# Design, Synthesis, and Characterization of pH-Resettable Photoswitches Mimicking the GFP Fluorophore Structure

Nicola Ferrara, Germano Giuliani, Marios Maimaris, Salvatore Prioli,

Madushanka Manathunga, Lluís Blancafort, Jérémie Léonard, Andrea

Cappelli, Massimo Olivucci, Marco Paolino

► To cite this version:

Nicola Ferrara, Germano Giuliani, Marios Maimaris, Salvatore Prioli, Madushanka Manathunga, et al. Design, Synthesis, and Characterization of pH-Resettable Photoswitches Mimicking the GFP Fluorophore Structure. Journal of Physical Chemistry B, 2025, 129 (11), pp.2845-2855. 10.1021/acs.jpcb.4c07003. [hal-05078998]

HAL Id: hal-05078998

https://hal.science/hal-05078998v1

Submitted on 29 Aug 2025

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<figure><img src="image_1.png" /></figure>
Design, Synthesis, and Characterization of pH-
Resettable Photoswitches Mimicking GFP Fluorophore

Structure

Nicola Ferrara,<sup>a,§</sup> Germano Giuliani,<sup>a,§</sup> Marios Maimaris,<sup>b,§,†</sup> Salvatore Prioli,<sup>a</sup> Madushanka Manathunga,<sup>c,¥</sup> Lluís Blancafort,<sup>d</sup> Jérémie Léonard,<sup>b</sup> Andrea Cappelli,<sup>a</sup> Massimo Olivucci,<sup>a,b</sup> and Marco Paolino.<sup>a,*</sub>

<sup>a</sup> Dipartimento di Biotecnologie, Chimica e Farmacia, Università di Siena, Via A. Moro 2, 53100 Siena, Italy;

<sup>b</sup> Institut de Physique et Chimie des Matériaux de Strasbourg, Université de Strasbourg & CNRS UMR 7504, Strasbourg, France;

<sup>c</sup> Chemistry Department, Bowling Green State University, USA;

<sup>d</sup>Institute of Computational Chemistry and Catalysis and Department of Chemistry, University of Girona, C/ M. A. Capmany 69, 17003 Girona, Spain;

<sup>†</sup>Present address: Dipartimento di Fisica, Politecnico di Milano, Piazza Leonardo da Vinci 32, 20133 Milano, Italy;

<sup>¥</sup>Present address: ATTMOS Inc., 325 E Grand River Ave Ste 328, East Lansing, Michigan 48823, United States.

<footnote><sup>§</sup>These authors have equally contributed to the research.</footnote>

<footnote>Corresponding author: E-mail paolino3@unisi.it</footnote>
**Abstract**

Light-controlled molecular switches based on the double bond isomerization can represent classical binary systems in logic gates. Here, starting from the biomimetic photoswitch **1** and combining computational and experimental techniques, we designed the insertion of a third control element (the “reset button”) proposing an appropriate structural modification capable to alter the electronic distribution within the molecule. Thus, the substitution on the pyrrolidinone nitrogen atom of **1** with methane sulfonic (in **2a**) or a toluene sulfonic (in **2b**) functional group furnished molecules capable of alternating between two stable equilibrium forms by light irradiation. The addition of KOH deprotonates the phenolic moiety of the molecular photoswitches providing systems in which the Z isomer becomes thermally unstable and spontaneously evolves into the starting E isomer. Re-establishing the initial phenolic form (e.g. by addition of acetic acid), the two molecules re-establish its photochemical properties. These molecules represent a prototype of a potentially pH-resettable photoswitch with possible applications in pH-sensitive organic materials.
# 1. Introduction

Owing to the ease of its application, light is used in biological and materials science as a selective tool to chemically control complex process.<sup>1-4</sup> Since the last century were developed light-driven molecular switches, molecules capable of reversible interconversion between two or more states as a result of irradiation with precise wavelengths.<sup>5,6</sup> A large number of molecular photoswitches based on structures such as azobenzene, diarylethenes, stilbenes and other have been developed and involved in many studies to control phenomena such as ion complexation, catalysis, folding/unfolding of peptides and photopharmacology.<sup>7-12</sup> Recently, molecular photoswitches were used for the design of logic gates useful for the construction of memory elements capable of preserving information in a write-read-erasable form.<sup>13-17</sup> However, the introduction of new photoswitches accessible through simple synthetic routes and with different properties such as size, polarity, isomerization mechanism and regeneration capacity appear to be an interesting target to grow this basic technology and apply it to more complex systems.

