Off States of Photoswitchable Probes in Biological

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Triggering On/Off States of Photoswitchable Probes in Biological Environments Silvio Osella, and Stefan Knippenberg J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b13024 • Publication Date (Web): 02 Mar 2017 Downloaded from http://pubs.acs.org on March 2, 2017

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Journal of the American Chemical Society

Triggering On/Off States of Photoswitchable Probes in Biological Environments

Silvio Osella,‡ Stefan Knippenberg

Division of Theoretical Chemistry and Biology, School of Biotechnology, Royal Institute of Technology, SE-10691 Stockholm, Sweden.

AUTHOR EMAIL ADDRESSES: [email protected] (SO); [email protected] (SK)

‡

Current address: Centre of New Technologies, University of Warsaw, Banacha 2C, 02-097

Warsaw, Poland.

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Abstract The use of hybrid systems for which the change on properties of one component triggers the change in properties of the other is of outmost importance when ‘on/off’ states are needed. For such a reason, azobenzene compounds are one of the most used probes due to their high photoswitching efficiency. In this study, we consider a new derivative of azobenzene interacting with different lipid membrane phases as a versatile fluorescent probe for phase recognition. By means of a multiscale approach, we found that the cis and trans conformers have different positions and orientations in the different lipid membranes (DOPC for the liquid disordered phase and DPPC for the gel phase), and these have a profound effect on the optical properties of the system, for both one and two photon absorption. In fact, we found that the cis state is the ‘on’ state when the probe is inserted into the DOPC membrane, while it is in the ‘off’ state in the DPPC membrane. This behaviour enhances the selectivity of this probe for phase recognition, since the different environments will generate different response on the same conformer of the probe. The same effect is found for the fluorescence anisotropy analysis, for which the trans (cis) isomer in DOPC (DPPC) presents a fast decay time. Due to the ‘on/off’ effect it is possible to screen the different membrane phases via fluorescence decay time analysis, making this new probe versatile for phase detection.

KEYWORDS Azobenzene, Photoswitching, Lipid Bilayer, Optical Properties, Fluorescence Lifetime, Molecular Dynamics, QM/MM.


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INTRODUCTION The ability of different molecular systems to change their properties under the control of light paved the way to a vast research field that has found extensive applications in areas ranging from molecular nanotechnology to molecular probes and organic electronics.1,2 In particular, the ability of switching properties in a reversible manner is of crucial importance when ‘on/off’ states are needed, with a high stability and reproducibility, such as for organic field effect transistors (OFET), biosensors, photonic and nanoscale device applications.3-7 In this view, one of the most used compounds is azobenzene and its derivatives. In fact, this molecule shows an extremely high photostability and an ease of functionalization that makes it virtually applicable for any kind of analysis and application.8-10 The kernel of the wide use of azobenzenes relies in the reversible trans to cis isomerization of the N=N bond upon photoexcitation, and the stability of the cis metastable state. Understanding this mechanism has been a hot topic for many years, and although progresses have been made, there is still no uniform view on the mechanistic details of this process.11-13 From the experimental point of view, there is no clear interpretation of the excited state dynamic spectroscopy, mainly due to the lack of spectral resolution in spite of temporal resolution, resulting in a loss of vibrational structure of the excited states, which provides fingerprints of the potential energy surface along the normal modes.14 As in the case of photochromism within naphtyridene and quinoline derivatives,15 where the potential energy surfaces of the first excited singlet and triplet states are decisive for the excited state dynamics as well as the nonadiabatic crossing between the different electronic states, from a computational point of view the description of the mechanism for the trans/cis isomerization of azobenzene is challenging.16,17 In addition, the embedding of the molecule in different environments (either solvent or biological) can lead to different pathways. The accepted description of the isomerization process for azobenzene considers the presence of two



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possible pathways, concerning the torsion along the C-N=N-C dihedral or the inversion along the C-N=N angle. It has been suggested that in viscous media the second prevails, while in non-viscous solvents the torsion is favoured, but most probably a mixed mechanism involving both pathways takes place, and only in limiting cases one prevails on the other.18 In addition, a three state model should be considered for the process, with absorption of a photon from the S0 state of the trans isomer into the bright ππ* second singlet S2 state, followed by a decay into the dark nπ* S1 state and finally by a decay either through a conical intersection (S0/S1 CI) or through weak fluorescence, to the S0 state of the cis isomer.19 Interestingly, the trans to cis quantum yield is higher for the nπ* absorption than for the ππ* absorption, in violation of the Kasha rule were they should be the same. If the fluorescence pathway can be controlled by functionalization of the azobenzene molecule through molecular design, the use of this system for biosensing can be enhanced. In particular, fluorescent probes are of high interest for lipid membrane phase recognition, since it is possible to experimentally differentiate between the phases trough fluorescence spectroscopies, microscopies and fluorescence lifetime imaging (FLIM) techniques.20-22 The phase in which the lipid membrane is present is crucial to recognise between a healthy and an ill cell. Three different membrane phases are present in nature: solid gel (So), characterized by low lateral diffusion and high order parameter (degree of ‘anti’ conformation of the alkyl tails); liquid ordered (Lo), with high diffusion and high order parameter; liquid disordered (Ld), with high diffusion and low order parameter. These three phases can be obtained by varying temperature of a single phospholipid membrane (such as DPPC, in the So phase at room temperature and in the Ld phase over 39 °C) or by mixing different classes of lipids such as phospholipid, sphingholipid and cholesterol in various ratios.23,24 The most common procedure used for the phase recognition is to insert a fluorescent probe into the membrane vesicle, and the difference in orientation of the transition dipole moment 4 ACS Paragon Plus Environment

