Cover Page. The handle holds various files of this Leiden University dissertation

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1 Cover Page The handle holds various files of this Leiden University dissertation Author: Limburg, Bart Title: Photocatalytic redox reactions at the surface of liposomes Issue Date:

2 Chapter 4 Kinetics and mechanism of photoinduced electron transfer at the surface of liposomes 4 In this Chapter we elucidate the kinetics and mechanism of the photocatalytic reduction of on the one hand methyl viologen (MV2+), and on the other hand 5,5'dithiobis(2-nitrobenzoic acid) (H2DTNB). A comparison was made between the photoreduction when the porphyrin photosensitizer is adsorbed at the surface of liposomes to that in homogeneous aqueous solutions. The rate of the oxidative quenching of negatively or positively charged porphyrin photosensitizers is decreased by several orders of magnitude upon adsorption to edmpccl:dmpc or NaDMPG liposomes, respectively, with charges opposite to that of the porphyrin. Therefore, the process that limits the photoreduction quantum yield is different when the reaction occurs at the surface of liposomes, compared to when it occurs in homogeneous solution. For the photoreduction of MV2+ static quenching of the negatively charged photosensitizer limits the overall quantum yields of the reaction in homogeneous solution, which is overcome using positively charged edmpccl:dmpc liposomes. For the photoreduction of DTNB2, the rate of oxidativequenching is so low at the surface of the liposome that the excited state of the porphyrin is, instead, reductively quenched by the electron donor TEOA. In addition, besides the acceptors the charged photoproducts are repulsed from the membrane, which limits product inhibition. For instance, the rate of reaction of the photoproduct MV + with the oxidized photosensitizer was so low in the presence of liposomes that we could not obtain any value for the rate constant. This chapter is to be submitted as a full paper: B. Limburg, M. Hilbers, A.M. Brouwer, E. Bouwman, S. Bonnet, in preparation. 89

3 Chapter Introduction Artificial photosynthesis is a challenging goal to achieve. In order to produce solar fuels by extracting electrons from water, photochemical charge separation reactions must be linked to redox catalysis. By using light in the initial charge separation, highly reactive intermediates are generated that can react with each other to regenerate the ground state of the system, thus producing only heat. For this reason, no system is known to date that can perform artificial photosynthesis in homogeneous solution. The only reported systems that are able to link photochemical water oxidation to the production of a fuel are based on devices employing semiconductors. 1-4 These solid-state devices work well because the electron transfer is directional, which effectively separates intermediates and thus inhibits for a great part the energy-wasting charge recombination reactions. However, the use of solid surfaces complicates the mechanistic analysis of these systems. Also, because the chemistry occurs at surfaces, only a relatively small area is used for chemistry, which might decrease the overall light-to-fuel efficiency. For these reasons, and also because many new molecular oxidation and reduction catalysts have been reported in the literature that were not available decades ago, several groups 5-7 recently revisited the use of liposomes as a supramolecular scaffold on which artificial photosynthesis can be performed At the nanoscale, liposomes have properties of a heterogeneous surface at which directional electron transfer may occur. At the macro scale, however, liposomal samples can be treated as a homogeneous solution, thus allowing easier mechanistic studies. In Chapter 3 of this thesis, 22 we have described how liposomes influence the quantum efficiency of two different photocatalytic systems where photoelectron transfer occurred either in homogeneous solution, or at the surface of liposomes. The liposomal systems showed significantly improved kinetics compared to their homogeneous analogues. However, a detailed kinetic study was lacking. Herein, we report a thorough kinetic analysis of these systems and explain in details the effect of the liposomes on the mechanism of the photoreaction. 90

4 4.2 Photocatalytic reduction of MV Dependence of the quantum yield of photoreduction on the concentration of methyl viologen 4.2.1a Introduction The kinetics of the photocatalytic reduction of methyl viologen (MV 2, Scheme 4.1) by cysteine sensitized by [1] 4 (Scheme 4.1) have been studied in detail in the past. 23 In order to study the effect of positively charged liposomes on the kinetics and mechanism of the photoreduction, we first analyzed the steady-state UV-vis spectroscopy data obtained in Chapter 3 with a kinetic model. Due to the good overlap between the spectrum of the light from the LED and the absorbance spectrum of the porphyrin, and because the probability of photon absorption is close to 1 due to the high optical density, we assume that every photon that enters the cuvette is absorbed. Therefore, the total number Θ of excitations since t = 0 is equal to the total number of photons emitted by the LED since t = 0, which is proportional with irradiation time. The derivative of Θ vs. time gives θ, the rate of formation of the excited state photosensitizer *1 4 (equation 1). The quantum yield of the formation of MV + (; >?) can be numerically calculated from the rate of formation of MV + divided by θ, see equation 2. A7 CD? = AB A( : E = A7 CD? AF (1) =; >? (2) 4.2.1b Photon absorption We described in Chapter 3 that ; >? depends on the concentration of MV 2+, and that this dependence is significantly different in homogeneous solution compared to that in liposomes-containing samples. These differences were explained by the liposome-induced destabilization of the 1:1 {H MV } ground-state (GS) complex that would otherwise form in homogeneous solution. In the kinetic model described below, the photosensitizer is allowed to form a {H MV } ground-state complex with the electron acceptor, characterized by an equilibrium constant K. The absorption of a photon by either 1 4 or {H MV } is defined as a fraction of the total incoming photon flux. The corresponding rates, noted θ a and θ b, for the absorption of a photon by 1 4 or {H MV }, respectively, are given by equations 3 and 4 (see also Appendix B). 91

5 Chapter 4 Scheme 4.1. The compounds used in this = ; M : :OPQR S? T (4) In equations 3 and 4, M is the fraction of uncomplexed porphyrin 1 4. We assume that the concentration of MV 2+ is much higher than that of 1 4 (which was experimentally realized), and we assume the change in absorbance by the formation of a complex does not alter the probability of absorption significantly (see Appendix B) c Kinetic model To verify the hypothesis that absorption of a photon by GH MV L does not lead to product formation, and to quantify the effect of liposomes addition on the kinetics of the different elementary steps of the photoreaction the kinetic model shown in Scheme 4.2 was postulated. The definition of the rates (r) shown above reaction arrows is given in equations 5 14, where U VW, U, and U 8 are secondorder rate constants, and the other rate constants are first-order. 92

