Photodynamic activity of sapphyrins

The present invention includes a method to produce singlet oxygen from molecular oxygen generated by reaction with a sapphyrin compound excited at an absorbing wavelength to form a triplet species. Specifically, the sapphyrin compound is an alkylated sapphyrin, most preferably diprotonated 3,8,12,13,17,22-hexaethyl-2,7,18,23-tetramethylsapphyrin. Generation of the triplet species may be accomplished in an organic solvent, of which the most preferable solvents are chloroform, methanol or acetonitrile. Also encompassed within the present invention is a method to selectively produce singlet oxygen in an aqueous environment. A sapphyrin compound is incorporated within a membranous vesicle, the vesicle is illuminated with exciting light and the resultant excited triplet state sapphyrin compound reacts with molecular oxygen to produce singlet oxygen. Singlet oxygen was not generated external to the vesicle where the sapphyrin compound is present in an aqueous medium.

This application is a continuation-in-part of the co-pending application, 
U.S. Ser. No. 07/320,293, filed Mar. 6, 1989, now U.S. Pat. No. 4,935,498, 
issued June 19, 1990. 
BACKGROUND OF THE INVENTION 
This application relates to two other patent applications filed on an even 
date herewith: SAPPHYRINS, DERIVATIVES, AND SYNTHESES (by Sessler and Cyr 
Ser. No. 07/454,298) and PHOTODYNAMIC VIRAL DEACTIVATION WITH SAPPHYRINS 
(by Matthews, Sessler, Judy, Newman and Sogandares-Bernal Ser. No. 
07/454,300). 
The present invention relates to a photochemical method of producing 
singlet oxygen using sapphyrin compounds to generate a triplet excited 
state species which then interacts with molecular oxygen The resultant 
singlet oxygen is highly reactive and is potentially useful for a variety 
of medical applications. Discovery of the sapphyrin molecules as 
photosensitizers for singlet oxygen production appears likely to lead to 
efficient, localized, and selective in vivo therapy, especially in the 
emerging field of photodynamic tumor or viral therapy. 
Investigations relating to the present invention were supported by Texas 
Advanced Research Program grant number 1581 and by the Biotechnology 
Resources Program of the NIH (RR 008866). 
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 .pi.-electrons periphery. Recently, the "texaphyrin" family of expanded 
porphyrins was introduced and shown to have useful photosensitizing 
properties ((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.. Chem. Commun. 1989, 314: (d) 
Maiya, B. K.; Harriman, A.; Sessler, J. L.; Hemmi, G.; Murai, T.; Mallouk, 
T. E. J. Phys. Chem. in press). This work is now extended to include 
"sapphyrin", a pentapyrrolic 22 .pi.-electron "expanded porphyrin" first 
prepared by the groups of Johnson and Woodward (Reported by R. B. Woodward 
at the Aromaticity Conference, Sheffield, U.K. 1966) a number of years ago 
but essentially unexplored in the years since ((a) Broadhurst, M. J.; 
Grigg, R. J. Chem. Soc., Perkin Trans. 1 1972, 2111; (b) Bauer, V. J; 
Clive, D. L. J.; Dolphin, D.; Paine III, J. B.; Harris, F. L.; King, M. 
M.; Lodger, J.; Wang, S.-W. C.; Woodward, R. B. J. Am. Chem. Soc. 1983, 
105, 6429). The sapphyrins possess two properties which 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 light strongly at a wavelength of about 680 nanometer 
(nm). The present invention relates to the synthesis of a sapphyrin 
molecule and investigation of its photophysical properties in various 
solvents. The most stable form of sapphyrin under such conditions is the 
diprotonated conjugate diacid (SAP.sup.2+) in which all five N-atoms are 
protonated and we have concentrated on this molecule, the structure of 
which is given in FIG. 1. 