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of the probe is of utmost importance to discriminate between different membrane phases. If the interaction between the probe and the head of the membrane is weak, the transition dipole moment is randomly oriented in the membrane. A strong interaction leads to a more anisotropic orientation of the transition dipole moments of the probes. As a result of photoselection, those species with a transition dipole moment oriented parallel to the electric field vector are preferentially excited. Hence, especially in the latter cases, any change in direction of the transition moment during the lifetime of the excited state will cause this anisotropy to decrease, inducing a partial or total depolarization of fluorescence. In this paper, we study an azobenzene derivative (hereafter called HBC-azo) designed to enhance the ‘on/off’ effect triggered by the trans to cis photoswitch reaction, by means of theoretical calculations. The molecule has been designed in order to have a strong interaction with the polar head of the lipid membrane and, at the same time, to have a high affinity with the hydrophobic tails. For such a reason, a hydroxyl group is added in the para position of one phenyl ring to enhance the polar interaction, and the second phenyl ring is substituted by a hexabenzocoronene (HBC, C42H18) moiety, to interact with the alkyl tails of the membrane (see Figure 1). HBC has been chosen since it is known that small graphene nanoflakes orient themselves parallel to the bilayer normal, enhancing the hydrophobic interactions.25,26 With this design, different orientations of the trans and cis isomers in a lipid membrane are foreseen. For the phase recognition, it would be good to have a probe whose sensitivity to membrane phases is versatile and switchable so that it can be used in more than one membrane phase and can be activated by light. For such a reason, our HBC-azo probe in both its trans and cis isomers has been inserted into two different membranes, namely DOPC for the Ld phase and DPPC for the So phase, and the HBC-azo probe has been proven to be a highly promising



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candidate for versatility as it has a prospected ability to switch and to discriminate between the different membrane phases.

Figure 1. Chemical structures of the azobenzene used in the study. For clarity, hydrogen atoms are not shown.

METHODS All MD calculations have been performed with the GROMACS 5.0.6 software.27 Two different lipid bilayers have been considered: DOPC for the Ld phase and DPPC for the So phase. A pre-equilibrated DOPC lipid bilayer made of 126 lipid molecules, 63 in each leaflet, solvated with 4584 water molecules and neutralized with NaCl salt (18 Na+ and 18 Cl- ions) has been used. The pre-equilibrated DPPC membrane in its solid gel phase is made of 124 lipid molecules, 62 in each leaflet, solvated with 3450 water molecules and 14 Na+ and 14 Clions; both bilayers were oriented parallel to the xy plane of the simulation box. The probe, inserted in the water phase of the simulation box for the two different membranes, enters the membrane after a simulation time of 30 (65) ns for the trans (cis) isomer. To ensure convergence of the results, 400 ns long simulations were performed for both trans and cis isomers of the probe in both DOPC and DPPC membranes. The HBC-azo molecule studied is described using the GROMOS 43A1-S3 force field28 and parameters for the azo group were taken from literature.29 Partial charges of the isomers have been obtained with the Electrostatic Potential (ESP) scheme which provides an accurate model for the simulation of 6 ACS Paragon Plus Environment

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lipid bilayer systems,30 as implemented in the Gaussian09 program.31 The membranes were modeled using the united atom Berger lipid force field,32 and TIP3 parameters were used for water solvent. The equation of motion time step was kept fix at 2 fs with the Linear Constraint Solver (LINCS) algorithm,33 and the Particle Mesh Ewald (PME) method was employed to compute Coulomb interaction, with a cut-off of 1.2 nm for both electrostatic and van der Waals interactions.34 All simulations were performed at 300 K using the Nosé−Hoover thermostat, Parrinello−Rahman anisotropic pressure coupling to 1 bar with 5 ps time constant and compressibility 4.5 × 10−5 bar-1, in the canonical NPT ensemble. The geometries of the trans and cis isomers of the HBC-azo probe were optimized by means of Density Functional Theory (DFT) calculations, using the CAM-B3LYP functional35 and the cc-pVDZ Dunning basis set36 implemented in the Qchem 4.3 software.37 Time dependent density functional theory (TDDFT) calculations were performed at the same level of theory to study the optical properties of the probe in vacuum. As a proof of principle, the photoswitch mechanism has been calculated, too, using the same functional and the 6-31G Pople basis set.38 For the trans and cis isomers of the HBC-azo probe, 30 uncorrelated snapshots were extracted from both DOPC and DPPC MD simulations. A cylindrical cutoff of 2 nm for the membrane and a semispherical cutoff of 1.5 nm for the solvent has been considered, in order to obtain the input structures for the QM/MM calculations within the electrostatic embedding method as implemented in the Dalton2016 package of programs.39,40 In this scheme, the system is separated into two portions: the probe is described at the QM level of theory through the CAM-B3LYP functional and Dunning’s cc-pVDZ basis set, while the environment (membrane and water molecules) are described using the above mentioned molecular mechanics GROMOS force field. For these 30 snapshots, optical properties of the four lowest excited states have been computed. In particular, One-Photon Absorption (OPA) spectra have 7 ACS Paragon Plus Environment


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been computed from linear response calculations, while Two-Photon Absorption (TPA) spectra are calculated from the first residue of the quadratic response function. This methodology has been previously validated in the study of optical properties of the wellknown Laurdan probe and its new derivative, C-Laurdan, in a DOPC membrane.41