6 Scheme 4.2. Oxidative quenching reaction scheme with static and dynamic quenching. The photosensitizer forms 1:1 complexes with MV 2+ in the ground state in an equilibrium with constant K. These complexes have their own photocatalytic pathway as portrayed on the right side (absorption of a photon occurs with ). The left side shows the dynamic oxidative quenching pathway (absorption of a photon occurs with ). The fate of CysSH is ignored in this model. The subscript indices are defined as follows: d (decay), oq (oxidative quenching), cr (charge recombination), esc (escape), rr (reductive regeneration), pi (product inhibition), ra (rearrange). X < =U < P H T (5) X VW U VW PMV TP H T (6) X Y U Y PGH Z :MV LT (7) X \!Y U \!Y PGH Z :MV LT (8) X U PH Z TPCysSHT (9) X 8 U 8 PH Z TPMV T (10) X a < U a < P GH MV LT (11) a a X VW U VW P GH MV LT (12) a X Y U a Y P GH MV LT (13) a a X U PGH Z MV LT (14) Two distinct photochemical pathways can lead to the production of MV +. First, absorption of a photon by the free porphyrin 1 4 leads to formation of the excited state *1 4. The excited state can either decay to the GS by internal 93

7 Chapter 4 conversion with a corresponding rate X <, or be dynamically oxidized by MV 2+ with rate X VW, which leads to formation of the encounter complex {H Z :MV }. This encounter complex can then recombine the charges (rate X Y ) or the products of oxidative quenching can escape from the encounter complex (rate X \!Y ), leading to the formation of the photoproduct MV + and of the oxidized photosensitizer 1 3. To complete a photochemical turnover, 1 3 reacts with CysSH to reductively regenerate the GS porphyrin (rate X ). The oxidized photosensitizer can, however, also be reduced by the photoproduct MV + ; this product inhibition reaction occurs with the rate X 8. The second photochemical pathway begins with absorption of a photon by the GS complex {H MV }, which generates the excited state {H MV }. As with the first pathway, this excited state can decay to the GS (rate: X a < ) or separate the charges (rate: X a VW ) leading to the charge-separated complex {H Z MV }. This complex is notably different from the encounter complex {H Z :MV }(note, vs. :, respectively), because the reactions leading to {H Z MV } are fast enough to not let nuclear reconfiguration occur. The complex {H Z MV } can then recombine the charges with rate X a Y, or rearrange to form the encounter complex {H Z :MV } (rate X a ). From thereon, the same reactions as in the first pathway occur. Furthermore, in this model, we assume that CysSH dimerizes quantitatively after donating an electron and thereafter reduces a second molecule of MV 2+ without a second photon being absorbed. Intuitively, this hypothesis means that for each CysSSCys molecule produced, two electrons are transferred to two molecules of MV 2+, with the absorption of a single photon. By applying the steadystate approximation to the intermediate species *1 4, 1 3, to the encounter complex {H Z :MV }, to the excited complex {H MV }, and to the chargeseparated complex {H Z MV }, the rate equation 15 was obtained. The factor 2 thus originates from the fate of CysSH, which reduces a second molecule of MV 2+ after the first oxidation by 1 3 \!Y. The escape yield constant b Y (equation 16) is the probability that the photoproduct MV + escapes from the encounter complex instead of performing a charge recombination reaction. cpmv T U PCysSHT =@ 2 c# U PCysSHT+U 8 PMV T \!Y em +(1 M) f gh PQR S? Tf k b Y f gh iqr S? j fl gh l l f k f gh fl 1 f l m f 1 l n (15) 94

8 b \!Y Y = f o3m (16) f o3m f m 4.2.1d Fitting the data obtained in homogeneous solution The experimental data in homogeneous aqueous solution obtained in Chapter 3, i.e., for the evolution of the quantum yield of MV + formation at t = 0 as a function of the concentration in MV 2+, were fitted with formula 17, a mathematical equivalent of equation 15 at t = 0. ; >? APQR? T A( : E 2 p - q : iqr S? j OiQRS? j :OPQR S? T PQR S? Tr :OPQR S? T st (17) u f k f gh ; s fl gh l l f k f gh fl 1 f l m f 1 l (18) In equation 17, u is the ratio of rate constants U < U VW, and s is the combined probability of charge separation of the excited complex GH MV Land rearrangement of the charge separated complex GH Z MV L, see equation 18. The quantum yield was experimentally determined at t = 0, where the concentration of [MV + ] is negligible, therefore the term including [MV + ] in equation 15 is equal to 1. The parameter c 0 in equation 17 was thus equal to b \!Y Y. The fit of equation 17 to the plot of ; >? vs. [MV 2+ ] is shown in Figure 4.1; the corresponding values for the fitting constants are shown in Table 4.1. The fit Figure 4.1. Evolution of the initial quantum yield of MV + formation vs. the concentration in MV 2+ in homogeneous solution and in the presence of positively charged edmpccl:dmpc liposomes. The data obtained from Chapter 3 was fitted using equation 17. Conditions: CysSH (8.3 mm), Na41 (3.3 µm) in phosphate buffer (10 mm, I = 50 mm, ph = 7.0), T = 298 K, λirr = 420 nm, θ = 13 nmol s 1. 95

