J. Phys. Chem. 1990, 94, 3597-3601 for this reaction for late transition metals. Note that barriers for reactions 22 and 23 are energetically mediated by ?r donation from the newly formed C-C double bond to the metal and, for reaction 22, relief of cyclopropane strain energy. This may explain why reaction 24 is nor observed for the late transition metals and also why barriers for reaction 22 are not apparent. Theoretical considerations indicate that processes like reactions 22-24 can occur with little or no barrier for metals where the bonding primarily involves a metal d ~ r b i t a l . ~ ' Calculations ,~~ suggest that this is true for Sc+-Cr+ but not for Mn+ and later transition metals, where the bonding has mainly s character.* This suggests that concerted pathways for reactions 22-24 should occur with larger barriers for later transition metals than for early transition metals. If this suggestion is true, then the upper pathway in Scheme I may not be a viable route to products at their thermodynamic threshold. An alternative mechanism for reaction 22 that involves a nonconcerted radical pathway is also shown in Scheme 1. This involves formation of 111, either directly from reactants or via Mn-C bond cleavage in 1. Formation of 111 should be only -0.4 ( 6 2 ) Reactions 22 and 24 are different from reaction 23 in that the metal-carbon bond formed is a T bond rather than a u bond. Direct formation of a metal-carbon T bond is not symmetry-allowed. Thus, for these reactions to occur in a concerted manner, interactions between the u and T bonds presumably occur.
3597
eV endothermic, assuming the Mn-C bond strength in Ill is equal to Do(Mn+-CH3). Movement of the radical center results in IV, which has an energy compared to reactants of 1.8 eV Do(H2CMn+-C2H4). Since Do(Fe+-C2H4) Z 1.4 eV,6' Do(Co+-C2H4) = 2.0 f 0.3 eV, and Do(Ni+-C2H4) = 2.1 eV,63 formation of IV is probably near thermoneutral (C0.4 eV endothermic). If the radical center is low-spin-coupled (quintet spin) to the nonbonding electrons on the metal, IV can collapse immediately to 11, which can then lose ethene to form ground-state products. If the electrons are high-spin-coupled (septet spin), the spin-orbit interactions may be necessary for the eventual formation of the Mn-C T bond in 11. In either case, intermediate I1 is lower in energy than IV due to formation of the M-C T bond. Overall, since reaction 12 is endothermic by 0.9 eV, this nonconcerted reaction pathway is thermodynamically plausible since it does not appear to lead to a barrier in excess of the reaction endothermicity. In the case of ethylene oxide, similar energetics hold, except that if the initial insertion is into a C-0 bond, the energy of intermediates I and Ill can be lowered by donation from the lone-pair electrons of 0 to Mn+. Acknowledgment. This research is funded by the National Science Foundation. CHE-89 17980. (63) Hanratty, M. A.; Beauchamp, J. L.; Illies, A. J.; van Koppen, P. A. M.; Bowers, M. T . J . Am. Chem. SOC.1988, 110, 1-14.
I n Vitro Photodynamic Activity of Diprotonated Sapphyrin: A PP-?r-Eiectron Pentapyrroiic Porphyrin-like Macrocycle Bhaskar G. Maiya,t Mike Cyr,t Anthony Harriman,*,*and Jonathan L. Sessler*'t Department of Chemistry and Center for Fast Kinetics Research, The University of Texas at Austin, Austin, Texas 78712 (Received: July 3, 1989; In Final Form: October 10, 1989)
Optical and IH N M R spectroscopy indicates that diprotonated 3,8,12,13,17,22-hexaethyl-2,7,18,23-tetramethylsapphyrin (SAP2+), a 22-*-electron pentapyrrolic porphyrin-like macrocycle, dimerizes at quite low concentration in polar solvents such as acetonitrile or methanol but exists in a monomeric form in chloroform. Fluorescence quantum yields, excited singletand triplet-state lifetimes, quantum yields for formation of the triplet state and of singlet molecular oxygen 02('Ag), and rate constants for triplet quenching by ground-state oxygen have been determined in each of the above solvents. It IS seen that, in acetonitrile solution, both monomer and dimer species appear to retain similar photodynamic properties, both species exhibiting modest efficienciesfor generation of 02(IAg) upon illumination with visible light. The pentapyrrolic macrocycle binds in the form of an aggregate to human serum albumin and to liposomes, and under such conditions, the quantum yields for formation of triplet state and of 02(1$)are minimal. The suitability of sapphyrin as a photosensitizer for in vivo photodynamic therapy is discussed in terms of its in vitro photodynamic properties and by comparison to previously studied tetrapyrrolic macrocycles.