RESULTS AND DISCUSSIONS Optoelectronic properties of the HBC-azo probe Before the insertion on the trans and cis isomers of the HBC-azo probe into the different embeddings (DOPC for the liquid disordered phase and DPPC for the solid gel phase), the optoelectronic properties in vacuum of the two isomers are considered. We found that the stability of the trans isomer is higher than for the cis isomer, by 0.73 eV, confirming that the cis isomer is a metastable state, as found in previous studies on various azobenzene derivatives.42 The Natural Transition Orbitals (NTO) analysis of the absorption spectra of the two isomers shows similar features, with the first excited state related to a nπ* transition for both isomers, defined as a ‘ghost state’ in a previously reported study on the photoswitching mechanism of azobenzene.19 Although this state is a dark state for the trans isomer, it is bright for the cis isomer, with an absorption peak at 460 nm and a transition related to the HOMO-3 to LUMO+3 orbitals. Hence, for the cis isomer the S1 state is not a pure nπ* state, but a mix of nπ* and ππ* states, due to the non orthogonality of the n and π orbitals resulting from the change in geometry from the trans to the cis isomer. The first strong bright state is S3, at 370 and 358 nm for the trans and cis isomer, respectively. This state is related to a ππ* transition which has a main contribution from the HOMO to LUMO molecular orbitals. The S2 state is a weakly bright ππ* state that is present as a shoulder of the S3 peak for both isomers, with energy lying at 383 and 378 nm for the trans and cis isomer, respectively, and is related to a mix of HOMO-1 to LUMO and HOMO to LUMO+1 transitions. Details of the 8 ACS Paragon Plus Environment

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NTO orbitals are reported in the Supporting Information. The wave function character of the excited states is evaluated with the so-called Λ-overlap parameter proposed by Peach et al.,43 in which a value larger than 0.5 corresponds to a localized excitation, while a small value (~0.3-0.4 or smaller) indicates the presence of charge transfer character. We found a value of

Λ = 0.32 for the S1 state of the trans isomer reflecting a charge transfer character of this state, while for all other states for trans and cis isomer Λ is higher than 0.52, depicting localized excitations. This analysis explains the difference in the S1 state obtained for the two isomers: while for trans S1 has an excited state character with zero oscillator strength, the same state for the cis isomer presents a more localized character with Λ = 0.71 and an oscillator strength of 0.07, making this state a bright state. The analysis of the triplet states show that they are low lying states for both isomers (see Table 1). In particular T1 is lying at 677 and 779 nm for the trans and cis isomer, respectively, and is related to a HOMO to LUMO transition for the trans and to a HOMO-3 to LUMO+3 transition for the cis isomer. The first S1 state is found at an energy above the T4 and T3 states for the trans and cis isomer, respectively, and is quite high in energy which might only give rise to a competitive pathway for non-radiative decay involving the triplet states through intersystem crossings and high singlet-triplet coupling elements, which are expected to have a major distortion of the molecular geometry of the probe with respect to the energy differences between the states. State 1

2

3

4

HBC trans T1 1.83 H→L [0.65] T2 1.98 H-4 → L [0.30] T3 2.25 H-3 → L [0.62] T4 2.69

T1

T2

T3

S1

HBC cis 1.59 H-3 → L+3 [0.72] 1.94 H-1 → L+1 [0.79] 2.67 H → L+2 [0.67] 2.69 (0.07) 9

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5

S1

6

S2

7

S3

8

S4

H → L+2 [0.53] 2.87 (0.00) H-4 → L [0.32] 3.24 (0.02) H-1 → L [0.52] 3.35 (0.48) H→L [0.65] 3.69 (1.33) H-1 → L+1 [0.67]

T4

S2

S3

S4

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H-3 → L+3 [0.71] 2.72 H-1 → L+2 [0.70] 3.28 (0.001) H → L+1 [0.66] 3.46 (0.01) H→L [0.82] 3.87 (0.02) H → L+2 [0.66]

Table 1. Low lying singlet and triplet excited states of the HBC-azo probe in the trans and cis isomers in vacuum. Values in brackets refer to the Λ-overlap Peach parameter, in parenthesis to the oscillator strength.

Although the photoswitching mechanism of the HBC-azo probe in different environments is a complex and interesting subject, it is however not the aim of the current study and thus, we are in the current section engaged to give a prove of principle and shed light into the excited state dynamics of the molecule in vacuum. As reported above, starting from the trans isomer, the absorption of a photon from the ground state to the first bright ππ* excited state is obtained considering the S2 singlet excited state, followed by a decay into the dark nπ* S1 state. To simplify the study, we start the analysis of the photoisomerization from the FranckCondon absorption of a photon to the S1 state of the trans conformer and follow the changes in the CNNC torsion angle along the S1 potential energy surface (PES), as reported in Figure 2. Calculations were performed using the CAM-B3LYP functional and the 6-31G Pople basis set. The energy difference between the Franck-Condon point and the one of the optimized S1 geometry amount to 0.8 eV for cis and 0.7 eV for trans conformers of HBC-azo probe. This 10 ACS Paragon Plus Environment

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twisting along the CNNC dihedral is slow compared to the decay from the S2 to the S1 state, and it drives the molecule towards the conical intersection between S0 and S1 at around 90°. At this point the geometry is strongly distorted, with the main contribution arising from the symmetric and asymmetric CNN and NNC bending distortions. From this crossing seam, the distorted molecule can either continue the torsional path following the S0 PES leading to the cis isomer, or come back to the S0 trans minimum. The same scenario is found if the analysis is started from the absorption of a photon from the cis isomer, with similar dihedral and bending distortions in the S0-S1 crossing region. Although this is a qualitative view on the photoisomerization process, it gives some insight into the mechanism and the possibility, for this new designed probe, to photoswitch between the two conformers in a similar way as obtained for azobenzene. To have a full, quantitative description of the mechanism and to have a deepen insight into the S0-S1 crossing region, (TD) DFT cannot be used anymore and highly accurate multireference methods such as CASSCF and CASPT2 should be considered, at the expense of a considerable computational extra cost. Yet, our qualitative view of the mechanism is in agreement with high level ab-initio calculation performed on the azobenzene molecule,19 hence ensuring that our HBC-azo probe can undergo the photoswitch isomerization.