9 Chapter 4 only converged if s was equal to 0, which confirms that excitation of the GS complex {H MV } does not lead to any product as observed by Rougee et al. 23 From the fit we also obtained a value for the ground state binding constant K of Z M : \!Y, and a value for b Y of 0.25, which are comparable with Rougee s reported values of Z M : and ~0.5, respectively. 23 Furthermore, a value for u of M was obtained, meaning that, as an example, at a MV 2+ concentration of 0.67 mm, 97% of the excited states *1 4 are oxidatively quenched by MV 2+ at t = 0 (b VW <, equation 19). In homogeneous solution, oxidative quenching is very efficient compared to non-productive decay of the photosensitizer. b < VW = r PQR S? T = f ghiqr S? j f gh PQR S? Tf k (19) 4.2.1e Fitting the data obtained in the presence of charged liposomes The addition of positively charged liposomes made of a 1:1 mixture of edmpccl and DMPC lipids leads to the adsorption of 1 4 to the surface of the liposomes (see Chapter 3). As a change in mechanism was not expected to occur, equation 17 was also used to fit the evolution of ; >? as a function of the concentration in MV 2+ for samples containing liposomes. As shown in Figure 4.1 a good fit was obtained as well. The values for the fitting constants are shown in Table 4.1. From this fit a value K = 0 was obtained, confirming our hypothesis from Chapter 3 that the adsorption of the 1 4 on the surface of a charged liposome prevents the formation of GS complexes between 1 4 and MV 2+. Furthermore, the numerical values u = \!Y M and b Y = 0.66 were obtained. At a MV 2+ concentration of 0.67 mm, only b VW < =43% of the excited state *1 4 are oxidatively quenched at the surface of the positively charged liposomes (equation 19), compared to 97% in homogeneous solution (see Section 4.2.1d). The probability of escape from the encounter complex is, however, higher at the surface of liposomes than in homogeneous solution (0.66 compared to 0.25, respectively). The lower quantum yield of oxidative quenching b VW < in presence of the liposomes is likely caused by a decrease in the rate constant U VW. Compound 1 4 is indeed adsorbed to a positively charged surface, and the bicationic nature of methyl viologen predicts a more difficult approach of the electron acceptor from the excited state of the photosensitizer because of electrostatic repulsion with the surrounding surface. In contrast, the rate constant of the non-radiative decay, U <, is not expected to change very much upon adsorption of 1 4 to the liposome surface as non- 96

10 radiative decay is an intramolecular process. In summary, at low concentrations of MV 2+, the production of MV + proceeds more slowly in presence of liposomes due to a slower rate of oxidative quenching compared to homogeneous conditions. However, as the photochemically-unproductive {H MV } complexes cannot form in the presence of liposomes, the reaction is more efficient at higher concentrations of MV 2+ than in homogeneous solution. In addition, the probability that the initial photoproducts after oxidative quenching escape from the encounter complex is higher in the presence of liposomes, which also increases the overall quantum yield for the production of MV +. Table 4.1. Numerical values obtained by fitting of the data presented in Figure 4.1 in homogeneous solution and in the presence of positively charged liposomes using equation 17. constant homogeneous liposomal c u (M) s 0 - K M Dependence of the quantum yield of photoreduction on the concentration of CysSH 4.2.2a At t = 0, in the absence of photoproduct According to equation 15, the quantum yield of the reaction should not depend on the concentration of CysSH at t = 0. In order to validate this conclusion of equation 15, the photoreduction of MV 2+ in homogeneous solution or in the presence of liposomes, was studied again at different concentrations of CysSH. A mixture of MVCl 2 (0.67 mm), CysSH (1.7 mm 8.3 mm), Na 41 (3.3 µm), with or without liposomes made of a 1:1 mixture of DMPC and edmpccl (83 µm:83 µm) and prepared similarly to Chapter 3 in a phosphate buffer, was deaerated by bubbling argon through the solution. The absorbance at 605 nm was monitored while the solution was irradiated from the top using a violet LED (420 nm). Figure 4.2a and b show the evolution of the amount of photoproduct MV + in the absence and presence of charged liposomes as a function of the total number of excited states produced since t = 0 (Θ). The initial quantum yield ; >? for the production of MV + at Θ 0 (i.e., the initial slope of the traces in Figure 4.2), was found identical for all concentrations of CysSH investigated, thus indicating that 97

11 Chapter 4 equation 15 correctly describes the quantum yield of MV + formation at t = 0 for both the homogeneous and liposomal systems. Figure 4.2. Evolution of the amount of MV + as a function of the amount of excited states formed since t = 0 (Θ) a) in homogeneous solution, and b) at the surface of liposomes consisting of 1:1 edmpccl:dmpc (83 µm:83 µm). Conditions: [MVCl2] (0.67 mm), Na41 (1.7 µm) in 3 ml phosphate buffer (10 mm, I = 50 mm, ph = 7.0), λirr = 420 nm, θ = 13 nmol s 1. The grey areas around the traces depict the area of the standard deviation for a set of at least two experiments b Kinetic model at t > 0, in the presence of photoproduct While the traces in Figure 4.2 have the same initial slopes, the difference between the different concentrations of CysSH becomes evident at t > 0. Clearly, the slope of these traces (and thus the quantum yield of photoreduction) was higher if more CysSH was present next to some photoproduct MV +. In presence of photoproduct MV + the fate of CysSH after its initial oxidation cannot be approximated as a simple quantitative dimerization anymore. 24 As described by Rougee et al. 23 the model shown in Scheme 4.3 can be used to describe the fate of CysSH after it reductively quenches the excited photosensitizer. The rates in Scheme 4.3 are defined by equations Using the steady-state approximation, a combination of Scheme 4.2 with Scheme 4.3 leads to the global rate equation Equation 26 agrees with the finding that as more photoproduct is formed a higher concentration of CysSH leads to an increase in ; >?: if the concentration of MV + approaches 0, the terms depending on [MV + ] become 1, whereas if the concentration of MV + becomes significant, the quantum yield of the reaction will become lower, and will decrease with CysSH concentration. 98

12 Scheme 4.3. Reaction mechanism describing the fate of CysSH after initial oxidation by 1 3. Taken from reference 23. The rates of the elementary reactions are indicated next to the reaction arrows. The subscript indices are defined as follows: rr (reductive regeneration) pi2, pi3 (product inhibition 2 or 3), dp (deprotonation), di (dimerization), cl (cleavage), 2e (2 nd electron). cpmv T c# b VW < b Y f k f k f 4S PQR? T X 8 =U 8 PMV TPCysSH T (20) X < U < PCysSH T (21) X 8Z U 8Z PMV TPCysS T (22) X <8 U <8 PCysS TPCysS T (23) X Y U Y PCysSSCys T (24) X \ U \ PCysSSCys TPMV T (25) \!Y U PCysSHT U PCysSHTU 8 PMV T f k4 f So P 5 TiQR S? j f k4 f So P 5 TPQR S? Tf 4S f So PQR? TPQR S? Tf 4S f mƒ PQR? T (26) 4.2.2c The influence of liposomes on the kinetics of the elementary reactions In order to determine what the influence of positively charged liposomes is on the kinetic parameters in equation 26, the data from Figure 4.2 were smoothed using a FFT filter, and derived numerically. The resulting evolution of ; >? as a function of [MV + ] was plotted and fitted with equation 27 (Figure 4.3). 99

