Polymer bound pyrrole compounds, IX. Photophysical and singlet molecular oxygen photosensitizing properties of mesoporphyrin IX covalently bound to a low molecular weight polyethylene glycol

ELSEVI ER

Journal of Photochemistryand PhotobiologyB: Biology41 ( 19971 53-59

Polymer bound pyrrole compounds, IX. Photophysical and singlet molecular oxygen photosensitizing properties of mesoporphyrin IX covalently bound to a low molecular weight polyethylene glycol Pilar Ab6s a, Carme Artigas a, Sonia Bertolotti b, Silvia E. Braslavsky b, Pere Fors':, Kamil Lang c, Santi Nonell a.,, Francisco J. Rodrfguez c, Mafia L. Ses6 c Francesc R. Trull c.i "h~stitutQufmic de Sarriia. Unirersitat Ramnn Llull. Via Augusta 390. E-08017 t~arcelona. Catalunya. Spain h Mttt-PInnck.hlstitutfttr Strahlenchemie. Stiftstrasse 34-36. D-45470 Mlllhe,:n a.d. Ruhr. Germany Departament de Qufmica Orgdnica. Universitat de Barcelona. c~ Martf i Franqut>s I. E-08028 Barcelona. Catalunya. Spain

Received2 June 1997;accepted I August 1997

Abstract

The photophysical and singlet molecular oxygen photosensitizing properties of two amphiphilic systems consisting of ~ r p h y r i n bound to one polyoxyethylene chain through ester or amide bonds (MPEGE and MPEGA, respectively) have been determined in aqueous and organic solvents. Steady-state and time-resolved UV-Vis-NIR absorption and emission, as well as chemical trapping studies indicate LI~ both systems are essentially monomeric in CH2CI.,, their properties resembling those of mesoporphyrin dimethyl ester. In aqueous .solutions the photoproperties show a remarkable dependence on the concentration which is attributed to aggregation phenomena. Below 10 v,M the process can be described as a monomer--dirner equilibrium with constant K = ( 1.6 + 0.5) × I 0~' in water. The absorption, fluorescence, and singlet oxygen production characteristics of both monomer and dimer have been determined. © 1997 Elsevier Science S.A. Keywords: Porphyrin; Polyethyleneglycol;Amph!philic;Aggregation;Fluorescence;Singletmolecularoxygen: Photodynamic

1. Introduction

Ampbiphilic porphyrins are attracting considerable attention in view of their potential use in light-induced processes as broad as photodynamic therapy, PDT [ 1-3 ] electron transfer [4,5] and energy transfer [6]. As part of our investigations on polymer-bound pyrrole compounds suitable for the above applications [7-11 ], we were interes',ed in designing a system soluble in a broad range of solvents, i.e., from apolar organic solvents to water. The bare porphyrin ring is highly hydrophobic causing porphyrins to aggregate in water as a result of weak 1r-~r interactions holding the macrocycles together [ 12-14]. Solubility in aqueous media has been enhanced by preparation of glycoside derivatives [ 3 ], coupling to dextran [ 15] and to phosphorylcholine [ 16], functionalization as cationic [ 17] and anionic [ 18] derivatives, complex formation with cyclodextrins [ 19,20], solubilization in micellar media [ 21 ], incorporation into lipid bilayers * Corresponding author. Tel.: + 34 3 203 89 00: Fax: + 3 43 205 62 66: e-mail:nonel@iqs,url.es : Deceasedon June 28,1997, at the age of 43. 1011-1344/97/$17.00 © 1997ElsevierScienceS,A. All fightsreserved PIIS lOl I - 1344(97)00080-8

[221, immobilization on sepharose [23], and functionalization as polyhydroxy [24] or fluorinated [6] derivatives. A tess explored approach is the use of hydrophilic polyether side chains [25,26]. Polyethylene glycol derivatives of hemin [271 and hematoporphyrin [28] have been reported, though their synthesis produced poorly characterized preparations rather than pure compounds. Following the at~ove rationale, we decided to investigate two systems consisting of mesoporphyrin covalently bound to one polyoxyethylene chain of low molecular weight (M, = 2 000 ) through an ester (MPEGE) or an amide (MPEGA) linkage ( Scheme ! ). The synthesis of these compounds has been reported elsewhere

McOOC

Com~s~

x

MFDME

0

I~FEGA

.NH

I~C~

O

COXR

Scheme 1.

CH3

54

P. Abtk et aL / Journal of Photochemisto" and Phombiology B: Bhdot,v 41 ¢1997) 53-59

[ 29 ]. In this paper we describe the absorption, photophysical, and photosensitizing properties of both systems and discuss them in terms of aggregation phenomena.