In a parallel study (Judy, M. M.; Mathews, J. L.; Boriak, R.; Skiles, H.; 
Cyr, M.; Maiya, B.G.; Sessler, J. L. Photochem. Photobiol. to be 
submitted), the present inventors have found that SAP.sup.2+ acts as an 
effective in vitro agent for the photodynamic inactivation (PDI) of herpes 
simplex virus (HSV). At a SAP.sup.2+ concentration of 60 .mu.M and with a 
light intensity of 10 J cm.sup.-2, a five logarithm killing of HSV is 
effected under conditions where little or no dark activity is detected. 
Singlet oxygen, directly or indirectly, appears to be the inactivating 
agent. 
PDI-based purifications also appear to lend themselves to ex vivo 
photosensitized blood purification procedures. One method would be to 
generate matrix-supported PDI sapphyrin systems. A sapphyrin molecule 
would be attached to a solid matrix, such as polystyrene beads. Blood 
would then be irradiated, for example, while being passed through the 
sapphyrin-coated beads. Clearly, beads which do not absorb in the 680-730 
nm range should be used since the sapphyrin compounds absorb in the 
680-690 nm region. Carboxylated sapphyrins might be good candidates for 
attachment to amino-functionalized matrices. For example, sapphyrin 2 
could be converted to an acid chloride after partial ester hydrolysis, 
then reacted with a pendant amino group (e.g., aminomethylated partially 
cross-linked Merrifield-type polystyrene) to form an amide linkage to the 
matrix. 
SUMMARY OF THE INVENTION 
The present invention includes a method to produce singlet oxygen from 
molecular oxygen generated by reaction with a sapphyrin compound excited 
at an absorbing wavelength to form a triplet species. Specifically, the 
sapphyrin compound is an alkylated sapphyrin, most preferably diprotonated 
3,8,12,13,17,22-hexaethyl-2,7,18,23-tetramethylsapphyrin. Generation of 
the triplet species may be accomplished in an organic solvent, of which 
the most preferable solvents are chloroform, methanol, acetonitrile or 
similar solvents. 
Also encompassed within the present invention is a method to selectively 
produce singlet oxygen in an aqueous environment. A sapphyrin compound is 
incorporated within a membranous vesicle, the vesicle is illuminated with 
exciting light and the resultant excited triplet state sapphyrin compound 
reacts with molecular oxygen to produce singlet oxygen. Singlet oxygen was 
not generated external to the vesicle where the sapphyrin compound is 
present in an aqueous medium. 
The present invention further includes a means to moderate the production 
of singlet oxygen in an organic solvent. This involves adding to the 
solution in which singlet oxygen is being generated an appropriate amount 
of a protein such as serum albumin or a membranous material such as a 
liposome to which the sapphyrin compound will bind. The reaction between 
molecular oxygen and the bound sapphyrin compound in the presence of 
exciting light is greatly diminished.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
This invention demonstrates the generation of singlet oxygen from reaction 
of molecular oxygen with an excited state triplet species of a sapphyrin 
compound. This discovery demonstrates a safe and efficient photosensitizer 
for use in the photodynamic inactivation of envelope viruses such as HIV-1 
and other pathogens in blood without harm to normal blood components. 
The physical state of SAP.sup.2+ depends markedly upon the environment. In 
organic solvents of low polarity, SAP.sup.2+ exists as a monomer but with 
extensive ion-pairing with its Cl.sup.- counterions ((a) Austin, E.; 
Gouterman, M. Bioinorg. Chem. 1968, 90, 2735: (b) Harriman, A.; Richoux, 
M.-C. J. Photochem. 1984, 27, 205). 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 .pi.-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 O.sub.2 (.sup.1 .DELTA..sub.g). However, in aqueous media 
SAP.sup.2+. aggregates and deposits onto the surface of albumin and 
liposomes. These aggregates exhibit no useful photodynamic activity. 