13 Chapter 4 Figure 4.3. Plot of the numerically calculated quantum yield of MV + production as a function of concentration in photoproduct MV + for the photoreduction of MV 2+ by CysSH catalyzed by 1 4- under blue light irradiation and at different concentrations of CysSH, a) in homogeneous solution, and b) in the presence of 1:1 edmpccl:dmpc liposomes (0.17 mm). The fits were obtained by a global fit of the data using equation 27. The data from which the quantum yield of MV + production was obtained are shown in Figure 4.2. Conditions are equal to those in Figure 4.2. ; >? p : P T P T PQR? T PQR? T P T P T PQR? T *ˆPMV T (27) α f 4 ; β f k ; γph T f 4Sf mƒ f 4S f So iqr S? j f f 4S O 1 f k4 f So PQR S? T ; M U ŒŒ (28) PCysS T O 1 PCysSHT (29) P? T p : 2 : f gh iqr S? j b :OPQR S? T f gh PQR S? Tf Y k \!Y (30) In equation 27, the first term is another mathematical expression of equation 26, but we added an extra term, ˆPMV T, which was not reported by Rougee et al. 23 and is not part of the photocatalytic model. This term describes the monoexponential decay of MV + that occurs also in the absence of light irradiation (see Figure B.1 in Appendix B). MV + has indeed been reported to be unstable in aqueous solution in which it can dimerize, or form a complex with MV In homogeneous solution, the rate constant for this exponential decay (U <<, dd = dark decay) was determined to be s 1 (see Figure B.1 in Appendix B). We thus assume that MV + forms a complex with MV 2+, which would explain the first-order decay pathway. The fitting constants, u, s and ˆ are defined in equation

14 From the value of ˆ (see Table 4.2) we obtained a value for U << of s 1 that is comparable to the value obtained in the dark, indicating that MV + decays via the same dark pathway also under photocatalytic conditions. The concentration of CysS was calculated using its deprotonation constant K a and the constant ph of 7.0 ([H + ] = ) according to equation 29. The fitting parameter p : in equation 27 is the initial quantum yield, i.e., when [MV + ] equals 0, and depends (equation 30) on the percentage of free photosensitizer 1 4 (i.e., the porphyrin that is not complexed with MV 2+ ), the quantum yield of oxidative quenching b VW <, and the cage-escape yield b \!Y Y. From the fit in Section 4.2.1d-d, where PMV T=0, the value for p : with [MV 2+ ] = 0.67 mm was calculated to be 0.18 and 0.57 in homogeneous solution and at the surface of liposomes, respectively. Table 4.2. Fitting constants for the fits of the data shown in Figure 4.3 using equation 11. constant homogeneous liposomal c < 5 u (M) s ˆ (M 1 ) From Figure 4.3, it is clear that in presence of liposomes the initial quantum yield of MV + formation is not only higher than in homogeneous solution, but also that this quantum yield decreases much slower when more photoproduct MV + is formed. This trend is clearly observed by comparing the values for, u and s ( ), as discussed below. The fit could not differentiate between the constants or s; however, the reactions from which the rate constants in s originate, i.e., reactions between intermediates of CysSH oxidation and the photoproduct MV +, do not occur at the surface of the liposome, and are thus not expected to differ very much between the homogeneous or the liposomal sample. Therefore, the two very similar values from both fitting procedures were taken as s. As expected, the value for u does not vary significantly between homogeneous solutions or samples containing positively charged liposomes, as the reactions from which the rate constants in u originate, i.e., deprotonation of CysSH + and reaction of CysSH + with MV + also occur in homogeneous solution. The striking difference between homogeneous and liposome-containing samples becomes clear when comparing in Table 4.2. This constant represents the ratio between the rate constants of product inhibition (k pi, reaction of 1 3 with MV + ) vs. that of reductive regeneration (k rr, reaction of 1 3 with CysSH), see Scheme 4.2. In 101

15 Chapter 4 homogeneous solution, the ratio is 690, thus product inhibition (r pi) is the major component of the decrease in photoreduction quantum yield with increasing concentration of MV +. In contrast, at the surface of liposomes was found too low to obtain a reproducible value; a good fit could be obtained with < 5. In the presence of positively charged liposomes, product inhibition (r pi) is decreased several orders of magnitude due to electrostatic repulsion of MV + by the positively charged surface of the liposomes. The decrease of the quantum yield by an increase in the concentration of MV + is thus solely caused by product inhibition occurring in homogeneous solution (i.e., reactions between oxidized intermediates of CysSH, and MV + ) Conclusion on the effect of positively charged liposomes on the photocatalytic reduction of MV 2+ by CysSH. From the kinetic study it appears that the addition of positively charged liposomes has a large effect on multiple elementary reactions in the photocatalytic reduction of MV 2+ by CysSH. The most obvious reason for the change in kinetics was postulated in Chapter 3: the addition of liposomes prevents the formation of the photocatalytically inactive 1:1 GS complex between the photosensitizer 1 4 and the electron acceptor MV 2+. This effect is visualized by the value of the equilibrium constant for the formation of these complexes, which from our fitted data in Section 4.2.1d was M 1 in homogeneous solution and essentially 0 M 1 in the presence of liposomes (Section 4.2.1e). However, kinetic data fitting with the model shown in Scheme 4.2 and Scheme 4.3 also demonstrates that besides this already reported effect 22 the kinetics of the elementary reactions changed dramatically. When the photosensitizer is bound at the surface of a positively charged liposome, the rate of oxidative quenching of *1 4 by the electron donor CysSH is almost two orders of magnitude lower than in homogeneous solution. Because of this, the quantum yield of formation of the photoproduct MV + at low MV 2+ concentration is lower in presence of liposomes than in homogeneous solution. However, both in homogeneous solution and in presence of liposomes the photocatalytic reduction of MV 2+ is strongly inhibited by the formation of the photoproduct MV +. Two of the possible productinhibition reactions always occur in homogeneous solution (see Scheme 4.3), and their rates thus do not change much upon addition of liposomes. However, once oxidized, the photosensitizer 1 3 reacts several orders of magnitude slower with the photoproduct MV + when liposomes are present, probably due to electrostatic repulsion of MV + away from the membrane. In turn, the reaction of 1 3 with CysSH proceeds with a greater efficiency in the presence of liposomes. Product 102