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Figure 2. Summary of the photoisomerization path from the lowest nπ* S1 state starting from the trans (right) and cis (left) conformers. Red stars indicate the energy of the Franck-Condon absorption on the S1 state. Dotted lines refer to the mixing region of S1 and S0 states.

MD Analysis of the Probe in Different Membrane Phases Before analyzing the MD trajectories for both the HBC-azo trans and cis isomers in the lipid bilayers, convergence criteria have to be assessed. The probe is considered equilibrated when the positions of all parts constituting the probe (HBC moiety, N=N bond and OH group) do not change its average position in the membranes. In addition, the orientation of the probe in the membrane is also evaluated by means of analysis of the angle between the transition dipole moment and the normal to the membrane. For both trans and cis isomers in the DOPC membrane, this convergence of position and orientation is reached after 250 ns of MD simulation time. Hence, all results presented in this and in the following sections will refer to a simulation time which is comprised between 250 and 400 ns. Different simulation windows are considered for the probes in the DPPC solid phase membrane; the equilibration of both orientation and position of the probe is reached after 90 ns for the trans isomer and after 200 ns for the cis isomer. Details of the equilibration procedure are reported in Figure S1-S4 of the Supporting Information. We stress here that the convergence of only the position or orientation of the probe in an environment does not assure the equilibration of the probe and that both parameters should be taken into account for this analysis. The plots in Figure 3 report the density of the different equilibrated components of the system for the HBC-azo isomers in the different membrane phases under investigation in this study, DOPC (Ld) and DPPC (So).


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Figure 3. Mass density profiles along the z axis for the different components of the simulated box in the DOPC (top) and DPPC (bottom) membranes: DOPC/DPPC (black), water (cyan), hydroxyl group (red), N=N bond (blue) and HBC core (green). Zero value is set at the bilayer center. All curves for the probes are fitted with a Gaussian function and enhanced by a factor of 100.

Considering the average distance of the phosphorous and nitrogen atoms present in the head of the DOPC and DPPC membranes, we estimate a thickness of 4.24 nm for the DOPC and 4.8 nm for the DPPC membranes, in good agreement with reported data.44,45 The water penetration depth, defined as the distance between 50% water molecules density and the bilayer center along the bilayer normal, amounts to 2.25 nm for DOPC and 2.33 nm for



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DPPC. To have a deeper understanding on the position of the isomers in the membranes, the HBC-azo molecule is analysed in its different components: HBC moiety, azo bond (N=N) and the hydroxyl group. From the plots in Figure 3, it is possible to see the different behaviour of the trans and cis isomers of the different membranes. In fact, the trans isomer seems to be only slightly affected by the membrane phase; in both DOPC and DPPC membranes, the position of the different components of the probe are lying at a similar distance from the bilayer centre. The HBC moiety slightly moves and is located at 0.9 and 0.83 nm from the bilayer centre for DOPC and DPPC membranes; more interestingly, the N=N azo group is lying at 1.36 nm in DOPC and at 1.23 nm in DPPC, with a deepening into the aliphatic part of the membrane of 0.13 nm going from Ld to So phase. A similar behaviour is found for the hydroxyl group, which is located at 1.75 nm in DOPC and at 1.58 nm in DPPC, thus following the same trend as the azo group, with a deepening of 0.17 nm. As a result, only small variation in position of the HBC-azo trans isomer occur in the different membrane phases. A different scenario arises for the HBC-azo cis isomer. Now, all the components of the probe experience a different position depending on the phase of the membrane. In particular, the HBC moiety, lying at 1.0 nm in the DOPC membrane, is deepened at 0.73 nm in DPPC. A much strong change in position is found for the azo and hydroxyl groups; the azo group shifts from 1.73 nm to 0.81 nm going from DOPC to DPPC, while the hydroxyl group is shifted from 1.83 to 1.39 nm going from DOPC to DPPC. These differences in positions are a strong indication of the different interaction of the azo and hydroxyl groups with the membranes: in DOPC, they interact strongly with the polar part of the membrane, while in DPPC they are buried into the aliphatic tails. We can thus already foresee a different response in optical properties from this probe depending on its conformation and on the environment.


