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Photophysics of the Singlet Oxygen Sensor Green Chromophore: Self-Production of O Explained by Molecular Modelling 1

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Marco Marazzi, Vanessa Besancenot, Hugo Gattuso, HenriPierre Lassalle, Stephanie Grandemange, and Antonio Monari J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b04383 • Publication Date (Web): 24 Jul 2017 Downloaded from http://pubs.acs.org on July 24, 2017

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The Journal of Physical Chemistry

Photophysics of the Singlet Oxygen Sensor Green Chromophore: Self-Production of 1O2 Explained by Molecular Modelling Marco Marazzia,b, V. Besancenotc, Hugo Gattusoa,b,*, Henri-Pierre Lassallec, Stéphanie Grandemangec, Antonio Monaria,b,* a

Université de Lorraine Nancy, Theory-Modeling-Simulation SRSMC, Vandeouvre-lès-Nancy,

France. c

b

CNRS, Theory-Modeling-Simulation SRSMC, Vandeouvre-lès-Nancy, France.

Université de Lorraine Nancy and CNRS, CRAN, Vandeouvre-lès-Nancy, France.

Corresponding Author *send correspondence to: [email protected], [email protected]

ABSTRACT. We report a combined computational and experimental study in order to rationalize the behavior of a well known singlet oxygen (1O2) probe, i.e. the chromophore of the Singlet Oxygen Sensor Green: a fluoresceine-based sensor. In particular we evidence that the presence of an intramoleculer charge transfer state that is no more present upon reaction with 1O2 explains the fluorescence enhancement observed in presence of reactive oxygen species. Furthermore we also unequivocally show the photophysical pathways leading to fluorescence enhancement of fluoresceine upon irradiation with UVA lights and also in the absence of any

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oxygen activator. More specifically we evidence that the presence of a possible intersystem crossing upon population of higher energy singlet electronic excited states will lead to the population of the fluoresceine triplet manifold and hence to the self-production of 1O2.

INTRODUCTION Singlet oxygen (1O2) is very well known reactive oxygen species (ROS), which is able to efficiently oxidize a large variety of organic compounds.1–4 As such it has also been demonstrated that 1O2 has a strong biological significance. Indeed, oxygen activation in biological tissues and cells leads to the oxidation of different biological macromolecules such as nucleic acids,5–8 protein aminoacids,9,10 or unsatured and polyunsaturated lipids.11 The accumulation of 1O2 and the consequent oxidative stress produced may lead to cellular apoptosis. For this reason, 1O2 is also routinely used in antibacterial, antiviral and even anticancer therapy, in particular in the field of photodynamic therapy (PDT).12–20 Application of 1O2 in depollution and water treatments has also been reported successfully.21 Although the energy difference between ground state triplet and singlet oxygen is extremely modest the direct activation of molecular oxygen is precluded by the spin-forbidden singlet-triplet transition. As such, singlet oxygen is produced indirectly by activation of a sensitizer,22,23 usually absorbing in the visible or near infrared region and undergoing intersystem crossing and hence populating its triplet manifold.4 Subsequently energy transfer from the sensitizer’s triplet toward molecular oxygen will occur resulting in the production of 1O2.


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O

O

HO

Cl COOH

a) Fl CH3

HOOC

O

O

HO

Cl COOH

O O

b) FlEndo CH3

HOOC

Figure 1) Molecular structures of the a) the fluoresceine-based chromophore (Fl) present in the SOSG probe and b) of the product of its reaction with 1O2 (FlEndo) leading to the formation on an endoperoxide on the anthracene moiety. Due to its relevant biological and chemical role, the quantitative detection of 1O2 is crucial. Infrared emission at 1268 nm is regarded as a key signature of the presence of singlet oxygen, however such a direct measurement is hampered by technical difficulties preventing its large applications.24–28 Conversely, the use of fluorescent probes has proven extremely efficient in the dosage of 1O2:29–33 in this strategy singlet oxygen is measured by the change of emission properties of a given dye in presence of 1O2. The aforementioned change in the optical properties may arise from different reasons, for instance the reaction of the probe with 1O2 will induce a change in its emissive properties.