2. Materials and metbgds 2. I. Chemicals

CH:CI: was freshly distilled from K2CO3 and stored over molecular sieves. H_,O was deionized by a Millipore system. D_,O (99%). ethanol, ethylene glycol and dimethyl formamide. DMF, were from Merck. MPDME, MPEGE, and MPEGA were prepared following literature procedures [ 29]. Bilirubin IXa (BR5 containing ca. 5% of each of the symmetrical isomers was purchased from Sigma. Uric acid (UA) was from Aldrich. meso-Tetra-(4-sullbnatophenyl)-porphine (TPPS) was from Porphyrin Products. All compounds were used as received. 2.2. Methods

UV-Vis spectra were recorded on a Perkin-Elmer Lambda 5 spectrophotometer. Fluorescence spectra were recorded with an Aminco SPF-500 spectrofluorometer. Fluorescence quantum yields were determined by comparing the area under the emission spectrum for optically-matched solutions of MPEGE. MPEGA. and MPDME (the latter in CH:CI:, for which qbf=O.12 was assumed [30]) and correcting for refraction index differences [ 31 i. In CHzCI_, the absorbance was 0.090 + 0.002 and the excitation wavelength A¢,, = 399 nm ( maximum of the Soret band 5. In H_,O the quantum yields were determined over a wide range of concentrations and excitation wavelengths ( see Results and Discussion). The fluorescence decay kinetics were determined by the single photon counting technique exciting at 354_+ 12 nm and observing at 6 2 6 + 12 and 6 6 7 + 12 nm I321. Singlet molecular oxygen 0_4 lag) quantum yields. ¢ba. were determined using both a chemical acceptor and timeresolved near-infrared emis,&,n (TRNIRS. Oxygen-saturated CHzCI_, solutions containing the same concentration of MPEGE, MPEGA. and MPDME (96 ttM) and 0.5 mM BR were irradiated at room temperature in a merry-go-round apparatus (Applied Photophysics) using the 365 nm line of a 200 W medium pressure mercury lamp ( selected by a combination of a heat-absorbing 5 cm water filter and a 365 nm interference filter). Gentle bubbling of solvent-presaturated oxygen was kept throughout the irradiation. The initial rate of BR disappearance was calculated from the plot of loss of absorbance at 452 nm versus time at conversions below 15%. Blank experiments with BR alone proved the stability of this quencher under the irradiation conditions. Similar experiments were conducted in water using TPPS as reference sensitizer and 0.05 mM UA as quencher. The disappearance of UA was monitored at 290 nm and required the presence of light, a sensitizer, and oxygen, as shown by blank experiments

in which either component was excluded in turn. It could also be prevented by the addition of 0.1 M NAN3, a specific O2 (aAg) quencher. For the TRNIR experiments, the samples were irradiated with ! 5-ns laser pulses at 355 nm (JK lasers, Nd:YAG system 20005 as described elsewhere [33]. The laser fluence was varied with a neutral density ,filter and monitored with a pyroelectric detector-based energy meter ( Laser Precision Corp. RJ7610) being always kept below I mJ cm-2. The emission arising from the cuvette was monitored at right angles to the excitation beam with a liquid N2-cooled germanium diode ( North Coast EO-817P5 after being filtered by a silicon plate and a 1270 nm interference filter. The output of the diode was fed to a Biomation 4500 digital oscilloscope and computer stored and analyzed. The amplitude of the signal extrapolated to the center of the laser pulse, 1(0). was taken as a measure of the singlet oxygen concentration produced by the laser pulse. The quantum yield for singlet oxygen production, q}.~,was determined by comparing the slopes of plots o f / ( 0 ) versus the laser fluence, El...... obtained lbr optically-matched solutions of the compounds and a reference sensitizer, i.e., MPDME in CH:CI_, (assumed to have @.x= 0.57 as in benzene 134 ] ) and TPPS in D.,O ( qba = 0.65 [ 35.36 ] 5.

3. Results and discussion 3. I. Absorption spectra

The absorption spectra of the present chromopolymers depend on the solvents used, with two clearly distinct situations. In organic solvents, such as CH_,CI2, EtOH and DMF, both MPEGE and MPEGA appear to be essentially monomeric at least below 100 IxM, as deduced from the linearity of Beer-Lambert plots. The positions and absorption coefficients of the bands are indistinguishable from those of MPDME. In contrast, the spectra in water are concentration dependent in the range 0.1-100 p,M (Fig. I ). Increasing the concentration produces the onset of new bands shifted to the blue in the Soret region and to the red in the Q region. This is characteristic of cofacial aggregates as shown profusely in the literature [37--41 ]. This type of aggregation is also supported by theoretical studies on similar porphyrins [42]. Below I0 I.zM, isosbestic points can be observed at 374 and 420 nm which suggest an equilibrium between monomeric and dimeric species [4]. lsosbestic points can no longer be observed above this concentration, suggesting the presence of higher aggregates. The absorption spectra below 10 p,M have been analyzed usiag the monomer--dimer ( M - D ) equilibrimn model, described by equations ( i ) through ( 3 ) : A(A)=rM(A)'[ MI+~D(A)'[ DI

( 1)

2M=D

(2)

K=[D]/[M]-"

55

P. Ah~is et aL /Jounml t~fPhomchentistt3" and Photohioh,gy B: Bi,,h,gy 41 (1997) 53-59

!.5

80

6O '~_.,.