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To gain insight into the different orientations of the conformers in the different membrane phases, the analysis of the angle distribution between the transition dipole moment (t.d.m.) and the normal to the membrane is performed. We recall here that the t.d.m. considered for this analysis is a vector parallel to the N=N bond direction for both trans and cis isomers, and is related to the S2 and S1 excited states, respectively. Interestingly, a similar angle distribution is found for the trans and cis isomers in both membranes, with angles of 102° and 14° for the trans in DOPC and 12° and 100° for the same isomer in DPPC. The difference in between the two peaks is the intensity: in DOPC the most intense peak is at 102°, while in DPPC, it is at 12° (see Figures S3-S4). The same angle of 43° is found for the cis isomer in both membrane phases. The analysis of the torsional angles is thus needed to be able to understand the different position of the two isomers in the different membrane phases. For the torsion distribution, two different dihedrals are considered: the first is the angle between the normal to the membrane and the long vector of the molecular axis, considered from the nitrogen atom to the bottom of the HBC group (hereafter named β), while the second considers the angle between the normal to the HBC plane and the z-axis (normal-to-z-axis angles). This analysis, reported in Figure 4, shows the different behaviour of the two conformers in the different membranes. The trans isomer presents the same normal-to-z-axis angle value of 95° in both membranes, and a similar β angle of 53 and 58° in DOPC and DPPC, respectively. As already found for the position of the cis isomer, also the orientation presents strong differences depending on the membrane phases. While the normal-to-z-axis angle value of 92° in DOPC is decreased to 78° in DPPC, the β angle strongly changes depending on the membrane phase, with value of 9° in DOPC that is enhanced up to 84° in the DPPC membrane. These results explain the different positions found for the cis conformer in the two membranes: in DOPC, the probe is more parallel to the membrane tails, with an ‘upright’ orientation of the azo group, while in DPPC the probe is rotated of almost 15 ACS Paragon Plus Environment


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90° along the z axis of the membrane, leading to the deeper positioning of the azo group (representative structures are reported in Figure S5 and S6 of Supporting Information). This double different orientation of the trans and cis isomers within the same membrane and of the cis isomer in different membrane phases, will lead to different optical properties and, in turn, to a switch of states responsible for the phase detection.

Figure 4 β torsion (top) and normal-to-z-axis (bottom) torsion angle distribution of the two probe isomers (red for trans and black for cis, respectively) in the DOPC (Ld) and DPPC (So) membranes. Solid lines refer to the isomers in the DOPC membrane, while dashed lines point at the isomers in DPPC. Inserts show two representative orientations of the two probes in the membrane; the green thick arrow refers to the β angle vector, while the red square refers to the plane considered for the normal-to-z-axis torsion analysis. 16 ACS Paragon Plus Environment

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QM/MM From the 30 uncorrelated snapshots extracted from the MD simulations, electrostatic embedding QM/MM calculations were performed in order to obtain the One Photon and Two Photon Absorption spectra. The convergence of the OPA spectra is reached already with 20 snapshots, but to enhance the statistic, a total amount of 30 snapshots has been considered for the following analyses (Figure 5).

Figure 5. CAM-B3LYP One Photon Absorption (OPA) spectra of the HBC-azo trans (top) and HBC-azo cis (bottom) isomers in DOPC (Ld) (black lines), DPPC (So) (red lines) 17 ACS Paragon Plus Environment


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membranes and in vacuum (dotted lines). The absorption spectra were generated for each snapshot with Lorentzian broadening and then averaged over the 30 snapshots.

The OPA properties of the trans and cis isomers in the two different membrane phases reflect the results obtained from the MD simulations. In fact, the HBC-azo trans isomer, for which the position and orientation in the two different membranes is similar, presents also a similar absorption spectra, independent of the different environments. For this conformer, the first low lying excited state (at 577 and 602 nm in DOPC and DPPC, respectively) is an nπ* dark state associated to a HOMO-3 to LUMO transition in both DOPC and DPPC membranes. The first bright state is S2, which is present as a shoulder of the more intense peak from the S3 state in Figure 5. S2, peaking at 405 nm in DOPC and 403 nm in DPPC is associated with a HOMO-1 to LUMO transition, while the strong bright peak at 385 (386) nm in DOPC (DPPC) of the S3 state is associated to a HOMO to LUMO transition for both membranes. At higher wavelengths values of 344 nm for both membranes, is found the S4 state which is associated to a HOMO-1 to LUMO+1 transition. To have an indication of the nature of those excited state, and in particular to the delocalization of the wave function, the excitations were analyzed with the Λ-overlap parameter. All four excited states of the HBC-azo trans isomer present a Λ-overlap value larger than 0.52, indicating the localized character of the excited states. A different scenario is found for the HBC-azo cis isomer in the two different membrane phases. Now, the S1 excited state is allowed, with weak absorption strength and spread over an absorption window of more than 300 nm in DOPC, from 470 to 800 nm, and a similar window of 330 nm, from 525 to 858 nm, in DPPC. The transition associated to the S1 state is a mix of nπ* and ππ* states, and refers to a HOMO-3 to LUMO in DOPC and to a HOMO-2