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Among the different commercial probes we may cite the commercially available Singlet Oxygen Sensor Green (SOSG) that experiences a strong fluorescence enhancement in presence of 1O2.34,35 SOSG is based on a fluoresceine derivative chromophore covalently bound to an anthracene moiety (Fl, Figure 1a).29 The recognition mechanism of SOSG is thought to be due to the reaction of Fl with 1O2 to produce an endoperoxide on the anthracene moiety (FlEndo, Figure 1 b). While the lowest lying excited state of Fl, of charge transfer nature is thermically quenched, the π−π* state of FlEndo is an efficient fluorophore. However, a stunning characteristic of Fl, is the fact that if the probe is irradiated in the UVA region, instead that in the visible light operating condition, one observe a very strong fluorescence enhancement. Furthermore it has been reported that Fl itself can eventually produce small amount of 1O2.36 This behaviour has been interpreted recalling the possible sensitization of 1O2 by the SOSG anthracene moiety triplet state.29 The hypothesis of a two-photon absorption (TPA) in the UVA region leading to Fl photoionization is also invoked.29 However such hypothesis would require the population, after TPA of a high energy excited state, and furthermore TPA being a non-linear phenomenon strong light intensity would be required. In this contribution we experimentally prove the fluorescence enhancement of Fl after irradiation at 360 nm with low intensity. Subsequently by molecular modelling we rationalize the behaviour of Fl and FlEndo, and in particular we prove the possibility of the occurrence of an intersystem crossing responsible for the self-production of singlet oxygen.

COMPUTATIONAL AND EXPERIMENTAL PROTOCOLS Computational Protocol.


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Fl and FlEndo ground and excited state properties have been calculated using density functional theory (DFT) and its time dependent extension (TD-DFT), respectively. Dynamical and vibrational properties effect on the absorption and emission spectra have been estimated using a Wigner distribution to sample the equilibrium geometry region. In that strategy the final spectrum is obtained as the convolution of individual transitions from different representative geometries selected via a proper exploration of the potential energy surface around equilibrium thanks to its Hessian matrix (i.e. the harmonic vibrational frequencies). For the reader’s convenience we remind that such a strategy, allows to efficiently take into account the vibrational induced red shift of the absorption spectrum especially due to large amplitude lowfrequency out-of-plane vibrations; moreover a good reproduction of the band shape is usually achieved.8,37–39 In addition to the Wigner based sampling one can rely either on snapshots extracted from a classical molecular dynamics trajectory, even if then one loses the quantum description of the potential energy surface. A complementary solution is the explicit calculation of Franck-Condon coupling as proposed by Santoro and coworkers, even though this strategy is more adapted to high-frequency vibrational modes.40,41 Fl and FlEndo ground state geometries have both been optimized at DFT level using B3LYP42 and 6-31G(d) basis set withj the Gaussian09 code43. The calculation of vibrational frequencies has confirmed the minimum nature of the optimized geometry and allowed to obtain the Wigner distribution. Remarkably, although the system is complex being composed of three independently bound fragments the sterical hindrance of the fused rings leads to only one relevant conformer. Indeed any other arrangement than having the two peripheral groups perpendicular to the aromatic core will lead to significant sterical clashes. Note that harmonic frequencies of about 10 cm-1 have been found corresponding to out-of-plane vibration of the


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conjugated rings. Environment effects have been taken into account by using the polarizable continuum model (PCM) to describe water solution. Excited state spectra have been obtained using different functionals, the hybrid B3LYP,42 the long range-corrected CAM-B3LYP44 and the meta-hybrid M06-2X45; the 6-31+G(d) basis set has been used. To better reproduce experimental spectra shapes individual vertical transitions have been convoluted with a Gaussian function of full-width at half-length (FWHL) of 0.2 eV. The nature of the different excited states has been analysed using the natural transition orbital formalisms (NTO),46 the φS index47–49 has also been used to quantify the charge transfer nature of the different states. Excited state topological analysis has been performed post-processing Gaussian output with the Nancy_EX suite of codes.47,48 The comparison of the absorption spectrum obtained using different functionals is reported in ESI (Figure S1). It is noticeable that CAMB3LYP and M06-2X gives exactly the same position of vertical transitions and the same band shape. On the contrary B3LYP gives a much red shifted spectrum with the most intense band being shifted of about 60 nm. For this reasons, and also since the photophysics will depend crucially on the presence of charge-transfer excited states we relied on CAM-B3LYP for the calculation of dynamically corrected absorption and emission spectra. All the spectra presented in the main text are obtained using CAM-B3LYP. Subsequently, the first singlet excited state (S1) of both Fl and FlEndo have been optimized at CAM-B3LYP/6-31+G(d) level to avoid the bad description of charge-transfer state using hybrid functionals. Harmonical vibrational frequencies have been obtained providing a Wigner distribution, used to calculate the emission spectra at CAM-B3LYP/6-31+G* level, solvent relaxation has been taken into account via the state specific equilibrium PCM.50 Wigner distribution has been obtained post-processing Gaussian09 output by the Newton-X code.51


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Finally to estimate the probability of intersystem-crossing triplet energies as well as singlet/triplet spin-orbit coupling have been calculated at the Franck-Condon region using the TD-DFT implementation52 of Dalton53,54 based on the single residue of the quadratic response.