16 inhibition between the photosensitizer and the photoproduct can thus be prevented by electrostatic repulsion of the charged molecules MV 2+ and MV + from the positively charged lipid bilayer. 4.3 Photocatalytic reduction of DTNB Electrochemical study of the two-electron reduction of DTNB 2 to NTB 2 Unlike the reduction of MV 2+ to MV +, which involves the transfer of only one electron, the reduction of 5,5'-dithiobis(2-nitrobenzoate) (DTNB 2 ) to 2-nitro-5- thiolatobenzoate (NTB 2 ) is a two-electron process (Scheme 4.4). The first electron is accepted into the σ* orbital of the S-S bond of DTNB 2, thus lengthening the S-S bond. 26,27 The resulting disulfide radical anion (DTNB 3 ) is in a thermodynamic equilibrium with a thiyl radical NTB and the thiolate NTB 2. 26,27 Scheme 4.4. The two-electron reduction of DTNB 2 to NTB 2. After the first reduction, a thermodynamic equilibrium between disulfide radical anion and thiyl radical + thiolate exists. 26,27 To find out if the equilibrium is biased towards the disulfide radical anion side, or towards the thiyl and thiolate side, electrochemistry was performed in phosphate buffer at ph=7. Under electrochemical conditions the disulfide DTNB 2 can be irreversibly reduced at E pc = 0.7 V vs. Ag AgCl in phosphate buffer (Figure 4.4, scan 1). The reduction does not show a re-oxidation peak, confirming that the disulfide radical anion is unstable, and reacts quickly to form NTB 2 and NTB. The latter is further reduced at this same potential to form the second molecule of NTB 2. The rate of such cleavage of the S-S bond in the disulfide radical anion has been shown to be in the order of 10 8 s 1 or higher for similar aryl disulfides, 27 thus explaining the irreversible nature of the electrochemical reduction. Upon scanning towards more positive potentials, the electrochemically generated NTB 2 can be oxidized to NTB reversibly at E ½ = V vs. Ag AgCl (Figure 4.4, 103

17 Chapter 4 scan 2). This result highlights the much higher stability of the thiyl radical NTB compared to that of the disulfide radical anion DTNB 3. The equilibrium constant corresponding to the cleavage of the disulfide radical anion bond is thus biased towards the side of the thiyl and thiolate under these conditions. In contrast, after oxidation of electron-rich alkyl thiols such as cysteine 28,29 or glutathione, 30 the corresponding thiyl radical readily reacts with a second thiol/thiolate with a rate constant in the order of 10 8 M 1 s The disulfide radical anion formed has a much lower rate constant for cleavage of the S-S bond (~10 5 s 1 ) 30 thus giving an equilibrium constant for the formation of the disulfide radical anion in the order of 10 3 M 1, favoring the disulfide radical anion at relatively low thiol concentrations (in the order of mm). 30 Thus, it is the electron-poor nature of the disulfide DTNB 2 that is responsible for the fast S-S bond cleavage observed after its one-electron reduction. Figure 4.4. Cyclic voltammogram of H2DTNB (1 mm) in phosphate buffer (0.1 M, ph = 7.0). The first (grey) and second (black) scan are shown separately. A glassy carbon electrode was used as electrode, a platinum electrode was used as auxiliary and a Ag AgCl electrode was used as reference. Scanning was started at the point marked by the arrow in the positive direction at a scan rate of 0.1 V s 1. The small oxidation wave present in scan 1 is explained by the fact that in phosphate buffer at ph 7.0, H2DTNB decomposes very slowly to form NTB Under photocatalytic conditions employing meso-tetra(4-(nmethylpyridinium)porphyrinato zinc chloride (2Cl 4, see Scheme 4.1) as photosensitizer and triethanolamine (TEOA) as electron donor (ED), the transfer of a single electron to DTNB 2 leads to the formation of a NTB radical and of NTB 2-. Due to the limited oxidative potential of NTB, the thiyl radical cannot 104

18 react with any of the starting reagents (i.e., 2 4+, TEOA or DTNB 2 ) and thus must react with other intermediates to prevent the build-up of NTB radicals, which was never observed in our reaction conditions. For this reason, more information was required on this photocatalytic system in order to be able to describe the difference in reactivity between homogeneous conditions and liposomecontaining samples Transient absorption spectroscopy in homogeneous solution. In order to obtain knowledge about the kinetics of the photocatalytic reduction of DTNB 2, transient absorption spectroscopy experiments were realized. The excited state of the photosensitizer *2 4+ has been thoroughly studied in the past. 32 Upon irradiation in the Soret band the positively charged zinc porphyrin 2 4+ is excited into its S 2 state, after which internal conversion occurs to the first singlet excited state S 1. The porphyrin can then relax to the ground state by internal conversion, emission of a photon, or can undergo intersystem crossing to the triplet state T 1 with a quantum yield of This T 1 state is long-lived and decays non-radiatively via internal conversion. We monitored the disappearance of this triplet state in order to study the effects of the presence of DTNB 2 or of NaDMPG liposomes on the photochemistry of the porphyrin. Figure 4.5a shows an overlay of the transient absorption spectra of a solution of 2Cl 4 (3.3 µm) in phosphate buffer (10 mm, I = 50 mm, ph = 7.0) at different time offsets (λ ex = 445 nm, 0.2 mj per pulse, 5 Hz). The spectrum shows ground-state bleaching at 442 nm, a T 1-T n absorbance band at 483 nm and a broad T 1-T n absorbance band from 630 to above 800 nm. After formation these three bands mostly decay (80%) at an equal rate with a lifetime τ of 32(2) µs, with a small additional contribution (~20%) of a longerlived species with a lifetime τ of 0.25(3) ms (Figure 4.5b). In the following we neglected this secondary decay pathway and focused on the faster decay process. The value of the rate constant of decay U < of 3.2(3) 10 4 s 1 is higher than what the literature reports in similar media. 32,33 We ascribe the low lifetime of the T 1 in our conditions to the method of deaeration: argon was bubbled through the solution for 15 minutes prior to measurement, which we assume does not lead to a completely deoxygenated solution. We tested this assumption by performing a different method of deaeration. We freeze-pump-thawed our solutions at least three times (or as many times as needed before evolution of gasses during thawing stopped). Under completely deaerated conditions, the kinetics of decay became one order of magnitude slower (U < = 2.5(2) 10 3 s 1 ), and a second order process indicating triplet-triplet annihilation (TTA) became apparent (see Figure 105