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to LUMO transition in DPPC. In addition, the absorption peaks of excited states at shorter wavelength (i.e. S2-S4) are red shifted by ̴ 10 nm in the liquid disordered phase (DOPC) compared to the solid gel phase (DPPC). The transitions associated to the excited states differ also in the two environments. The second excited state peaks at 413 nm in DOPC and at 403 nm in DPPC, and is related to mixed HOMO to LUMO (transition probability of 55%) and HOMO-1 to LUMO (45%) transitions in the Ld phase, while it relates to the HOMO-1 to LUMO transition for the So phase. S3, at 394 nm (DOPC) and 383 nm (DPPC) relates again to a HOMO to LUMO (45%) and HOMO-1 to LUMO (55%) mix transitions in the first environment, while it relates to the HOMO to LUMO transition in DPPC. Finally, the band which is related to the excitation into S4 peaks at 350 (341) nm in DOPC (DPPC) and relates to a HOMO-2 to LUMO transition in the Ld phase, and to a HOMO to LUMO+2 transition in the So phase. Also for this case, the Λ-overlap value is higher than 0.5, suggesting a localized character of the wave function for these excited states. In addition, in the DOPC membrane, the excited states of the cis isomer are red shifted by ̴ 10 nm compared to the trans isomer. As a result, for this isomer it is possible to see a different effect of the environment depending on the phase of the membrane: in DOPC, the absorption of S2-S4 excited states is red shifted compared to the same states in DPPC, but for S1 the opposite effect is present, with an overall blue shift of the absorption in DOPC compared to DPPC and different transitions related to the different environments. Apart from the S1 excited state for the HBC-azo cis conformer, the absorption is mainly confined in a borderline region between UV and visible absorption range of the light. To aim at the possible use of the HBC-azo probe for experiments in living organisms, the use of near-IR or IR light wavelength is preferred, in order to decrease the damage caused by the use of UV photons. This result can be accomplished by the study of the Two Photon Absorption (TPA) microscopy. In addition to the use of less intense photons, the intensity of 19 ACS Paragon Plus Environment


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the laser used is more localized, and a higher 3D resolution is obtained, as well as a higher penetration depth in tissues compared to OPA techniques.46,47 Moreover, this fluorescence microscopy is based on non-linear optics properties, from which the two photon absorption cross section is the most important. The cross section (in GM units) is computed as:

(1)

where α is the fine constant, a0 the Bohr radius, t0 the atomic unit for time, Γ the Lorentzian broadening (here considered constant with a value of 0.1 eV), ω the OPA excitation energy and δ the two-photon absorption strength in au. The averaged TPA values for the HBC-azo trans and cis conformers in the Ld and So phases are reported in Figure 6, and span a range of 30 GM.


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Figure 6. Two-photon absorption cross section (in GM) for HBC-azo trans (solid lines) and cis (dotted lines) isomers in the DOPC (Ld) (top) and DPPC (So) (bottom) environments. The different colors represent different excited states: S1 black, S2 red, S3 green and S4 blue.

As already found for the previous analysis, also for the TPA cross section the HBC-azo trans isomer is not sensitive to the different environments surrounding the probe, since its position and orientation are similar in both Ld and So phases. On the other hand, the strong different position of the cis isomer in the two membranes leads to a strong difference in TPA cross section values. The big increase in GM values for the S4 state of the cis isomer in DPPC (between frames 14 and 17, see Figure 6) is due to a change by 15° in orientation of the conformers, which is sufficient to change the orientation of the transition dipole moment and, thus, enhance the TPA cross section. The full TPA spectra for both isomers are reported in Figure S7 of the Supporting Information, and the absorption ranges from 650 to 850 nm for both probes. Interestingly, in the range 780-850 nm, the cross section for the cis isomer is higher in DOPC, while when the same TPA absorption range is considered in DPPC, the cross section is higher for the trans isomer. This is related to the different TPA for the different excited states considered. In both membrane phases, S1 and S2 states for both conformers are dark, while S3 and S4 are bright. Interestingly, while in DOPC the S3 TPA values are higher for the cis isomer, in DPPC these values are higher for the trans isomer. We can thus describe this different behavior as binary ‘on/off’ states depending on the membrane phase. Starting with the trans isomer inserted in the Ld phase (i.e. DOPC) the TPA microscopy will lead to low signal, while the photoswitch to the cis isomer will activate the ‘on’ state and enhance the TPA values. On the contrary, the same trans isomer in a So phase (i.e. DPPC) leads to the ‘on’ state with strong TPA absorption, and the photoswitch to the cis isomer will deactivate the signal, making this HBC-azo probe a versatile candidate for phase 21 ACS Paragon Plus Environment


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detection, due to the different interaction of the cis isomer depending on the different membrane phases.

Fluorescence decay time and anisotropy In a first order approximation, it is possible to compute some fluorescence properties considering the absorption transition dipole moment to be parallel to the emission transition dipole moment. In such a way, properties such as fluorescence and rotational decay times can be predicted and also MD simulations done on the ground state can be considered to take into account the environment effects. With this approximation, we compute the inherent radiative lifetime of the HBC-azo trans and cis isomers, which depends on the spontaneous emission rate by:

1⁄

(2)

This relation can be expanded as:48

! ℏ

#

,

(3)

where $%& and %& are the transition dipole moment and the transition frequency of the probe, respectively; ' is the permittivity in vacuum, c the light speed constant in vacuum and

ℏ the reduced Plank constant. The radiative lifetime is an intrinsic property of the probe, and is not influenced by the environment.49 Yet, in any environment (solvent or biological) the lifetime of the probe is strongly dependent on its surrounding, and in particular on the dielectric properties of the medium. Consequently, considering the environment, the energy of the emitted photon can be renormalized through the substitution ' → ') ' and * → * ⁄+ , with ') is the relative permittivity and n the refractive index of the medium. The spontaneous emission rate can now be rewritten as

) → +

(4) 22

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Were + √' ) is the refractive index of the environment. The values of the radiative decay time for both HBC-azo trans and cis isomers in the different environments are reported in Table 2. HBC-azo Trans

HBC-azo Cis

S1

S2

S3

S4

S1

S2

S3

S4

3358

18.3

0.67

0.19

7.2

315

31.3

11.6

-./0 2436

13.3

0.48

0.14

5.2

228

22.7

8.42

-//0 1877

10.2

0.37

0.11

4.0

176

17.5

6.49

Table 2. Decay times for the first four excited states of the HBC-azo probe in its trans and cis isomers. refers to the decay time in vacuum, while the other data reported refer to the probe into the surrounding. All decay times reported are in ns.