Singlet Oxygen Sensor Green (SOSG) Experiments: 5 mM stock solution of SOSG (Molecular Probes, USA) was prepared in methanol according to manufacturer's instructions. Immediately before use, 1µM working solution was prepared in sterile injection water. SOSG solution were irradiated with 365 nm UV lamp and fluorescence emission spectra (λex=504nm, λem=525nm) were recorded using a Perkin Elmer LS55 fluorimeter. Absorbance spectra were recorded using a Perkin Elmer Lambda 35.

RESULTS AND DISCUSSION The calculated absorption and emission spectra of Fl and FlEndo are reported in Figure 2 together with the experimental absorption spectrum in water and methanol (Figure 2c). The measured absorption spectrum in water shows a band in the visible ~525 nm with a tail extending to around 550 nm, a more intense band is also present in the UV region. Notice that the visible band is the most sensitive to the environment and is blue-shifted in methanol, this aspect may indeed be due to the presence of charge transfer states whose energy is more sensitive to the solvent polarity. As far as the calculated spectra are concerned, we notice that our protocol is able to correctly reproduce the optical properties of both the original dye and of the endoperoxide adduct. In particular we notice the presence of absorption maximum in the visible


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range (~425 nm) that matches the experimental results and is due to the excitation to the bright π−π* state (S2). This feature is common for both chromophores, i.e. Fl and FlEndo with the latter only presenting a slightly red-shifted absorption (less than 5 nm). Although it is less interesting for common applications, we also notice a very intense maximum in the UV range (~250 nm) for both species, even if in the case of FlEndo its intensity is reduced by a factor of five. In addition, and more interestingly, we also notice the presence of a lower intense band in the UVA region centered on 350 nm.

0.7

1

Fl EndoFl

Fl EndoFl

0.9

0.6

a)

0.5

0.8

b)

0.7 0.6

φ

0.4

f

0.5

0.3 0.4 0.3

0.2

0.2

0.1 0.1

0 250

300

350

400

450

0 400

500

500

600

λ(nm)

700

800

900

λ(nm) 0.14

SOSG Water SOSG Methanol

0.12

c) 0.1

0.08

f

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0.06

0.04

0.02

0 250

300

350

400

450

500

550

λ(nm)

Figure 2) Calculated absorption (a) and emission (b) spectra of Fl and FlEndo, and the experimental absorption spectrum of SOSG in water and methanol (c). Calculated spectra have been obtained as convolution of structures extracted from Wigner distribution around the S0 (absorption) and S1 (fluorescence) minima, respectively.


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As far as the band shapes are involved, we may observe that the band in the visible region is quite broad, and in the case of Fl we notice a very evident shoulder at around 470 nm, that disappears for FlEndo. On the other hand, both in the case of Fl and FlEndo we also observe a shoulder in the blue region centred around 370 nm. The difference between the two species is, as expected, much enhanced in the case of the emission (fluorescence) spectrum reported in Figure 2b. Indeed, while FlEndo gives a very intense emission band in the visible and more specifically green region of the spectrum, FlEndo only gives a weak and extremely broad band extending up to the infrared region. Hence, we have been able to correctly recover the fluorescence enhancement caused by the 1O2 reaction’s products and to confirm the role played by the endoperoxide adduct. Notice that, compared to the experimental results29 our values of the fluorescence are slightly blue-shifted, but this effect can be ascribed to a non-optimal treatment of the solvent reorganization, already observed in similar systems, and should not change the main conclusion, i.e. emission enhancement by FlEndo.

S1

S1

Φs 0.47

Φs 0.83

b) FlEndo

a) Fl

S2

S2

Φs 0.43

Φs 0.86

Figure 3) Natural transition orbitals (NTO) describing the S0àS1,S2 transition for Fl (a) and FlEndo (b).


