Photobleaching efficiency parallels the enhancement of

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Photobleaching efficiency parallels the enhancement of membrane damage for porphyrazine photosensitizers Thiago T Tasso, Jan C. Schlothauer, Helena C Junqueira, Tiago A Matias, Koiti Araki, Erica Liandra-Salvador, Felipe C.T. Antonio, Paula Homem-de Mello, and Maurício S. Baptista J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b05991 • Publication Date (Web): 06 Sep 2019 Downloaded from pubs.acs.org on September 7, 2019

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Journal of the American Chemical Society

Photobleaching efficiency parallels the enhancement of membrane damage for porphyrazine photosensitizers Thiago T. Tassoa*, Jan C. Schlothauera, Helena C. Junqueiraa, Tiago A. Matiasb, Koiti Arakib, Érica Liandra-Salvadorc, Felipe C. T. Antonioc, Paula Homem-de-Melloc and Mauricio S. Baptistaa* aDepartment

of Biochemistry, Chemistry Institute, University of São Paulo, São Paulo, 05508-000, Brazil. Department of Fundamental Chemistry, Chemistry Institute, University of São Paulo, São Paulo, 05508-000, Brazil. cCenter of Natural Sciences and Humanities, Federal University of ABC, Santo André, 09210-580, Brazil. b

ABSTRACT: Photostability is considered a key asset for photosensitizers (PS) used in medical applications as well as for those used in energy conversion devices. In light-mediated medical treatments, which are based on PS-induced harm to diseased tissues, the photoinduced cycle of singlet oxygen generation has always been considered to correlate with PS efficiency. However, recent evidence points to the fundamental role of contact-dependent reactions, which usually cause PS photobleaching. Therefore, it seems reasonable to challenge the paradigm of photostability versus PS efficiency in medical applications. We have prepared a series of Mg(II) porphyrazines (MgPzs) having similar singlet oxygen quantum yields and side groups with different electron-withdrawing strengths that fine tune their redox properties. A detailed investigation of the photobleaching mechanism of these porphyrazines revealed that it is independent of singlet oxygen, occurring mainly via photoinduced electron abstraction of surrounding electron rich molecules (solvents or lipids), as revealed by the formation of an air-stable radical anion intermediate. When incorporated into phospholipid membranes, photobleaching of MgPzs correlates with the degree of lipid unsaturation, indicating that it is caused by an electron abstraction from the lipid double bond. Interestingly, upon comparing the efficiency of membrane photodamage between two of these MgPzs (with the highest and the lowest photobleaching efficiencies), we found that the higher the rate of PS photobleaching the faster the leakage induced in the membranes. Our results therefore indicate that photobleaching is a necessary step towards inflicting irreversible biological damage. We propose that the design of more efficient PS for medical applications should contemplate contact-dependent reactions as well as strategies for PS regeneration.

mechanistic alternatives.7 Synergistically coupling 1O2 production with other actions such as the release of chelating agents, coupling of PS to proteins favoring type I reactions, or developing photochemotherapeutic agents that work under low oxygen concentrations, are examples of novel promising strategies in the area of photomedicine.8–10

INTRODUCTION Photobleaching causes a loss of absorption of chromophores under light exposure. Therefore, many phenomena that require light absorption and the subsequent photoinduced action of photosensitizers (PSs), such as natural or artificial photosynthesis, dye-sensitized photovoltaic cells and optogenetics, suffer from photobleaching.1–3 Accordingly, PSs are usually designed to experience the minimum possible level of photobleaching. The same principles have been applied in photomedicine in the development of PSs against proliferative or infective diseases. However, there is no definitive evidence showing that photobleaching is as detrimental in photomedicine as it is in other areas.4 Indeed, it is commonly accepted that the more stable the PS the better it will perform, mainly because it can endure more cycles of singlet oxygen (1O2) production and efficiently trigger the consequent type II oxidation processes.5

For most PSs, photobleaching proceeds via an oxygendependent pathway, i.e., involving the intermediacy of 1O2, which can add to macrocycle double bonds.11 Arnaut and coworkers have shown that photostability can be enhanced by attachment of electron-withdrawing groups to the macrocycle, increasing the oxidation potential and imposing a barrier for 1O2 attack.12,13 Other PSs photobleach through direct-contact reactions of their excited states with surrounding molecules. In principle, these reactions can cause the neutralization of a specific biological target, but there is still a lack of knowledge about their mechanisms and consequences.14,15 For example, Bacellar and co-workers have showed that PSs have to be physically bound to the membranes in order to cause membrane leakage.16 The generation of membrane pores was shown to depend on the accumulation of lipid truncated aldehydes,

Although hundreds of “improved” photosensitizers have been planned and prepared based on these premises, it has been challenging to develop considerably more efficient Photodynamic Therapy (PDT) protocols based solely on this principle.6 Therefore, it is imperative to look for other

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which are formed by the PS-induced electron abstraction either directly from the double bond of the lipid or from hydroperoxides that were previously generated by the ene reaction (Scheme 1). Since an electron transfer reaction between the PS and the biological target can cause PS photobleaching, we put forward the hypothesis that photobleaching can be beneficial to the outcome of the photosensitized oxidation reactions.

