Tricationic Porphyrin Conjugates: Evidence for Chain-Structure

Oct 27, 2009 - The transient spectrum in ethanol 140 μs after the laser flash (solid red diamonds) is included. The porphyrin concentration was 7.5 Î...
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J. Phys. Chem. B 2009, 113, 16695–16704

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Tricationic Porphyrin Conjugates: Evidence for Chain-Structure-Dependent Relaxation of Excited Singlet and Triplet States Joa˜o Nuno Silva,†,‡,§,| Francisco Bosca,⊥ Joa˜o P. C. Tome´,# Eduarda M. P. Silva,# Maria G. P. M. S. Neves,# Jose´ A. S. Cavaleiro,# Larry K. Patterson,|,∇ Paulo Filipe,† Jean-Claude Mazie`re,‡,§,| Rene´ Santus,O and Patrice Morlie`re*,‡,§,| Faculdade de Medicina de Lisboa, Hospital de Santa Maria, Clinica UniVersita´ria de Dermatologia, 1600 Lisboa, Portugal, INSERM, ERI12, F-80054 Amiens, France, UniVersite´ de Picardie Jules Verne, Faculte´ de Me´decine et de Pharmacie, EA 4292, F-80036 Amiens, France, CHU Amiens, Laboratoire de Biochimie, F-80054 Amiens, France, UniVersidad Polite´cnica de Valencia, Instituto de Tecnologia Quı´mica, 46022 Valencia, Spain, UniVersidade de AVeiro, Departamento de Quı´mica, 3810-193 AVeiro, Portugal, UniVersity of Notre Dame, Radiation Laboratory, Notre Dame, Indiana 46556, and Muse´um National d’Histoire Naturelle, RDDM, Photobiologie, F-75231 Paris, France ReceiVed: August 17, 2009; ReVised Manuscript ReceiVed: October 8, 2009

Conjugates of 5-(4-carboxyphenyl)-10,15,20-tris(4-methylpyridinium-4-yl)porphyrin (P-H) are promising photoactive agents for medical applications. As their ultimate efficacy will depend on the behavior of initial excited states, photophysical parameters have been determined with conventional steady-state absorption and fluorescence as well as time-resolved femto- and nanosecond spectroscopies. The fluorescence quantum yield of P-H and P-H conjugated to uncharged groups increases from ∼0.03 in pH 7 buffer to ∼0.05 in Triton X100 micelles (TX100) and in ethanol and to 0.12 in sodium dodecyl sulfate (SDS) micelles. Corresponding 1 S1 lifetimes are ∼5-10 ns. In buffer, an equilibrium between P-H monomers and small-size aggregates is observed. Conjugation with poly-S-lysine (P-(Lys)n) results in fluorescence quenching in all solvents. Structural reorganization of conjugates bearing a Di-O-isopropylidene-R-D-galactopyranosyl or a R/β-D-galactopyranosyl group occurs in ethanol (k ∼0.15 ps-1) after 1S1 state solvation (∼700 fs). Relaxation of bulky P-(Lys)n polypeptide chains takes place on a longer time scale in all solvents (k e 0.01 ps-1) with enhanced internal conversion. Triplet state (3T1) transient spectra of all derivatives in PBS, SDS, TX100, and ethanol exhibit a strong absorbance with a broad maximum in the 460-475 nm region and minor maxima at ∼540, 630, and 690 nm. In ethanol, energy transfer from the P-H 3T1 state to β-carotene provides an estimate of ε ∼40 000 M-1 cm-1 at 460 nm for the P-H 3T1 state. Using triplet meso-tetraphenylporphyrin as an actinometer, the P-H triplet quantum yield (ΦT) is estimated to be ∼0.50 in all solvents. This high ΦT leads to effective singlet oxygen production in buffered solutions. 1. Introduction Tetrapyrrole derivatives are ubiquitous in nature. Owing to their ability to complex a large variety of metal ions and to their extended and intense absorbance over the whole range of solar light, they have stimulated numerous basic and applied studies over several decades.1 Currently, they are the focus of ongoing investigation in several domains of public concern, in particular that of solar energy conversion as components of photochemical solar cells2 as well as in biomedical applications.3 Consequently, extensive research is currently under way to synthesize new derivatives and to characterize structural factors which confer unique spectroscopic and photochemical properties under differing microenvironmental conditions, e.g., those encountered in nanostructures2 and in microheterogeneous assemblies of * Corresponding author. Mailing address: INSERM ERI12, Laboratoire de Biochimie, CHU Amiens - Hoˆpital Nord, place Victor Pauchet, 80054 Amiens Cedex 1, France. E-mail: [email protected]. † Faculdade de Medicina de Lisboa. ‡ INSERM. § Universite´ de Picardie Jules Verne. | CHU Amiens. ⊥ Universidad Polite´cnica de Valencia. # Universidade de Aveiro. ∇ University of Notre Dame. O Muse´um National d’Histoire Naturelle.

biological interest.4 Our research focuses on the development of new photoactive substances suitable for therapeutic applications in dermatology. Regulatory approval has already been obtained for the treatment of malignant diseases by photodynamic therapy (PDT) involving tetrapyrrole derivatives.3,5,6 In dermatology, δ-aminolevulinic acid and its methyl ester (Metvix)sthe precursors of protoporphyrin IX, a potent natural photosensitizersare currently used topically as pro-drugs in the clinical treatment of various benign or malignant cutaneous lesions. These prodrugs are rather insoluble in water.6 To counter this disadvantage, the synthesis of water-soluble derivatives of porphyrins and chlorins has been undertaken. In addition to enhancing water solubility, conjugating amino acids, peptides, and sugars to porphyrins is of interest, since such groups play a key role in cell metabolism and recognition, thereby facilitating specific targeting of photosensitizers to diseased cells or micro-organisms.7,8 Along these lines, effective photosensitizers have been synthesized whose water solubility is improved by conjugating polypeptide, dicyclohexylurea, and galactopyranosyl side chains to cationic meso-tetra-substituted porphyrins.8-10 Among these are examples in which a poly-S-lysine chain, a dicyclohexylureidooxy group, or a Di-O-isopropylidene-D-galactopyranosyl moiety has been conjugated to the 5-(4-carboxyphenyl)-10,15,20tris(4-methylpyridinium-4-yl)porphyrin (P-H) skeleton. The