During the past years we have used the biomimetic strategy to prepare light-driven molecular switches. Indeed, we have shown several positively charged *N*-alkylated or *N*-protonated indanylidene pyrroline Schiff bases (NAIPs and NHIPs respectively)<sup>18-27</sup> as biomimetic structures which mimic the reactivity of the retinal chromophore of visual pigments with a regioselective subpicosecond C=C double bond photoisomerization. More recently, still using the biomimetic approach, we synthesized a second family of light-driven molecular switches and motors bearing negative charge.<sup>28-31</sup> The anionic photoswitches have been prepared by taking in account the fluorophore of the Green Fluorescent Protein (*p*-HBDI)<sup>32-34</sup> by transforming it from an efficient emitter (inside the protein) to an ultrafast molecular photoswitch (Figure 1).
<figure><img src="image_2.png" /><figcaption>**Figure 1.** Ultrafast photoswitches designed to mimic the isomerization of the chromophore of the Rhodopsin (up) and of the *p*-HBDI fluorophore (down).</figcaption></figure>

For this purpose, previously we decided to modify the *p*-HBDI structure introducing an ethylenic bridge into the structure and replace the imidazolone with a pyrrolidinone moiety.²⁸ The ethylenic bridge prevents dissipation of the absorbed photon energy through rotation about the single aryl–alkene bond. The pyrrolidinone moiety enables an accessible synthesis, enhances the translocation of the indanylidene negative charge along the molecular framework upon light absorption and could favor the *E/Z* rearrangement due to its larger flexibility with respect to the imidazolone moiety. This photoswitch is thermally stable at room temperature and the photoisomerization of both diastereomers occurs on a sub-picosecond timescale.²⁸

Here we report two new pH-resettable photoswitches designed starting from the previously reported GFP mimic compound 1. Preliminary computational investigations predicted the effects of the substituents to unable rational design of resettable photoswitches, which were studied experimentally to characterize their photomodulable “on-off” (*E/Z*) behavior and the resettable capability through protonation/deprotonation.

## 2. Results and Discussion

### 2.1 Design
The photoisomerization ability of the prototype molecule **1** from the *E* to *Z* form and *vice versa* seems to be related to its photoacid behaviour. In the excited state, **1** is capable to reversibly relocate the negative charge from the indene moiety to the pyrrolidinone head.²⁸ This feature leads to the reorganization of electrons that allows the exocyclic double bond to become a single bond, free to rotate.

In order to design a molecule useful as reversible photoswitch we decided to change the weak charge delocalization inside the molecules and alter the thermal energy barriers under controlled conditions. Thus, we choose to introduce an electron-withdrawing group on the pyrrolidinone head in order to promote and stabilize the electron density towards this portion of the molecule. The pyrrolidinone nitrogen atom of compound **1** can be selected as a possible point of substitution. In fact, the *N*-substitution intuitively should not sterically hinder the photoisomerization of the exocyclic double bond and electronic redistribution in the structure. In addition, the nitrogen derivatization is similarly present in the natural chromophore in which the same atom is replaced with a methylene-carbonyl group (residue of Gly67 of the protein after the biosynthesis).³⁵ Therefore, sulfonyl groups (-SO₂R) were selected as substituent of the nitrogen atom due to their electron-withdrawing effect and the large stability at different pH, with the aim to promote the delocalization of the negative charge from phenolic to carbonyl oxygen through controlled pH variation without illumination. Two *N*-sulfonyl derivatives **2a,b** were designed using mesyl and tosyl substituents, respectively (Figure 1).

In order to rationalize this choice, preliminary computational studies were performed on the simplest mesylated compound **2a** used as model to explore the effect of the *N*-sulfonylation. The comparison with the unsubstituted parent photoswitch **1** in the same computational conditions already provides a first level of understanding.

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>
**Figure 2.** Geometries and relative ground state energies of neutral (protonated) derivatives of compounds **1** and **2a**, calculated for the entire rotation of the C₇a-C₁-C₃'-C₄' dihedral angle. a) Protonated derivative **1** with absorbance energy for 0° and 180°. b) Protonated derivative **2a** with relative absorbance energy for 0° and 180°. For both compounds, the *E,Z* isomers and *P,R* helicity configurations are plotted on the x-axis.