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RESULTS AND DISCUSSION Synthesis and photophysical studies. The magnesium(II) porphyrazines (Pz) shown in Figure 1 were synthesized in a single-step by cyclization of the respective dinitriles (cis/trans mixture) in the presence of Mg(OBu)2 under reflux. While the meta-substituted phenyl rings of CF3Pz confer high solubility in most organic solvents, the para-substituted Pzs are more symmetric and thus soluble mainly in THF. In this sense, CF3Pz was purified by column chromatography, while the purification of the other Pzs was achieved by successive recrystallizations from THF/MeOH. 1H NMR, 13C NMR and MALDI-TOF mass spectrometric analysis were used for the characterization of the starting materials and products, as shown in the Supporting Information (SI). Substituents ranging from strong electron-withdrawing (CF3) to electron-donating groups (MeO) were chosen in order to modulate the Pz electronic properties.

Scheme 1. Oxidation pathways of unsaturated lipids via contact-dependent and independent reactions between the PS and the lipid.

The Pzs showed characteristic * absorption bands in the red (Q band,  ~ 105 M-1cm-1) and blue regions (Soret band) of the spectrum, with MeOPz showing an additional band at 490 nm, arising from a charge transfer transition from the substituents to the porphyrazine ring (Figure S1 in the SI).17 The Q band position red-shifts from 630 nm for CF3Pz to 649 nm for MeOPz following the order CF3 < F < Cl < Br < MeO, with the same trend observed for the fluorescence emission maxima (Figure 2, Table 1). The values of the Stokes shifts (ca. 17 nm) are in agreement with those found for other porphyrazines18,19, and reflect the high rigidity of the macrocyclic ring. Pzs are usually efficient fluorophores, but susceptible to the influence of the substituents. Fluorescence quantum yields (F) varied from 0.18 for CF3Pz to 0.05 for BrPz, because of the enhancement of the intersystem crossing imposed by the bromine atoms, as evidenced by the increase in the singlet oxygen production (see below). F was even smaller for MeOPz (0.007) due to an alternative route of excited state deactivation, which is a photoinduced charge transfer from the peripheral groups to the Pz ring (see below).

In this work, we aim to test this hypothesis with wellcontrolled model systems. We designed a series of magnesium porphyrazines (Figure 1) substituted with symmetrically distributed electron-donating or electronwithdrawing phenyl groups, in order to obtain PSs with tunable levels of photostability. We initially showed that the photobleaching mechanism of these molecules is independent of 1O2 and involves an electron abstraction from surrounding molecules. Importantly, by comparing the photoinduced disruption of phospholipid membranes with the photobleaching rate of the PSs, we challenge the paradigm that photobleaching is necessarily correlated with low PS efficiency.

To define the HOMO and LUMO gap for this Pz series, TDDFT calculations using the B3LYP functional were performed. The HOMO and LUMO are centered on the porphyrazine ring and their energy gap correlates well with the red shift order observed in their solution spectra (Figure S2). Electron-donating groups tend to destabilize both the HOMO and LUMO of the macrocycle. However, a stronger destabilization of the HOMO is responsible for the decrease in the energy gap from CF3Pz to MeOPz, explaining the consequent red shift in the spectrum.

Figure 1. Molecular structure of the MgPzs.


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Table 1. Photophysical parameters obtained for the Pzs in THF solution. abs / nm (log 

emiss / nm

F a

 b

CF3Pz

630 (4.96)

648

0.177 ± 0.001

0.34 ± 0.07

FPz

634 (4.87)

651

0.110 ± 0.001

0.34 ± 0.02

ClPz

638 (4.96)

655

0.11 ± 0.01

0.48 ± 0.04

BrPz

639 (4.92)

656

0.053 ± 0.005

0.64 ± 0.06

MeOPz

649 (5.03)

666

0.007 ± 0.002

0.07 ± 0.01

Pzs

Cresyl violet in MeOH as reference (F = Methylene blue ( = 0.52) and 1,9-dimethylmethylene blue ( = 0.71) in EtOH as references. a

Figure 2. Normalized absorption (solid lines) and fluorescence emission (dashed lines) spectra of CF3Pz (black), FPz (red), ClPz (blue), BrPz (pink) and MeOPz (green) in THF.

0.57).b

Photostability studies were conducted by irradiating and following the absorbance change over time of the Pzs (Figures 3a, S4). Photobleaching was irreversible and was characterized by absorbance loss in all Pz bands (300 to 700 nm), accompanied by a small absorbance increase in the 700 to 800 nm region (Figure 3a). Absorption of Pzs in the NIR have been previously attributed to a macrocyclebased radical species (this will be further discussed below).24 Photobleaching quantum yields (pb) of Pzs were calculated by using the values of the photobleaching rate (kPB) taken from the fitting of the absorption decay curves to a kinetic model, which was developed to allow better fittings to the experimental data (see SI – section 1.1). As clearly seen in Figure S5, the data fit with the kinetic model is a lot better than the fit using a mono-exponential function. It is worth noting that photobleaching varied substantially within the Pz series (Figure 3b). pb values decreased with the increase in the electron donating ability of the substituents, as seen in Table 2. Using the Hammett sigma parameter (), which accounts for both inductive and resonance effects of substituent groups,25 it was possible to find a linear relationship between the substituents electronic nature and the porphyrazine photostability, as shown in Figure 3c. A higher  value indicates a more electron-withdrawing (or less electrondonating) nature of the group, so, clearly, CF3 is the most electron-withdrawing group of the series. Therefore, if one wants to minimize photobleaching of the Pz, one should choose electron-donating substituents, suggesting that an electron donation to the macrocycle is involved in the photo-induced degradation of the Pzs. MeOPz is an outlier since it showed no detectable photobleaching under the conditions used in the experiment. Although it has the strongest electron donating group in the series, the absence of photodegradation cannot be solely explained by the redox properties of the excited state, but it is mainly a consequence of the intramolecular suppression mechanism of the excited state, which is in accordance with the photophysical data shown above (see Figure S6 for additional discussion).