Introduction Macrocyclic ligands capable of complexing metal cations are finding an increasing number of applications in biomedical research, most specifically as photosensitizers in photodynamic therapy and as targets for magnetic resonance imaging processes. One approach to extending the range of compounds available for such studies involves the use of expanded porphyrins in which the basic ring structure is enlarged beyond the normal 18-*-electron periphery. Recently, the "texaphyrin" family of expanded porphyrins was introduced and shown to have useful photosensitizing properties.' We now extend this work to include "sapphyrin", a pentapyrrolic 22-*-electron "expanded porphyrin" first prepared by the groups of Johnson and Woodward2 a number of years ago *To whom correspondence should be addressed.
'Department of Chemistry.
'Center for Fast Kinetics Research.
but essentially unexplored in the years since.3 The sapphyrins possess two properties that make them of potential interest for biomedical applications. First, they contain an unusually large central cavity which could provide an effective means of complexing large lanthanide cations for use in magnetic resonance imaging. Second, they absorb strongly around 680 nm. This latter ( I ) (a) Sessler, J. L.; Murai, T.; Lynch, V.;Cyr, M. J . Am. Chem. SOC. 1988, 110, 5586. (b) Sessler, J. L.; Murai, T.; Lynch, V. Inorg. Chem. 1989, 28, 1333. (c) Harriman, A.; Maiya, B. K.; Murai, T.; Hemmi, G.; Sessler, J. L.; Mallouk, T. E. J . Chem. SOC.,Ckem. Commun. 1989, 314. (d) Maiya,
B. K.; Harriman, A,; Sessler, J. L.; Hemmi, G.; Murai, T.; Mallouk, T. E. J . Phys. Chem. 1989, 93, 81 1 I . (2) Woodward, R. B. Presented at the Aromaticity Conference, Sheffield, U. K., 1966 (see ref 3a). ( 3 ) (a) Broadhurst, M. J.; Grigg, R. J . Chem. Soc., Perkin Trons. I 1972, 21 11. (b) Bauer, V . J.; Clive, D. L. J.; Dolphin, D.; Paine 111, J. B.; Harris, F. L.; King, M. M.; Loder, J.; Wang, S.-W. C.; Woodward, R. 8 . J . Am. Chem. SOC.1983, 105, 6429.
0022-3654/90/2094-3597$02.50/00 1990 American Chemical Society
3598 The Journal of Physical Chemistry, Vol. 94, No. 9, 1990
/
\
M
I
2
I
+
Figure 1. Structure of the diprotonated sapphyrin conjugate diacid (SAP2+) derivative used in this work.
property implies that the sapphyrins might be useful as photosensitizers if their photodynamic properties are similar to those of analogous tetrapyrrolic pigments. Since nothing is known of these properties, we have repeated the synthesis of a sapphyrin molecule and investigated its photophysical properties in organic solvents. We have found that the most stable form of sapphyrin under such conditions is the diprotonated conjugate diacid (SAP2+) in which all five N atoms are protonated, and we have concentrated on this molecule, the structure of which is given in Figure 1 . I n a parallel study,4 we have found that SAP2+ acts as an effective in vitro agent for the photodynamic inactivation (PDI) of herpes simplex virus (HSV). At a concentration of 60 pM and with a light intensity of 10 J a five-logarithm killing of HSV is effected under conditions where little or no dark activity is detected. Experimental Section 3,8,12,13, I7,22-HexaethyI-2,7,18,23-tetramethylsapphyrin (SAP) was prepared by using a modification of the general method of Bauer et al.3band purified by extensive column chromatography. It was converted into the more stable conjugate diacid SAP2+by treatment with dilute HCI. Solvents were spectroscopic grade and were used as received. Human serum albumin was obtained from Sigma Chemical Co. and defatted according to the method of Chens Aliquots of albumin in water at pH 7 containing 0.01 M NaCl were titrated with solutions of SAP2+ in CH,OH, and the treated albumin solutions were dialyzed against neutral aqueous NaCl solution. Liposomes were prepared by the general method of Jori et a1.6 For the positively charged liposomes, an ethanol solution ( 5 cm3) containing L-a-phosphatidylcholine (egg yolk) (63pmol), stearylamine ( 1 8 pmol), and cholesterol (9 pmol) was purged continuously with N 2 while the ethanol was slowly evaporated. Water (5 cm3) at pH 7.2 (2 mM) phosphate buffer was added, and the mixture was vortexed for 5 min at I O “C. Aliquots of an ethanol solution of SAP2+ were injected, and the ethanol was removed by purging with N2. The mixture was then vortexed for a further 5 min and homogenized in a sonicator for 20 min. Negatively charged liposomes were prepared similarly except that diacetyl phosphate was used in place of stearylamine. Absorption spectra were recorded with a Hewlett-Packard 8450A diode array spectrophotometer, and fluorescence spectra were recorded with a Perkin-Elmer LS5 spectrofluorimeter. Fluorescence spectra were fully corrected for the instrumental response,’ and fluorescence quantum yields were calculated with respect to tetraphenylporphyrin (H2TPP) in benzene* after making corrections for changes in refractive index.9 Singlet-excited-state (4) Judy, M. M.; Mathews. J. L.; Boriak, R.; Skiles, H.; Cyr, M.; Maiya, B. G.; Sessler, J. L. Photochem. Photobiol., submitted for publication. ( 5 ) Chen, R. F. J . Biol. Chem. 1967, 242, 173. (6) Jori, G.; Tomio, L.; Reddi, E.; Rossi, E.; Corti, L.; Zorai, P. L.; Calzavara, F. Br. J . Cancer 1983, 48, 307. (7) Argauer, R. J.; White, C. E. Anal. Chem. 1964, 36, 368. (8) Brookfield, R. L.; Ellul, H.; Harriman, A,; Porter, G . J . Chem. Soc., Faraday Trans. 2 1986, 82, 2 19.
Maiya et al. lifetimes were measured by the time-correlated single-photon counting technique using a mode-locked, synchronously pumped, cavity-dumped dye laser (A = 590 nm, time resolution 280 ps) as excitation source.l0 Error limits are given in the text. Flash photolysis studies were made with a Q-switched, frequency-doubled (A = 532 nm, 280 mJ) or -tripled (A = 355 nm, 80 mJ) Quantel YG481 Nd:YAG laser (pulse 12 ns). An averaging procedure was used to increase the signal-to-noise ratio in which the signals from 30 laser pulses were averaged for each determination. The concentration of SAP2+in each solvent was selected to optimize the presence of monomer or dimer, as described in the text, and all solutions were purged thoroughly with N2. Transient species were monitored by optical absorption spectroscopy, spectra being recorded point by point, using a pulsed high-intensity Xe arc as probing beam and a high radiance monochromator (slit widths 0.5 mm). Absorbance changes were recorded at a fixed wavelength and analyzed with a PDP 11/70 minicomputer. Difference molar extinction coefficients for the triplet excited state of SAP2+ in each solvent were determined by the complete conversion methodll using high laser intensities. In each case, the laser intensity was varied over a wide range by using metal screen filters, and complete conversion was apparent from attainment of a plateau region. Triplet-excited-statequantum yields were measured with respect to zinc tetraphenylporphyrin in benzene, for which the molar extinction coefficientf2(et) at 470 nm is 74000 M-’ cm-l and the quantum yield for formation of the triplet excited statei3(at)is 0.83. Yields of 0 2 ( ’ A g ) were determined by monitoring its characteristic phosphorescence at 1270 nm with a Ge photodiode.14 Solutions of SAP2+or the reference compound in each solvent were matched to possess an identical absorbance at 355 or 532 nm within the range 0.05-0.25and were saturated with 0,; at least four different absorbances were used for each set of experiments. The solutions were irradiated with single 12-11s pulses delivered with a frequency-doubledor -tripled, Q-switched Quantel YG48 1 Nd:YAG laser. The energy of the laser was attenuated with metal screen filters, and the observed 02(’Ag)luminescence intensity was measured as a function of laser power. For CD30D solutions, free-base tetrakis(3-hydroxypheny1)porphyrin (@a = 0.57)was used as reference materialIs with both 355- and 532-nm laser excitation. The reference compound used for CD,CN