by first invertsuch a simple correlation exists for this series of molecules between the ZFS parameters on the one hand ?Since the diagonalized zero-field (ZF) tensor-characterized by the three parameters X , Z-is traceless (i.e. the and phototoxicity on the other will require measuresum X + Y + Z = 0), the Z F splittings may be character- ments on the entire series of derivatives. Nevertheless, ized equally well by the two independent parameters D it seemed worthwhile to explore the properties of the and E . where D = -(3/2) Z and E = 1/2(X - Y). The hydroxy derivatives since they are inactive as skin magnitude of the zero-field splitting (ZFS) parameters will depend on how one chooses to label the principal axes sensitizers (Musajo et al., 1967). Barring ionization, of the molecule. If for example the out-of-plane axis is one might expect the 8-hydroxy and -methoxy derivadesignated as the -? axis, then for the lowest n,n* triplet tives to show similar triplet state reactivity in the pyrstate of most planar aromatic hydrocarbons, D 2 31EI con- one region of the molecule. Indeed, measurements of sistent with a common convention. Further, if it is assumed the ZFS parameters showed almost identical values that D is positive, then the sublevel associated with the energy Z will lie lowest. Based on this axis convention for the hydroxy and methoxy derivatives. However, it has been found in fact (Harrigan and Hirota, 1972) that Song and coworkers (Song et al., 1975) have shown for coumarin D < 31EI. If we use the same convention in that the 5- and 8-hydroxy derivatives undergo dissocomparing the ZFS parameters of the psoralens we find ciation of the hydroxyl proton in the excited state that D remains essentially constant whereas E changes markedly in going from coumarin ( E = -0.042 cm- ') to in the presence of an organic base. Consistent with 8-MOP ( E = -0.056cm-'). On the other hand, if one these workers, we find the triplet energy of the anion chooses the x axis as the out-of-plane axis then E remains of 8-hydroxypsoralen is similar to that of the neutral virtually constant while D increases from D = 0.1 14 cm-' species while the quantum yield of intersystem crossfor coumarin to D = 0.136 cm-' for 8-MOP. To avoid this ing appears lower in the anion. By carrying out uniqueness problem we have chosen to use the axis-independent parameter D* as a composite measure of the mag- ODMR experiments in EGW plus pyridine we have nitude of the zero-field splittings; (D*)2 = (D' + 3E2) = observed the three zero-field transitions of the anion (3/2XXz + Y 2 + Z 2 ) .The precision of the measurement of and calculate D* = 0.120cm-'. Thus the ODMR exthese transitions is better than 0.001 cm-'. Much more periments show that the triplet state electronic strucprecise measurements are possible using additional signal ture of the anion is substantially changed from that averaging. $In general such comparisons should be restricted to of the neutral molecule. However, as expected, the classes of molecules having similar molecular symmetry. triplet electronic structure of the neutral 8-hydroxy
below were carried out as previously reported (Moore and Kwiram, 1974). Except where noted. all measurements were made at about 1.5 K in (1:l) ethyleneglycol-water (EGW) glasses. 8-Methoxypsoralen (8-MOP) was obtained from Sigma Chemical Company and shown to be free of luminescent impurities in the excitation (3M-366 nm) and emission (46S500 nm) regions of interest. 8-Hydroxypsoralen was synthesized from 8-MOP using the method of Schonberg and Sina (1950). Final purification involved sequential thin layer chromatography, first on acid silica gel type G and then neutral type H silica gel, in both cascs using 3:l ether. cyclohexane as the developing solvent. Purified in this way 8-hydroxypsoralen (mp 246°C) exhibits no extraneous luminescence or ODMR signals. Psoralen and 4.4-dimethylisopsoralen were generously provided by Professor P.4. Song of Texas Tech University.