The in vivo photodynamic properties of SAP.sup.2+ will depend, therefore, 
upon its site of localization. In a non-polar membrane, such as found in 
mitochondria, SAP.sup.2+ should exist as a monomer ion-pair and be 
capable of producing O.sub.2 (.sup.1 .DELTA..sub.g) at modest rates upon 
illumination under aerobic conditions. Localizing SAP.sup.2+ at a more 
polar site, perhaps near an interface, will favor dimerization of the 
macrocycle but this will not affect its photodynamic activity. Any 
SAP.sup.2+ that resides in the aqueous phase will aggregate onto the 
surface of serum albumin or intact cells and will be photochemically 
inert. Thus, photodynamic action appears restricted to the inside of cells 
and membranes. This is a useful property for a photosensitizer, especially 
if it can be allied to selective incorporation into infected cells, since 
it ensures minimum destruction of the transporting proteins. The other 
useful property shown by SAP.sup.2+ 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 .pi.,.pi. interaction and, particularly, the exciton coupling 
energy depends upon the mutual separation distance and orientation of the 
two macrocycles within the dimer (Gouterman, M.; Holten, D.; Lieberman, E. 
Chem. Phys. 1977, 25, 39). 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 photodynamic 
inactivation (PDI) activity (Judy, M. M.; Mathews, J. L.; Boriak, R.; 
Skiles, H.; Cyr, M.; Maiya, B. G.; Sessler, J. L. Photochem. Photobiol. to 
be submitted) of this hitherto little studied system. 
The following procedures, preparations, and analytical methods were 
employed in the determination and use of the present invention. 
Materials 
3,8,12,13,17,22-Hexaethyl-2,7,18,23-tetramethylsapphyrin (SAP) was prepared 
using an improvement of the general method of Bauer et al. (Bauer et al. 
J. Am. Chem. Soc. 1983, 105, 6429) and purified by extensive column 
chromatography. It was converted into the more stable conjugate diacid 
SAP.sup.2+ by treatment with dilute HCl. Solvents were spectroscopic 
grade and were used as received. Human serum albumin was obtained from 
Sigma Chem. Co. and defatted according to the method of Chen (Chen, R. F. 
J. Biol. Chem. 1967, 242, 173). Aliquots of albumin in water at pH 7 
containing 0.01 M NaCl were titrated with solutions of SAP.sup.2+ in 
CH.sub.3 OH and the treated albumin solutions were dialyzed against 
neutral aqueous NaCl solution. Liposomes were prepared by the general 
method of Jori et al.(Jori, G.; Tomio, L.; Reddi, E.; Rossi, E.; Corti, 
L.; Zorai, P. L.; Calzavara, F. Br. J. Cancer 1983, 48, 307). For the 
positively-charged liposomes, an ethanol solution (5 cm.sup.3) containing 
L-.alpha.-phosphatidyl choline (egg yolk) (63 .mu.mols), stearylamine (18 
.mu.mols) and cholesterol (9 .mu.mols) was purged continuously with 
N.sub.2 while the ethanol was slowly evaporated. Water (5 cm.sup.3) at pH 
7.2 (2 mM) phosphate buffer was added and the mixture vortexed for 5 mins 
at 10.degree. C. Aliquots of an ethanol solution of SAP.sup.2+ were 
injected and the ethanol removed by purging with N.sub.2. The mixture was 
then vortexed for a further 5 minutes and homogenized in a sonicator for 
20 mins. Negatively-charged liposomes were prepared similarly except that 
diacetylphosphate was used in place of stearylamine. 
Absorption Spectra 
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 (Argauer, R. J.; White, C. E. 
Anal. Chem. 1964, 36, 368) and fluorescence quantum yields were calculated 
with respect to tetraphenylporphyrin (H.sub.2 TPP) in benzene (Brookfield, 
R. L.; Ellul, H.; Harriman, A.; Porter, G. J. Chem. Soc., Faradav Trans. 2 
1986, 82, 219) after making corrections for changes in refractive index 
(Nakashima, N.; Meech, S. R.; Auty, A. R.; Jones, A. C.; Phillips, D. J. 
Photochem. 1985, 30, 207). Singlet excited state lifetimes were measured 
by the time-correlated single photon counting technique using a 
mode-locked, synchronously -pumped, cavity-dumped dye laser (lambda =590 
nm, time resolution 280 ps) as excitation source (O'Connor, D. V.; 
Phillips, D. "Time Correlated Single Photon Counting" Academic Press: 
London, 1984). 