solutions was benzophenone16 (@Pa= 0.37)with excitation at 355 nm, whereas for CD2C12solutions the ethyl ester of Rose Bengal (ap1 = 0.61) was used as standard” with excitation at both 355 and 532 nm. In each case, 50 individual signals were averaged and analyzed with the PDP 11/70 minicomputer, the initial luminescence intensity being extrapolated to the center of the laser pulse by standard computer least-squares fitting procedures. Results and Discussion Spectroscopic Properties. The IH NMR spectrum of SAP2+ in CDCl3 solution showed well-resolved peaks at 11.66 and 1 1.70 ppm (s, 4 H, meso H), 4.71and 4.55ppm (m, 12 H, methylene H),and 4.23,4.12,and 2.25ppm (s, 30 H, methyl H). The meso H signals observed for SAP2+(Figure 2) are ca. 1 and 0.6 ppm downfield compared to the corresponding signals for free-base octaethylporphyrin18 and the hydrofluoroacetate salt of di(9) Nakashima, N.; Meech, S. R.; Auty, A. R.; Jones, A. C.; Phillips, D. J . Photochem. 1985, 30, 207. (IO) O’Connor, D. V.; Phillips, D. Time Correlated Single Photon Counting; Academic Press: London, 1984. ( 1 I ) Bensasson, R. V.; Land, E. J.; Truscott, T . G. Flash Photolysis and Pulse Radiolysis: Contributions to the Chemistry of Biology and Medicine; Pergamon Press: Oxford, 1983. (12) Pekkarinen, L.; Linschitz, H . J . A m . Chem. SOC.1960, 82, 2407. (13) Hurley, J . K.; Sinai, N.; Linschitz, N . Photochem. Photobiol. 1983, 38, 9. (14) Rodgers, M. A. J.; Snowden, P. T. J . Am. Chem. Sor. 1982, 104, 5541. (15) Bonnett, R.; McGarvey, D. J.; Harriman, A,; Land, E. J.: Truscott, T. G.;Winfield, U.-J. Photochem. Photobiol. 1988, 48, 271. (16) Chattopadhyay, S. K.; Kumar, C. V.; Das, P. K. J . Photochem. 1985, 30, 8 1 (17) Lamberts, J. J . 41.;Neckers, D. C . Tetrahedron 1985, 4 1 , 2183.
The Journal of Physical Chemistry, Vol. 94, No. 9, I990 3599
Photodynamic Activity of Diprotonated Sapphyrin
4+
Figure 3. Structure of the SAP2+ ordered dimer formed in polar organic solvents, as inferred from N M R studies in CD$N solution. T h e two pentapyrrolic macrocycles a r e held in a n offset coplanar arrangement with an interplanar separation of ca. 0.57 nm.
12
11
10
-2
-4
-6
-8
-10
-12
6, PPm Figure 2. Portions of the 'H N M R spectra of SAP2+ as recorded in different solvents. The signals observed between IO and 12 ppm and
between -4 and -12 ppm a r e assigned to meso H and pyrrolic N-H protons, respectively.
protonated ~ctamethylporphyrin,~~ respectively. Most probably, these shifts relate to the larger ring size of the 22-a-electron SAP2+ molecule relative to the 18-*-electron tetrapyrrolic macro cycle^.'^ Indeed, even larger downfield shifts have been reportedzo for the peripheral protons of some 26- and 34-a-electron macrocycles. The spectrum recorded for SAP2+also shows three singlets upfield of TMS, located at -4.33, -4.66, and -4.99 ppm, which are assigned to the pyrrolic protons residing in the cavity in the center of the macrocycle (Figure 2). The pattern observed for these latter resonances (2:1:2) indicates that the five central protons are nonequivalent, quite unlike the case observed with the corresponding tetrapyrrolic macrocycles,20under these conditions. This suggests that these central protons interexchange on slow time scales, possibly because the increased cavity size keeps them spatially remote. The larger size of the SAP2+induces a further upfield shift in the central proton resonances than found in the corresponding porphyrin analogues. The solvent has a marked influence on the 'H NMR spectrum observed for SAP2+. Whereas both meso H and pyrrolic N-H signals are sharp and well-resolved in CDCI, solution, the corresponding signals in CD3CN and CD,OD solutions are broad and shifted (Figure 2). In CD3CN solution, the meso H resonances are observed at 10.65 and 10.36 ppm and the pyrrolic N-H resonances appear at -9.58, -9.93, and -10.16 ppm with the pattern changing to 1 :2:2. In C D 3 0 D solution, the meso signals appear as a very broad peak centered at ca. 1 1.1 ppm, and the pyrrolic N-H signals are lost due to rapid exchange with the solvent. These spectral changes are consistent with the SAP2+ macrocycle existing as a monomer in CDCI, solution but in a dimer in CD,CN solution.2' Stacking of the macrocycle planes introduces strong * , A interaction between adjacent rings which affects the ring currents and induces significant upfield shifts.22 The observed change in the pattern of the pyrrolic N-H resonances (Figure 2) infers that the dimer possesses an ordered structure23