<figure><img src="image_5.png" /></figure>

**Figure 3.** Geometries and relative ground state energies of anionic compounds **1** and **2a**, calculated for the entire rotation of the C₇a-C₁-C₃'-C₄' dihedral angle. a) Anionic derivative **1** with absorption energy at 0° and 180°. b) Anionic derivative **2a** with relative absorbance energy at 0° and 180°. For both compounds, the *E,Z* isomer and *P,R* helicity configurations are plotted on the x-axis. The intensity of the red squares shown in the figure, is proportional to the negative charge of the pyrrolidinone and indene moieties (the relative percentage of charge is also reported).

Two different forms of compounds **1** and **2a** have been selected for the computational study: the neutral form (with protonated phenyl oxygen) and the anionic form (bearing a delocalized negative charge). A complete characterization of prototype compound **1** in anionic form has been previously reported using the REKS (RE-LC-ωPBE/6-31(+)G*) and SI-SA-REKS (SSR-LC-ωPBE/6-31(+)G*) methods employing ensemble density functional theory.²⁸ Here the calculations were performed using the density functional theory (DFT) calculations in connection with the CAM-B3LYP functional³⁶ and 6-31G* basis set as implemented in the GAUSSIAN 16,³⁷ in MeOH as implicit solvent. DFT and TD-DFT provide a computationally efficient approach, and CAM-B3LYP was selected to avoid the well known charge transfer error of TD-DFT.³⁸ Firstly, the protonated form of **1**, not computed before, has been investigated. The *E-P,R* isomer is the most stable, while *Z-P,R* isomer was found 1.7 kcal/mol above. The transition states (TSs) of the isomerization reaction from *E-P,R* to *Z-P,R* in clockwise or counter clockwise directions were found at about 55.8 kcal/mol (Figure 2a). The ground state energy surface for compound **1** in anionic form is shown in Figure 3a. The transition state occurring in the double bond isomerization between *E-P,R* (the most stable isomer) and *Z-P,R* (found 2.9 kcal/mol above) has been computed with a barrier of 34 kcal/mol. To convert *Z-P,R* to *E-P,R* (both in clockwise or in counter clockwise direction) an energy barrier of 30.6 kcal/mol must be overtaken. The energy profile is in good
agreement with the one obtained with REKS,²⁸ the main difference being that Z-P,R is more stable than with REKS (2.9 with CAM-B3LYP vs 6.0 kcal/mol with REKS), resulting in a somewhat lower barrier for the Z-P,R to E-P,R conversion with REKS (34 kcal/mol with CAM-B3LYP vs 26.8 kcal/mol with REKS). In addition, the Z-M,R structure, which is a shallow minimum with REKS, could not be located at the CAM-B3LYP level. Thus, the anionic form of compound **1** shows TSs with lower energy than in its neutral form. However, as previously observed, the computed interconversion energy barrier both in neutral and anionic form are sufficiently high to prevent thermal interconversion in solution at room temperature.

Optimization of the S₀ equilibrium structures of the *N*-substituted *p*-HDBI-like compound **2a** in neutral form results in *E*-P,R and *Z*-P,R isomers. Similarly to compound **1**, also in **2a** the *E*-P,R isomer is the most stable while the *Z*-P,R isomer (the only *Z* isomer present here) was found at about 1.7 kcal/mol above (Figure 2b). The conversion of *E*-P,R to *Z*-P,R can occur through TSs that overcome a high barrier of 44.8 (or 42.8) kcal/mol, which allow both *E/Z* isomerization and P/M helicity inversion. Comparing the energy of compounds **1** and **2a** in a neutral form, it is clear that the presence of sulphonyl substituent on the nitrogen does not significantly alter the energy differences between the two equilibrium structures (the energy of the *Z* isomers are 1.7 kcal/mol higher than the energy of the *E* isomers).