10.1021/jp907930w  2009 American Chemical Society Published on Web 10/27/2009

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Figure 1. Names and chemical structures of 5-(4-carboxyphenyl)10,15,20-tris(N-methylpyridinium-4-yl)porphyrin derivatives under study. P-H: 5-(4-Carboxyphenyl)-10,15,20-tris(N-methylpyridinium-4-yl)porphyrin Tri-iodide. P-Me: 5-(4-Methoxycarbonylphenyl)-10,15,20tris(N-methylpyridinium-4-yl)porphyrin Tri-iodide. P-DDC: 5-[4(N,N′-Dicyclohexylureidooxycarbonyl)phenyl]-10,15,20-tris(Nmethylpyridinium-4-yl)porphyrin Tri-iodide. P-OGal: 5-[4-(1,2:3,4Di-O-isopropylidene-R-D-galactopyranosyl-6-oxycarbonyl)phenyl]10,15,20-tris(N-methylpyridinium-4-yl)porphyrin Tri-iodide. P-Gal: 5-[4-(R/β-D-Galactopyranosyl-6-oxycarbonyl)phenyl]-10,15,20-tris(Nmethylpyridinium-4-yl)porphyrin Tri-iodide. P-(Lys)n: Porphyrin-polyS-lysine conjugate.

resulting molecules have recently been shown to be of potential interest in dermatological PDT, as they are effective in treating rapidly proliferating human skin cells in Vitro (ref 11 and unpublished observations). To elucidate the markedly differing photobiological effectiveness of the water-soluble tricationic porphyrin conjugates compared to the parent compound or its methyl ester, a study of their photophysical properties has been carried out. The microenvironmental-dependent interaction of the various conjugated groups with the porphyrin ring in singlet and triplet excited states has been investigated in buffer, ethanol, and negatively charged or neutral micelles in which the first excited singlet and triplet states are formed in significant yield. Special attention has been paid to buffered aqueous solutions in which the positively charged P-H skeleton facilitates solubility. Additionally, such charge may alleviate stacking interactions between hydrophobic porphyrin macrocycles as well as static quenching of the excited states which is frequently encountered with tetrapyrrole derivatives. Indeed, a yield of at least 0.5 for 1O2 formation has been obtained in buffered aqueous solutions. The species 1O2 is the major cytotoxin produced by PDT. 2. Materials and Methods 2.1. Chemicals. The synthesis of P-H and of the five derivatives (P-R) under study (Figure 1) has been previously described.9,12,13 Stock solutions of porphyrins (500 µM) were prepared in a water-dimethylsulfoxide mixture (1/1 v/v). The concentration of the polylysine conjugate stock solution was determined in 1% SDS assuming the same molar absorbance as that of the parent compound at the absorbance maximum of the Soret band.11 meso-Tetrasulfonatophenylporphyrin (TPPS4) was supplied by Porphyrin Products (Logan, UT). L-Histidine (His) and meso-tetraphenylporphyrin (TPP) were purchased from

Silva et al. Sigma Chemical Co. (St Louis, MO). Triton X100 (TX100), sodium dodecyl sulfate (SDS), absolute ethanol, toluene, and dimethylsulfoxide (spectroscopic grade solvents) were supplied by Merck (Darmstadt, Germany). All other chemicals used in this work were of the purest available grade and were used without further purification. The phosphate buffer (10 mM, pH 7) or phosphate buffer saline (PBS) were prepared in water purified with a reverse osmosis system from Ser-A-Pure Co. 2.2. Spectroscopic Equipment. UV/vis absorption measurements were performed with either a UVIKON 943 or a Shimadzu UV-2101PC spectrometer. Fluorescence spectra were recorded with an SLM Aminco-Bowman (series 2) (Bioritech, Chamarande, France) equipped with software for emission spectra correction. Solutions for fluorescence measurements were prepared with an absorbance e0.05 at the excitation wavelength (usually 407 nm). Fluorescence quantum yields (ΦF) were determined using TPP as a reference (ΦF ) 0.12 in toluene).14 Porphyrin fluorescence lifetimes were measured in solutions whose absorbance was ∼0.5 and in optical cells whose light path was 5 mm. Lifetimes were determined with a HoribaJobin Yvon NanoLed single photon counting system using 200 ps laser pulses excitation at 373 nm and monitoring emission at 654 nm. The IBH software library provided emission decay analysis. 2.3. Laser Flash Spectroscopy. Femtosecond transient absorbance measurements were conducted at the Radiation Laboratory of the University of Notre Dame (Notre Dame, IN) using a Clark 6MXR 2010 laser system and an optical detection system provided by Ultrafast Systems (Helios). The source for the pump and probe pulses is the fundamental emission at 775 nm (1 mJ/pulse, fwhm ) 130 fs, 1 kHz repetition rate). A second harmonic generator provides laser pump pulses at 387 nm (3.20 eV, 130 fs, 5 µJ/pulse, 2 mm diameter beam). Five percent of the fundamental is diverted through a sapphire crystal to create a white light continuum (∼450-750 nm) for monitoring transient absorbance at times after the pump pulse, determined by an optical delay. Measurements are carried out in a magnetically stirred sample cell (2 mm light path). A time window of 1.6 ns with a maximum step resolution of 7 fs for transient observation was used.15 Nanosecond laser flash experiments were performed with the third harmonic (λexc ) 355 nm) of a Quantel pulsed Nd:YAG spectrum laser system. Samples were contained in 10 mm × 10 mm light path cells made of Suprasil quartz and were deaerated for at least 20 min with dry nitrogen or, when desired, saturated with dioxygen prior to the experiments. Decays were generally recorded after automatically averaging 6-8 traces, whereas for transient spectrum acquisition absorbance (generally 5 nm intervals) resulted from averaging 3-4 data at each wavelength. All laser spectroscopic measurements were conducted at room temperature with solutions of the porphyrins whose absorbance was ca. 0.3 (387 nm) and 0.4 (355 nm). 2.4. Photosensitized Histidine Degradation. Dioxygen- and air-saturated 10 mM phosphate buffer solutions (pH 7) containing 500 µM His and 5 µM TPPS4 or the porphyrin under study were irradiated with increasing light doses at 365 nm. Histidine destruction was monitored by HPLC using a Whatman Partisil 10/25 SCX cation exchange column and 15 mM NH4H2PO4 whose pH was adjusted to 2.3 by addition of phosphoric acid as a mobile phase.16 Irradiations in a 1 × 1 cm cuvette (2.5 mL) were performed as detailed before.16 Chemical actinometry based on the photoreduction of ferrioxalate by the UV radiation was carried out according to Parker.17