Research Note
85
Table 1. D* values of selected compounds; zf transition frequencies of the psoralens Compound
D* (cm-l)
benzene
0.158
de Groot et al. (1969)
naphthalene
0.105
McGlynn et al. (1969)
anthracene
0.077
McGlynn et al. (1969)
indole
0.125
Harrigan and Hirota (1973)
coumarin
reference
0.124
Graber et al. (1969)
0.128
Kirkiacharian et al. (1972)
transition
frequencies (GHz)
8-methoxypsoralen
0.142
1.46,
3.35,
4.81
psoralen
0.141
1.49,
3.27,
4.76
4,4'-dimethylisopsoralen
0.135
1.88,
2.77,
4.65
8-hydroxypsoralen
0.141
1.41,
3.34,
4.75
8-hydroxypsoralen anion
0.120
1.55,
2.55,
4.10
derivative is very similar to that of the 8-methoxy derivative. In light of this similarity one might predict photoactivity by 8-h ydroxypsoralen under microenvironmental circumstances favoring the neutral molecule, and indeed, Song et a!. (1975) have found 8-hydroxypsoralen to be weakly photomutagenic to certain bacteria. Current work is underway to investigate the relative populations of the anion and neutral species in various in uiuo environments using the ODMR technique. While the rather high D* values of the active compounds suggest a localized triplet state, adequate theoretical descriptions are not generally available. However, Song et al. (1975), using the configurational analysis technique of Baba e t a / (1969) have calculated the lowest triplet of psoralen to be approximately 40% localized at the ethylenic unit of the pyrone moiety. Additionally, as has recently been shown in the case of conjugated enones (Jones et a/., 1973), the zero-field splittings increase substantially in x,n* triplets as a result of coupling with nearby localized n,x* states. Such mixing is expected to be a sensitive function of the energy gap between the n,x* and the 7c,x* states and experiments are underway to elucidate these coupling mechanisms in psoralens. We have demonstrated that in contrast to the results of conventional spectroscopic measurements, the ODMR technique provides a powerful means for distinguishing between the different psoralen derivatives. The next crucial task is that of establishing an unequivocal relationship between the ZFS parameters, (and hence the electronic structure) and the photoreactivity of the psoralens. The triplet state of the psoralens is of central importance in understanding the phototoxic reactions of these molecules with DNA. The ODMR technique is ideally suited, given any accessible triplet state, for
investigating the structure and microenvironment of reaction products, metabolites, and their complexes with DNA and other cellular macromolecules. Indeed, the ODMR technique has considerable potential as a tool for studying the binding of chemical carcinogens to DNA and preliminary experiments along these lines have been initiated in our laboratory. We have already demonstrated that by exploiting the dual domain feature of ODMR (the microwave transitions. defining a unique fingerprint, provide the resolution while the sensitivity is comparable to that of optical spectroscopy) the technique is analytically useful to sub-nanomolar concentrations. SUMMARY
Psoralen and its derivatives are implicated in a variety of photobiological processes including skinsensitization in mammals, the experimental photochemotherapy of psoriasis, and photomutagenesis in bacteria. Although the various derivatives differ markedly in photoactivity, their excited triplet states as characterized by conventional luminescence spectroscopy are very similar and closely resemble that of the parent compound, coumarin, which is inactive as a skin sensitizer. Employing a more sensitive probe of triplet electronic structure, we have utilized optical detection of magnetic resonance to measure the zero-field splittings in the triplet state of several psoralens and find a striking variation among the derivatives. D*,taken as a composite measure of the dipole-dipole interaction of the unpaired electrons in the triplet state was found to be anomalously large, greater than 0.140 cm-', for the very active compounds, psoralen and 8-methoxypsoralen, while D* = 0.124 cm-' for the inactive, though smaller, coumarin molecule. Although 8-hydroxypsoralen has a large D* value its
86
T . A. MOORE,A. B. MONTGOMERY and A. L. KWIRAM
inactivity as a skin-sensitizer may be explained by the dissociation of the hydroxyl proton. The resulting (excited) triplet state anion has D* = 0.120 cm-'.
Acknowledgment-This work was supported by Institutional Cancer Grant IN-26 from the American Cancer Society.