Pz macrocycles usually show high singlet oxygen quantum yields (), which justifies their use in medical applications.  of Pzs were obtained by measuring the 1O2 phosphorescence decay at 1270 nm (Figure S3) and comparing to MB and DMMB in ethanol as references (see SI). The values (Table 1) clearly followed the trend based on the presence of heavy atoms, increasing from FPz (0.34) to BrPz (0.64), which showed the highest yield among the Pzs. The efficiency was lowest for MeOPz (0.071), in agreement with its excited states being deactivated mainly by non-radiative pathways. Photobleaching. There are many photobleaching mechanisms that can be generically classified as oxygendependent and -independent mechanisms. Oxygen dependent photodegradation usually involves oxidation by 1O and is common for porphyrinoids12,13,20 and other 2 chromophores such as BODIPY21. The attachment of electron-withdrawing groups to the ring increases its oxidation potential, resulting in a greater photostability12,13,21. The oxygen-independent mechanism usually involves direct-contact reactions between the excited state of the PS and neighboring molecules, which can be the dye itself (dye-dye mechanisms)14,22 or preferably a biological target that can be key to the cellular response.23 An initial electron or hydrogen transfer reaction leads to the formation of radicals that undergo further reactions. Although very likely to occur, less attention has been paid to these reactions compared with the oxygen-dependent photodegradation mechanisms.



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(Table S1). A possible involvement of residual water in the degradation mechanism was discarded, because the photobleaching rates in anhydrous (water content < 0.002%) and water-containing solvent were essentially the same (Figure S9). pb was plotted as a function of polarity and nucleophilicity, showing no clear correlation (Figure S10), suggesting that a nucleophilic attack by the solvent on the Pz ring is not a probable mechanism involved in photodegradation. However, good correlations are observed with Gutmann’s donor number26 and the coordination ability (aTM)27 of the solvents (Figure 4b), parameters that reflect the interaction between a Lewis base (donor) and a Lewis acid (acceptor). In the work of Mielcarek and collaborators,28 the photobleaching reported for free-base (2H) amino-substituted Pzs was 70 times slower than that for the Zn(II) and Mg(II) complexes. The authors gave no explanation for these observations, nor for the strong photobleaching observed. However, their results reinforce that solvent coordination to the central metal can help promote the electron transfer process, increasing photobleaching. It is indeed likely that solvents with high donor number strongly coordinate to the Mg(II) center of the porphyrazine, stabilizing the donor-acceptor pair for the electron transfer reaction to proceed. Toluene, for instance, shows a very weak coordination ability and thus the driving force must be highly endergonic, explaining the very low rate of photobleaching in this solvent. Interestingly, the photostability of Pzs in biological environments was also studied by inserting them into phospholipid vesicles. When incorporated in vesicles of DMPC, a phospholipid containing only saturated chains, the pb values decreased sharply for all Pzs, as shown in Table 2 (data with other lipids are shown below).

Figure 3. (a) Spectral changes for CF3Pz in air-saturated THF solution under Q band irradiation. (b)Absorbance decay with time upon irradiation at the Q band for MeOPz (green), FPz (red), ClPz (blue), BrPz (pink) and CF3Pz (black) in THF. (b) Correlation between the photostability of the Pzs and the Hammett sigma value of the substituents (m for the metasubstituted CF3Pz and p for the para-substituted Pzs).

Pzs photodegradation is not affected by oxygen, i.e., when oxygen is removed, pb values are very similar to those observed in air-saturated solutions (Table 2), even though oxygen quenches Pzs triplets efficiently (Figure S7). Therefore, the results in the presence of oxygen rule out the involvement of triplet species and suggest that the photodegradation occurs directly in the singlet excited state by an electron-transfer reaction with molecules that are in the solvation sphere of the Pzs singlet excited state. Note also that the triplet-triplet absorption of CF3Pz does not return to zero, in either air saturated or argon purged solutions, indicating the formation of a long-lived intermediate species upon excitation (Figure S7). Besides, the fact that the photodegradation rates follow the firstorder kinetic model, as calculated for CF3Pz in THF (Figure S8), indicates that Pzs do not tend to engage in dye-dye photobleaching mechanisms (dye-dye mechanisms would require second or higher order kinetics).

Classical photolysis data suggest that Pzs photobleaching takes place through a reductive mechanism by accepting an electron from neighboring donors (D), without interference of type II mechanism or dye-dye reactions. Thus, the fundamental step is the photoinduced formation of the Pz radical anion, which was previously detected by spectroscopic techniques.23 Interestingly, electron paramagnetic resonance (EPR) measurements of irradiated pyridine and THF CF3Pz solutions showed the presence of a well-defined singlet signal with g = 2.03, absent in the non-irradiated solutions (Figure 5, S11). This signal, with no hyperfine coupling, is characteristic of a single electron radical, which is delocalized over the porphyrazine ring.29 Table S2 contains the values of spin density calculated for all MgPzs, showing that the free electron is delocalized over the whole Pz ring, but with no charge density in the magnesium ion.