with the two macrocycles partially overlapping each other as shown in Figure 3. For SAPZ+in CD,OD solution, however, the spectra may be distorted by rapid exchange with the solvent such that the observed changes cannot be assigned unambiguously to dimerization of the macrocycle. Evidence from optical absorption spectra indicates, however, that SAP2+ dimerizes at low concentration in both CHJN and C H 3 0 H solutions but not in CHC13. The single-crystal X-ray structure of the hydrated monocation form of the sapphyrin used in this study has been e ~ t a b l i s h e d and , ~ ~ it was shown that the unit cell contained two sapphyrin subunits. These were found to stack in a face-to-face but head-to-head fashion similar to the structure given in Figure 2. The interplanar separation was found to be 0.34 nm, and the center-to-center parallel displacement was 0.46 nm. In dilute CHC1, solution, the optical absorption spectrum of SAP2+exhibits an intense Soret band at 456 nm (log e = 5.65) and two weaker Q-type bands at 624 (log e = 4.26) and 676 nm (log t = 4.41). The spectrum is in good quantitative agreement with that described by Bauer et al.3bfor a related diprotonated sapphyrin derivative, and it resembles spectra for other conjugate diacids derived from tetrapyrrolic macro cycle^.^^ The observed absorption spectrum is consistent with the high symmetry of the macrocycle, and each of the three major bands is assigned to a (a,**)transition. The compound was found to follow Beer's law over a wide concentration range M). Thus, in accordance with the NMR studies, SAPz+appears to exist in CHCI, solution in a monomeric form. The strong electrostatic repulsion between molecules will help to minimize aggregation of the macrocycle in nonpolar solvents. Because of the low polarity of the solvent, it is probable that extensive ion pairing occurs between the SAP2+ macrocycle and the two chloride counterions. Similar ion pairing is known to occur in related diprotonated porphyrin conjugate diacids in nonpolar solvents.25 In more polar solvents, SAP2+ dimerizes at relatively low concentration. Beer's law is not obeyed in either C H 3 0 H or CH3CN solutions, and following the treatment given by Pasternack,26dimerization constants of (1.2 f 0.3) X IO4 and (1.5 f 0.3) X IO4 M-' were derived in C H 3 0 H and CH3CN solutions, respectively. Detailed analyses of these concentration-dependent absorption profiles indicated that with concentrations of SAP2+ below 1 X IO-, M intermolecular association was restricted to dimerization.26 In CH,OH solution at very low concentration, there is a pronounced blue shift in the Soret band (A = 422 nm; log e = 5.68) together with a modest red shift of 4 f 1 nm in the Q bands. These spectral shifts are not consistent with stabilization of (a,..*) transitions by the more polar solvent, and instead, they are attributed to increased dissociation of the salt in CH30H solution. At higher concentration, the Soret band becomes (23) (a) Abraham, R. J.; Eivazi, F.; Pearson, H.; Smith, K. M. J . Chem.
(18) Broadhurst, M. J.; Grigg, R.; Johnson, A. W. J . Chem. Soc., Chem. Commun. 1969, 1480. (19) Kniibel, G.; Franck, B. Angew. Chem., Inf. Ed. Engl. 1988,27,1170. (20) Janson, T. R.; Katz, J . J. The Porphyrins; Dolphin, D., Ed.; Academic Press: New York, 1979; Vol. IV, Chapter 1. (21) Doughty, D. A,; Dwiggins Jr., C. W. J . Phys. Chem. 1969,73,423. (22) Snyder, R. V.; La Mar, G . N. J . Am. Chem. Soc. 1977, 99, 7178.