On the other hand, in the anionic form of **2a** both *Z* isomers (*Z*-M,R and *Z*-P,R which differ in their helicity) were found at about 3-4 kcal/mol above the *E*-P,R isomer (Figure 3b). The first TS, connecting the *E*-P,R to the *Z*-M,R isomer, determines the first activation energy of 23.7 kcal/mol. From this point, the molecule can reach again *E*-P,R isomer passing through the same TS placed now at 19.4 kcal/mol. Alternatively, following the isomerization coordinate, a second TS connecting *Z*-M,R to *Z*-P,R (not optimized here) can be found. This TS controls the helicity inversion from *M* to *P* and is expected to be very low. For comparison, the barrier calculated for helicity inversion in anion **1**, at the REKS level, is 1.8 kcal/mol.²⁸ Then, from the most stable *Z*-P,R isomer, the molecule could move towards *E*-P,R overcoming an energy barrier of 21 kcal/mol to get to a further TS connecting *Z*-P,R to *E*-P,R located at 23.8 kcal/mol.

The comparison of the energy profiles of the photoswitch **1** and compound **2a** in anionic form shows interesting differences. The presence of the sulfonyl substituent slightly decrease the energy gap between the *E*-P,R and *Z*-P,R of **2a** compared to **1**, but the most important result is the significant decrease in energy of the transition states of **2a** which are almost halved with respect to the TSs of the same molecule in neutral form and considerably lower than the TSs of **1** in anionic form (34 kcal/mol for TSs of **1** vs about 24 kcal/mol for TSs of **2a**). For this reason, the interconversion on S₀ from *Z* to *E* isomers would require the overcoming of a barrier of 31.1 kcal/mol.
kcal/mol for compound **1** (not observed in solution at room temperature) and only 19-21 kcal/mol for the new compound **2a**.

The anionic molecules **1** and **2a** possess a negative charge that can be delocalized within the structure. In **1** the charge is principally delocalized on the indene portion (about 75%) in both *E*-P,R and *Z*-P,R isomers. This charge moves towards the lactam portion in the TSs until reaching on it a distribution of about 60%. The relocation of the charge favors the stretching of the exocyclic double-bond (C1=C3'). In fact, the bond length changes from 1.36 Å in the *E* and *Z* equilibrium structures until 1.47 Å in the TSs structures. The presence of the sulfonyl group in **2a** seems to significantly alter the negative charge distribution. In fact, already in the equilibrium structures *E*-P,R and *Z*-P,R of **2a**, the negative charge is more distributed within the two heterocyclic rings (38% in the pyrrolidinone moiety of *E*-P,R-**2a** and 35% in *Z*-P,R-**2a**). The negative charge moves almost completely on the pyrrolidinone head in the TS structures of **2a** (80% in *E*-P,R-**2a** and 83% in *Z*-P,R-**2a**). The higher charge delocalization towards the lactam moiety alters the character of the double bond C1=C3' which is slightly elongated (1.40 Å) already in the *E*-P,R and *Z*-P,R and reaches 1.48 Å in the TSs structures.

Thus, the effect of the sulfonyl substituent does not seem to modify significantly the neutral form, but on the other hand, considerably changes the electronic distribution in the anionic form and drastically reduces the energy of the TSs structures. These effects could favor an *E/Z* thermal interconversion of the *N*-mesylated molecule in the anionic form without modifying the photochemical property in neutral form.
<figure><img src="image_6.png" /><figcaption>a)</figcaption></figure>

Dihedral angle $\theta = C_{7a}-C_{1}-C_{3'}-C_{4'}$

Pyramidalization angle $\psi = C_{2}-C_{7a}-C_{1}-C_{3'}$

<figure><img src="image_7.png" /><figcaption>b)</figcaption></figure>

$r_{1-3'} = 1.37 Å$

$\theta = 8^{\circ}$

$\psi = 180^{\circ}$

<figure><img src="image_8.png" /></figure>

$r_{1-3'} = 1.41 Å$

$\theta = 24^{\circ}$

$\psi = 178^{\circ}$

<figure><img src="image_9.png" /></figure>

$r_{1-3'} = 1.43 Å$

$\theta = 71^{\circ}$

$\psi = 147^{\circ}$

<figure><img src="image_10.png" /></figure>

$r_{1-3'} = 1.43 Å$

$\theta = 83^{\circ}$

$\psi = 174^{\circ}$

<figure><img src="image_11.png" /><figcaption>c)</figcaption></figure>

**Figure 4.** Computational characterization of the photochemical *E/Z* isomerization mechanism of anionic **2a** (TD-CAM-B3LYP/6-31G*, details see text). (a) Definition of the bond stretch, dihedral rotation and pyramidalization coordinates $r_{1-3'}$, $\theta$ and $\psi$. (b) Structures of optimized critical points. (c) Energy profile along the isomerization coordinate obtained with linear interpolation in internal coordinates between the critical points.