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From a photophysical point of view the previous behavior can be rationalized and confirmed by inspecting the natural transition orbitals of the two species describing the lowest lying excited states (Figure 3), whose corresponding transition energies and oscillator strengths for the vertical transitions are given in ESI. In Figure 3a) we report the NTO for the Fl chromophore, one immediately sees that the S1 state is characterized by an extended charge-transfer with the electron density withdrawn from the electron donor anthracene to the fluoresceine core. On the contrary the S2 state is local and has an evident π−π* character. These qualitative observation are also confirmed by the fact that value of the φS index being of 0.4 for S1 and of 0.8 for S2. Indeed, we remind the reader that values of φS close to 0 mean a very low spatial overlap between the ground and excited state electron densities, and hence are indicative of charge transfer, while values close to 1 are observed for local excitation leading to a high overlap of the electronic densities. The charge transfer state being characterized by a much lower oscillator strength than the π−π* state (ratio of about 1/10) it is responsible for the long-wavelength shoulder observed in the absorption spectrum of Fl. On the other hand, and because of the breaking of anthracene conjugation induced by the endoperoxide formation, the charge transfer is no more available in the case of FlEndo. The NTO describing the S1 state (Figure 3b) are indeed indicative of a local π−π* state, as well as the value of φS. Furthermore, it appears evident that FlEndo’s S1 is characterized by the same electron density reorganization observed for the S2 state in Fl. Hence the two bright states are correlated and should be characterized by the same electronic and optical properties, as it is confirmed by the great similarity between the absorption spectra of the two chromophores.


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The situation is on the other hand more complex in the case of emission. Indeed, even if in both chromophores the bright π−π* state will be populated upon excitation with visible light, in the case of Fl, and coherently with Kasha’s rule, the π−π* state will experience internal conversion and will non-radiatively populate the charge transfer state (S1). Due to its charge transfer nature the S1 state will be almost dark and the preferential relaxation pathways will be through non-radiative phenomena. This hypothesis has also been confirmed by the fact that the charge-transfer is always the lowest lying excited state both at Franck-Condon and at the π−π* equilibrium geometry. On the other hand, in the case of FlEndo, absorption in the visible wavelengths will result in the population of the π−π* S1 state, that after relaxation to its minimum geometry will lead to fluorescence emission. Hence we have rationalized the photophysical and photochemical principles allowing the use of SOSG as an efficient 1O2 probe. Indeed, and as correctly suggested by previous works,29 the quantitative reaction with the photoactivated singlet oxygen transforms the non-emissive Fl in the emissive FlEndo, by suppressing the non-radiative relaxation induced by the presence of the charge transfer from anthracene to the fluoresceine core. Hence, 1O2 can be dosed simply following the fluorescence enhancement upon irradiation. The previous argument holds only when the irradiation is performed at visible wavelengths, i.e. resulting in the excitation of the S2 state of Fl. However, certain photosensitizers leading to singlet oxygen production may have absorption in the UVA region. Because of the relatively high reactivity of 1O2 a temporal and/or spatial separation of the oxygen activation and the probe excitation light sequence would be difficult and certainly will hamper the routinely use of singlet oxygen probes, hence the same pulse should be used for production and detection.


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This is for instance the case of the biological and environmentally relevant benzophenone, or its derivative the well-known drug ketoprophene, whose carbonyl n-π* and π−π* transitions absorb at around 370 nm. Furthermore, as observed in Figure 2, both Fl and FlEndo present absorption maxima, even if less intense, in the UVA region and are characterized by shoulders in the absorption band at around 370-380 nm. Hence, we investigate the photophysical and photochemical pathways opened upon irradiation with shorter wavelengths and their effect in the 1

O2 detection ability.

Figure 4) Experimental fluorescence enhancement observed for different irradiation time of a water solution of SOSG upon UV irradiation.

In Figure 4 we report the time evolution of the fluorescence enhancement of the SOSG chromophore (Fl) in water solution subjected to irradiation at 365 nm. One may easily conclude that, even in absence of other sensitizers, the steady and regular increase in fluorescence intensity could be related to the production of FlEndo. However, in order for the reaction to occur