2.5~ /,,~

40

....... lOlxM ....... 50~M ~,"

~--

0.5

o 0.0. 300

400

500

600

700

300

Wavelength / nm Fig. I. Concentration-normalizedabsorptionspectraof MPEGA in the concentration range 2.5 to 50 ttM. Inset: Absorbance-concentrationprofile at 399 nm in H,O and DMF. The solid line in water is the curve litted using Eq. (4) (see textL cT=IMI+2IDI

509

6~

700

Wavelength / nm Fig. 2. Calculated absorptionspectrumof MPEGA monomerand dh'nerin water. Solid line: monomer spectrum in DMF. CIo~,edcircles: calcular~ed monomer spectrum in w'ater ~Eq. ! 4 ). floating ~..iand ev, ","..dues). Do.shed line:calculateddimer spectrumin waterusing Eq. (4) withfixedext t DMF I ~alues.

(3)

where ¢~t and ¢t, stand for the monomer and dimer absorption coefficient, respectively. Combination of the above equations affords Eq. (4) for the dependence of the solution absorbance at wavelength A with the stoichiometric concentration Or: A(A)=~'cT+

400

e~,t(A)--~

•

_

4K

14)

Fitting Eq. (4) to data sets A ( A ) versus C-rin the Soret region using the Lavenberg-Marquardt algorithm [43] yielded the absorption spectrum of the monomer and dimer and an equilibrium constant K = ( 1.6 + 0.5 ) × I 0 ~', identical for both porphyrins. This value is somewhat smaller than that observed for the parent mesoporphyrin-lX ( K = 2 . 7 × i0 ~' ['44] ) and similar to those observed for other non-ionic porphyrins (e.g.. K = 7.5 × I 0 ~ for ethylenediamine porphine [ 45 ] ). This suggests that the polyoxyethylene chain is effective at providing the desired solubility but not as much at preventing aggregation. Outside the Soret region, the above numerical method tailed to produce consistent results. To analyze those regions we assumed that the monomer spectrum could be assimilated to the one observed in mixtures of organic solvents with water, e.g. ethylene glycol [46], or in neat polar solvents such as DMF where linearity of the Beer-Lambert plots is observed [47]. The validity of this assumption was confinned by the agreement between both sets of data in the Soret region. The monomer absorption coefficients in DMF were therefore used to calculate the dimer spectrum and equilibrium constant from the data in water using Eq, (4) with fixed EM(A). The results are shown in Fig. 2. 3. 2. F l u o r e s c e n c e s p e c t r a a n d k i n e t i c s

The fluorescence results are in line wi,h those observed using absorption spectroscopy. In CH2CI: :he polymer-bound samples behave indistinguishably from "¢IPDME, i.e., iden-

tical fluorescence spectrum and quantum yield, and morroexponential decay kinetics with lifetime 11.2_+0.4 ns in airsaturated solutions. In water the spectrum, quantum yield, and kinetics are strongly concentration and excitation-wavelength dependent. At O. I p.M these properties resemble lhose of the monomer, i.e., same emission spectrum and monoexponential kinetics with lifetime 19.7 + 0.2 ns. The lifetime increase relative to the value in CH_,CI2 is due to tire lower oxygen concentration in water [ 48 I. At higherconcentrations the fluorescence spectrum shows additional bands whose relative amplitudes are sensitive to the excitation wavelength (Fig. 3). Concomitant with this, a ~ c o n d component is increasingly needed to fit the decay kinetics data, with lifetime !.5-+ 0.2 ns. The relative amplitudes of the two components are also sensitive to the observation wavelength. Clearly. the results above lit the aggregation model, with monomer and dimer having not only different absorption properties but also different fluorescence spectra, yields, and lifetimes. Similar results have been reported for hematopor-

6080[ ....... oA 25 ~M ~M(~,, riM((~~ == = 450 58O 580rim) riam) m)

":2: 600

650

700

750

Wavelength / n m Fig. 3. Fluorescencespectrumof MPEGA in water at 0.! and 25 ~.M. For each spectrum,the excitationwavelengt.his indicatedin parenthesis.