After the study of the ground-state energy profile of the *E/Z* isomerization, the photochemically induced process was studied for the anionic form **2a** with linear response time-dependent functional theory (TD-DFT), with the CAM-B3LYP functional and the 6-31G* basis set, using Gaussian16, in MeOH as implicit solvent. Following previous computational work for similar systems, we postulate that the isomerization involves passage through a conical intersection (**CI**) and focus our investigation on the energy profile involving this structure.<sup>22,28,39–47</sup> Structural changes were monitored following the $C_{1}-C_{3'}$ bond length ($r_{1-3'}$), the dihedral angle $\theta$, and the pyramidalization angle $\psi$ at $C_{1}$, where a value of $\psi = 180^{\circ}$ corresponds to planarity (see Figure 4a).

Starting from the ground state minimum (Franck-Condon structure, labeled **FC**), excited state optimization leads to structure $S_{1}$-min. Initially, the $S_{1}$ energy decreases from 78 kcal/mol to 69 kcal/mol. Excitation induces a stretch of the $C_{1}-C_{3'}$ bond from 1.37 Å at **FC** to 1.41 Å at $S_{1}$-min,
while retaining essentially the planarity, as seen from the small changes of the dihedral and pyramidalization angles (see Figure 4b). Using an implementation of the double-Newton-Raphson CI optimization algorithm⁴⁸ in a modified version of Gaussian, we have approximately characterized the CI by locating a point of near degeneracy between S₀ and S₁ (energy gap 0.1 kcal/mol) with a relative energy of 64 kcal/mol. At the surface crossing point, the central bond is stretched to 1.43 Å and rotated to θ = 71°, and there is partial pyramidalization at C₁ to 147°. The energy profile for the isomerization is completed with linear interpolations in internal coordinates between FC and S₁-min, this point and surface crossing point, and further to the Z isomer (see Figure 4c). There is a small barrier of 9 kcal/mol to access surface crossing point from S₁-min. The interpolation also shows that decay from surface crossing point can lead to the Z isomer, completing the isomerization. We have also located a second excited-state minimum where the central bond is rotated to θ = 83° and the planarity at C₁ is retained (ψ = 174°). Its relative excited-state energy is 58 kcal/mol and the energy gap to S₀ is 20 kcal/mol. This structure, labeled S₁-trap, may act as population trap on the excited state before the decay to the ground state.

Overall, higher-level calculations would be necessary to provide a more accurate description of the potential energy surface. However, the current approximate characterization provided by TD-DFT confirms the potential of our molecules as a light-induced switch. The results also suggest that access to the CI requires intramolecular rotation around the double bond and pyramidalization, and that population can be trapped on the excited state at S₁-trap after double bond rotation, before there is enough pyramidalization at C₁ to access CI.

## 2.2 Synthesis

On the basis of the computational predictions, compounds 2a,b were prepared according to the reaction shown in the scheme 1.
<figure><img src="image_12.png" /><figcaption>Scheme 1.</figcaption></figure>

**Reagents:** (i) n-Butyl Lithium, ClSO₂R, THF dry; (ii) BBr₃, CH₂Cl₂.

The previously reported compound 3²⁸ was reacted with mesyl chloride or tosyl chloride in presence of n-Butyl Lithium as base to obtain the intermediate **4a** and **4b** respectively. Then, the intermediate **4a,b** were treated with BBr₃ to obtain the phenols **2a,b** as E/Z mixture (E/Z ratio for each compound are reported in the experimental section).

The structures and the *E/Z* configuration of compounds **2a,b** were investigated by means of different NMR techniques. In particular, NOE experiments were also used to discriminate between *E* and *Z* form of each compound. In fact, the predominant forms of compounds **2a,b** showed contacts between H-4' and H-7 allowing to identify the *E* configuration, while the minor forms displayed contacts between H-4' and H-A possible in the *Z* configuration. ¹H and ¹³C NMR spectra of compounds **2a,b** were entirely assigned by means of homonuclear ¹H-¹H COSY,⁴⁹ ¹H-¹H-NOESY,⁵⁰ and heteronuclear ¹H-¹³C-HSQC,⁵¹ ¹H-¹³C-HMBC⁵² 2D correlation experiments.