Soc., Chem. Commun. 1976, 698. (b) Abraham, R. J.; Barnett, G. H.; Hawkes, G. E.; Smith, K. M. Tefrahedron 1976, 32, 2949. (c) Abraham, R. J.; Evans, B.; Smith, K. M. Tetrahedron 1978, 34, 1213. (24) Sessler, J. L.; Cyr, M.; Lynch, V. Unpublished results. (25) (a) Austin, E.; Gouterman, M. Bioinorg. Chem. 1968.90.2735. (b) Harriman, A,; Richoux, M.-C. J . Phofochem. 1984, 27, 205. (26) Pasternack, R. F. Ann. N.Y. Acad. Sei. 1973, 206, 614.
3600 T h e Journal of Physical Chemistry, Vol. 94. No. 9, 1990
I 4
t
Maiya et al.
n
‘I A II -4mi
350
.
,
450
.
, 550
.
,
.
650
, 750
tm
I
350
450
Wavelength (nm)
(27) Gouterman, M.; Holten, D.;Lieberman, E. Chem. Phys. 1977, 25, 39.
, 550
.
,
.
,
.
650
750
850
Wavelength (nm)
Figure 4. Absorption spectra recorded for SAP2+ in CH3CN solution: (a) [SAP2+] = 5 triplet-state difference spectra are shown in (c) and (d). respectively.
broadened and the Q bands are slightly red-shifted, although no new absorption bands appear. Ion pairing between macrocycle and counterion is expected to be less important in CH3CN than in CHCI, solution, and again, this is evidenced by the position of the Soret band at very low concentration ( A = 448 nm; log t = 5.65). In this solvent, increasing the concentration of SAP2+is accompanied by a slight red shift (ca. 3 nm) in the two Q bands and by a pronounced splitting of the Soret band (Figure 4a,b). This splitting arises from strong exciton coupling2’ between the .Ir-electron systems of stacked macrocyclic rings in a dimeric form of the compound. The NMR spectral shifts are in agreement with strong intermolecular x , interactions ~ in CH3CN solution and, in addition, suggest the ordered structure displayed in Figure 3. On this basis, it is clear that intermolecular interaction between SAP2+molecules increases in the order CHCI3 < C H 3 0 H < CH,CN, as suggested also by N M R spectral shifts and from derived dimerization constants. The strong intermolecular interaction found with high concentrations of SAP2+ M) in CH3CN solution corresponds to an exciton coupling energy2’ (V) of ca. 1055 cm-I. Accepting a parallel structure for the dimer (Figure 3), the interplanar separation distance is calculated to be 0.57 f 0.05 nm2’ Similar splitting of the Soret band was observed for SAP2+molecules deposited on the surface of human serum albumin ( V = 880 cm-I) and both negatively ( V = ca. 500 cm-I) and positively charged ( V = I265 cm-I) liposomes. The spectra observed in such cases were broader and much less well-resolved than found in CH3CN solution and are assigned to aggregates rather than ordered dimers. It seems likely that such aggregated forms of SAP2+will abound under the in vivo conditions associated with photodynamic therapy, and consequently, it is important to determine the photodynamic properties of the aggregates as well as of the monomer and of the ordered dimer. Photophysical Properties. The photophysical properties of SAP2+ were determined in dilute CHCI, solution, where it is considered that the macrocycle exists as a monomer but with extensive ion pairing with the CI- counter ion^.^^ Fluorescence emission can be detected readily; there is reasonably good mirror
.
,
.