REFERENCES
Baba, H.. S . Suzuki snd T. Takemura (1969) J . Chem. Phys. 50. 2078-2086. de Groot, M. S., I. A. M. Hesselmann and J. H. van der Waals (1969) Mol. Phys. 16. 45-60. Epstein, J. H. and K. Fukuyama (1975) Photochem. Photobiol. 21. 325-330. Graber, D. R., M. W. Grimes and A. Haug (1969) J . Chem Phys. 50. 1623-1626. Harrigan, E. T., and N. Hirota (1973) Chem. Phys. Letters 22. 29-32. Igali, S., and R. C. von Borstel (1974) Abstracts, Second Annual Meeting, American Society for Photobiology, July 22-26, 1974, University of British Columbia, Vancouver, B.C., p. 99. Jones, C. R., A. H. Maki and D. R. Kearns (1973) J . Chem. Phys. 59. 873-878. Kirkiacharian, B. S., R. Santus and C. Helhe (1972) Photochem. Photobiol. 16, 455458. Kwiram, A. L. (1972) In M T P International Review of Science, Series I, Physical Chemistry, Vol. IV, (Edited by C. A. McDowell), pp. 271-315. University Park Press and Butterworth, Baltimore and London. Mantulin, W. W., and P. S. Song (1973) J . Am. Chem. Soc. 95. 5122-5129 (and references therein). McGlynn, S. P., Azumi and M. Kinoshita (1969) Molecular Spectroscopy of the Triplet State, p. 362. Prentice-Hall, Englewood Cliffs, NJ. Moore, T. A., and A. L. Kwiram (1974) Biochemistry 13. 5403-5407. Musajo, L.. and G . Rodighiero (1962) Experientiu 18. 153-200. Musajo, L., and G. Rodighiero (1970) Photochern. Photobiol. 11. 27-35. Parrish, J. A., T. B. Fitzpatrick, L. Tanenbaum and M. A. Pathak (1974) New England J . Med. 291. 1207-1211. Schonberg, A., and A. Sina (1950) J . Aiii. Chem. Soc. 72. 48264827. Song, P. S., W. W. Mantulin. D. Mclnturff, I. C. Felkner and M. L. Harter (1975) Photochem. Photobiol. 21. 317-324. Song, P. S., C.-A. Chin, I. Yamazuki and H. Baba (1975) Intern. J . Quantum Chem. QBS No. 2,
,
1-8.
Pergamon Press. Printed in Great Britain
RESEARCH NOTE
TRIPLET ELECTRONIC STRUCTURE AND PHOTOREACTIVITY OF 8-METHOXYPSORALEN* THOMAS A. MOORE,ALANB. MONTGOMERY and ALVINL. KWIRAMt Department of Chemistry, University of Washington, Seattle, WA 98195, U.S.A. (Received 17 November 1975; accepted 23 February 1976)
troscopy, our measurements strongly suggest that there is a unique feature of the triplet electronic structure of the very active derivatives which distinguishes them from the less active derivatives and from the parent coumarin.
INTRODUCTION
The photochemical reactions of furocoumarin derivatives (see Fig. 1) with D N A have received widespread attention recently. These derivatives, especially 8-methoxypsoralen (8-MOP) are implicated in skin
photochemotherapy of psoriasis (Parrish et al., 1974). However, the derivatives differ markedly in their photosensitivity. Musajo and Rodighiero (1970) established a correlation between the photoreactivity of a variety of psoralen derivatives with DNA and their phototoxic potency in uiuo. The photochemical reactions of interest are most likely the cycloaddition to the DNA pyrimidine bases by the psoralen (Musajo and Rodighiero, 1970), a reaction which occurs via the psoralen excited triplet state (Mantulin and Song, 1973). Utilizing luminescence spectroscopy and theoretical techniques Song and coworkers (Mantulin and Song, 1973) have investigated the excited states of coumarin and the psoralen derivatives. It is somewhat surprising to find, based on these data, that the common photophysical parameters which characterize the triplet state (i.e. energy, quantum yield, and lifetime) are similar for most of the derivatives and do not show any correlation with the wide range of phototoxicities represented among the psoralen derivatives. Consequently, we have undertaken a detailed characterization of the excited triplet state of 8-MOP and several other derivatives utilizing optical detection of magnetic resonance (ODMR) and we report herein the zero-field splitting (ZFS)parameters for these compounds. In contrast to the results of optical spec-
6:0.142
8-methoxypsoralen
state
sac.
k sac:'
I
I
T
cm-'
relative kinetic const.
'k 0.5
Niol
A
I
I
u psoralen
I
1
I
I
I
0.3
0.1
1.46 GHz 4.0
*Presented at the First Chemical Congress of the North American Continent, Mexico City, Mexico, 4 December 1975, paper no. 102, BMPC session. Preliminary results presented at the 29th Northwest Regional Meeting, American Chemical Society, Cheney, Washington, 14 June 1974, abstract no. 234. t A u thor to whom correspondence should be addressed. 83
0.25
0.1
Figure 1. Lifetimes, relative kinetic constants and zerofield splittings for the lowest triplet state of S-methoxypsoralen. The labels x , y and z are arbitrary. k' is the relative radiative rate constant, N(0) is the relative steady state population and A is the relative populating rate constant. 7 and k are the state lifetime and rate constant, respectively.