The assigned ¹³C spectra of **2a,b** were checked in a comparative analysis respect to ¹³C spectrum of compound **1** (Figure 5) in order to experimentally evaluate the effects of the *N*-sulphonyl substituents on the electron distribution in the molecular orbital.
<figure><img src="image_13.png" /><figcaption>**Figure 5.** Effects of the *N*-sulfonyl substitution on the ¹³C NMR chemical shift values of the compounds **2a,b** with respect to compound **1**. ¹³C NMR spectra were recorded in deuterated methanol at 100 MHz. Chemical shift values (δ) are reported for compound **1**, while increments (Δδ) are given for **2a** and **2b**. In blue the value related to the *E* isomers and in red to the *Z* isomer.</figcaption></figure>

The introduction of sulfonyl groups into the structure of the prototype photoswitch **1** alters the electronic distribution of the entire molecule. In fact, the signals in the ¹³C NMR spectrum of compounds **2a,b** (in both *E/Z* isomeric forms) are shifted with respect to the signal of compound **1** (Figure 5). The most susceptible carbons were found in the pyrrolidinone head. In particular, the carbonyl carbon C-2' significantly shift upfield (-6.3 ppm in *E-2a* and -7.3 ppm in *E-2b*), as well as the carbon C-4' (-4.3 ppm in *E-2a* and -4.5 ppm in *E-2b*) and C-3' (-4.0 ppm in *E-2a* and -4.2 ppm in *E-2b*) suggesting a greater electronic density on them with respect to the same carbons in the compound **1**. On the other hand C-5', near to the nitrogen atom, shift downfield (+2.6 ppm in *E-2a* and +3.4 ppm in *E-2b*). In addition, a significant effect was found on the carbon C-1 which together with C-3' forms the isomerizable double bond. In fact, in both *E-2a,b* the value of C-1 signal shift of +5.3 ppm with respect to the C-1 in compound *E-1* suggests an electronic depletion. The comparative NMR studies confirm the greater electronic delocalization on the pyrrolidinone head in the sulfonated compound **2a,b**.

**2.3 Photophysical Properties.**

The absorption spectrum of the parent compound **1** (*E* isomer) strongly depends on the protonation/deprotonation state.⁵³ Its absorption maxima ($\lambda_{max}$) in methanol or DMSO and at neutral
or anionic (obtained by addition of KOH in excess) forms are reported in Table 1 in comparison with the $\lambda_{max}$ of the new N-substituted compounds **2a,b** (E/Z mixture at thermostationary state) obtained in the same conditions. In Table 1 are also reported the theoretical computed $\lambda_{max}$ values obtained by vertical excitation of the equilibrium structures reported above in figure 2. These $\lambda_{max}$ values cannot be directly compared to the experimental data because they have been obtained by simulation in implicit solvent. However, computed values give indication of the absorption maxima variations in the UV-vis spectrum. In fact, it is interesting to note that the $\lambda_{max}$ of **1** and **2a** shift to longer wavelengths in the anion as compared to the neutral molecules. Moreover, **2a** both in neutral and anionic form is more red-shifted than **E-1** suggesting an electronic effect of the substituent on the conjugated structure.