Flash Photolysis 
Flash photolysis studies were made with a Q-switched, frequency-doubled 
(lambda=532 nm, 280 mJ) or tripled (lambda=355 nm, 80 mJ) Quantel YG481 
Nd-YAG laser (pulse 12 ns). The laser intensity was attenuated with 
neutral density filters. An averaging procedure was used to increase the 
signal-to-noise ration in which the signals from 30 laser pulses were 
averaged for each determination. The concentration of SAP.sup.2+ 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 
N.sub.2. 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. 
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 SAP.sup.2+ in each solvent were determined by 
the complete conversion method (Bensasson, R. V.; Land, E. J.; Truscott, 
T. G. "Flash Photolysis and Pulse Radiolysis: Contributions to the 
Chemistry of Biology and Medicine" Pergammon Press: Oxford, 1983) using 
high laser intensities. In each case, the laser intensity was varied over 
a wide range using metal screen filters and complete conversion was 
apparent from attainment of a plateau region. Triplet excited state 
quantum yields were measured with respect to zinc tetraphenylporphyrin in 
benzene, for which the molar extinction coefficient (Pekkarinen, L.; 
Linschitz, H. J. Am. Chem. Soc. 1960, 82, 2407) (e ) at 470 nm is 74,000 
M.sup.-1 cm.sup.-1 and the quantum yield for formation of the triplet 
excited state (Hurley, J. K.; Sinai, N.; Linschitz, N. Photochem. 
Photobiol. 1983, 38, 9) (.PHI..sub.t) is 0.83. 
Yields of O.sub.2 (.sup.1 .DELTA..sub.g) were determined by monitoring its 
characteristic phosphorescence at 1270 nm using a Ge photodiode (Rodgers, 
M. A. J.; Snowden, P. T. J. Am. Chem. Soc. 1982, 104, 5541). Solutions of 
SAP.sup.2+ or the reference compound in each solvent were matched to 
possess an identical absorbance at 355 or 532 nm within the range 
0.05-0.25 and were saturated with O.sub.2 ; at least 4 different 
absorbances being used for each set of experiments. The solutions were 
irradiated with single 12 ns pulses delivered with a frequency-doubled or 
tripled, Q-switched Quantel YG481 Nd-YAG laser. The energy of the laser 
was attenuated with metal screen filters and the observed O.sub.2 (.sup.1 
.DELTA..sub.g) luminescence intensity was measured as a function of laser 
power. For CD.sub.3 OD solutions, free-base 
tetrakis(3-hydroxyphenyl)-porphyrin (.PHI..sub..DELTA. =0.57) was used as 
reference material (Bonnett, R.; McGarvey, D. J.; Harriman, A.; Land, E. 
J.; Truscott, T. G.; Winfield, U.-J. Photochem. Photobiol. 1988, 48, 271 ) 
using both 355 and 532 nm laser excitation. The reference compound used 
for CD.sub.3 CN solutions was benzophenone (Chattopadhyay, S. K.; Kumar, 
C. V.; Das, P. K. J. Photochem. 1985, 30, 81) (.PHI..sub..DELTA. =0.37) 
with excitation at 355 nm whereas for CD.sub.2 Cl.sub.2 solutions the 
ethyl ester of Rose Bengal (.PHI..sub..DELTA. =0.61) was used as standard 
(Lamberts, J. J. M.; Neckers, D. C. Tetrahedron 1985, 41, 2183) 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. 
Spectroscopic Properties 
The .sup.1 H-nmr spectrum of SAP.sup.2+ in CDCl.sub.3 solution showed well 
resolved peaks at 11.66 and 11.70 ppm (s, 4H, meso-H), 4.71 and 4.55 ppm 
(m, 12H, methylene-H) and 4.23, 4.12 and 2.25 ppm (s, 30H, methyl-H). The 
meso-H signals observed for SAP.sup.2+ (FIG. 2) are ca. 1 and 0.6 ppm 
downfield compared to the corresponding signals for free-base 
octaethylporphyrin (Broadhurst, M. J.; Grigg, R.; Johnson, A. W. J. Chem. 