56

P. Ab6s et al. / Journal of Photochemistry and Photobiology B: Biology 41 (1997) 53-59

phyrin aqueous solutions [49,50] and for porphyrin c [51], The individual q~f values for both monomer and dimer have been deduced analyzing the concentration dependence of the obset'ved 'Pf (see inset in Fig. 4) Thus, for an equilibrated mixture of monomeric and dimeric species, each one absorbing a fraction oftbe total incident light and fluorescing independently with its own quantum yield (qbfM and ~ro, respectively; adapted from [511), the observed quantum yield exciting at wavelength A is: qbf(A)= eM(A)-IM] "q~fM+ eD(A)'[DI A(A) A(A)

"~fo

Rearrangement ef this equation era(A)" [M] +eD(A)" [D] yields:

(5)

using

A(A) =

(6)

(~f( A ) = O f D .j~_((~f M __ (~f D ) e M ( A ) ' [ M ]

A(A)

Thus, a plot of the measured q~f versus the fractional absorbance of the monomer (calculated from the stoichiometric concentration CTdata using the absorption coefficients at the excitation wavelength (355 nm) and equilibrium constant determined above, i.e., em(355)=3.04X 104 M -u cm -~, ~D(355) = 8 . 5 0 × 104M -I c m - ~, and K = 1.6× 106) should yield a straight line. From the slope and intercept the ~- for monomer and dimer are determined. This plot is shown in Fig. 4 for MPEGA. Above ca. 10 ItM, i.e., at low fractional absorption by the monomers, the points deviate significantly from linearity, thus confirming the presence of higher aggregates with little, if any, fluorescence. Below 10 ItM, where the fraction of light absorbed by the monomers is largest, the predicted linear relationship is observed, which is taken as a further support for the interpretation in terms of monomer-dimer equilibrium. From the intercept and slope (see Eq. (6)) we deduce qb~ = 0.1 ! and Ofo = 0.07. The value |or the monomer is in

0.09

~f

excellent agreement with that determined for MPDME in organic solvents, i.e., q~f= 0.12 in benzene [ 301. Observation of dimer fluorescence with lower quantum yield than the monomer is in line with similar results for hematoporphyrin [52,53] and contrasts with the behaviour of related phthalocyanines [ 54 ] and benzoporphyrins [ 551 whose dimers are non fluorescent. The same results are obtained lot MPEGE ( ~tM = 0. I0 and qbro= 0.08).

3.3. 02(I A~,)Sensitizatimt The ability of MPEGE and MPEGA to sensitize the formation of O2(tAg) in CH,CI 2 was quantified by using a chemical acceptor and TRNIR. T~e rate of BR photo-oxygenation under steady-state i~lumination conditions (see Materials and Methods) is identical to that shown by MPDME (Fig. 5). Thus, MPEGE and MPEGA produce 02(tag) with the same quantum yield as the parent MPDME. TRNIR experiments (not shown) confirm this result, again in line with the lack of aggregation found in organic solvents. As in CIt2CI2, the ~ a values in water were determined using both uric acid as chemical acceptor (H20 solutions, irradiation at 365 nm) and TRNIR (D20 solutions, irradiation at 355 nm). The latter showed that the 02(~Ag) decay kinetics were in all cases monoexponential with lifetime 65 + 5 Its, in agreement with published values for D20 [ 56 ]. This excludes any significant quenching of 02(~Ag) by the monomer or the aggregates at the concentrations used. Fig. 6 shows a typical plot of the 02(nag) phosphorescence intensity at 1270 nm extrapolated back to the center of the laser pulse as a function of the incident laser fluence for both MPEGE and the reference TPPS. Such plots were used to calculate the cPa values at each concentration ( see Materials and Methods). The inset in Fig. 7 shows a systematic decrease in qba upon increasing the concentration of MPEGA. For a given concentration the UA phomoxidation experiments afforded a slightly higher qba than the TRNIR ones. Since the monomers absorb a larger fraction of light at 365 than at 355 nm, as deduced

5

0.05 oos o

o

0.01

.

0

~

04+i

1'o Cr:~

4.0

0

01.1

0'.2

0'.3

0.4

(eM(355) X [M]) /A(355)

Z

[ • MPEGA 3 . 5 t m MPDME

Fig. 4. q~+of MPEGA solutions in air-saturated H,O as a function of the fraction of light absorbed by the monomer (see Eq. (6)). Departure from linearity at concentrations higher than ca. I0 mM ( i.e.. leftmost part of the plot i is attributed to the formation of non-fluorescem higher aggregates that compete for light absorption. Inset: q~fof MPEGA solutions as a function of

Fig. 5. Photo-oxygenation of 0.50 mM bilirubin in CH,CI, by O:( 'A N)

sensitizerconcentration:Ac~,= 355 nm.

photosensitizedby MPEGAand MPDME.

!

0

,

i

30

,

t

,

60

t

90

,

120

Time / min

P. Ab6s et aL /Journal of Phot¢whemistryand Phowbiedogy B: Biology 41 (I997153-59

8.0

t~y!mV ,0tl

60 tK2 ;~

4.0 00~

~

I " MPEGEl0 ltM

~ ,n..c,

2.0 ~ 0.0

0 '2;0

4~

6~

800

Elascr I pJ x cm"~ Fig. 6. Laser fluencedependence of the singlet oxygen phosphorescence intensity at 1270 nm extrapolatedback to the center of the laser pulse, for MPEGE and the referenceTPPS in D,O. Inset: typical phosphorescence trace for MPEGE. from the monomer and dimer absorption spectra, this observation suggests that the monomer's quantum yield for 02('Ag) generation is larger than the dimer's one. It is also interesting to no,re that the steady-state UA method allows the use of lower sensitizer concentrations than TRNIR. which is an advantage for the study of systems with large dimerization equilibrium constants. The concentration dependence can again be interpreted using the monomer--dimer equilibrium, each species absorbing a fraction of the total incident light and generating O_,(tA~) independently with its own quantum yield (qbaM and qbaD, respectively) 1511. As for the fluorescence yield data, the Oa observed can be discussed using Eq. (7): qba(X) = qbao + (Oa M_ qbao) eM(A)'[MI