In order to further understand the mechanism of photodegradation of Pzs, photobleaching rates of CF3Pz were obtained in different organic solvents, taking advantage of its improved solubility compared to the other Pzs. As shown in Figure 4a, the photobleaching rate of CF3Pz increases in the order toluene < acetone < acetonitrile < THF < pyridine. From toluene (pb = 4.2 x 106) to pyridine ( -4 pb = 4.4 x 10 ), the pb values increased by two orders of magnitude, indicating a strong solvent effect


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Journal of the American Chemical Society experiment led to an apparent degradation of the macrocycle into smaller, less conjugated fragments as indicated by depletion of the NIR peaks and appearance of new bands at 412 and 438 nm (Figure S13), suggesting that the absence of the oxidation peak is caused by subsequent chemical reactions.30 These results are in agreement with those reported by Schröder et al. for a resonance-stabilized organic radical anion.30 Although this anion radical is stable both in the solid state and in solution, the precursor molecule also shows a single irreversible electrochemical reduction in cyclic voltammetry measurements.30 MALDITOF-MS spectra for both irradiated and non-irradiated solutions of CF3Pz are rather similar, and the high abundance of the molecular ion species [M] (m/z 1488.1) in the negative mode (Figure S14) corroborates with the proposed CF3Pz structure as the main photobleaching product for this Pz. Theoretical calculations were used to obtain additional information about this intermediate species. One-electron reduction of the Pz ring was shown to break the degeneration of the HOMO-LUMO transition, i.e., Q band splits into two transitions (one higher and other lower in energy – Table S3 and Figure S15), agreeing with the absorbance changes around 700 nm observed during photobleaching and electrochemical reduction of CF3Pz (Figure 5b).

105)

Table 2. Photobleaching quantum yields (pb x for the Pzs in THF solution and in DMPC liposomes. Pzs

THF (air)

THF (argon)

DMPC (air)

CF3Pz

15 ± 2

14 ± 3

1.3 ± 0.3

FPz

2.5 ± 0.1

2.6 ± 0.1

1±1

ClPz

3.9 ± 0.2

6.1 ± 0.9

0.8 ± 0.4

BrPz

4.8 ± 1

6±3

0.7 ± 0.1

MeOPz

**

**

**

Irradiation at the Q band (74 mW, pulse frequency of 10 Hz).**No photobleaching was detected under the experimental conditions used in this work.

The stability of CF3Pz●− was tested in aerated THF solution by monitoring its absorption band (ca. 700 nm) over time. This species is stable for more than one week in solution, even in the presence of water in the medium (Figure S16). Molecular oxygen (Ered = 0.33 V)31 does not seem to oxidize the Pz radical to form superoxide anion radical (O2) since CF3Pz●− is stable in the presence of oxygen and regeneration of the original macrocycle was never observed. 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ, Ered = +0.50 V)32, a stronger oxidant compared with molecular oxygen, could not oxide the CF3Pz radical either. Ceric ammonium nitrate (CAN, Ered = +1.70 V)33, however, completely depleted the band around 700 nm (Figure S17), indicating that with enough oxidizing potential, it is possible to oxidize CF3Pz. Since no bands appeared in the visible region, the oxidation of CF3Pz species must lead to degradation of the Pz ring (resulting in smaller products with little or no conjugation). These data are also in agreement with the spectroelectrochemical results above.

Figure 4. (a) Absorbance decay with time upon irradiation in the Q band of CF3Pz in pyridine (black), THF (pink), acetonitrile (blue), acetone (red) and toluene (green). (b) Relationship between the CF3Pz photobleaching quantum yield (pb) and the solvent donor number and coordination ability (inset).

Surprisingly, this EPR peak is stable at room temperature even in the presence of oxygen (Figure S11), explaining the relatively long-lived species observed in the transient absorption measurements discussed above (Figure S7). Further electrochemical investigations (spectroelectrochemical and cyclic voltammetric) agrees with this interpretation. Indeed, when a reduction potential of -0.7 V (vs. Ag/AgNO3 10 mM in acetonitrile) was applied to a CF3Pz solution in pyridine containing 0.1 M tetrabutylammonium perchlorate (TBAP), its absorption spectrum started to change in a way similar to that observed during irradiation of the solution at 630 nm, as shown in Figure 5. All typical absorption bands of CF3Pz decreased in intensity, followed by the surge of new absorption bands at 702 and 765 nm. This process is irreversible, in agreement with the cyclic voltammetry measurement of CF3Pz in deaerated pyridine, which shows a single irreversible reduction process at -0.9 V vs. Ag/AgNO3 (Figure S12). Oxidation of the reduced species by sweeping back to -0.2V in the spectroelectrochemistry

In summary, several data from different techniques (transient absorption, EPR, spectroelectrochemistry MALDI-TOF-MS and theoretical calculations) all agree with the formation of a π-radical Pz derivative species (MgPz) as the intermediate in the photodegradation pathway. Although porphyrin and phthalocyanine radicals tend to be extremely unstable in solution (especially in aerated environments)34, MgPz shows up to be fairly stable even in the presence of oxygen. This long-lived radical species is interesting especially to other research areas, such energy conversion devices, and certainly deserves further investigation. However, we consider that



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experiments of liposomes having membrane-incorporated CF3Pz shows that triplets are not suppressed by the lipid double bonds (Figure S20), in agreement with the electron abstraction step being a consequence of the direct reaction with the singlet excited state of CF3Pz, as observed in bulk solutions (Table 2). These results clearly show the influence of biological targets (lipid double bonds) on the photodegradation rate of these compounds. The feasibility of a photoinduced electron-transfer between the lipid unsaturation and the CF3Pz excited state is discussed in terms of driving force (G) in the SI section.

further studies with MgPz are out of the scope of this paper. The trend observed in the plot of Figure 3c can now be rationalized based on this proposed mechanism. Electrondonating groups attached to the Pz ring considerably increase the energy of the Pz’s LUMO (see Figure S2) and decrease electron affinity (Table S2), which disfavors the reduction of the macrocyle thermodynamically. The opposite is observed for substituents that withdraw electronic density from the ring, explaining the higher photobleaching rate observed for CF3Pz. Indeed, the spinorbital energy calculated for CF3Pz is the lowest (~−4eV) in the MgPzs series (Figure S18), indicating that the trifluoromethyl groups largely stabilize the additional electron.