Soc., Chem. Commun. 1969, 1480) and the hydrofluoroacetate salt of 
diprotonated octamethylporphyrin (Bauer et al. J. Am. Chem. Soc. 1983, 
105, 6429) respectively. Most probably these shifts relate to the larger 
ring size of the 22 .pi.-electron SAP.sup.2+ molecule relative to the 18 
.pi.-electron tetrapyrrolic macrocycles (Knubel, G.; Franck, B. Angew. 
Chem. Int. Ed. Enol. 1988, 27, 1170). Indeed, even larger downfield shifts 
have been reported (Janson, T. R.; Katz, J. J. "The Porphyrins" Dolphin, 
D. Ed. Academic Press: New York. 1979, Vol. IV. Chap. 1) for the 
peripheral protons of some 26 and 34 .pi.-electron macrocycles. The 
spectrum recorded for SAP.sup.2+ 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 
(FIG. 2). The pattern observed for these latter resonances (2:1:2) 
indicates that the five central protons are non-equivalent, quite unlike 
the case observed with the corresponding tetrapyrrolic macrocycles 
(Janson, T. R.; Katz, J. J. "The Porphyrins" Dolphin, D. Ed. Academic 
Press: New York. 1979, Vol. IV. Chap. 1), under 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 SAP.sup.2+ 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 .sup.1 H-nmr spectrum observed 
for SAP.sup.2+. Whereas both meso-H and pyrrolic N-H signals are sharp and 
well resolved in CDCl.sup.3 solution, the corresponding signals in 
CD.sub.3 CN and CD.sub.3 OD solutions are broad and shifted (FIG. 2). In 
CD.sub.3 CN 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 -0.16 
ppm with the pattern changing to 1:2:2. In CD.sub.3 OD solution, the 
meso-H signals appear as a very broad peak centered at ca. 11.1 ppm, and 
the pyrrolic N-H signals are lost due to rapid exchange with the solvent. 
These spectral changes are consistent with the SAP.sup.2+ macrocycle 
existing as a monomer in CDCl.sub.3 (FIG. 2a) solution but as a dimer in 
CD.sub.3 CN solution (Doughty, D. A.; Dwiggins Jr., C. W. J. Phys. Chem. 
1969, 73, 423). Stacking of the macrocycle planes introduces strong 
.pi.,.pi. interaction between adjacent rings which affects the ring 
currents and induces significant upfield shifts (Snyder, R. V.; La Mar, G. 
N. J. Am. Chem. Soc. 1977, 99, 7178). The observed change in the pattern 
of the pyrrolic N-H resonances (FIGS. 2a, 2b, and 2c) infers that the 
dimer possesses an ordered structure ((a) Abraham, R. J.; Eivazi, F.; 
Pearson, H.; Smith, K. M. J. Chem. Soc.. Chem. Commun. 1976, 698, 699: (b) 
Abraham, R. J.; Barnett, G. H.; Hawkes, G. E.; Smith, K. M. Tetrahedron 
1976, 32 2949: (c) Abraham, R. J.; Evans, B.; Smith, K. M. Tetrahedron 
1978, 34, 1213) with the two macrocycles partially overlapping each other 
as shown in FIG. 3. For SAP.sup.2+ in CD.sub.3 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 indicate, however, 
that SAP.sup.2+ dimerizes at low concentration in both CH.sub.3 CN and 
CH.sub.3 OH solutions but not in CHCl.sub.3. 