<table><caption>**Table 1.** $\lambda_{max}$ value of the E-1, (Z-1 in brackets) in neutral and anionic forms compared with the $\lambda_{max}$ value of the compounds **2a,b** in neutral and anionic forms in methanol and DMSO, and predicted values in gas-phase.</caption><thead><tr><th rowspan="3">Compound</th><th colspan="2">Methanol (1.70 D)</th><th colspan="2">DMSO (3.96 D)</th><th colspan="2">Computed<sup>d</sup></th></tr><tr><th>Neutral</th><th>Anionic<sup>c</sup></th><th>Neutral</th><th>Anionic<sup>c</sup></th><th>Neutral</th><th>Anionic</th></tr><tr><th>λ<sub>max</sub> (nm)</th><th>λ<sub>max</sub> (nm)</th><th>λ<sub>max</sub> (nm)</th><th>λ<sub>max</sub> (nm)</th><th>λ<sub>max</sub> (nm)</th><th>λ<sub>max</sub> (nm)</th></tr></thead><tbody><tr><td><b>1</b></td><td>320[324]<sup>a</sup></td><td>351[359]<sup>a</sup></td><td>320[322]<sup>a</sup></td><td>381[386]<sup>a</sup></td><td>286[288]<sup>d</sup></td><td>350[351]<sup>d</sup></td></tr><tr><td><b>2a</b> (9/1)<sup>b</sup></td><td>337</td><td>387</td><td>340</td><td>442</td><td>304[304]<sup>d</sup></td><td>370[380]<sup>d</sup></td></tr><tr><td><b>2b</b> (8/2)<sup>b</sup></td><td>341</td><td>392</td><td>344</td><td>446</td><td></td><td></td></tr></tbody></table>

The neutral form of the E-1 in solution does not display significant solvatochromism, with $\lambda_{max}$ values remaining at 320 nm in both solvents.²⁸ A similar behavior was observed also for the newly synthesized compounds **2a,b**. However, the N-sulfonyl substitution causes a significant red-shift of the $\lambda_{max}$ values by about 17-21 nm in methanol and about 20-24 nm in DMSO with respect to the unsubstituted compound E-1 (Table 1). By contrast, the anionic form of the reference compound E-1 is characterized by a significant red-shift of the $\lambda_{max}$ values with respect to the neutral form and displays solvatochromism ($\lambda_{max}$ = 351 nm in methanol, and 381 nm in DMSO). The N-sulfonyl derivatives compounds **2a,b** in anionic forms are stable and show even larger red-shift and solvatochromism, with $\lambda_{max}$ values red-shifted by about 50 nm in MeOH and 100 nm in

<footnote><sup>a</sup> $\lambda_{max}$ values of E and (in brackets) Z isomers; <sup>b</sup> E/Z composition of the thermostationary state at room temperature determined by ¹H NMR; <sup>c</sup> generated by addition of KOH to the neutral solutions; <sup>d</sup> computed values obtained with TD-DFT (CAM-B3LYP functional and 6-31G* as basis set): $\lambda_{max}$ values of E-P,R and (in bracket) Z-P,R isomers.</footnote>
DMSO, with respect to the corresponding neutral forms. The difference in λ<sub>max</sub> red-shift by about 50 nm between the two solvents may be due to the solvent's capability to separate the ion-pairs. In fact, it has been reported that the counterions form solvent separated ion-pairs in methanol and less separated ion-pairs may be possible in the aprotic solvents (e. g. DMSO).<sup>22</sup> Aprotic solvents seem to favor a better stabilization of S<sub>1</sub> with respect to S<sub>0</sub> or, alternatively, a lesser stabilization of the S<sub>0</sub> with respect to S<sub>1</sub> in comparison with protic solvents (e. g. through formation of a solvent-solute adducts).

The red-shift effect with respect to the compound **1** is already present with the mesyl group (**2a**). The introduction of the aromatic residues (tosyl) confers a further slight red-shift effect.

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

**Figure 6.** Comparison of the normalized UV-vis absorption spectra of compounds E-1, **2a,b** in neutral and anionic forms registered in methanol (A) and DMSO (B). The anionic forms are obtained by addition of an excess of KOH to the solutions.
The pronounced red-shift of **2a,b** with respect to the parent compound **1** could be caused by a higher stabilization of the negative charge on pyrrolidinone oxygen due to the electron withdrawing effect of the sulfonyl group capable of modulating the conjugation in the molecules generating a further charge separation at the nitrogen level.

In line with parent compound **1**, compounds **2a,b** did not show significant emission. This behaviour suggests that as for **1** also for the *N*-sulfonylated compounds **2a,b** the emission is not the preferential phenomenon for the relaxation from the excited state. This sets the basis for the characterization of these molecules as molecular photoswitches.