Figure 5. Changes in the absorption and EPR spectra (inset) of CF3Pz in pyridine before (black line) and after (red line) application of -0.7V to -1.0V (a) or irradiation at 632 nm for 20 min (b).

Photostability vs. membrane damage efficiency. The photoinduced efficiency of cell death induced by a PS is often associated with membrane damage, since subtle damages in membranes, either the cytoplasmic membrane or those of intracellular organelles, have important consequences for cells, defining survival or death outcomes.7,35 In order to correlate membrane damage with photobleaching efficiency, we performed experiments in several membrane mimic systems. First, we investigated the photobleaching rates of CF3Pz in the presence of lipids with different degrees of unsaturation and compared them with the rate of membrane leakage. As shown in Figure 6 and Table S4, the rate for CF3Pz photobleaching in POPC (a monounsaturated lipid) is about two orders of magnitude higher than in DMPC (a saturated lipid) under the same conditions, indicating that the unsaturated C=C bond act as electron donor in the photoinduced redox process that lead to Pz bleaching. This is further confirmed by the rate in DOPC (lipid with two unsaturation sites, one in each carbon chain), which showed a ca. three times higher value than in POPC vesicles. Note also that irradiation of CF3Pz in the presence of unsaturated lipids leads to the formation of CF3Pz in both air saturated and deaerated solutions (Figure S19). Triplet-triplet absorption

Figure 6. Photobleaching kinetics for CF3Pz (0.5 mol%) incorporated in the membrane of liposomes of (a) DMPC, POPC and (b) DOPC under irradiation with red LEDs (620 – 625 nm).

Aiming to correlate photobleaching with membrane leakage, we compared the membrane damage efficiencies of two PSs (CF3Pz and FPz) with similar photophysical properties (absorption maxima, molar absorption coefficients, fluorescence and 1O2 quantum yields − Table 1), but distinct photobleaching efficiencies (Table 2). The results of the membrane damage experiments are shown in Figure 7. When liposomes containing 0.25 mol% of Pz were irradiated, liposome-entrapped carboxy-fluorescein (CF) release was clearly faster for CF3Pz than for FPz. The release rate, taken from the linear fit of the curves from 0 to 30 min, was ca. 4 times higher for CF3Pz (3.1%/min) compared to FPz (0.8%/min). The same experiment was


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Journal of the American Chemical Society membrane leakage and in agreement with previous literature.16 By using a series of Pz molecules, our data clearly indicate that PSs having faster bleaching are more effective at causing irreversible damage in membranes (Figure 8). CF3Pz is able to oxidize molecules, not only by the action of diffusing species such as 1O2, O2 etc, but also via direct reaction with suitable target molecules located within the contact volume. While the reaction with oxygen is basically dependent on the velocity of diffusion of the reactants (quantum yields in the order of 0.3-0.6), the rate of type I contact-dependent oxidation of the double bond depends on the presence of the photosensitizer in physical contact with the target and in the proper geometry. This results in comparably lower photobleaching quantum yields (10-4-10-5). Even though being relatively rare, these contact-dependent reactions are the ones that trigger membrane leakage. In the case of the Pz, it is likely that the contact-dependent reaction occurs within the solvation sphere of the PS (no diffusion), since it occurs within the lifetime of the singlet excited state (~1 ns)38. It is interesting to observe that membrane binding is not the only factor affecting membrane leakage, since other PSs that are fully integrated into membranes, but only generate 1O , do not cause membrane permeation.39 Porphyrazines 2 do so because they are stronger oxidizing agents and consequently able to abstract an electron from the lipid double bond.40,41 Nevertheless, this concept can now be used to design and develop optimized PSs, which ideally could specifically oxidize and inactivate intracellular molecular targets (Figure 8).

reproduced with a very low Pz concentration in the membrane (0.06 mol%) in order to follow the absorption changes of the PSs (Figure 7b and inset). During the first 100 min of experiment, the rate of CF release was similar for both Pzs since the Q band absorbance values indicated no significant bleaching until this point. Proceeding with the irradiation, CF3Pz outperforms FPz in terms of causing membrane leakage and it is possible to see a marked decrease of the CF3Pz Q band absorbance with an increase of the 700 nm absorption, indicating formation of the Pz species. Therefore, the higher photobleaching rate of CF3Pz improves its efficiency in causing membrane leakage. In order to visualize the progress of membrane oxidation and leakage, CF3Pz was incorporated into giant unilamellar vesicles (GUVs) of DOPC loaded with sucrose in a glucose medium and monitored by phase-contrast microscopy. As shown in Figure 7c, in the first minutes of irradiation, the shape of the GUV is highly deformed as the membrane tries to accommodate the oxidized lipid formed via the photosensitization by CF3Pz. Membrane buds are then formed and expelled from the GUV, while it resumes its circular shape with smaller diameter, due to lipid loss. From this point forward, the membrane starts to lose its contrast, indicating pore formation and exchange between the inner content (sucrose) and the external solution (glucose). The progress of phase contrast loss was monitored as a function of time (Figure S21) and, after 34 min of irradiation, the GUV membrane was only barely visible. FPz also induces membrane fluctuations and bud formation in the first few minutes of irradiation, but contrast loss was not observed even after 35 min, indicating no membrane leakage (Figure 7d). This agrees with the hypothesis that PS needs to photobleach in order to make a membrane leak and those that photobleach faster are the ones that cause faster irreversible membrane damage.