In dilute CHCl.sub.3 solution, the optical absorption spectrum of 
SAP.sup.2+ exhibits an intense Soret band at 456 nm (log .epsilon.=5.65) 
and two weaker Q-type bands at 624 (log .epsilon.=4.26) and 676 nm (log 
.epsilon.=4.41) (see FIGS. 4a and 4b). The spec quantitative agreement 
with that described by Bauer et al. (Bauer et al. J. Am. Chem. Soc. 1983, 
105, 6429) for a related diprotonated sapphyrin derivative and it 
resembles spectra for other conjugate diacids derived from tetrapyrrolic 
macrocycles ((a) Austin, E.; Gouterman, M. Bioinorg. Chem. 1968, 90, 2735: 
(b) Harriman, A.; Richoux, M.-C. J. Photochem. 1984, 27, 205). The 
observed absorption spectrum is consistent with the high symmetry of the 
macrocycle and each of the three major bands is assigned to a (.pi.,.pi.*) 
transition. The compound was found to follow Beer's law over a wide 
concentration range (0-10.sup.-3 M). Thus, in accordance with the nmr 
studies, SAP.sup.2+ appears to exist in CHCl.sub.3 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 SAP.sup.2+ macrocycle and the two chloride 
counterions. Similar ion-pairing is known to occur in related diprotonated 
porphyrin conjugate diacids in non-polar solvents ((a) Austin, E.; 
Gouterman, M. Bioinorg. Chem 1968, 90, 2735: (b) Harriman, A.; Richoux, 
M.-C. J. Photochem. 1984, 27, 205). 
In more polar solvents, SAP.sup.2+ dimerizes at relatively low 
concentration. Beer's law is not obeyed in either CH.sub.3 OH or CH.sub.3 
CN solutions and, following the treatment given by Pasternack (Pasternack, 
R. F. Ann. N.Y. Acad. Sci. 1973, 206, 614), dimerization constants of 
(1.2.+-.0.3).times.10.sup.4 and (1.5.+-.0.3).times.10.sup.4 M.sup.-1, 
respectively derived in CH.sub.3 OH and CH.sub.3 CN solutions. Detailed 
analyses of these concentration dependent absorption profiles indicated 
that with concentrations of SAP.sup.2+ below 1.times.10.sup.-3 M 
intermolecular association was restricted to dimerization (Pasternack, R. 
F. Ann. N.Y. Acad. Sci 1973, 206, 614). In CH.sub.3 OH solution at very 
low concentration, there is a pronounced blue-shift in the Soret band 
(lambda=442 nm; log .epsilon.=5.68) together with a modest red-shift of 
(4.+-.1) nm in the Q-bands. These spectral shifts are not consistent with 
stabilization of (.pi.,.pi.*) transitions by the more polar solvent and, 
instead, they are attributed to increased dissociation of the salt in 
CH.sub.3 OH solution. At higher concentration, the Soret band becomes 
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 CH.sub.3 CN than in CHCl.sub.3 solution and, again, this is 
evidenced by the position of the Soret band at very low concentration 
(lambda=448 nm; log .epsilon.5.65). In this solvent, increasing the 
concentration of SAP.sup.2+ is accompanied by a slight red-shift (ca. 3 
nm) in the two Q-bands and by a pronounced splitting of the Soret band 
(FIG. 4A, (a) and 4B(b). This splitting arises from strong exciton 
coupling (Gouterman, M.; Holten, D.; Lieberman, E. Chem. Phys. 1977, 25, 
39) between the .pi.-electron systems of stacked macrocyclic rings in a 
dimeric form of the compound. The nmr spectral shifts are in agreement 
with strong intermolecular .pi.,.pi. interactions in CH.sub.3 CN solution 
and, in addition, suggest the ordered structure displayed in FIG. 3. On 
this basis it is clear that intermolecular interaction between SAP.sup.2+ 
molecules increases in the order CHCl.sub.3 &lt;CH.sub.3 OH&lt;CH.sub.3 CN, as 
suggested also by nmr spectral shifts and from derived dimerization 
constants. 
The strong intermolecular interaction found with high concentrations of 
SAP.sup.2+ (&gt;10.sup.-5 M) in CH.sub.3 CN solution corresponds to an 
exciton coupling energy (Gouterman, M.; Holten, D.; Lieberman, E. Chem. 