Tricationic Porphyrin Conjugates

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TABLE 1: Absorbance (Absorption Maxima and Extinction Coefficients) and Fluorescence (Emission Maxima, Quantum Yield, and Lifetime) Parameters of P-R absorbance P-H

P-Me

P-DDC

P-OGal

P-Gal

P(Lys)n

ethanol bufferd TX100e SDSf PBSg ethanol buffer TX100 SDS PBS ethanol buffer TX100 SDS PBS ethanol buffer TX100 SDS PBS ethanol buffer TX100 SDS PBS ethanol buffer TX100 SDS PBS

fluorescence

λmax, [ε × 10-3] (nm, [M-1 cm-1])

solvent 426 [190] 421 [244] 422 [255] 426 [239] 421 [236] 425 [184] 421 [227] 423 [239] 426 [229] 421 [229] 425 [239] 421 [268] 425 [285] 426 [257] 421 [257] 425 [217] 421 [248] 424 [276] 426 [251] 422 [258] 425 [231] 421 [258] 422 [273] 426 [243] 421 [256] 426 [245] 423 [190] 423 [217] 427 [239] 423 [184]

518 [14,4] 518 [14,8] 519 [15,8] 519 [16,4] 519 [14,2] 517 [14,0] 518 [13,8] 517 [15,6] 518 [15,6] 519 [14,2] 517 [18,2] 518 [16,7] 517 [18,9] 520 [17,2] 519 [15,5] 517 [16,2] 518 [15,8] 517 [18,3] 518 [17,0] 519 [16,5] 517 [17,7] 518 [15,8] 518 [17,1] 519 [16,3] 518 [15,8] 518 [26,0] 521 [17,4] 520 [19,5] 520 [21,2] 521 [17,0]

555 [6,9] 557 [6,5] 556 [7,0] 556 [7,4] 557 [6,2] 553 [6,1] 556 [5,8] 553 [6,2] 555 [6,1] 557 [5,8] 554 [7,8] 556 [6,9] 553 [8,1] 556 [7,9] 556 [6,4] 553 [7,6] 556 [6,8] 553 [7,5] 555 [7,6] 553 [6,8] 554 [7,6] 556 [6,7] 554 [7,3] 556 [7,4] 556 [6,8] 554 [14,0] 556 [9,0] 555 [10,0] 556 [10,7] 556 [9,0]

592 [5,5] 583 [6,8] 584 [7,0] 585 [6,7] 583 [6,2] 592 [5,1) 583 [6,2] 587 [5,9] 589 [5,9] 583 [6,2] 592 [6,7] 583 [7,4] 588 [7,2] 589 [7,1] 584 [7,4] 592 [6,1] 583 [7,3] 587 [6,8] 589 [6,8] 583 [7,3] 592 [6,5] 583 [7,1] 584 [7,3] 588 [6,7] 583 [7,1] 592 [12,5] 588 [8,1] 588 [9,1] 590 [9,1] 589 [8,0]

648 [2,2] 639 [2,1] 641 [2,2] 645 [2,4] 639 [2,0] 648 [1,8] 639 [1,8] 643 [1,9] 647 [2,1] 639 [1,8] 648 [2,4] 639 [2,3] 644 [2,7] 646 [2,4] 640 [1,9] 648 [2,2] 639 [2,4] 643 [2,3] 647 [2,3] 639 [2,1] 648 [2,4] 639 [2,1] 640 [2,2] 645 [2,4] 639 [2,1] 649 [6,5] 646 [3,2] 647 [3,7] 648 [3,5] 648 [3,2]

λmaxa (nm)

λmaxb (nm)

ΦF

663 716 667 663 717 659 716 657 658 716 660 715 658 661 716 659 716 718 661 717 659 719 719 663 717 660 721 659 662 718

722 658 (shoulder) 718 722 657 (shoulder) 720 658 (shoulder) 718 723 655 (shoulder) 721 664 (shoulder) 720 721 653 (shoulder) 721 656 (shoulder) 659 723 655 (shoulder) 721 658 (shoulder) 659 724 657 (shoulder) 723 662 719 721 665

0.049 0.030 0.041 0.125 0.031 0.041 0.032 0.064 0.125 0.031 0.038 0.030 0.069 0.118 0.027 0.036 0.027 0.068 0.110 0.024 0.031 0.031 0.033 0.125 0.030 0.014 0.011 0.019 0.053 0.010

τFc (ns) 8.9 3.9 6.1 9.7 9.7 4.6 9.3 10.6 9.4 4.5 10.5 9.9 8.7 4.3 10.1 10.1 7.8 4.5 10.6 10.5 3.9 (08%)-8.1 (92%) 1.4 (16%)-6.3 (84%) 1.3 (13%)-7.1 (87%) 1.7 (13%)-8.8 (87%)

a Main emission band. b Minor emission band. c 1S1 lifetime measured by fluorescence decay. d pH 7, 10 mM phosphate buffer. e 0.3% Triton X100 in pH 7, 10 mM phosphate buffer. f 1% SDS in pH 7, 10 mM phosphate buffer. g 150 mM NaCl in pH 7, 10 mM phosphate buffer.