84
T. A. MOORE,A. B. MONTGOMERY and A. L. KWIRAM
ing the populations of x and y, thus creating a population difference between x and z, and then sweeping through the 3.35 GHz transition and observing the concomitant change in phosphorescence intensity. Note that sublevel x plays only a minor role in determining the photophysical parameters of the triplet state. At temperature greater than approximately 4 K where spin-lattice relaxation will assure equilibrium populations of the three states, the observed phosphorescence is approximately 6% from x, 31% from z and 63% from y. In Table 1 we present for comparison the general ZFS parameter? D* for a selection of aromatic hydrocarbons and heterocyclic compounds together with the psoralens and coumarin. All of these comRESULTS AND DISCUSSION pounds have lowest triplet states of n. n* symmetry. Figure 1 presents the kinetic parameters as well One expects D* to decrease as the size of the delocaas the magnetic resonance parameters for the lowest lized n-system increases since the zero-field splitting excited triplet state of 8-MOP. The kinetic parameters is dominated in these systems by the dipole-dipole were obtained using the techniques described preinteraction of the two unpaired electrons.$ This trend viously (Moore and Kwiram, 1974). Spin lattice relaxis clearly evident in comparing D* for benzene, naphation processes were assumed to be slow compared thalene, and anthracene. The value, D* = 0.124cm-', with the lifetime of the state and were neglected. The for coumarin is typical for a molecule of its size. In emission was monitored at the origin of the phoscontrast to the parent coumarin which is not photophorescence spectrum and thus these kinetic partoxic, psoralen and 8-MOP have unusually high D* ameters characterize the relatively pure electronic values, 0.141 and 0.142 cm-I, respectively. transitions. The labels for the three spin states are 4,4'-Dimethylisopsoralen which is somewhat less phoarbitrary since the principal axes and state assigntotoxic than psoralen has D* = 0.135 cm-'. ments are not determined in the zero-field experiment. The measured D* values are consistent with the Although three transitions were observed, the transisuggestion (Mantulin and Song, 1973) that the lowest tion at 3.35 GHz is relatively weak due to the similartriplet state of psoralen has a rather localized distriity in radiative and kinetic properties of sublevels z bution of the unpaired electrons, and that this in turn and y. The 3.35 GHz transition was observed in a could facilitate triplet state reactivity. Whether in fact transient double resonance experiment by first invertsuch a simple correlation exists for this series of molecules between the ZFS parameters on the one hand ?Since the diagonalized zero-field (ZF) tensor-characterized by the three parameters X , Z-is traceless (i.e. the and phototoxicity on the other will require measuresum X + Y + Z = 0), the Z F splittings may be character- ments on the entire series of derivatives. Nevertheless, ized equally well by the two independent parameters D it seemed worthwhile to explore the properties of the and E . where D = -(3/2) Z and E = 1/2(X - Y). The hydroxy derivatives since they are inactive as skin magnitude of the zero-field splitting (ZFS) parameters will depend on how one chooses to label the principal axes sensitizers (Musajo et al., 1967). Barring ionization, of the molecule. If for example the out-of-plane axis is one might expect the 8-hydroxy and -methoxy derivadesignated as the -? axis, then for the lowest n,n* triplet tives to show similar triplet state reactivity in the pyrstate of most planar aromatic hydrocarbons, D 2 31EI con- one region of the molecule. Indeed, measurements of sistent with a common convention. Further, if it is assumed the ZFS parameters showed almost identical values that D is positive, then the sublevel associated with the energy Z will lie lowest. Based on this axis convention for the hydroxy and methoxy derivatives. However, it has been found in fact (Harrigan and Hirota, 1972) that Song and coworkers (Song et al., 1975) have shown for coumarin D < 31EI. If we use the same convention in that the 5- and 8-hydroxy derivatives undergo dissocomparing the ZFS parameters of the psoralens we find ciation of the hydroxyl proton