Phys. 1977, 25, 39), v of ca. 1055 cm.sup.-1. Accepting an orthogonal 
structure for the dimer (FIG. 3), the interplanar separation distance is 
calculated to be 0.57.+-.0.05 nm (Gouterman, M.; Holten, D.; Lieberman, E. 
Chem. Phys. 1977, 25, 39). Similar splitting of the Soret band was 
observed for SAP.sup.2+ molecules deposited on the surface of human serum 
albumin (v=880 cm.sup.-1) and both negatively- (v=ca. 500 cm.sup.-1) and 
positively-charged (v=1265 cm.sup.-1) liposomes. The spectra observed in 
such cases were broader and much less well resolved than found in CH.sub.3 
CN solution and are assigned to aggregates rather than ordered dimers. It 
seems likely that such aggregated forms of SAP.sup.2+ 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 SAP.sup.2+ were determined in dilute 
CHCl.sub.3 solution, where it is considered that the macrocycle exists as 
a monomer but with extensive ion-pairing with the Cl.sup.- counterions 
((a) Austin, E.; Gouterman, M. Bioinorg. Chem. 1968, 90, 2735: (b) 
Harriman, A.; Richoux, M.-C. J. Photochem. 1984, 27, 205). Fluorescence 
emission can be detected readily; there is reasonably good mirror-symmetry 
with the Q-bands, good correlation between corrected excitation spectrum 
and ground-state absorption spectrum and a Stokes shift of 360 cm.sup.-1. 
The fluorescence quantum yield (.PHI..sub.f) is only 0.05.+-.0.01 and the 
excited singlet state lifetime (.tau..sub.s =1.2.+-.0.1 ns) is short. The 
triplet excited state, which shows strong absorption at 460 nm 
(.epsilon.=85,000.+-.6,000 M.sup.-1 cm.sup.-1) is formed in appreciable 
yield (.PHI..sub.t =0.54.+-.0.06) and has a relatively long lifetime 
(.tau..sub.t =60.+-.5 .mu.s) in N.sub.2 -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 
non-radiative process does not involve vibrational relaxation via the 
pyrrolic N-H bonds since exchanging these labile protons with D.sup.+ 
ions does not affect the photophysical properties. Instead, the efficient 
internal conversion process is believed to arise, in part, from 
ion-pairing with the Cl.sup.- counterions, as found with phosphorus(V) 
porphyrins (Harriman, A. J. Photochem. 1983, 23, 37). 
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 SAP.sup.2+ to CHCl.sub.3 but the halogen atoms can be expected to 
exert some spin-orbit coupling effect on the non-radiative deactivation 
process of the triplet state (McGlynn, S. P.; Azumi, T.; Kinoshita, M. 
"Molecular Spectroscopy of the Triplet State Prentice-Hall, New York, 
1969). Despite any such perturbations, the triplet state reacts 
quantitatively with molecular oxygen upon aeration of the solution 
(k.sub.1 =1.0.+-.0.1.times.10.sup.9 M.sup.-1 s.sup.-1). 
EQU (SAP.sup.2+).sup.* +O.sub.2 .fwdarw.SAP.sup.2+ +O.sub.2 (.sup.1 
.DELTA..sub.g) (1) 
The quantum yield for formation of singlet molecular oxygen 
(.PHI..sub..DELTA.) formed from this reaction in CD.sub.2 Cl.sub.2 
solution was determined to be 0.28.+-.0.07. Thus, the triplet energy 
conversion efficiency is ca. 50%, which is low with respect to the 
corresponding porphyrins and phthalocyanines where conversion factors of 
ca. 75% are normal (This work does not address the aggregation state of 
the triplet species and it is possible that the dimer dissociates upon 
excitation into the singlet state). 