(7)

A(A) 0.40 IDA

• TRNIR (355 nm) ta Uric Acid (365 nm)

0.30

0.20

0.10

G~.M 0.%

' 01.i ' 01.2 ' 01.3 ' 01.4 ' 0.5

(eM(2Q x [M]) / A(~,) Fig. 7. ~b.~of MPEGA solutions in air-saturated D20 as a function of tl " fraction of light absorbed by the monomer (see Eq. (7)). For TRNIR, A,,¢=355 nm, eM(355)=3.04x104 M -~ cm -t. eD(355)=8.50x 10"* M - ~cm- ~:for uric acid, A¢~.= 365 nm. -%t( 365 ) = 4.98 x l0 t M - ~cm - J, Co(365) = 1I. 1x 10"~M - ~cm - ~.Departurefromlinearityat concentrations higberthan ca. 10p,M ( leftmostpart of the plot) is attributedto the formation of non-sensitizing higher aggregates. Inset: qb.xof MPEGE solutions as a functionof MPEGAconcentration.

57

Titus, a plot of the measured ~ versus the fracfiooa! a b s o ~ ance of the monomer should yield a straight line. This is indeed the case (Fig. 7) and the presence of non-sensRizing higher aggregates above ca. I0 IxM is confirmed. The agree.. ment between the UA and TRNIR dat.a is especia||y rewarding. From the intercept and slope of the linear part of the p ~ ( see Eq. (7) I we deduce qb_,.~:=0.50 and ~ = 0 . 1 7 . Similar results are obtained for MPEGA ( ~ M = 0 . 5 2 am] qbaD=O.16). The value for the monomer is again in good agreement with that determined for MPDME in organic solvents, i.e., qbz=0.57 in benzene [341. The moderate but distinctly non-zero ability of the dimers to sensitize 02(JAg) fits the corcept developed by Tanielian eta[. in which the dimer's q~z reflects the extent of rr-stacking between the two adjacent macrocyclic rings [ 57 ]. Thus, compounds that form closely stacked dimers show little or no ,sensitizing abi|Ry, reflecting an efficient non-radiative relaxation of the excited singlet state to the ground state. In this category fall nmcrocycles such as sodium pheophorbide a and, b [57[ and some t-butyl-substituted Zn (II) phthalncyanines whose dimers are consistently non fluorescent [54l. in the opposite extreme are sensitlzers such as TPPS, whose four symmetrL',dly spaced negative charges favour a dimer loosely spaced and therefore with high qbaD, which is experimentally confirmed [57]. The porphyrins studied in this work display intermediate behaviour, suggesting with some degree of steric hindrance induced by the polyethylene glycol chains. To this category belong also ionic porphyrins with the charges located away fom the macrocycle, e.g. hematoporphyrin [52,~.57]. Preliminar experiments in pH 7.5 aqueous Tfis buffer show a ca. ten-fold decrease in the dimerization equilibrium constant with a concomitant increase in qbaD,pointing to a specific interaction between "Iris and the MPEGA/ MPEGE systems that keeps the two macrocycles further apart.

4. Conclusions Binding of mesoporphyrin to a polyoxyethylene chain through an amide (MPEGA) or an ester (MPEGE) linkage renders efficient singlet oxygen photosensitizers with solubility ranging from apolar organic solvents to water. Both compounds are monomeric in organic solvents at least below 100 IxM, agd form aggregates in water. Below ca g0 o,M aggregation in ware="produces mainly dimeric species with fluorescence and O2(tag) sensitizing ability comparable to those of the monomers. Higher aggregates, formed above this concentration, are no longer fluorescent nor sensitize 0_4 'Ag). Acknowledgements This work is part of the Generalkat de Catalunya- CICYT research program QFN94-4613-C02 Post- and ,eredoctoral fellowships from the Spanish Ministerio de Edncaci6n y

58

P. AbtSs et aL /Journal o f Photochemistry and Photobiology B: Biology 4111997) 53-59

C i e n c i a t o K.L., P.F, a n d M . L . S . , a s w e l l a s t r a v e l a i d s f r o m i.he S p a n i s h M i n i s t e r i o d e E d u c a c i 6 n y C i e n c i a a n d the D e u t sche Akadcmi~he Austauschdienst (Acciones lntegradas H A 9 4 - 5 9 A a n d H A 9 5 - 1 2 3 ) are t h a n k f u l l y a c k n o w l e d g e d . W e t h a n k M s . E. Hiittel a n d S. POrting for a s s i s t a n c e w i t h the f l u o r e s c e n c e a n d O-,( ~ A ) k i n e t i c m e a s u r e m e n t s in M U l h e i m .