3. Results and Discussion 3.1. Properties of the First Singlet Excited State for P-H and Conjugates. Steady-State Results. Table 1 gives the main parameters for absorbance spectra of P-R in ethanol, pH 7 phosphate buffer, and PBS. Additionally, data from anionic (SDS) and neutral (TX100) buffered (pH 7) micellar solutions are included, as these systems provide microheterogeneous media which mimic hydrophobic microenvironments potentially relevant to specific interactions between photosensitizers and biological structures. The large excess micelle concentration used here (1% SDS and 0.3% TX100) ensures that the porphyrins (5 µM) were essentially present as monomers.18,19 In all solvents, the absorbance spectra show the characteristic features of porphyrins with strong Soret band (1S0 f 1S2 transition) maxima at ∼420 nm (ε ∼230 000-250 000 M-1 cm-1) and four Q bands, with the weakly absorbing first Qx band of the 1S0 f 1S1 transition (ε ∼2000 M-1 cm-1 at ∼650 nm) (Figure 2A). In all solvents, the absorbance follows the Beer-Lambert law up to concentrations of at least 10 µM. Comparison of data in Table 1 demonstrates that the absorbance maxima are less sensitive to conjugation than molar absorptivity. However, for all derivatives, absorbance maxima are shifted to the blue by a few nanometers in buffer and PBS. Fortunately, more specific information concerning solvent and conjugation effects on electronic states of the cationic porphyrin can be obtained by fluorescence spectroscopy. In ethanol, the fluorescence spectrum for all derivatives is composed of two distinct bands corresponding to the 1S1 f 1S0 transition with the prominent emission at λmax ∼660 nm, as is generally observed with monomeric porphyrins in organic solvents (Figure 2B). The 350 cm-1 Stokes shift is rather large as compared to uncharged porphyrins or chlorins for which values of ∼100 cm-1 are generally observed in short chain alcohols.20 These porphy-

rins obey the Kasha rule with no 1S2 f 1S0 emission as expected from substituted free base porphyrins.14,21 By contrast, a broad emission spectrum with a long emission tail extending beyond 800 nm is observed in buffer and PBS with all derivatives except for the polylysine conjugate. The fluorescence λmax is shifted to the red by ∼50 nm, whereas the higher energy emission band attributed to monomers appears as a shoulder, independent of ionic strength (Table 1 and Figure 2B). However, the excitation spectra for fluorescence of all the conjugates in buffer are independent of emission wavelength and quite similar to their corresponding ground state absorbance spectra. It is of note that the closely related 5-[4-(5-carboxy-1-butoxy-)phenyl]-10,15,20tris(4-methylpyridinium-4-yl)porphyrin does not exhibit such dramatic changes in fluorescence spectral shape and in Stokes shift under aqueous conditions.22 The marked changes in the fluorescence emission of all compounds except P-(Lys)n in changing solvent from ethanol to pH 7 buffer is consistent with the presence of an equilibrium between monomers and presumably small-size porphyrin aggregates and/or noncovalent dimers. Such behavior has been observed in neutral buffered aqueous solutions with this class of positively charged porphyrins.23 The solvation shell of highly polar water molecules around the positive charges of the N-methylpyridinium side chain may not support sufficient long-range electrostatic repulsion between isolated macrocycles to prevent hydrophobic interactions, despite the good water solubility of the tricationic porphyrins and overall compliance with the Beer-Lambert law. Altogether, the results add support to the contention that aggregation of cationic porphyrins involves both electronic and structural components.23 Such loose stacking interactions would perturb singlet state energy levels enough to favor radiation-less conversion processes. As a result, a shortening of the 1S1 lifetime and a lower fluorescence quantum yield may be observed, as is commonly

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Figure 2. (A) Absorbance spectrum of 5 µM P-H in solution in 10 mM phosphate buffer (pH 7) (open blue squares), in 1% SDS containing 10 mM phosphate buffer (pH 7) (open green circles), and in ethanol (open red diamonds). Spectra were recorded at 22 °C in a 1 cm light path optical cell. (B) Fluorescence spectra of 0.1 µM P-H in 10 mM phosphate buffer (pH 7) (open blue squares, λexc ) 421 nm), in 1% SDS containing 10 mM phosphate buffer (pH 7) (open green circles, λexc ) 426 nm), and in ethanol (open red diamonds, λexc ) 426 nm). The fluorescence of P-H in SDS is multiplied by 0.4. Fluorescence spectrum of 0.1 µM P-(Lys)n in 10 mM phosphate buffer (pH 7) (solid blue squares, λexc ) 421 nm). Optical paths were 1 and 0.4 cm in excitation and in emission, respectively. Spectra were recorded at 22 °C with 4 nm slits in both light paths. The absorbance of the solutions was 0.02-0.03 at the excitation wavelength.

the case with porphyrin aggregates.24 In the P-(Lys)n conjugate, however, the strong electrostatic repulsion between the heavily positively charged polylysine side chains appears large enough to potentially weaken interactions which lead to stacking of the solvated porphyrin rings. Both sharper spectral resolution and increased τF are observed (Figure 2B and Table 1). Similarly, in micellar SDS solutions, the strong electrostatic interaction of the polylysine chain with SDS micelle head groups and the large micelle excess concentration should serve to suppress ring-ring interactions, allow solubilization of P-(Lys)n in the buffered aqueous phase, and lead to a higher fluorescence quantum yield (ΦF ∼0.05) (Figure S1 in the Supporting Information and Table 1). It is observed that the ΦF of the other tricationic porphyrins in SDS is twice that of P-(Lys)n. This is probably due to the nonionic character of their conjugated side chain allowing deeper access of the macrocycle into the Stern layer as opposed to P-(Lys)n where the polylysine chain would tend to localize the porphyrin at the water-Stern layer interface.25 Additionally, the enhanced water solubility of P-(Lys)n may cause the micelle-water distribution of this molecule to favor the water phase more than do the other conjugates. However, the positive (Lys)n group will interact electrostatically with the SDS micellar surface favoring solubilization. In TX100 micelles (Figure S2 in the Supporting Information), there are two possible domains for the incorporation of amphiphatic