19 Chapter 4 B.2 in Appendix B). The relatively short lifetime of the porphyrin in solution deaerated by bubbling argon (τ = 32(2) µs) compared to freeze-pump-thawed solution (τ = 0.41(1) ms) thus indicates a significant amount of quenching of the T1 state by residual 3O2 in the former case. However, since the amount of dissolved 3O2 is assumed to be constant over the range of experiments deaerated by argon bubbling, the experimental first-order rate constant for the decay of T1 is the addition of the two (constant) decay rate constants: that for internal conversion, and that of dioxygen quenching, according to the formula U< = U8Y + U= P ZO T. Figure 4.5. a) Transient absorption spectrum of a homogeneous solution of 2Cl4 (3.3 µm) in phosphate buffer (10 mm, I = 50 mm, ph = 7.0), T = 293 K, deaerated by bubbling argon through the solution for 15 minutes. Pump laser intensity 0.2 mj per pulse (λex = 445 nm, 5 Hz, pulse-time-width = 3-5 ns). b) Time traces of the transient absorption spectra shown in a, and their global fit as biexponential decay. The residuals are shown on top Transient absorption spectroscopy at the surface of liposomes. Samples containing liposomes cannot be deaerated by freeze-pump-thaw methods, as this method leads to the destruction of liposomes as indicated by precipitation of the lipids upon thawing. Therefore, transient absorption spectra were obtained using deaerating by bubbling argon. In the presence of negatively charged liposomes consisting of NaDMPG at a low bulk lipid concentration (0.17 mm, Zave = 100 nm, PDI = 0.09, for preparation, see Chapter 3) the Soret band of 24+ was red-shifted to 450 nm as described in Chapter 3. The transient absorption spectrum also shows a shifted ground-state bleach band (452 nm), a shifted T1-Tn band at 494 nm, and a similar broad T1-Tn absorbance band from 630 to above 800 nm (Figure 4.6a). Besides the observed shifts, the decay of the 106

20 T1 state was considerably faster compared to homogeneous conditions, and could not be fitted with a mono-exponential function. The addition of higher concentrations of liposomes led to an decrease of the decay rate of the T1 state, and at a NaDMPG concentration of 8.3 mm the data could be fitted by a monoexponential function again, with a lifetime of 83 µs. Our interpretation of the much shorter lifetime observed in the presence of liposomes is that the adsorption of 24+ to the surface of the negatively charged lipid bilayer leads to a much increased local concentration of the dye, which increases the probability of triplet-triplet annihilation (TTA) decay as it is a second-order process (see below).34,35 In other words, increasing the lipid bulk concentration of the NaDMPG liposomes decreases the local concentration of 24+, resulting in an increase of the lifetime of T1. Figure 4.6. a) Transient absorption spectrum of a solution of 2Cl4 (3.3 µm) and NaDMPG as liposomes (0.17 mm, Zave = 100 nm, PDI = 0.09) in phosphate buffer (10 mm, I = 50 mm, ph = 7.0), T = 293 K. Pump laser intensity 0.2 mj per pulse (λex = 445 nm, 5 Hz, pulse-time-width = 3-5 ns). b) time-traces of the transient absorption spectra shown in a, and their global fit as a mixed first and second order decay (equation 32). The residuals are shown on top. The differential equation best describing the decay of the T1 state at the surface of liposomes is given by equation 31. This general model takes into consideration both the first-order decay rate constant U<, which thus includes also quenching by dioxygen (see Part 4.3.2), and the TTA rate constant U. This differential equation can be solved to give equation 32, as described in Appendix B. AP 6 T A( *U< PT: T * U PT: T (31) 107

21 Chapter 4 PT : T= Y f k f k \ k Y f "\ k : (32) We assumed that the unimolecular decay (U < ) does not depend significantly on the bulk concentration of NaDMPG and thus that the decay caused by TTA is the reason for the increase in lifetime with increasing NaDMPG concentration. The dye environment will be qualitatively similar for all lipid concentrations tested as the loading of the porphyrin on NaDMPG is smaller than 10 mol% in all cases, which justifies this assumption. A global fit of the kinetic traces at different NaDMPG concentrations was performed using equation 32 (see Figure 4.7a), with a shared U < and p -. A value of U < = 1.2(1) 10 4 s 1 was found that was slightly lower than that determined for the homogeneous system (3.2(3) 10 4 s 1, see Section 4.3.2). Thus, the adsorption of the sensitizer to negatively charged liposomes does not significantly alter the rate of unimolecular decay compared to homogeneous solution. In addition, the rate constant for TTA, U, was found to be inversely proportional to the concentration of NaDMPG, as shown by the linear relation found in the plot of U as a function of [NaDMPG] 1 (Figure 4.7b). As the concentration of NaDMPG increases, the local concentration of 2 4+ at the membrane will decrease, in turn decreasing U. According to those results, the Figure 4.7. a) Time traces for the decay of the T1 state of 2 4+ at the surface of NaDMPG liposomes, as probed at A494 for 6 different bulk concentrations of NaDMPG (0.17 mm, 0.42 mm, 0.83 mm, 2.1 mm, 8.3 mm and 17 mm). The data-points were globally fitted by equation 32, keeping kd constant for each fit. b) plot of ktta as a function of 1/[NaDMPG]. Each rate constant ktta was obtained from the fits of the time-dependent decay in a. Conditions: 2Cl4 (3.3 µm) in phosphate buffer (10 mm, I = 50 mm, ph = 7.0), T = 293 K. Pump laser intensity 0.2 mj per pulse (λex = 445 nm, 5 Hz, pulse-timewidth = 3-5 ns). 108