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activation of 1O2 is necessary, and since no other photosensitizers are present this should be accomplished by Fl itself. In order to rationalize the process we have calculated the spin orbit coupling (SOC) between the excited states that may be populated upon absorption at 365 nm. Strikingly enough for an organic compound we observe that the S4 state presents an extremely high SOC with the first triplet state (T1), its value exceeding 240 cm-1. Even if the energy difference between the two states (~2 eV) is relatively high the value of the coupling is sufficient to allow the opening of a relaxation pathways leading to intersystem crossing and triplet population. In addition we also observe the possible intersystem crossing pathway happening from the S5 state to the quasidegenerate T3 or T4. Indeed the SOC between the singlet and the two triplets is of 11 cm-1, while the singlet-triplet energy difference amounts to only 0.6 eV. Notice, that the previously given ordering of the states is the one at the Franck-Condon geometry. In reality due to vibrational and dynamical effects and to the small energy gap, exchanges in the S4, T3, T4 state order can happen leading to different state population, or more likely to a complex equilibrium between the different states. The singlet-triplet energy difference and the corresponding SOC for the six lowest excited states are given in ESI. The proposed photophysical mechanism is illustrated in Figure 5. Upon absorption in the UVA region, at the Franck-Condon region, we observe the possible population of the S4 or S5 states that, due to the high SOC, allows the opening of two photophysical pathways: either remaining on the singlet manifold and proceeding through internal conversion to the non-radiative relaxation from S1 or populate the triplet manifold after intersystem crossing (S4,5 à T1,2,3). In presence of oxygen the T1 state will transfer its energy to produce 1O2, which in turn will react with Fl to produce FlEndo. The accumulation of FlEndo will than induce the fluorescence


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enhancement as previously illustrated.29,36 Our results propose a complementary explanation to the self-production of 1O2 by SOSG, and more generally by fluoresceine based probes. In particular, we univocally prove the possible, and rather efficient, or at least competitive, population of the triplet manifold that could indeed lead to singlet oxygen activation.

Fl

FlEndo SOC ~ 10 cm-1 ΔE ~0.6 eV T3,4

S4,5,n S2 π π S1 CT

ISC SOC ~ 200 cm-1

IC

T1

Sn S1 π π

3O

2

1O

2

Fluorescence S0

S0

Figure 5) Schematic representation, via the Jablonski diagram of the fluoresceine photophysics, illustrating the possible population of the triplet manifold upon UVA irradiation, followed by the activation of 1O2. Solid arrows indicate radiative phenomena (absorption or emission) while dashed lines represent non radiative decays.

CONCLUSIONS We have performed a state-of-the-art study of the optical and photophysical properties of a commercial and widely used singlet oxygen probe. In particular we have been able to correctly reproduce the absorption properties of the Fl dye and of its derivative FlEndo, obtained by reaction with 1O2. Emission has also been correctly reproduced and in particular we have been able to model the strong fluorescence enhancement of FlEndo compared to Fl. This property has


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been ascribed to the presence of a charge-transfer excited state in the case of Fl that indeed quenches luminescence. In the case of FlEndo, on the contrary, the charge-transfer state is no more accessible. Furthermore, we have been able to prove experimentally and computationally the capacity of Fl to self-activate 1O2 upon excitation in the UVA range. By calculating both the singlet and triplet excited states manifolds properties, and in particular their spin-orbit couplings, we have clearly seen that UVA opens the way to a possible intersystem crossing pathways leading to Fl triplet population and consequently oxygen activation. Those channels become available due to the population of either a singlet excited state characterized by a very high SOC (>200 cm-1), or a singlet states presenting reasonable SOC (~10 cm-1) and a low energy difference with the nearby triplets. In the future we plan to extend the present work performing non-adiabatic molecular dynamics in order to access the triplet population quantum efficiency and time scale. Our results may also lead to the proposition of SOSG chemical modification and functionalization to suppress the population of the intersystem crossing leading states and hence eliminating the self-production of singlet oxygen.

ASSOCIATED CONTENT Supporting Information. Comparison of the absorption spectrum of fluoresceine obtained with different functionals and the with the static and dynamic (Wigner distribution) approach, vertical excitation energies and oscillator strength, singlet triplet energy difference and corresponding SOC values, optimized geometries for S0 and S1. The following files are available free of charge ESI_fluoresceine-rev.pdf.


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AUTHOR INFORMATION The authors declare no competing financial interests. ACKNOWLEDGMENT We gratefully acknowledge support from the COST action “MOLIM: Molecules in Motion”, and Lorraine region under the “Idea” project. M. M. is thankful to the French and Austrian National Research Agencies (ANR and FWF, respectively) for a grant under the “DeNeTheor” project.

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Page 24 of 24

TOC GRAPHICS

do FlEn S n

Singlet Oxygen Produc2on Fl

π

nce sce ore Flu

-1

0 cm ~ 1 V SOC 0.6 e ΔE ~

S 1 π

T 3,4

S 0 3 O 2

-1

S 4,5,n

cm ISC ~ 200 SOC

π S 2 π T S 1 C

T 1 IC

1 O 2

Fluorescence Enhancement

S 0


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Photophysics of the Singlet Oxygen Sensor Green ... - ACS Publications

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