Silva et al. molecules.26 The long hydrophilic tail of P-(Lys)n must extend into the aqueous phase, maintaining the porphyrin ring in the region of the micellar surface, while the other conjugatess depending on the hydrophobic character of their side chainssare probably distributed at different depths among the extended hydrophilic poly(oxyethylene glycol) headgroups of the TX100 palisade layer whose thickness is ∼2.5 nm.26 This region exhibits a much higher microviscosity than that of ionic micelles in which the Stern layer is only a fraction of a nanometer thick.25 This may explain the variable fluorescence lifetimes and yields in TX100 micelles. It should be noted that P-OGal and P-Gal are very sensitive to microenvironmental changes. The fluorescence of P-Gal is more markedly quenched in neutral TX100 micelles as compared to anionic SDS micelles in which the positive charges of the macrocycle favor distribution at the micelle-water interface. By constrast, the 4-(R/β-D-galactopyranosyl-6-oxycarbonyl) tail favors incorporation into the neutral TX100 micelles. On the other hand, the replacement of adjacent OH groups by more rigid and less hydrophilic O-isopropylidene bridges strongly enhances the 1S1 lifetime of P-OGal in both micellar systems. The inherent heterogeneity of the conjugated polylysine chain9 is probably responsible for the complex fluorescence decay of P-(Lys)n in micelles and ethanol. At this stage, ultrafast spectroscopic investigation may lead to a better understanding of the distinct properties of excited singlet states of conjugates in the various solvents. Study of Excited Singlet States by Ultrafast (Femtosecond) Spectroscopy. Time-resolved ultrafast absorption spectroscopy of the lowest excited singlet state of P-H and its conjugates is of practical interest, since it may reveal specific interactions of the different conjugated groups with the positively charged porphyrin ring in the 1S1 state or with solvent molecules. Given that the fluorescence lifetimes of these derivatives in most solvents are of the order of several nanoseconds, there exists a spectroscopic window extending to tens of picoseconds following light absorption during which a quasi-stable 1S1 state is reached. During this interval, transient absorption from the 1S1 state to upper excited singlet states (1Sn) of the molecules may be examined to obtain information on the type of interactions mentioned above. Figure 3A presents transient absorbance difference spectra observed for P-H in buffer, ethanol, SDS, and TX100 micelles ∼2 ps after excitation in the Soret band with a 0.13 ps, 387 nm laser light pulse. The spectral shape and the initial absorbance difference spectra are practically independent of solvent and are very similar for transient species obtained with P-Me, P-DDC, P-OGal, and P-Gal (Figure S3 in the Supporting Information). This is consistent with rather similar absorbance cross sections of these porphyrins not only for the 1S0 f 1S2 (Table 1) but also for the 1S1 f 1Sn transitions. An apparent absorbance maximum is observed at ∼455 nm because of the strong contribution of bleaching in the Soret band resulting from the depletion of ground state molecules by the femtosecond laser pulse. In the case of the polylysine conjugate, the transient absorbance is also solvent-independent though somewhat less so, but its maximum is shifted to 475 nm (Figure 3B). In Figure 3A and B, the strong apparent negative absorbance changes in the 630-680 nm region are due to the contribution of the 1S1 f 1S0 laser induced fluorescence arising from the so-called Qx bands of the porphyrin ring21,27 on which is superimposed the bleaching of the weak Qx bands and the absorbance of excited transient Sn species. It may be noted that the order of increasing intensity of the negative bands in the various solvents is qualitatively the same as that of ΦF given in Table 1. The 387

Tricationic Porphyrin Conjugates

Figure 3. (A) Transient absorbance difference spectra obtained ∼2 ps after femtosecond 387 nm laser flash photolysis of air-saturated solutions of P-H in 10 mM phosphate buffer (pH 7) (open blue squares), 1% SDS containing 10 mM phosphate buffer (pH 7) (open green circles), 0.3% TX100 containing 10 mM phosphate buffer (pH 7) (open black triangles), and ethanol (open red diamonds). (B) Transient absorbance difference spectra observed after femtosecond 387 nm laser flash photolysis of air-saturated solutions of P-(Lys)n in 10 mM phosphate buffer (pH 7), ∼0.3 ps (open blue squares), ∼2 ps (blue squares with × in middle), and 210 ps (solid blue squares) after laser flash, and in 1% SDS in 10 mM phosphate buffer (pH 7) (open green circles) and ethanol (open red diamonds), ∼2 ps after laser flash. Inset: kinetics of growth of absorbance difference at 625 nm in buffer. (C) Transient absorbance difference spectra observed with air-saturated solutions of P-H in 10 mM phosphate buffer (pH 7) at ∼0.9 ps (open blue squares), ∼1.4 ps (blue squares with × in middle), and 10 ps (solid blue squares), after laser excitation. Inset: kinetics of growth of transient absorbance at 605 nm in buffer.

nm light pulse directly populates high lying vibronic states in the 1S2-1S3 region. Their relaxation leads to 1S1 within ∼200 fs, as previously estimated for dye molecules.28 Hence, the negative absorbance in the 660 nm region due to the 1S1 f 1S0 fluorescence appearing within the experimental time response (data not shown). In contrast to the steady-state emission spectrum, where the fluorescence λmax of P-H in buffer is shifted by ∼50 nm, the fluorescence λmax observed by femtosecond spectroscopy is similar to that found in the three other