References I I I D. Kessel, Hematoporphyrin and HpD: photophysics, photochemistry and photothempy. Photocbem. Photobiol.. 39 ( 1984 ) 851-859. 121 G. Oenbrink. P. Jiirgenlimke and D. Gahel. Accumulation of porphytins in cells: influence of hydrophobicity, aggregation and protein binding. Photochem. Photobiol.. 48 ( 1988 ) 451 --456. I31 G. F011ing.G. Schr'00er and B. Franck. Water-Soluble Porphyrin Diglycosides with Photosensitizing Properties. Angew. Chem. Int. Ed. Engt,. 28 (1989) 1510-1521. 141 W.I. Wtfite. in D. Dolphin (Editor). The Porphyrins. vol. 5. Academic Press. N.Y.. 1978. p. 303. [5] J. Deisenhofer. O. Epp. K. Miki. R. Huber and H. Michel. X-ray structure analysis of a membrane protein complex. Electron density map at 3 ,~ resolution and a model of the chromophores of the photosynthetic reaction center of Rhodopseudomonas viridis. J. Mol. Bi~l., 80 (1981)) 385-398. 16i M. Nango, K. iida. T. Kawakita. M. Matsuura. Y. Harada. K. Yamashita, K. Tsuda and Y. Kimura. Characterization of amphipatic fl-orina~ed pmphyrin derivatives. J. Chem. Sot.. Chem. Colnmun.. ( 1992 ) 545-547. 171 E. Giralt, J.M. Ribo and F.R. Troll. Polymer bound pyrrole compounds. Reversible anchoring of bilirubin and biliverdin to a polystyrene matrix. Tetrahedron Lett.. 25 (1984) 4145--414,5. [81 P. Casuin. E. Giralt. C. P~rez. J.M. Rib6, N. Siscart and F.R. Trufl, Polymer hot.rid pyrrole compounds. 11. Synthesis and properties of polystyi'ene-bound linear tetrapyrrole pigments and related 5( I H)pyrromethenones, Tctrahedron. 43 i 1987) 2593-2608. [91 M. Feliz. E. Gimlt, J,M. Rib6 and F.R. Trail. Intrapolymeric interactions of bile pigments covalently anchored to an insoluble polystyreneba:,ed resin, as determined by gel phase. ~~C-NMR. Makromol. Chem., 189 (1988) 1551-1559. 1101 S. Nonell. M.L. Se~. D. M,'irlire. S.E. Braslavsky and F.R. Trull. Polymer bound pyrrole compounds-Vl. Photophysical properties of monomer~c m,~dcls for pol',,styrene-bound porphyrins. Photochem. Photobiol.~ 53 ( l~tk31) 185-193. l I 11 P. Castfin. S. Serra. M.L. Ses~.. 2.M. Rib6 and F.R. Troll. Polymer bound pyrrole compounds. V. On the optical properties of an insoluble polystyrene matrix bearing di- and tetrapyrroles covalently anchored. Reactive Polymers, 16 (1992) 297-310. 1121 D.C. Barber and D.G. Whitten. Metalation of surfactant porphyrins at anionic interfaces in micelles and reversed micelles: dramatic effects of chain length and atropoisomer structure on reactivity, J. A m. Chem. Soc.. 109 ( t987t 6842-6844. 1131 D.C. Barber. R.A. Freitag-Beesion and D.G. Whitten. Atropoisomerspecific t'ormation of premicellar porphyrin J-aggregates in aqueous surfactant solutions. J. Phys. Chem.. 95 ( 1991 ) 4074-4086. 1141 P. Leighton. J.A. Cowan, R.J. Abraham mid J.K.M. Sanders, Geometry of porphyrin-porphyrin interactions, J. Org. Chem., 53 (t988) 733-740. [ 151 O. Nakajima, H. Mizoguchi. Y. Hashimoto and S. lwasaki, Non-ionic water-soluble dextran-coupled tetraphenylporphyrin derivatives. J. Am. Chem. Soc.. 114 (1992) 9203-9205. [ 161 Y. Matsushita. E. Hasegawa. K. Eshima and E. Tsuchida. Synthesis of amphiphilic porphinato iron complexes having phosphorylcholine groups.Chem. Lett.. (1983) !387-13b'O. [171 R.K. Pandey. N.W. Smith, F.-Y. Shiau. T.J. Dougherty and K.M.

Smith. Synthesis of cationic porphyrins and chlorins. J. Chem. Soc.. Chem. Commun., ( 1991 ) 1637-1639.