X 10”
M, (b) [SAP2+]= 2
M. The corresponding
X
symmetry with the Q bands, good correlation between corrected excitation spectrum and ground-state absorption spectrum, and a Stokes shift of 360 cm-I. The fluorescence quantum yield (aPf) is only 0.05 f 0.01, and the excited-singlet-state lifetime ( T ~= 1.2 f 0.1 ns) is short. The triplet excited state, which shows strong absorption at 460 nm (t, = 85000 f 6000 M-’ cm-I), is formed in appreciable yield (@,= 0.54 f 0.06) and has a relatively long lifetime ( T ~= 60 f 5 ps) in N2-saturated solution. However, internal conversion from the first excited singlet to the ground state accounts for ca. 40% of the overall photon balance. This rapid nonradiative process does not involve vibrational relaxation via the pyrrolic N-H bonds since exchanging these labile protons with D+ ions does not affect the photophysical properties. Instead, the efficient internal conversion process is believed to arise, in part, from ion pairing with the CI- counterions, as found with phosphorus(V) porphyrins.28 The triplet excited state is much shorter-lived than observed for the corresponding tetrapyrrolic macrocycles, possibly because of interaction with the halogenated solvent. Laser flash photolysis studies gave no indication of redox ion intermediates arising from electron transfer from triplet SAP2+to CHCI,, but the halogen atoms can be expected to exert some spin-orbit coupling effect on the nonradiative deactivation process of the triplet state.29 Despite any such perturbations, the triplet state reacts quantitatively with molecular oxygen upon aeration of the solution ( k , = (1.0 f 0.1) x 109 M-* s-1): (SAP2+)* O2 SAP2+ 0 2 ( l A g ) (1)
+
-
+
The quantum yield for formation of singlet molecular oxygen (GA) formed from this reaction in CD2CI2solution was determined to be 0.28 f 0.07. Thus, the triplet energy conversion efficiency is ca. SO%, which is low with respect to the corresponding porphyrins and phthalocyanines where conversion factors of ca. 75% are normal.30 Similar photophysical measurements were made in C H 3 0 H solution under conditions where SAP2+exists as an equilibrium mixture of monomer and dimer species. In very dilute solution, (28) Harriman, A. J . Photochem. 1983, 23, 37. (29) McGlynn. S . P.; Azumi, T.; Kinoshita, M. Molecular Spectroscopy
ofrhe Triplet State; Prentice-Hall: New York, 1969. (30) Keir, W . F.; Land, E. J.; MacLennan, A . H.; McGarvey, D. J.; Truscott. T. G. Photochem. Photobiol. 1987. 46, 587
Photodynamic Activity of Diprotonated Sapphyrin the monomer fluoresces at 685 nm, which corresponds to a Stokes shift of 280 cm-I, with ar = 0.06 f 0.02, and possesses a singlet-excited-state lifetime of 2.7 f 0.3 ns. Increasing the concentration of SAP2+results in a progressive decrease in fluorescence quantum yield and a red shift in the peak maximum. The dimer fluorescence maximum occurs at 710 nm with af = 0.015 f 0.007 and 7,= 0.8 f 0.3 ns. Thus, dimerization is accompanied by a red shift in the fluorescence maximum of ca. 500 cm-' and a marked decrease in both fluorescence quantum yield and lifetime. Under conditions of high dilution where the monomer species abounds, it was not possible to obtain accurate estimates for the quantum yields for formation of triplet state or O2(IA ). At higher concentration of SAP2+ in N,-saturated C H 3 6 H solution ([SAP2+] = 9 X IO-5 M) where the dimer accounts for approximately 65% and 45% of the total absorbance at 355 and 532 nm, respectively, excitation of the monomer/dimer mixture resulted in formation of a triplet species3' which decayed via first-order kinetics with it = 80 f 5 p . Upon aeration of the solution, the triplet state reacted quantitatively with molecular oxygen ( k , = (3.0 f 1.0) X I O 9 M-' s-I) and, in CD,OD solution, produced, O2(IAg) with a quantum yield of 0.13 f 0.04. The fluorescence properties of SAP2+ in CH3CN are similar to those described above for C H 3 0 H solution. Thus, the monomer species fluoresces at 680 nm with af = 0.07 f 0.02 and 7, = 2.8 f 0 . 3 ns, whereas the dimer species fluoresces a t 7 1 0 nm with ar = 0.020 f 0.008 and i, = 0.8 f 0.2 ns. As seen from Figure 4b, the triplet difference absorption spectral profile in the Soret region depends strongly upon the concentration of SAP2+. The differences appear to relate to changes in the ground-state absorption spectrum due to exciton coupling effects associated with dimerization. In very dilute solution, where the ground-state monomer accounts for more than 95% of the total absorbance at both 355 and 532 nm, the triplet difference spectrum shows an intense absorption maximum at 480 nm (e, = 78000 f 9000 M-' cm-I). Under these conditions, the triplet state (a, = 0.35 f 0.05) is formed in lower yield than observed for the ion pair species in CHC13 solution although the triplet lifetime (7,= 50 f 10 ps) remains similar. In dilute CD3CN solution, the triplet reacts with molecular oxygen ( k , = (2.0 f 0.5) X IO9 M-' s-') to produce 0 2 ( ' A ) with a quantum yield of 0.19 f 0.06. In t H 3 C N solution, the dimer exhibits a much higher molar extinction coefficient at 355 nm than does the monomer species whereas the values are comparable at 532 nm. At high concentrations of SAP2+ ( [SAP2+] = 1 X M) in CH3CN solution, incident photons from laser excitation at 355 nm are absorbed preferentially (ca. 90%) by the ground-state dimer. Under such conditions in 0,-saturated CD3CN solution, generation of 0 2 ( 1 A g ) occurred with a quantum yield of 0.17 f 0.05. Excitation at 355 nm in N2-saturated CH3CN solution produces the triplet state3' which decays at the same rate as observed for the triplet in dilute solution. At high laser intensities conversion of the ground state into the triplet manifold is not quite complete, because of the high concentration needed to produce extensive dimerization, and the triplet difference molar extinction coefficient a t 4 8 0 nm was derived by extrapolation to be 52000 f 9000 M-' cm-I. Using this value, the quantum yield for formation of the triplet manifold upon excitation of the ground-state dimer was determined to be 0.39 f 0.10. Thus, the efficiency with which the triplet state (3 I ) This work does not address the aggregation state of the triplet species, and it is possible that the dimer dissociates upon excitation into the triplet state.