22 photophysical characterization of triplet excited-state photosensitizer molecules adsorbed at the surface of liposomes is thus strongly hindered by TTA processes that occur readily due to the high local concentration of the dye. 36 In our case, when the loading of the adsorbed dye at the lipid bilayer was above 1 mol% it became difficult to characterize the mono-exponential decay pathway of T 1 (U < ) as TTA became predominant Oxidative quenching of the T 1 state by DTNB 2 studied by timecorrelated spectroscopy To study the mechanism of photocatalytic reduction of DTNB 2 to NTB 2 by TEOA sensitized by 2 4+, the decay of the T 1 state of the photosensitizer (3.3 µm) was studied in homogeneous solution deaerated by freeze-pump-thawing in the presence of 1 mm H 2DTNB. Under such conditions, a mono-exponential decay was observed, and the lifetime of T 1 was substantially decreased (9.6 µs) compared to a solution deprived of DTNB 2 (0.41 ms). In contrast, reductive quenching by TEOA is not expected due to the low rate constant reported in the literature in a similar medium ( M 1 s 1 ). 37 In our experimental setup, quenching due to TEOA was difficult to measure due to irreversible reduction of Nevertheless, the decay trace (see Figure B.2 in Appendix B) does not indicate significant quenching of the T 1 state. The photoreduction of DTNB 2 by TEOA thus proceeds via an oxidative quenching mechanism. Such oxidative quenching of the T 1 state by DTNB 2 should produce radical molecules with clear UV-vis signatures. 38,39 However, neither the radical cation 2 3+, nor the disulfide radical anion (DTNB 3 ), nor the thiyl radical (NTB ), were observed by transient absorption spectroscopy. We attributed the absence of such signature to a low cage-escape yield (see also Section 4.3.8). By comparing the values of U < in the presence and absence of DTNB 2, the rate constant for the oxidative quenching of *2 4+ by DTNB 2 in homogeneous solution U VW was found to be 1.0(1) 10 8 M 1 s 1. In addition, when the quenching was studied in homogeneous solution deaerated by bubbling argon, a comparable value for U VW of 9.4(9) 10 7 M 1 s 1 was found. Thus, quenching can be studied in solution deaerated by bubbling argon, provided that the efficiency of quenching is high enough (i.e., higher than quenching by 3 O 2). At a DTNB 2 concentration of 1 mm the T 1 is oxidatively quenched with a quantum yield of 98%. Besides quenching the T 1 state, the presence of DTNB 2 also lowers the initial amount of the T 1 state that is formed, as indicated by the 4 times lower initial OD signal ( OD 483 = 0.04 vs in presence and absence of DTNB 2, respectively). This lower concentration of T 1 109

23 Chapter 4 indicates that addition of DTNB 2 also quenches an excited state that is formed prior to the T 1 state, as discussed in Section The decay of T 1 in the presence of DTNB 2 was also studied at the surface of NaDMPG liposomes. Due to the high rate of TTA at the surface of liposomes at high local concentration of 2 4+ we monitored the decay of the T 1 state at a high NaDMPG concentration (8.3 mm). Even at such high lipid concentration an increase in the decay rate constant upon addition of H 2DTNB was not observed (U < = 1.2(1) 10 4 s 1 vs. 0.8(4) 10 4 s 1 in the absence or presence of H 2DTNB, respectively). These results indicate that in presence of negatively charged liposomes the rate of oxidative quenching by DTNB 2 is very low at a DTNB 2 concentration of 1 mm. Thus, the oxidative quenching rate between porphyrin 2 4+ adsorbed to the surface of a negatively charged membrane, and in presence of negatively charged electron acceptors dissolved in the bulk aqueous phase, is decreased by several orders of magnitude compared to homogeneous conditions, which is similar to what we observed for the photoreduction of MV 2+ (Section 4.2.1e). This finding suggests that the formation of the photoproduct in the presence of liposomes is rate-limited by the quantum yield of quenching of the excited state by either DTNB 2 or TEOA, whereas in homogeneous solution, a different process must limit the quantum yield Quenching of the singlet excited state S 1 in homogeneous solution The first singlet excited state (S 1) of 2 4+ is generated from photon absorption prior to T 1 state formation. A decrease in the initial OD value in transient absorption of the T 1 state can thus be explained by quenching of the S 1 state. The S 1 state fluoresces with a quantum yield of 2%, 32 thus allowing monitoring of this state by time-resolved fluorescence spectroscopy. For this purpose, the fluorescence of 2 4+ at 610 nm was monitored by time-correlated single-photon counting. In homogeneous solution (phosphate buffer, 10 mm, I = 50 mm, ph = 7.0) at room temperature, the fluorescence intensity of 2 4+ decays monoexponentially with a lifetime of 1.3(1) ns, in agreement with the reported value. 32 Upon addition of DTNB 2 (1 mm), the decay becomes biexponential with lifetimes of 1.2 ns and ~0.2 ns (see Figure 4.8a). In Chapter 3, we showed that 2 4+ forms a 1:1 GS complex with DTNB 2, i.e., {2 4+ DTNB 2 }. Our interpretation of the biexponential decay of the fluorescence decay in the presence of DTNB 2 is that this 1:1 GS complex is not quantitatively quenched in the excited state, but that it fluoresces with a S 1 state lifetime of ~0.2 ns. From the pre-exponential factors from the fit of the data in Figure 4.8a the ratio of complexed vs. uncomplexed free sensitizer 2 4+ was 3.0. This ratio corresponds to a thermodynamic equilibrium 110