J. Phys. Chem. B, Vol. 113, No. 52, 2009 16699 solvents in which the fluorescence originates from the porphyrin monomer 1S1 state. This λmax remains constant during the experimental time window (1.6 ns) (Figure 3B and C). Similar results are obtained with the other derivatives (Figure S3 in the Supporting Information). It would appear that the formation of small aggregates of porphyrins favors effective internal conversion or migration of the excitation energy competing with transitions from the 1S1 state to upper excited states. Figure 3C demonstrates that transient spectral changes are observed within 1 ps following the 0.13 ps laser flash in buffer. The bandwidth of the 1S1 f 1S0 emission decreases, while absorbance growths are observed in the 440-460 nm (see also initial part of kinetics in Figure 4A) and 580-750 nm regions with the appearance of a new band exhibiting a maximum at about 615 nm. Assuming monomolecular processes, an average rate constant ks ) 1.5 ( 0.3 ps-1 can be estimated by kinetic analysis of data such as those shown in the inset of Figure 3C. No further change in the spectral shape is observed 1.5 ps after excitation. In the region of negative absorbance (e.g., ∼660 nm), after the instantaneous fluorescence burst, a decrease of the negative absorbance following similar kinetics is observed due to the overlapping 1S1 f 1Sn transitions induced by the probe beam (data not shown). Similar results are observed with all the derivatives in all solvents (see other examples with P(Lys)n and P-OGal in the insets of Figure 3B and of Figure S3 in the Supporting Information, respectively). These early spectral changes can be explained by the change of the dipole moment upon excitation of the porphyrin ring. The new local electric field generated by the excited porphyrin ring induces a reorientation of the surrounding polar solvent molecules, thereby creating a new H-bonding network surrounding the excited porphyrin-conjugated chain construct. This solvent relaxation decreases the energy of the 1S1 state and produces the change observed in the transient absorbance within less than 1 ps. In support of this explanation, it may be suggested that the 1/ks value is consistent with literature values for diffusive motion of water molecules in response to change in solute charge distribution.29 The same transient absorbance is observed with P-Me, P-DDC, P-OGal, and P-Gal after excitation in the Soret band; however, Figure 4A and B demonstrates that the structure of the conjugated group and the nature of the solvent can influence the decay of these transient species within a few picoseconds. In ethanol and buffer, no transient decay is observed for P-H, P-Me, and P-DDC on a 40 ps time scale (Figure 4A and B). On the other hand, in ethanol, transients from P-OGal and P-Gal conjugated to P-H by a longer and more flexible chain bearing a D-galactopyranosyl group decay by about 30% within 10 ps by a first-order process (decay rate constant ∼0.15 ps-1). These decays are wavelength-independent and are essentially absent in SDS and TX100 micelles (Figure 4A and B). It may be noted that conjugation with the polylysine chain induces an ultrafast decay of the singlet excited state species in all of the investigated solvents (Figure 4C). This decay occurs on a longer time scale than that observed with the conjugates P-OGal and P-Gal. It also follows more complex kinetics presumably due to the presence of a mixture of conjugates given the variable poly-S-lysine chain length. The contribution of main first-order processes characterized by rate constants varying from ∼0.01 ps-1 in TX100 to ∼0.003 ps-1 in ethanol and SDS and to 0.006 ps-1 in buffer can be determined from the data of Figure 4B. Despite marked decay of the transient species originating from P-(Lys)n within 200 ps (Figure 3B), no major change in the transient absorbance spectra are observed for other derivatives

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Figure 4. (A) Decays of transient absorbance at 454 nm after femtosecond 387 nm laser flash excitation of air-saturated solutions of P-H in 10 mM phosphate buffer (pH 7) (open blue squares) and ethanol (open red diamonds) and of P-OGal in ethanol (solid red diamonds). (B) Transient absorbance decay following femtosecond 387 nm laser flash photolysis of air-saturated solutions of P-OGal in 1% SDS containing 10 mM phosphate buffer (pH 7) (open green circles) and in 0.3% TX100 containing 10 mM phosphate buffer (pH 7) (open black triangles), of P-Gal in ethanol (open red diamonds), and of P-DDC in 10 mM phosphate buffer (pH 7) (open blue squares). Decays for P-OGal and P-DDC were measured at ∼450 nm, the region of apparent transient absorbance maximum for all compounds measured. For clarity of presentation, the P-Gal decay is given at 500 nm. (C) Decays of transient absorbance observed at 475 nm after laser flash excitation of air-saturated solutions of P-(Lys)n in 10 mM phosphate buffer (pH 7) (open blue squares), in 1% SDS in 10 mM phosphate buffer (pH 7) (open green circles), in 0.3% TX100 in 10 mM phosphate buffer (pH 7) (open black triangles), and in ethanol (open red diamonds).

in buffer or other solvents (data not shown). In ethanol, about 80% of the P-(Lys)n transient species disappear within 1.5 ns, consistent with the low fluorescence quantum yield (ΦF ) 0.01) reported in this solvent (Table 1). The absence of spectral changes during the decay of the transient species originating from the longer-lived 1S1 fluorescent state of P-OGal and P-Gal in ethanol suggests that these decays are not induced by photochemical change in the

Silva et al. porphyrin ring in the S1 state. In this less polar solventswhich forms weaker hydrogen bonds as compared to waterslaser excitation induces a more marked restructuring of the sphere comprised of the first solvent shell and the excited porphyrinconjugated chain construct. The formation of new intramolecular (within the excited porphyrin ring-side chain construct) and/ or intermolecular (within the sphere) hydrogen bond networks may well allow spatial reorganization of the D-galactosyl-bearing side chain, leading to increased energy loss by enhanced internal conversion to the ground state. In the case of P-(Lys)n, an effective excited singlet state quenching is observed on a much longer time scale in all of the solvents. It may be suggested that full structural reorganization of the excited porphyrin ring-polylysine construct subsequent to the solvation change could require more time and consume more energy. The long poly-S-lysine chain adopts a bulky random coil conformation in buffer and a helical conformation in ethanol.30 In micellar solutions, the bulky poly-S-lysine chain probably favors distribution of the charged porphyrin ring in the region of the water-micelle interface. However, the faster relaxation of the excited porphyrin-polylysine construct in TX100 micelles suggests interaction of the Lys residues with the micelle possibly via hydrogen bonding with the oxyethylene glycol groups of the detergent molecules (Figure 4C). 3.2. Triplet State Properties of the Tricationic Porphyrins. Porphyrins are type II photodynamic agents through activation of dioxygen molecules by energy transfer from their lowest excited triplet state (3T1) to the oxygen triplet ground state giving singlet oxygen (1O2). Type I electron transfer reactions, although much less frequent, are also possible because tetrapyrroles can donate electrons to strong electrophiles such as nitroimidazoles.31 In previous studies,11 we have shown that P-H and P-(Lys)n can produce 1O2 but the photodynamic activity of the other conjugates toward proliferating cultured human keratinocytes was found to be rather variable (unpublished observations). The characterization of triplet state parametersssuch as transient absorbance spectrum, lifetime, 3T1 quantum yield, and rate constant for 3T1 interaction with oxygensmay assist in understanding the contrasting photobiological activities of the conjugates under study. The 1S1 lifetimes of the order of ∼5 to ∼10 ns (Table 1) suggest that the triplet states of these tricationic porphyrins are populated with rate constants of ∼108 s-1, consistent with a 1S1(ππ*) f 3T1(ππ*) process. Triplet State Transient Spectra. Figure 5A gives the transient absorbance difference spectra recorded ∼2 µs after 355 nm nanosecond laser flash spectroscopy of P-H in deaerated PBS, SDS, and ethanol solutions. All transient species show strong absorbance with broad maxima in the 460-475 nm region and minor maxima at about 540, 630, and 690 nm corresponding to transitions between 3T1 and higher triplet levels 3Tn. Such behavior is generally observed with this class of molecules.32,33 The strong negative absorbance observed in the region of the Soret band results from the depopulation of ground state molecules by the exciting laser flash to produce molecules in the 3T1 state. The spectral shape is practically independent of solvents, conjugation, and time delays after the laser pulse. This behavior is exemplified with P-H (Figure 5A) and the polyS-lysine conjugate in buffer (Figure 5B). The quenching of the transient absorbance by dioxygen through pseudo-first-order kinetics is consistent with its triplet nature (Table S1 and Figure S4 in the Supporting Information). By contrast, the decay of the transient triplet absorption in N2-saturated solutions strongly depends on solvent and conjugation (Table 2). One may note the rather long triplet state lifetimes