[ 181 D. Praseuth. A. Gaudemer, J.-B. Verlhac, I. Kraljic, I. Sisso~f and E. Guill~', Photocleavage of DNA in the presence of synthetic watersoluble porphyrins, Photochem. Photobiol., 44 (1986) 717-724. [191 H. Hirai. N. Toshima. S. Hayashi and Y. Fujii. Complex tbrmation of water-soluble porphyrin with cyciodextrin, Chem. Lett.. ( 1983 ) 643646. [20[ M. Nango. M. Higuchi, H. Gondo and M. Hara, Characterization of water-soluble/bilayer active beta-cyclodextrin-linked porphyrin derivatives. J. Chem. Soc., Chem. Commun.. (1989) 1550-1553. [ 21 ] C.R. Lambert, E. Reddi, J.D. Spikes. M.A.J. Rodgers and G. Jori, The effects of porphyrin structure and aggregation state on photosensitized processes in aqueous and micellar media. Photochem. Photobiol., 44 (1986) 595-601. 1221 E. Hasegawa. Y.-i. Matsushita. M Kaneda. K. Ejima and E. Tsuchida, Liposomal heme as oxygen carrier under semiphysiologicalconditkm. Biochem. Biophys. Res. Commun.. 1()5 (1982) 1416-1419. 1231 S.L. Gibson. R.S. Murant and R. Hill'. Photosensitizing effects of hematoporphyrin derivative inmobilized on sepharose, Photochem. Photobiol., 45 (1987) 93-104. 1241 R. Bonnett. A.N. Nizhnik and M.C. Berenbaum. Second generation tumor photosensitizers: the synthesis of octa-alkyl chlorins and bactcriochlorins with graded amphiphilic charac'~er,J. Chem. Sot.. Chem. Commun.. (1989) 1822-1823. 1251 S. Zalipsky, C. Gilon an J A. Zilkha, Attachment ~,'fdrags to polyethylene glycols. Ear. Polym. J., 19 (1983) 1177-1183. [ 261 T. Aida. A. Takemura, M. Fuse and S. Inoue. Synthesis of a novel amphiphilic porphyrin carrying water-soluble polyether side chains of controlled chain length. Formation ofa col'acial molecular assembly in aqueous media.. J. Chem. Soc., Chem. Commun.. ( 1988 ) 391-393. 1271 K. Takahashi, A. Matsushima. Y. Saito and Y. Inada, Polyethylene glycol-modifiedhemin having peroxidase activity in organic solvents, Biochem. Biophys. Res. Commun., 138 ( 1986,J 283-288. [ 281 Y. Kodera. A. Ajima. K. Takahashi, A. Matsushima. Y. Saito and Y. luada. Polyethylene glycol-moditied hemaloporphyrin as photosensitizer, Photochem. Photobiol., 47 (1988) 221-223. 1291 M. Fontich. P. Fors, M.L. Ses~,and F.R.TrulI.Polymerbonndpyrrole compounds. VIII. Water-soluble pyrrole pigments carrying polyether side chains, Ear. Polym. J.. ( 1995 ). 1301 J.R. Darwent, P. Douglas. A. Harriman. G. Porter and M.~,. Richoux. Metal Phtbalocyaoines and Porphyrins as Photosensitizers for Reductkm of Water to Hydrogen. Coordination Chemistry Reviews, 44 ( 19821 83-126. [311 D.F. Eaton. Reference Materials l'or Fluc,resc:,nce Measurement. Pure and Appl. Chem.. 60 ( 19881 1107--I114. 1321 K. Hildenbrand and C. Nicolau, Nanosecond fluorescence anisotropy decays of 1,6-diphenyl- 1.3.5-hexatriene in membranes. Biochim. Biophys. Acta, 553 (1979) 365-377. 1331 G. Valdugu. S. Nonell, E. Reddi. G..tort ahd S.E. Bmslavsky. The production of singlet molecular oxygen by zinc(ll) phthalocyanine in ethanol a~ld in unilamellar vesicles. Chemical quenching and phosphorescence studies. Photochem. Photobiol., 48 (1988) I-5. [ 341 J.P. Keene, D. Kessel, E.J. Land. R.W. Redmond and T.G. Truscott, Direct detection of singlet oxygen sensitized by haematoporphyrin and related compounds. Photochem. Photobiol., 43 (1986) II 7-120. [ 351 J.B. Verlhac. A. Gaudemer and 1. Kraljic, Water-soluble porphyrins and metalloporphyrins as photosensitizers in aerated aqueous solutions. I. Detection and determination of quantum yield of formation of singlet oxygen. Nouv. J. Chim., 8 (1984) 401--406. [361 J. Davila and A. Harriman, Photoreactions of macrecyclic dyes bound to human serum albumin. Photochem. Photobiol., 51 (1990) 9-19. 1371 R.F. Pasternack. P.R. Huber. P. Boyd. G. Engasser. L. Francesconi. E. Gibbs. P. Fasella. G. Cerio Venturo and L.d. Hinds. On the aggregatio:: of meso-substituted water-solable porphyrins, J. Am. ('hem. Soc., 94 (1972) 451 I--4517. 1381 R.F. Pasteruack, L. Francesconi, D. Raft and E. Spiro, Aggregation