CONCLUSIONS A series of Mg(II) porphyrazine complexes with similar photophysical and tunable redox properties was prepared and studied both in bulk solution and in membrane mimetic systems. PS photobleaching was shown to occur by the oxidation of neighboring molecules, leading to the formation of a PS-derived anion radical species. Within membranes, PS photobleaching occurs through an electron abstraction from the lipid double bond, which causes irreversible membrane damage. Therefore, our data support a re-conception of the role of photobleaching in PDT by showing a positive correlation between the efficiencies of PS photobleaching and membrane leakage. Given an intracellular target, contact-dependent reactions can damage the biological target with a lot more precision compared with diffusive species such as 1O2 or other ROS. In addition, the use of drugs that have defined photobleaching rates can be crucial for the development of dosimetry in hypoxic regions, as well as for the clearance of the drug from the body.42,43 We hope our work will stimulate the development of new lead compounds for PDT, by designing molecules (e.g. metal complexes, phenothiazines, among others) with improved redox properties and also considering strategies that allow PS replenishment.

PS photobleaching was never thought to improve photoactivity,28-31 instead, improved PS efficiency has been usually associated with high 1O2 quantum yields. It is well documented though that 1O2 reacts with unsaturated lipids to form hydroperoxides, which alters membrane area and thickness but does not induce its permeabilization.36,37 Bacellar et. al. showed that membrane permeabilization is dependent on the accumulation of truncated lipid aldehydes, which are only formed by the direct-contact reactions of the excited state photosensitizer with either the lipid double bond or a lipid hydroperoxide.16 By abstracting electron/hydrogen from these targets, the PS is able to form peroxyls and alkoxyls radicals leading the carbon-carbon bond breakage and aldehyde formation.16 We also searched for lipid oxidation products originated from contact-dependent reactions with CF3Pz. As shown in Figure S22, liposomes incorporated with this PS induce much higher accumulation of malondialdehyde (MA), which is a short-chain aldehyde formed by C-C chain breakage of the phospholipid, compared to a control liposome (without Pz), explaining the higher rate of



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deactivated to the ground state; be converted to the triplet state (T1) via intersystem crossing (ISC); or engage in an electron-transfer process with surrounding molecules as solvent (solv) and unsaturated lipids. This irreversible process leads to photobleaching due to formation of a long lived reduced Pz species (Pz), which has low visible light absorption. The oxidation of lipids by Pzs through this contact-dependent reaction leads to the formation of membrane pores, resulting in membrane leakage. Therefore, photobleaching is a necessary step towards inflicting irreversible biological damage.

EXPERIMENTAL SECTION Materials and methods. Diethyl ether and tetrahydrofuran were refluxed over sodium and distilled prior to use for synthesis and photophysical measurements. All the other solvents and reagents were used without further purification. The phenylacetonitrile precursors were purchased from Sigma-Aldrich; 1palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) were purchased from Avanti Polar Lipids; and the solvents were analytical grade and acquired from Synth. 1H

NMR measurements were performed in a Bruker Avance III equipment operating at 300 MHz. For characterization of the porphyrazines, mass spectra were recorded in a Autoflex Speed-MALDI-TOF/TOF equipment in the positive mode, using 2,5-dihydroxybenzoic acid as matrix. Absorption spectra in the UV-Vis region were recorded in a Shimadzu UV-1800 spectrophotometer. Fluorescence spectra were recorded from 580 to 800 nm in a Cary Eclypse fluorometer, and the quantum yields were obtained using cresyl violet as reference (F = 0.57 in MeOH)44 according to the procedure described in ref. 45. The samples were excited at 570 nm, with excitation and emission slits of 5 nm. Singlet oxygen measurements were performed in an Edinburgh time-resolved phosphorescence spectrophotometer. A Hamamatsu R5509-72 photomultiplier detects the 1O2 phosphorescence at 1270 nm. The samples were excited at 630 nm using a OPO RAINBOW laser system from Quantel coupled to a Magic PrismTM, which can convert the Nd:YAG laser wavelength into a continuously tunable emission from 420 to 680 nm. The 1O2 quantum yields () were calculated from the equation below using the amplitudes (APz) of the mono-exponential decay function fitted to the 1O2 phosphorescence decay curves of each sample in THF, correlating with the quantum yields and respective values (Aref) for methylene blue ( = 0.52)46 and 1,9dimethylmethylene blue (DMMB,  = 0.71)47 in EtOH as references. By using the amplitude instead of the area under the decay curves, it is possible to minimize the error associated with using different solvents for the samples and references.

Figure 7. Plots of 5(6)-carboxyfluorescein release (%) from DOPC liposomes containing no Pz (black squares), CF3Pz (red circles) or FPz (blue triangles) at (a) 0.25 mol% and (b) 0.06 mol% under red light irradiation (max = 630 nm). Inset: Variation of the absorbance of the CF3Pz Q band (red circle) and 700 nm band (red star) and of the FPz Q band (blue triangle) and 700 nm band (blue cross) in the liposomal suspension during the experiment. Images of a DOPC GUV with CF3Pz (c) and FPz (d) 1 mol% with different times of irradiation with blue light (440-495 nm).