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Figure 5. (A) Transient absorbance difference spectra obtained 2 µs after 355 nm laser flash photolysis of N2-saturated solutions of P-H in PBS (open blue squares), 1% SDS containing 10 mM phosphate buffer (pH 7) (open green circles), and ethanol (open red diamonds). The transient spectrum in ethanol 140 µs after the laser flash (solid red diamonds) is included. The porphyrin concentration was 7.5 µM in all of the solutions. Inset: Reciprocal of the triplet lifetime of P-H measured at 620 nm (open black squares) and 460 nm (solid black circles) in ethanol as a function of the β-carotene concentration. Straight lines are the linear fit of data with y ) 97393 + 19890x with R ) 0.97228 at 620 nm and y ) 80288 + 14063x with R ) 0.99997 at 460 nm. The porphyrin concentration was 15 µM in all solutions. (B) Transient absorbance difference spectra observed after 355 nm laser flash photolysis of N2-saturated solutions of P-(Lys)n in PBS, 1.5 µs (open blue squares), 8 µs (blue squares with × in middle), and 19 µs (solid blue squares) after laser flash, and in N2-saturated solutions of 1% SDS containing 10 mM phosphate buffer (pH 7) (open green circles), 1 µs after laser flash. Inset: decays of transient absorbance at 460 nm in ethanol (open red diamonds), PBS (open blue squares), 1% SDS containing 10 mM phosphate buffer (pH 7) (open green circles), and 0.3% TX100 containing 10 mM phosphate buffer (pH 7) (open black triangles). The concentration of P-(Lys)n was 3.75 µM in all solutions.

TABLE 2: Triplet State Lifetimes of P-R in Various Solvents at 20 °C τDa (µs) compound

ethanol

PBSb

SDSc

TX100d

P-H P-Me P-DDC P-OGal P-Gal P-(Lys)ne

14.0 4.7 3.4 2.15 8.9 16.8-352.0

25.0 56.8 28.1 11.0 60.1 77.8-297.7

8.4 45.1 7.0 5.6 97.2 18.8-270.3

5.3 30.1 21.5 10.0 83.1 82.3-396.5

a Standard error on lifetimes: 10%. b 150 mM NaCl in pH 7, 10 mM phosphate buffer. c 1% SDS in pH 7, 10 mM phosphate buffer. d 0.3% Triton X100 in pH 7, 10 mM phosphate buffer. e Complex decay kinetics (see text).

of all the derivatives in PBS. In all the solvents, the shortest lifetime is observed with P-OGal bearing a di-O-isopropylidene-R-D-galactopyranosyl group. As mentioned above for the

femtosecond spectroscopic data, such a side chain increases electronic energy loss by imposing a rigid conformation on the porphyrin ring, at least in the excited states. Because of its intrinsic molecular heterogeneity, the transient originating from the poly-S-lysine conjugate shows complex decay kinetics illustrating the influence of structural factors in triplet state stabilization (inset of Figure 5B). In all solvents, a component with a very long lifetime is observed. While lifetimes of 300-400 µs may be expected in micelles where binding and concentration at the water-micelle interface may slow down triplet state deactivation, such very long-lived components are also recorded in ethanol and PBS. This behavior strongly suggests that the bulky conformation of the poly-S-lysine chains30 hinders fast collisional processes contributing to an increased rate of return from the 3T1 to the 1S0 state. These long-lived triplet states, coupled to their diffusion controlled O2 quenching rate, make possible prediction of photodynamic activity for the tricationic porphyrins in aqueous environments. Determination of Molar Extinction Coefficients. Triplet identification was confirmed using 7.5 µM concentrations of P-H and conjugates. In ethanol, the 3T1 states of these molecules act as energy donors to β-carotene (Car) whose lowlying 3T1 state energy is ∼7500 cm-1. The intersystem crossing yield of Car is exceedingly small, but the β-carotene 3T1 state (3Car1) can be populated by energy transfer from molecules with 3 T1 states of energy higher than that of 3Car1.34 By this means, using the so-called comparative method, a molar extinction coefficient of ∼100 000 M-1 cm-1 has been determined for 3Car1 at 520 nm in toluene. This wavelength has two advantages, since it is close to the absorbance maximum of 3Car1 and the Car ground state molar absorbance at 520 nm is very low.35,36 The present data have been obtained in ethanol, the only solvent common to Car and the conjugates. However, as a result of a 10 nm blue-shift of the 3Car1 transient spectrum in ethanol, the molar absorbance of the 3Car1 at 520 nm in this solvent can be estimated to be 75 000 M-1 cm-1 (Figure S5C in the Supporting Information). The demonstration of effective triplet energy transfer from the 3T1 state of P-H and the most photocytotoxic conjugates P-DDC and P-OGal to ground state Car is provided in Figure S5A and S5B in the Supporting Information, showing the growth of the 3Car1 absorbance in the presence of 10 µM Car. The inset of Figure 5A illustrates quenching of the 3T1 state of P-H by Car. The rate constants for the quenching of these 3T1 states are given in Table S2 in the Supporting Information. It can be seen that the energy transfer is most rapid for the P-DDC, conjugated with the amphiphatic N,N′dicyclohexylureido tail. Considering that saturation and inner filter effects are negligible (see Supporting Information) and assuming a collisional quenching efficiency of unity for the transfer from the long-lived 3T1 state of P-H to produce the Car triplet, the concentration of molecules in the first excited triplet state of P-H can be obtained from which molar extinction coefficients (εT) are determined. By this method, the molar extinction coefficient of the 3T1 state of the parent compound P-H at 460 nm, the wavelength of maximum absorbance, can be estimated to be ∼40 000 M-1 cm-1 (see Supporting Information). This is comparable to molar extinction coefficients previously reported for other porphyrins.20,32 Triplet Quantum Yield of P-H. With the 3T1 state molar absorbance of P-H in hand, it is possible to estimate the 3T1 formation quantum yield (ΦT) by the comparative method.36 meso-Tetraphenylporphyrin (TPP) can be used as an actinometer, since its ΦT is 0.8 in toluene for which ∆ε ) ε(3T1) -