Similar photophysical measurements were made in CH.sub.3 OH solution under 
conditions where SAP.sup.2+ exists as an equilibrium mixture of monomer 
and dimer species. In very dilute solution, the monomer fluoresces at 685 
nm, which corresponds to a Stokes shift of 280 cm.sup.-1, with .PHI..sub.f 
=0.06.+-.0.02 and possesses a singlet excited state lifetime of 2.7.+-.0.3 
ns. Increasing the concentration of SAP.sup.2+ 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 .PHI..sub.f 
=0.015.+-.0.007 and .tau..sub.s =0.8.+-.0.3 ns. Thus, dimerization is 
accompanied by a red-shift in the fluorescence maximum of ca. 500 
cm.sup.-1 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 O.sub.2 (.sup.1 .DELTA..sub.g). At higher 
concentration of SAP.sup.2+ in N.sub.2 -saturated CH.sub.3 OH solution 
([SAP.sup.2+ ]=9.times.10.sup.-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 species (This work does not address the aggregation 
state of the triplet species and it is possible that the dimer dissociates 
upon excitation into the singlet state) which decayed via first-order 
kinetics with .tau..sub.t =80.+-.5 .mu.s. Upon aeration of the solution, 
the triplet state reacted quantitatively with molecular oxygen (k.sub.1 
=3.0.+-.1.0.times.10.sup.9 M.sup.-1 s.sup.-1) and, in CD.sub.3 OD 
solution, produced O.sub.2 (.sup.1 .DELTA..sub.g) with a quantum yield of 
0.13.+-.0.04. 
The fluorescence properties of SAP.sup.2+ in CH.sub.3 CN are similar to 
those described above for CH.sub.3 OH solution. Thus, the monomer species 
fluoresces at 680 nm with .PHI..sub.f =0.07.+-.0.02 and .tau..sub.s 
=2.8.+-.0.3 ns whereas the d fluoresces at 710 nm with .tau..sub.f 
=0.020.+-.0.008 and .tau..sub.s =0.8.+-.0.2 ns. As seen from FIG. 4B(b), 
the triplet difference absorption spectral profile in the Soret region 
depends strongly upon the concentration of SAP.sup.2+. 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 (.epsilon..sub.t 
=78,000.+-.9,000 M.sup.-1 cm.sup.-1). Under these conditions, the triplet 
state (.PHI..sub.t =0.35.+-.0.05) is formed in lower yield than observed 
for the ion-pair species in CHCl.sub.3 solution although the triplet 
lifetime (.tau..sub.t =50.+-.10 .mu.s) remains similar. In dilute CD.sub.3 
CN solution, the triplet reacts with molecular oxygen (k.sub.1 
=2.0.+-.0.5.times.10.sup.9 M.sup.-1 s.sup.-1) to produce O.sub.2 (.sup.1 
.DELTA..sub.g) with a quantum yield of 0.19.+-.0.06. 
In CH.sub.3 CN 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 SAP.sup.2+ ([SAP.sup.2+ 
]=1.times.10.sup.-4 M) in CH.sub.3 CN solution, incident photons from 
laser excitation at 35 nm are absorbed preferentially (ca. 90%) by the 
ground state dimer. Under such conditions in O.sub.2 -saturated CD.sub.3 
CN solution, generation of O.sub.2 (.sup.1 .DELTA..sub.g) occurred with a 
quantum yield of 0.17.+-.0.05. Excitation at 355 nm in N.sub.2 -saturated 
CH.sub.3 CN solution produces the triplet state (This work does not 
address the aggregation state of the triplet species and it is possible 
that the dimer dissociates upon excitation into the singlet state) 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 at 480 nm was derived by extrapolation to be 
52,000.+-.9,000 M.sup.-1 cm.sup.-1. 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.+-.0.10. Thus, the efficiency with which 
the triplet state 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 CD.sub.3 CN (55%) and 
CDCl.sub.3 (50%) solutions. 
The photophysical measurements were extended to include studies of 
SAP.sup.2+ 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 O.sub.2 (.sup.1 .DELTA..sub.g). Indeed, the photophysics of such 
aggregated forms of SAP.sup.2+ are dominated by efficient internal 
conversion and the aggregates are poor triplet state photosensitizers. 
The references cited herein are incorporated for the reasons cited. 
Changes in the construction, operation, and arrangement of the various 
elements, steps and procedures described herein without departing from the 
concept and scope of the invention as defined in the following claims.