Figure 8. Major photo-induced processes of the MgPzs. The MgPz excited state formed upon light excitation (S1) can be


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∆ =

150 L of Tris buffer and 50 L of liposome suspension with or without Pz (control). A final row containing 140 L of Tris buffer, 50 L of liposome suspension and 10 L Triton X-100 (10%) was prepared for calculation of the percentage of release assuming these wells to represent 100% CF release. Irradiation of the plate was done using a red LED array (max = 630 nm) with irradiance of 6.6 mW/cm2. The CF fluorescence emission was read in a microplate reader (SpectraMax i3 from Molecular Devices) by exciting at 480 nm and reading the emission at 517 nm from bottom. Detection of malondialdehyde in photosensitized lipid membranes were performed according to the literature.49 Soy lecithin liposomes were prepared and irradiated similarly to the membrane leakage assays for 2 hours. The irradiated vesicles contained 0.5 mol% of CF3Pz or no CF3Pz as a control.

𝐴𝑃𝑧 ∆𝑟𝑒𝑓 𝐴𝑟𝑒𝑓

For the photobleaching studies, a Pz solution with maximum absorbance of 1.0 was irradiated at the Q band (74 mW, 10 Hz pulse frequency, 5-10 ns pulse width) using the OPO RAINBOW optical system described above, in 1 minute intervals for 10 to 20 minutes. At each interval, the solution was homogenized and the absorbance spectrum from 300 to 800 nm was registered. For better detection of the photobleaching process, a quartz cuvette of 700 L internal volume (1.0 x 0.4 cm) was used, with both irradiation and absorbance measurements performed with the 1.0 cm optical path. The photobleaching quantum yields 𝑃𝐵 were calculated using the equation below, where: kPB is the photobleaching rate in mol dm-3 s-1 calculated from the fitting of the photobleaching curves (see SI for further details), V is the irradiated solution volume in dm3, Np is the photon flux in einstein per second, and Abs is the initial absorbance of the porphyrazine Q band.

𝑃𝐵 =

Giant Unilamellar Vesicles (GUVs) were grown by the electroformation method following a previously reported procedure, using instead 0.2 M glucose and sucrose solutions and 2 mM DOPC in CHCl3 containing 0.5% of Pz.50 The GUVs were observed in a Zeiss Observer.D1 inverted microscope with phase contrast and 10x/40x objectives (A-PLAN 10x/0.25 Ph1 and EC Plan-NEOFLUAR 40x/0.75). Images and videos were recorded with an Axiocam MRm camera coupled to the microscope. A HXP fluorescent lamp and Zeiss filter set 05 (excitation filter: BP 365-440nm; beamsplitter: FT 460 nm; emission filter: LP 470 nm) were used for irradiating the GUVs.

𝑘𝑃𝐵𝑉 𝑁𝑝 (1 ― 10𝐴𝑏𝑠)

The photon flux (Np) was calculated from actinometry experiments using potassium tris(oxalato)ferrate(III) as actinometer according to the procedure described in ref. 48. For the oxygen-free experiments, the cuvette containing the Pz solution was bubbled with argon for 15 min and sealed with a septum prior to measurement. Photobleaching of the Pzs in the lipid vesicles was performed similarly to the procedure above, using instead two red LEDs (620-630 nm), each one positioned in the center of a transparent face of the quartz cuvette, with the power supply set to 0.7A.

Transient absorption measurements were performed in a flash photolysis instrument (Applied Photophysics Ltd, Surrey, United Kingdom) pumped by a nanosecond Nd:YAG laser (Spectra Physics, Stahnsdorf,Germany) with excitation wavelength at 355 nm. First-order decay kinetics were observed for the lowest triplet state. Transient absorption spectra (300–750 nm) were recorded by monitoring the optical density change at 10 nm intervals with signal averaging of 4 decays at each wavelength. For electron paramagnetic resonance (EPR) measurements, a solution of CF3Pz (0.5 M) in pyridine or THF was transferred to a flat cell and the spectra were recorded at room temperature on a Bruker EMX spectrometer equipped with a high sensitivity cavity and operating at 9.85 GHz and 100 KHz field modulation. This solution was then transferred to a cuvette (1.0 x 0.4 cm) and irradiated at 630 nm for 20 min in the set up used for the photobleaching studies. Immediately after irradiation, the EPR spectrum of the solution was measured again. Equipment settings: microwave power, 10 mW; modulation amplitude, 1.0 G; time constant, 82 ms; scan rate, 1.2 G/s; 4 scans per measurement.

Lipid vesicles were prepared by evaporating a CHCl3 solution of 2.8 mg of DOPC, POPC or DMPC with the MgPz (0.5% mol) under argon flow in a test tube. The films were hydrated for 15 min in deionized water and the tube was vortexed three times for 30 seconds or until complete detachment of the film from the glass. The lipids were extruded 11 times through a 100 nm pore membrane, and used immediately for the photobleaching assays using the same set up as for the solution experiments. For the membrane leakage assays, 5(6)-carboxyfluorescein (50 mM in 10 mM Tris buffer solution pH = 8.0) was encapsulated by the same procedure described above, using instead it in the solution for hydration of the lipid film. After extrusion, non-encapsulated CF was removed by column chromatography using Sephadex G-50 and Tris buffer (10 mM, NaCl 300 mM) as eluent. The liposome suspension collected (ca. 2 mL) was used on the same day. In a Costar 96-well black polystyrene plate, each well was filled with

Cyclic voltammograms were measured by using an Autolab PGSTAT30 potentiostat and a conventional threeelectrode cell consisting of a gold working electrode, a platinum wire as the auxiliary electrode, and a reference electrode made of Ag/AgNO3 (10 mM in acetonitrile; +0.503 V vs. NHE). Tetrabutylammonium perchlorate



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resultant green solid in THF/MeOH for several times until the supernatant become colorless. The powder was dried in an oven at 60oC for 24 hours prior to characterization. Yield: 10.8 mg, 17%. MALDI-TOF-TOF MS (m/z): Calcd. for [M+H]+: 1089.25. Found: 1089.34. 1H NMR in THF-d8 (, ppm): 8.37 (dd, J = 9 Hz, 16H), 7.38 (t, J = 9 Hz, 16H).