The Journal of Physical Chemistry, Vol. 94, No. 9, I990 3601 produces singlet oxygen (ca. 45%) observed for excitation of the ground-state dimer approaches that found for excitation of the corresponding monomer species in both CD3CN (55%) and CDCI, (50%) solutions. The photophysical measurements were extended to include studies of SAP2+bound to the surface of human serum albumin and to both negatively and positively charged liposomes. In each case, extensive aggregation of the macrocycle was apparent from optical absorption and emission spectra. Although quantitative measurements were restricted by the high levels of light scattering inherent with such samples, laser flash photolysis studies showed that these aggregates gave extremely low quantum yields for triplet-state formation and for subsequent production of 02('Ag). Indeed, the photophysics of such aggregated forms of SAP2+are dominated by efficient internal conversion and the aggregates are poor triplet-state photosensitizers. Concluding Remarks These studies have demonstrated that the physical states of SAP2+ depends markedly upon the environment. In organic solvents of low polarity, SAP2+ exists as a monomer but with extensive ion pairing with its CI- counter ion^.^^ Increasing the polarity of the solvent favors dissociation of the ion pair and dimerization of the macrocycle, the dimer appearing to possess an ordered structure with partial overlap of ?r-electron systems. Interestingly, both monomer ion pair and dimer exhibit similar photodynamic behavior and, upon illumination with visible light in aerated solution, give rise to modest quantum yields for formation of 02(1Ag).However, in aqueous media SAP2+aggregates and deposits onto the surface of albumin and liposomes. These aggregates exhibit no useful photodynamic activity. The in vivo photodynamic properties of SAP2+ will depend, therefore, upon its site of localization. In a nonpolar membrane, such as found in mitochrondria, SAP2+should exist as a monomer ion pair and be capable of producing O2('$) at modest rates upon illumination under aerobic conditions. Localizing SAP2+at a more polar site, perhaps near an interface, will favor dimerization of the macrocycle, but this will not affect its photodynamic activity. Any SAP2+that resides in the aqueous phase will aggregate onto the surface of albumin or intact cells and will be photochemically inert. Thus, photodynamic action is restricted to the inside of cells and membranes. This is a useful property for a photosensitizer, especially if it could be allied to selective incorporation into infected cells, since it ensures minimum destruction of the transporting proteins. The other useful property shown by SAP2+that has not been clearly demonstrated for any tetrapyrrolic macrocycle is the similarity in photodynamic activity shown by monomer and dimer species. Of course, the extent of ?r,?r interaction and, particularly, the exciton coupling energy, depends upon the mutual separation distance and orientation of the two macrocycles within the dimer.27 Since the medium will affect such parameters, it is possible that the photodynamic activity of the dimer will show a pronounced dependence upon the environment. These studies provide a background for the understanding of the high in vitro PDI activity4 of this hitherto little studied system. Acknowledgment. W e thank the Texas Advanced Research Program (Texas ARP No. 1581 awarded to J.L.S.) for financial support of this work. The CFKR is supported jointly by the Biotechnology Resources Program of the N I H (RROO886) and by the University of Texas at Austin. Registry No. SAP2+, 125921-22-2; 02.1782-44-1.