P. Ahr;s et aL / Journal af Phot¢~'hemtstry attd Photohiolo.o,y B: Bb,blg)" 41 (1997~ 53-59

of nickel(ll l. copper(ll ) and zinc(il) derivatives of water-soluble porphyrins, lnorg. Chem., 12 11973) 2606--261 I. 1391 O. Bilsen, J. Rodriguez. D. Holten. G.S. Girdam. S.N. Milam and K.S. Suslick, Observation of new low-energy fluorescent I ..... excited state in strongly coupled porphyrin dimers. J. Am. Chem. Soc_ 112 (1990) 4075-4077. 140] T. Shimidzu and T. lynda, Accordion-type aggregate of water-soluble me~o-tetraphenylporphyrin derivatives, Chem. Lett.. ( 1981 ) g53856. 1411 E. Ojadi, R. Seizer and H. Linscbitz, Properties of porphyrin dimers tbrmed by pairing cationic and anionic porphyrins, J. Am. Chem. Soc, 107 (1985) 7783-7784. [421 T.G. Gantchev. F. Beaudt3', J.E. Van Lier and A.G. Michel, Semiempirical molecular orbital studies of porphine and phthalocyanine derivatives, to simulate their intermolecular interactions. Int. J. Quantum. Chem., 46 ( 1993 ) 191-211). 143] P.R. Bevington. Data Reduction and Error Analysis for the Physical Sciences, McGraw-HilL New York, 1969. 1,141 J. Turay. P. Hambright and N. Datta-Gupta, intermolecularassociation of natural and synthetic water soluble porphyrins. J. Inorg. Nucl. Chem., 40 (1978) 1687-1688. [45] R.R. Das. R.F. Pasternack and R.A. Plane, Fast reaction kinetics of porphyrin dimerization in aquecus solution, J. Am. Chem. Soc.. 92 (1970) 3312-3316. 1461 O. Vald6s-Aguilera and D.C. Neckers, Aggregation phenomena in xanthepc dyes, Acc. Chem. Rcs. 22 ( 1989I 171-177. 1471 R. M~!:l/n Negri, A. Zalts. E.A. San Romfin, P.F. Aramendfa and S.E. Brasla,~sky, Carboxylated zinc-phthalocyanine, influence of dimerizatio~, on the spectroscopic properties. An absorption, emission, and thermal lensing study. Photochem. Photnbiol., 53 ( 1991 ) 317-322.

148l S.L. Marne. I. Carmichael and G.L. Hug. Handh'~k of Photochemistry. Marcel Dekker. New York. 1993 [491 A. Andreoni. R. Cubeddu. S. De Silvestfi. G. lot1. P. Lzporla ~ E Reddi, Time-resolved fluorescence studies of hem~toporphyrin i~ldif ferent solve'it systems. Z. Naturfor~h.. 38c ( 1983 ) 8.'~-g'~. [ 501 A. Andreoni. in D. Kesscl ( EditorL Ph~odynaaiic Tb~zrapyof Neoplasic Di~asc. Vol.2, CRC Press, Boc~ Ra~on. FL. 1990. p. 19|. 1511 L.E. Bennett, K.P. Ghiggino and R.W. Henderson. SingIct oxygen formation in monomeric and aggregated porphyrin c. J. Ph~ochem. Plaotobiol. B:Biol.. 3 (1989) 81-89. 152 [ A. Seret. E. Gandin and A. van der Vorst, Si~g!c~oxygen prod~zction by UV and visible photocxcited porphyrins under differem s ~ s of aggregation, Phorobiochem. Pb.olobiophys, 12 ( 1986t 25'.~--266. [ 531 C. Tanielian and G. Heinrich. Effect of aggregation on the hema~,porphyrin-:~ensitized pr,niuction of singlet molecular oxygen. Ph~ochem. PhotobioL, 61 (1995) 131-135. [54l D.A. Fernandez, J. Awrnch and L,E. Dicelio. Ph~ophysica! a~J aggregation studies of t-butyl-substituted Zn phehalocyanir~'s, Photin:hem. Pholobiol., 63 ( 1996; 784-792. 155 i B.M. Avelinc, T. Hasa:l and R.W. Redmond, The effects of aggregation, protein binding and cellular incorporation on fl~cph~t~hysical properties of henzoporphyrin tk*rivative mont,~cid ring A ( BPDMA ), J. Photochem. PhotobioL B:Biol.. 30 ( 1995l 16!-;.69. [561 A.A. Gorman and M.A.J. Rodgers, in J.C. 8caiane (Edit~'L CRC Handbook of Pholochemistry. VoL2. CRC Press. Boca Rafon. 1989, p. 229. 1571 C. Tanieli~'.n.C. Woiff and M Esch, Si~tglet oxygen production in water: aggregation and charge-transfer effects, J. Phys. Chem.. 100 ! 1996) 6555-6560,.