in the excited state that D remains essentially constant whereas E changes markedly in going from coumarin ( E = -0.042 cm- ') to in the presence of an organic base. Consistent with 8-MOP ( E = -0.056cm-'). On the other hand, if one these workers, we find the triplet energy of the anion chooses the x axis as the out-of-plane axis then E remains of 8-hydroxypsoralen is similar to that of the neutral virtually constant while D increases from D = 0.1 14 cm-' species while the quantum yield of intersystem crossfor coumarin to D = 0.136 cm-' for 8-MOP. To avoid this ing appears lower in the anion. By carrying out uniqueness problem we have chosen to use the axis-independent parameter D* as a composite measure of the mag- ODMR experiments in EGW plus pyridine we have nitude of the zero-field splittings; (D*)2 = (D' + 3E2) = observed the three zero-field transitions of the anion (3/2XXz + Y 2 + Z 2 ) .The precision of the measurement of and calculate D* = 0.120cm-'. Thus the ODMR exthese transitions is better than 0.001 cm-'. Much more periments show that the triplet state electronic strucprecise measurements are possible using additional signal ture of the anion is substantially changed from that averaging. $In general such comparisons should be restricted to of the neutral molecule. However, as expected, the classes of molecules having similar molecular symmetry. triplet electronic structure of the neutral 8-hydroxy
below were carried out as previously reported (Moore and Kwiram, 1974). Except where noted. all measurements were made at about 1.5 K in (1:l) ethyleneglycol-water (EGW) glasses. 8-Methoxypsoralen (8-MOP) was obtained from Sigma Chemical Company and shown to be free of luminescent impurities in the excitation (3M-366 nm) and emission (46S500 nm) regions of interest. 8-Hydroxypsoralen was synthesized from 8-MOP using the method of Schonberg and Sina (1950). Final purification involved sequential thin layer chromatography, first on acid silica gel type G and then neutral type H silica gel, in both cascs using 3:l ether. cyclohexane as the developing solvent. Purified in this way 8-hydroxypsoralen (mp 246°C) exhibits no extraneous luminescence or ODMR signals. Psoralen and 4.4-dimethylisopsoralen were generously provided by Professor P.4. Song of Texas Tech University.
Research Note
85
Table 1. D* values of selected compounds; zf transition frequencies of the psoralens Compound
D* (cm-l)
benzene
0.158
de Groot et al. (1969)
naphthalene
0.105
McGlynn et al. (1969)
anthracene
0.077
McGlynn et al. (1969)
indole
0.125
Harrigan and Hirota (1973)
coumarin
reference
0.124
Graber et al. (1969)
0.128
Kirkiacharian et al. (1972)
transition
frequencies (GHz)
8-methoxypsoralen
0.142
1.46,
3.35,
4.81
psoralen
0.141
1.49,
3.27,
4.76
4,4'-dimethylisopsoralen
0.135
1.88,
2.77,
4.65
8-hydroxypsoralen
0.141
1.41,
3.34,
4.75
8-hydroxypsoralen anion
0.120
1.55,
2.55,
4.10
derivative is very similar to that of the 8-methoxy derivative. In light of this similarity one might predict photoactivity by 8-h ydroxypsoralen under microenvironmental circumstances favoring the neutral molecule, and indeed, Song et a!. (1975) have found 8-hydroxypsoralen to be weakly photomutagenic to certain bacteria. Current work is underway to investigate the relative populations of the anion and neutral species in various in uiuo environments using the ODMR technique. While the rather high D* values of the active compounds suggest a localized triplet state, adequate theoretical descriptions are not generally available. However, Song et al. (1975), using the configurational analysis technique of Baba e t a / (1969) have calculated the lowest triplet of psoralen to be approximately 40% localized at the ethylenic unit of the pyrone moiety. Additionally, as has recently been shown in the case of conjugated enones (Jones et a/., 1973), the zero-field splittings increase substantially in x,n* triplets as a result of coupling with nearby localized n,x* states. Such mixing is expected to be a sensitive function of the energy gap between the n,x* and the 7c,x* states and experiments are underway to elucidate these coupling mechanisms in psoralens. We have demonstrated that in contrast to the results of conventional spectroscopic measurements, the ODMR technique provides a powerful means for distinguishing between the different psoralen derivatives. The next crucial task is that of establishing an unequivocal relationship between the ZFS parameters, (and hence the electronic structure) and the photoreactivity of the psoralens. The triplet state of the psoralens is of central importance in understanding the phototoxic reactions of these molecules with DNA. The ODMR technique is ideally suited, given any accessible triplet state, for