From Table 2, it is possible to see that S1 is a dark state for the trans isomer (decay time in the µs timescale), while the other states are allowed. Moreover, S3 is the first excited state with a decay time in the order of ps time scale decay, and is the first strong bright peak obtained from the absorption spectra. On the other hand, the first excited state for the cis isomer is bright, which is reflected in the short decay time being in the ns range. The S2, from the other hand, has a weak oscillator strength and has through the Einstein coefficients a longer decay time, on the order of hundreds of ns. The S3 state finally is a second strong bright peak and has a ns scale decay time. The effect of the inclusion of the permittivity of the environment upon the calculation of the decay time varies depending on the membrane phase; in the Ld phase (DOPC) the decay time is reduced compared to the vacuum, but this reduction is even more pronounced in the So phase, due to the difference in permittivity of the two membranes (values for the relative permittivity of the two membranes, of 1.9 and 3.2 23 ACS Paragon Plus Environment


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for DOPC and DPPC, respectively, have been taken from Electrostatic Force Microscopy experiments).50,51 Yet, this is a static picture that does not take into account the different conformations of the probe in the biological environments along the decay time. To have a dynamic view of the decay process and to enhance the statistic, the same snapshots considered for the QM/MM analysis are used, and the decay time is calculated for the probes in the different environments for each frame extracted (for a total of 30 frames). In this way, it is possible to study the effect of instantaneous change in orientation and position of the probe in the membrane, and to understand the different effect of the membrane phase onto the decay time. Results of this analysis, reported in Figure 7, show different decay times depending on both the nature of the conformer and the phase of the membrane (the S3 state is considered for the analysis).

Figure 7. Fluorescence decay time for the HBC-azo trans (black lines) and cis (red lines) isomers in the different membrane phases. Solid lines refer to the probes embedded in Ld (DOPC) phase, while dashed lines to the probes embedded in So (DPPC) phase.

Interestingly, with this analysis, which is directly comparable to the results of Fluorescence Lifetime Imaging experiments (FLIM), is possible to screen between the different decay times concerning both the different isomers and the membrane phases. In the DOPC Ld 24 ACS Paragon Plus Environment

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phase, the radiative decay time of the cis conformer is 3.61 ns, while for trans it is smaller, with value of 1.82 ns. The same situation is found for the probes in the DPPC So phase; the cis isomer has longer decay time of 5.41 ns while the trans has shorter time of 1.1 ns. Moreover, the radiative decay time for the cis isomer in different membrane phases is longer in DPPC than DOPC, while the opposite trend is present for the trans isomer, with longer decay time in DOPC than in DPPC. This difference is 0.65 ns for the trans conformer and 1.8 ns for the cis conformer, embedded in different membrane phases. As a result, we suggest hereby to perform the related experiments in order to confirm our simulations and to confirm that we are -within the approximation considered- not only able to detect different radiative decay times for the two isomers which are dependent on the phase of the membrane, but also that the differentiation of the decay time in the various environments depends on the conformation of the HBC-azo probe. This opposing behavior is another expression of the ‘on/off’ effect which is present for this probe; considering either the same isomer in different environments, or vice-versa different isomers in the same membrane phase, is possible to obtain different responses, thus enhancing the quality of this probe for phase detection in a versatile way. To unravel the nature of the different decay times for the trans and cis conformers, an additional analysis has been performed, considering now the ease of rotation of the probe in the different environments. This can be accomplished by studying the fluorescence anisotropy of the two isomers, by means of the wobbling in a cone model.52 In this model, the probe is considered free to rotate within a certain cone, when embedded in an environment. The rotational correlation function is thus obtained from the MD simulations performed, and within the model considered, these decays are fitted with a tri-exponential decay function:53

12 [56 exp− 2 ⁄6 + 5 exp− 2⁄ +5 exp− 2 ⁄ ] + 1=

(5)



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where the different ’s represent rotational decay parameters, and 1= is the residual anisotropy, that is related to the cone angle θ through: 6

1= > cos B1 + cos BC ∙ E0

(6)

where a small cone angle gives rise to large 1= and, vice-versa, 1= vanishes for isotropic rotational diffusion (like in a solvent). For the HBC-azo probe, this analysis has been performed considering the transition dipole moment vector for the computation of the rotational correlation function, which is related to the S2 excited state for the trans isomer and to the S1 state for the cis isomer. The quality of the fit was tested by the χ2 analysis (good fitting function leads to value of χ2 close to 1); our fit leads to χ2 value in the range of 1 ± 3·10-6, assuring the high quality of the function used. The results show once more the ‘on/off’ behavior of this probe in the different membrane phases: in DOPC, the averaged rotational decay time is 2.09 ns for the trans isomer and 25.51 ns for the cis isomer, while in DPPC the opposite scenario is present, with rotational decay time of 13.13 ns and 2.29 ns for the trans and cis conformers, respectively. This is directly related to the hindrance that the probes experienced in the different membrane phases. In fact, in the Ld phase, the flat trans isomer has more rotational freedom with respect to the rather voluminous cis isomer, due to the different orientation of the probes, while in the solid gel phase the cis has more freedom of motion. As a result, and as already seen for the TPA properties, in DOPC the trans isomer is the ‘on’ state and the cis isomer the ‘off’ state, while the opposite is found in DPPC. When the three decay times considered for the fitting function are analyzed, it is found that for the HBC-azo trans conformer in the DOPC membrane and the HBC-azo cis in DPPC, from which the total fluorescence anisotropy decay time is small, only one decay time is dominant, while for the other two conformers (cis in DOPC and trans in DPPC) all three decay times contribute to the total one. This explains the differences found for the two 26 ACS Paragon Plus Environment

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isomers in different environments: the HBC-azo trans isomer in DOPC experiences a strong hindrance that is expressed by freedom of rotation along one Cartesian axis only, while the cis isomer in the same membrane phase has more rotational freedom along the three axis, leading to an overall longer total decay time. The same explanation is true for the probe in the DPPC membrane, but now it is the HBC-azo cis isomer that experiences the highest hindrance, while the trans is more free to rotate along the three axis (see Table 3).