24 Figure 4.8. a) Time-dependent single-photon counting experiment in an homogeneous solution of 2Cl4 in presence (grey) or in absence (black) of H2DTNB. Inset: logarithmic plot of the data. b) Time-dependent single-photon counting experiment in a solution of 2Cl4 containing NaDMPG liposomes at different bulk lipid concentrations (from black to grey: 0.17, 2.1, 4.2, 8.3 and 17 mm). Common conditions: 2Cl4 (3.3 µm), H2DTNB (1 mm) in phosphate buffer (10 mm, I = 50 mm, ph = 7.0), λex = 445 nm, λmon = 610 nm. constant of complex formation of K = M 1 if we assume that the quantum yield of fluorescence is the same for the complex and for the uncomplexed photosensitizer. With the assumption that the complex {2 4+ DTNB 2 } does not yield any long-lived T 1 state upon excitation, the equilibrium constant could be calculated from the ratio of initial OD value (see 4.3.4) using equation 33, which gave K = M 1. š? š =1i œ žš Ÿ S5 j 1 PDTNB T (33) Pœ? T These two values agree reasonably well with the value obtained from steadystate UV-vis data as reported in Chapter 3 (K = M 1 ). In summary, in homogeneous solution the presence of DTNB 2 leads to both dynamic quenching of the T 1 state, and static quenching of the S 1 state The S 1 state in the presence of negatively charged liposomes. The emission of the singlet state of 2 4+ was also monitored in the presence of negatively charged NaDMPG liposomes (Z ave 107 nm, PDI = 0.12). At a NaDMPG bulk concentration of 0.17 mm, the S 1 state decays biexponentially with a short lifetime of ~0.3 ns and a long lifetime of 1.1(1) ns with equal contribution (Figure 4.8b). The long lifetime is comparable to the lifetime in homogeneous solution 111

25 Chapter 4 and the presence of a faster component is indicative of ground-state interactions of 2 4+ at such high loading (10 mol% of 2 4+ ). As the concentration of NaDMPG was increased to 2.1 mm, the fast component disappeared and the decay became almost identical to that of the homogeneous system (τ = 1.2(1) ns). Upon increasing the concentration of NaDMPG beyond 2.1 mm, the presence of a slower decay component (τ 9 ns) became apparent (Figure 4.8b inset). The origin of this component is unexplained, however its contribution to the overall decay is less than 2%, and thus it was neglected with regard to the decay component with a lifetime of 1.3 ns. The addition of 1 mm H 2DTNB to the sample with 8.3 mm NaDMPG liposome did not lead to any significant difference in the fluorescence lifetime (1.3 ns), concluding that at the surface of liposomes, DTNB 2 does neither statically, nor dynamically, quench the S 1 state of 2 4+ at this concentration Applying the MV 2+ model to the photoreduction of DTNB a Dependence on the concentration of DTNB 2 By time-resolved absorption and emission spectroscopy we have shown that in homogeneous solution the photocatalytic reduction of DTNB 2 is initiated by dynamic oxidative quenching of the triplet state T 1. Furthermore, the singlet state S 1 is statically quenched by DTNB 2, as indeed the GS complex {2 4+ DNTB 2- } was already postulated in Chapter 3 not to lead to product formation due to rapid charge recombination. This quenching pattern leads to a situation that is strikingly similar to the reaction discussed in Section for the photoreduction of MV 2+ by TEOA. Therefore, we fitted the data obtained in Chapter 3 for the dependence of the initial quantum yield of NTB 2 production (; Ÿ S5) on the concentration of DTNB 2 at a constant TEOA concentration (8.3 mm) using equation 34, a mathematical equivalent to equation 17 (see Figure 4.9a). ; Ÿ S5 = AiŸ S5 j A( : E =2 p - q : iš Ÿ S5 j + Oiš Ÿ S5 j :OPš Ÿ S5 T Pš Ÿ S5 Tr :OPš Ÿ S5 T st (34) u = f k f gh ; s fl gh l f l k f gh fl 1 f l m f 1 l (35) b \Y =q : iš Ÿ S5 j + Oiš Ÿ S5 j :OPš Ÿ S5 T Pš Ÿ S5 Tr :OPš Ÿ S5 T st (36) 112

26 Figure 4.9. a) Plot of the initial quantum yield of NTB 2 production (; Ÿ S5) vs. H2DTNB concentration at constant TEOA concentration (8.3 mm) during the photochemical reduction of H2DTNB by TEOA catalyzed by 1 4+ in homogeneous conditions. The dataset was taken from Chapter 3 and fitted with equation 34. b) Plot of the initial quantum yield of NTB 2 production as a function of TEOA concentration at constant H2DTNB concentration (0.67 mm). The new dataset was fitted with equation 48. Common conditions: 2Cl4 (3.3 µm), T = 298 K, blue light irradiation (450 nm, θ = 4.0 nmol s 1 ). The probability that the encounter complex {œ :DTNB Z L is formed via the two distinct photochemical pathways is defined as b \Y (equation 36). If we assume that static quenching (i.e., the second term of the addition in equation 34) does not lead to any product, i.e., if we set s = 0, a fit of the data gives a value for K of M 1. This value is lower than all other values obtained thus far for this equilibrium constant. When we, however, included the second term of the equation, i.e., allowed s > 0, and thus included excitation of the GS complex as a pathway leading to the formation of NTB 2, a good fit could be obtained by fixing the constant K at M 1 (value taken from Chapter 3). From this fit a value for the initial quantum yield of NTB 2 formation from absorption of a photon by the {2 4+ DTNB 2 } complex was obtained leading to 2 p - s = 0.46% at a TEOA concentration of 8.3 mm. From the fit a value for u of M was also obtained that fits the value obtained from transient absorption data in freezepump-thawed solution (U VW = 1.0(1) 10 8 M 1 s 1, U < = 2.5(2) 10 3 s 1, U < /U VW = M). During steady-state measurements, the residual dioxygen is consumed during the initial lag-phase of the irradiation by reaction of 1 O 2 with TEOA, thus giving a completely deoxygenated solution. The low value of u confirms that the dynamic oxidative quenching is highly efficient under constant 113