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TABLE 3: Triplet Formation Quantum Yields of P-H, P-DDC, and P-OGal in Various Solvents at 20 °C

of the tricationic porphyrins This is consistent with observations from the ultrafast spectroscopy above. The contributions of solvent and conjugation effects to the nonradiative 1S1 deactivation pathways can be assessed from knowledge of ΦT in Table 3 and ΦF and τF in Table 1. The measured mean 1S1 lifetime τF is the reciprocal of the sum of the rate constants (k) for competing first-order radiative (fluorescence (F)) and nonradiative (internal conversion (IC) and intersystem crossing (ISC)) deactivation processes; e.g., 1/ (kIC + kISC + kF).31,37 With P-H, P-DDC, and P-OGal, straightforward calculations clearly show that nonradiative processes are faster in aqueous solutions (Table 4). The interaction with the Stern layer of SDS micelles restores values close to those observed with less polar ethanol, consistent with the role of the hydrogen bond network on the excited state energy dissipation. It may be noted that the contribution of the conjugated chain to the rate of nonradiative pathways is much less apparent at longer time scales, after ultrafast equilibration and solvation of the 1S1 state. On the other hand, in ethanol, kIC, kISC, and kF values for P-(Lys)n are 8.7 × 107, 3.1 × 107, and 1.7 × 106 s-1, respectively. Since comparable values are obtained for P-(Lys)n in buffered solutions assuming ΦT ∼0.25, the almost doubled kIC values suggest that preferential interaction of the bulky poly-S-lysine helix or random coil with the hydrophobic porphyrin ring impedes the ISC pathway in these media. 3.3. Quantum Yield of 1O2 Formation by Tricationic Porphyrins in Buffered Aqueous Solutions. The formation of long-lived triplet states, quenched at a diffusion controlled rate by dioxygen (Table S1 in the Supporting Information) in buffered aqueous solutions, suggests the production of 1O2. The 1 O2 generation was detected using His as a specific 1O2 probe, since its photosensitized oxidation occurs via a type II photodynamic mechanism in buffered aqueous solutions.16,31 The quantity of 1O2 produced was assessed using a comparative method with meso-tetrasulfonatophenylporphyrin (TPPS4) as a reference water-soluble photosensitizer for which the quantum efficiency of 1O2 generation in pH 7 buffer has been reported.38,39 The 365 nm light dose dependence of His consumption, which at low light doses follows first-order kinetics, has been monitored by HPLC analysis of irradiated solutions containing TPPS4 or P-H or one of the five conjugates as a photosensitizer. The rate constants of His consumption have been normalized to the fraction of light absorbed at 365 nm, e.g., (1-10-A), with

ΦTa compound

ethanol

PBSb

SDSc

TX100d

P-H P-DDC P-OGal

0.55 0.56 0.55

0.58 0.56 0.50

0.55 0.45 0.56

0.46 0.42 0.55

a ΦT are given relative to that of P-H in ethanol taken as 0.55 (see text). b 150 mM NaCl in pH 7, 10 mM phosphate buffer. c 1% SDS in pH 7, 10 mM phosphate buffer. d 0.3% Triton X100 in pH 7, 10 mM phosphate buffer.

ε(1S0) ) 35 400 M-1 cm-1 at 460 nm (data not shown).14 To this end, a solution of TPP with an absorbance corresponding to that of 7.5 µM P-H at 355 nm was prepared in toluene. An equal population of singlet states of P-H and TPP under excitation with the same laser energy was assumed in the two solvents. The ΦT of P-H can subsequently be estimated to be ∼0.55 in ethanol based on the maximal transient absorbance difference of TPP and P-H triplets and a ∆ε value for P-H (∼40 000 M-1 cm-1) at 460 nm. Similar transient triplet absorbance spectra are observed with all of the derivatives in ethanol. As a result, P-H can be used as a secondary actinometer for the other derivatives. A ΦT value of 0.55 is probably valid for the other tricationic porphyrins except for the P-(Lys)n conjugate. For the latter, an estimate of ΦT ∼0.25 can be determined from data in the inset of Figure 5B after taking into account that, under the concentration conditions used, [P-H] ) 2[P-(Lys)n]. Similar spectra and bandwidths are observed in all the solvents. It may thus be assumed that the absorption coefficient of the triplet does not depend markedly on the solvent and is the same (∼40 000 M-1 cm-1). Given this assumption, the comparative method, with P-H in ethanol as the reference, allows an estimate of ΦT for all the studied solvents.36 No significant differences among ΦT values are observed with P-H and P-DDC and P-OGalsthe most photocytotoxic conjugatessgiven the expected experimental errors in such determinations (Table 3).36 It is assumed that the 3T1 state of P-R monomers is highly populated in buffer aqueous solutions despite the existence of monomer-aggregate equilibria. It can be seen that in all the cases ΦT + ΦF is