investigating the structure and microenvironment of reaction products, metabolites, and their complexes with DNA and other cellular macromolecules. Indeed, the ODMR technique has considerable potential as a tool for studying the binding of chemical carcinogens to DNA and preliminary experiments along these lines have been initiated in our laboratory. We have already demonstrated that by exploiting the dual domain feature of ODMR (the microwave transitions. defining a unique fingerprint, provide the resolution while the sensitivity is comparable to that of optical spectroscopy) the technique is analytically useful to sub-nanomolar concentrations. SUMMARY
Psoralen and its derivatives are implicated in a variety of photobiological processes including skinsensitization in mammals, the experimental photochemotherapy of psoriasis, and photomutagenesis in bacteria. Although the various derivatives differ markedly in photoactivity, their excited triplet states as characterized by conventional luminescence spectroscopy are very similar and closely resemble that of the parent compound, coumarin, which is inactive as a skin sensitizer. Employing a more sensitive probe of triplet electronic structure, we have utilized optical detection of magnetic resonance to measure the zero-field splittings in the triplet state of several psoralens and find a striking variation among the derivatives. D*,taken as a composite measure of the dipole-dipole interaction of the unpaired electrons in the triplet state was found to be anomalously large, greater than 0.140 cm-', for the very active compounds, psoralen and 8-methoxypsoralen, while D* = 0.124 cm-' for the inactive, though smaller, coumarin molecule. Although 8-hydroxypsoralen has a large D* value its
86
T . A. MOORE,A. B. MONTGOMERY and A. L. KWIRAM
inactivity as a skin-sensitizer may be explained by the dissociation of the hydroxyl proton. The resulting (excited) triplet state anion has D* = 0.120 cm-'.
Acknowledgment-This work was supported by Institutional Cancer Grant IN-26 from the American Cancer Society.
REFERENCES
Baba, H.. S . Suzuki snd T. Takemura (1969) J . Chem. Phys. 50. 2078-2086. de Groot, M. S., I. A. M. Hesselmann and J. H. van der Waals (1969) Mol. Phys. 16. 45-60. Epstein, J. H. and K. Fukuyama (1975) Photochem. Photobiol. 21. 325-330. Graber, D. R., M. W. Grimes and A. Haug (1969) J . Chem Phys. 50. 1623-1626. Harrigan, E. T., and N. Hirota (1973) Chem. Phys. Letters 22. 29-32. Igali, S., and R. C. von Borstel (1974) Abstracts, Second Annual Meeting, American Society for Photobiology, July 22-26, 1974, University of British Columbia, Vancouver, B.C., p. 99. Jones, C. R., A. H. Maki and D. R. Kearns (1973) J . Chem. Phys. 59. 873-878. Kirkiacharian, B. S., R. Santus and C. Helhe (1972) Photochem. Photobiol. 16, 455458. Kwiram, A. L. (1972) In M T P International Review of Science, Series I, Physical Chemistry, Vol. IV, (Edited by C. A. McDowell), pp. 271-315. University Park Press and Butterworth, Baltimore and London. Mantulin, W. W., and P. S. Song (1973) J . Am. Chem. Soc. 95. 5122-5129 (and references therein). McGlynn, S. P., Azumi and M. Kinoshita (1969) Molecular Spectroscopy of the Triplet State, p. 362. Prentice-Hall, Englewood Cliffs, NJ. Moore, T. A., and A. L. Kwiram (1974) Biochemistry 13. 5403-5407. Musajo, L.. and G . Rodighiero (1962) Experientiu 18. 153-200. Musajo, L., and G. Rodighiero (1970) Photochern. Photobiol. 11. 27-35. Parrish, J. A., T. B. Fitzpatrick, L. Tanenbaum and M. A. Pathak (1974) New England J . Med. 291. 1207-1211. Schonberg, A., and A. Sina (1950) J . Aiii. Chem. Soc. 72. 48264827. Song, P. S., W. W. Mantulin. D. Mclnturff, I. C. Felkner and M. L. Harter (1975) Photochem. Photobiol. 21. 317-324. Song, P. S., C.-A. Chin, I. Yamazuki and H. Baba (1975) Intern. J . Quantum Chem. QBS No. 2,
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