Trans (DOPC) Cis (DOPC) Trans (DPPC) Cis (DPPC)

56

6

5

5

1=

GH

0.09

0.002

0.03

0.14

0.22

2.11

0.66

2.09

0.04

0.44

-0.64

21.56

1.00

23.18

0.56

25.51

0.81

4.67

-0.37

7.14

-0.37

7.14

0.84

13.13

0.04

0.01

0.02

2.35

0.01

0.11

0.93

2.29

Table 3. Pre-exponential decay parameters β and rotational decay time τ for the trans and cis isomers in the two different environment: DOPC (Ld) and DPPC (So) membranes. The average decay time GH and 1= are also reported. All values for time are in the ns timescale.

To confirm this behavior, we can also consider the 1= parameter which is decisive for the cone angle magnitude. In agreement with the discussion above, the residual anisotropy is higher for the HBC-azo trans embedded in DOPC than for the cis isomer in this membrane (0.66 compared to 0.56, respectively) and for the HBC-azo cis isomer in DPPC than for trans in this phase (0.93 compared to 0.84, respectively). Such high values translate into a very small cone angle, hence confirming the high hindrance and anisotropy that these two conformers experience in the different environments. Once more, we can find in this behavior the different ‘on-off’ states for the two trans and cis conformers in the same environment. If we consider the shorter decay time as the ‘off’ state, it can be concluded that in the DOPC 27 ACS Paragon Plus Environment


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liquid disordered phase the trans isomer is in this ‘off’ state and the cis in the ‘on’ state. Changing the environment to the DPPC solid gel phase switches the states, with the trans isomer being now the ‘on’ state and the cis isomer the ‘off’ state, as previously found for the TPA properties and fluorescence decay times.

CONCLUSIONS In this work, a new azobenzene derivative probe with the working name HBC-azo has been designed for lipid membrane phase recognition. Through the use of different computational tools, it has been discovered that this new probe, named HBC-azo, is promising for phase recognition due to its photoswitch properties. The optoelectronic properties in vacuum have been studied; in particular for the photoswitching mechanism a prove of principle has been provided that the photoisomerization of the trans isomer starts from the first excited state and goes through a S0-S1 conical intersection that enables a decay to the ground state energy surface of the cis isomer (the same holds true for the photoisomerization from the cis to trans isomer). Next, the probe has been inserted into two different membranes, which represent different phases: DOPC has been chosen for its liquid disordered phase, while DPPC has been selected for its solid gel phase. By MD simulations, we observed different positions and orientations of the two conformers of the HBC-azo probe in the two considered membrane phases; although the trans isomer has similar position and orientation in both phases, the cis conformer shows strong differences, which lead to different responses in fluorescent experiments as the optical properties depend on both conformation and environment effects. To have insight into the optical behavior of the probe in the different embeddings, we performed one and two photon absorption spectra analyses by means of QM/MM calculations, and found that the optical properties are directly related to the position and orientation of the probe in the membrane. In fact, the trans isomer, which behaves in a similar


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way within the two membrane phases has similar OPA and TPA spectra, while the cis conformer is the one which controls the outcome of the optical properties. In fact, in DOPC membrane it acts as the ‘on’ state for OPA and TPA properties, while in DPPC, due to its different orientation and position, is the ‘off’ state. This should translate in different images from TPA microscopy and discriminate consequently between different membrane phases. Similar ‘on/off’ states are present for the fluorescent analysis. The dynamic radiative lifetime analysis of the HBC-azo conformers in the different membrane phases shows that it is possible to screen between different phases for the same isomer, and that the different isomers in the same phase lead to different response, making such probe versatile for phase detection. Finally, the fluorescence anisotropy is analyzed and different ‘on/off’ states are found also in this case. In particular, fast decay times (on the order of 3 ns timescale) are found for the trans isomer in DOPC and for the cis isomer in DPPC, while considerably longer decay times are found for the other conformer in the two membranes. Hence, once more there is an ‘on/off’ effect also for the anisotropy decay, related to the different hindrance that the two isomers experience in the different membranes. This work paves the way to the design and use of probes for phase recognition in a versatile manner, due to the presence of a photoswitch mechanism, which enhances the ‘on/off’ states of the probe in a selective way depending on the observable of interest.

Aknowledgements S.O. is grateful to the Center for Quantum Materials and Nordita for his current funding. The authors thank the Swedish Infrastructure Committee (SNIC) for the computational time granted within the medium allocations SNIC 2016/1-87, SNIC 1-415 and SNIC 1-465, as well as the small one SNIC 2015/4-44.



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Supporting Information Details of the Natural Transition Orbitals, MD convergence analysis, full TPA spectra and details of the anisotropy decay on the first excited state are reported. This information is available free of charge via the Internet at http://pubs.acs.org.

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