(Bu4NClO4, 0.10 M) was used as the electrolyte. UV-Vis absorption spectroelectrochemistry was performed by using a custom-built electrochemical cell consisting of a gold minigrid working electrode, the Ag/AgNO3 (10 mM) reference electrode used for cyclic voltammetry, and a platinum wire auxiliary electrode mounted inside a quartz cuvette with a path length of 25 μm. The application of potentials was controlled by an EG&G PAR 173 potentiostat, and the spectra were recorded with a HP 8453A spectrophotometer.

Synthesis of ClPz. Same synthetic and purification procedure as described for FPz, using 102 mg (0.34 mmol) of the chloro-substituted dinitrile. Yield: 10.8 mg, 10%. MALDI-TOF-TOF MS (m/z): Calcd. for [M+H]+: 1221.02. Found: 1221.06. 1H NMR in THF-d8 (, ppm): 8.32 (d, J = 8 Hz, 16H), 7.64 (d, J = 8 Hz, 16H).

The structure of the Pzs, unsubstituted Pz and phenyl groups in this work was investigated with Time Dependent-Density Functional Theory (TDDFT) as implemented in the Gaussian 09 package,51 using B3LYP and the 6-311G(d,p) basis set. After optimization of the structures, the HOMO (Highest Occupied Molecular Orbital) and LUMOs (Lowest Unoccupied Molecular Orbital) were calculated.

Synthesis of BrPz. Same synthetic and purification procedure as described for FPz, using 60 mg (0.15 mmol) of the bromo-substituted dinitrile. Yield: 6.0 mg, 10%. MALDI-TOF-TOF MS (m/z): Calcd. for [M+H]+: 1576.61. Found: 1576.71. 1H NMR in THF-d8 (, ppm): 8.25 (d, J = 8 Hz, 16H), 7.80 (d, J = 8 Hz, 16H).

Synthesis of the dinitriles. The well-known procedure reported by Linstead describes the use of 2 eq. of Na for 1 eq. of the acetonitrile for the formation of dinitriles.52 However, in this work, the addition of a 10% excess of Na in the reaction resulted in more reproducible yields, since oxidation of this metal by air and moisture may easily occur during handling. All dinitriles were obtained as a mixture of cis and trans configurations and no attempt was made to separate the isomers. The detailed synthetic procedure and characterization of these precursors are presented in the SI section.

Synthesis of MeOPz. Same synthetic and purification procedure as described for FPz, using 75 mg (0.26 mmol) of the methoxy-substituted dinitrile. Yield: 42 mg, 52%. MALDI-TOF-TOF MS (m/z): Calcd. for [M+H]+: 1186.42. Found: 1186.49. 1H NMR in THF-d8 (, ppm): 8.37 (d, J = 9 Hz, 16H), 7.15 (d, J = 9 Hz, 16H), 3.99 (s, 24H).

ASSOCIATED CONTENT Supporting Information. Synthetic procedure for the porphyrazines precursors; 1H NMR, 13C NMR and MALDITOF-TOF spectra of all compounds; UV-vis, fluorescence and singlet oxygen emission spectra for the porphyrazines in solution; HOMO and LUMO calculations; flash photolysis and EPR spectra for CF3Pz. “This material is available free of charge via the Internet at http://pubs.acs.org.”

Synthesis of CF3Pz. Magnesium turnings (5.10 mg, 0.21 mmol) and 1-butanol (3 mL) were refluxed until complete reaction of all magnesium (ca. 3 hours). The dinitrile (75 mg, 0.21 mmol) was added to the mixture and it was refluxed for 12 hours under magnetic stirring and covered from light. In the first hours the solution turns to dark green, however, a longer time is needed to consume most part of the dinitrile. The starting material is not fully consumed during reaction because the trans isomer cannot cyclize to form the porphyrazine ring. After cooling to room temperature, the mixture was filtered under vacuum and washed with methanol. The filtrate was collected, the solvent was removed and the crude product was purified on a silica gel column eluted with cyclohexane/CH2Cl2 (1:2). The dark green solid was dried in an oven at 60oC for 24 hours. Yield: 24.1 mg, 31%. MALDI-TOF-TOF MS (m/z): Calcd. for [M+H]+: 1489.23. Found: 1489.28. 1H NMR in CDCl3 (, ppm): 8.41 (d, J = 8 Hz, 8H), 8.31 (s, 8H), 7.81 (d, J = 8 Hz, 8H), 7.70 (t, J = 8 Hz, 8H).

AUTHOR INFORMATION Corresponding Authors *[email protected] *[email protected]

Author Contributions All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS The authors wish to thank Prof. Ohara Augusto and Dr. Edlaine Linares for the EPR measurements, Prof. Daniel Cardoso and Dr. Fernando Mattiucci for the transient absorption measurements and Prof. Frank H. Quina for revising the manuscript. We also thank FAPESP (scholarship grant No 2016/15916-9 and research grants 2012/50680-5 and 2017/23416-9), CEPID-Redoxoma, NAP-Phototech-USP, CNPq and CAPES (Finance Code 001) for the financial support.

Synthesis of FPz. Same synthetic procedure as described for CF3Pz, using 64 mg (0.24 mmol) of the fluorosubstituted dinitrile. Due to its very limited solubility in most organic solvents, the purification of this porphyrazine was achieved by first heating the crude reaction mixture in methanol, followed by filtration and methanol washings, and then recrystallization of the

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