**2.4 Photochemical behaviours.**

The photoisomerization characteristics of compounds **2a,b** were investigated by irradiation in a Pyrex NMR tube at room temperature (ca. 2.0 mg in 0.5 mL of CD₃OD or DMSO-d₆) at two different irradiation wavelengths (320 and 380 nm) for each solvent in neutral form. The isomeric compositions at the photostationary states are reported in Table 2 and were calculated using ¹H NMR spectroscopy by computing the area ratio of the signals of specific diagnostic protons. In particular, the signals of the protons H-7, H-6, H-4, H-3 and H-4' show in most cases isomer-specific signals. In the two derivatives, and independently of the solvent, the ¹H NMR spectrum of the *Z* isomer shows the proton signal of H-7 shifted downfield with respect to that of the *E* isomer, while the signals of the other diagnostic protons shifted upfield.

The irradiation at 320 nm of the neutral compounds **2a,b** in solution generates a photostationary state constituted by an excess of *Z* isomer (63-65% of *Z* isomer), while irradiation at 380 nm yields a photostationary state mainly constituted of the *E* isomer (20-26% of *Z* isomers). This behavior suggests that, as happens for **1**, the absorption spectrum of each *Z* isomer is red-shifted with respect to the corresponding *E* isomer. This makes the isomeric composition adjustable by the light wavelength.

Moreover, after irradiation the resulting mixtures in neutral form at photostationary states can be stored in the dark at room temperature for a few days without observing any significant change in *E/Z* composition or decomposition of the compounds. This behavior confirms the chemical stability of the compounds and is in line with the computational prediction which showed for compound **2a** in neutral form an energy barrier on S₀ (i. e. thermal) similar to that of **1** and thus, high enough to prevent the isomerization process at room temperature.
<table><caption>**Table 2.** Analysis of *E/Z* composition of compounds **2a,b** at the photostationary states obtained by irradiation at 320 and 380 nm in methanol-d₄ or DMSO-d₆.</caption><thead><tr><th rowspan="3">Compound</th><th colspan="2">CD<sub>3</sub>OD</th><th colspan="2">DMSO-d<sub>6</sub></th></tr><tr><th colspan="2">E/Z composition (±0.1)</th><th colspan="2">E/Z composition (±0.1)</th></tr><tr><th>320 nm</th><th>380 nm</th><th>320 nm</th><th>380 nm</th></tr></thead><tbody><tr><td>2a</td><td>1/1.70</td><td>1/0.25</td><td>1/1.63</td><td>1/0.28</td></tr><tr><td>2b</td><td>1/1.86</td><td>1/0.35</td><td>1/1.86</td><td>1/0.33</td></tr></tbody></table>

The composition of the *E/Z* mixture of **1** in the anionic form is modulated using irradiation wavelengths higher than those used for its neutral form.²⁸

When KOH is added to colorless solutions of neutral compounds **2a,b**, the solutions turn yellow (in methanol) or slightly orange (in DMSO), due to the important red-shift of the absorption spectra reported above (table 1) for the anionic forms. Surprisingly, the ¹H NMR spectra, registered under the same conditions using deuterated solvents, reveal in a few minutes the substantial disappearance of the Z isomers from the solutions (regardless of the photostationary *E/Z* mixture initially prepared with the neutral compounds) and the almost complete (>90%) regeneration of the E isomers. The irradiation of the solutions with light at wavelengths within the absorption range (from about 340 to 460 nm in methanol and from about 380 to 500 nm in DMSO) does not cause any change to the *E/Z* compositions detectable by ¹H NMR. However, the addition of acetic acid (AcOH) to restore the phenolic form of the photoswitches, causes an immediate discoloration of the solution that turns colorless and the ¹H NMR spectra show the E isomers (>90%) in phenolic form. Subsequent irradiation of the solutions at 320 nm yields a photostationary state constituted of an excess of Z isomer as previously observed at this wavelength. Using 380 nm light, the *E/Z* compositions are overturned, in line with the previous photostationary states (Figure 7, and Figures S1-S3). The change of pH (by addition of KOH and subsequently addition of AcOH) and the subsequent irradiation does not cause degradation of the compounds confirming the exceptional chemical stability of the systems and the possibility of their continuous reuse.
<figure><img src="image_16.png" /><figcaption>**Figure 7.** Evolution of the *E/Z* composition of the compound **2a** in methanol-*d*₄ monitored by ¹HNMR (from bottom to top) starting from the thermostationary state (TSS) and changing of the irradiation wavelengths between 320 nm (PSS 320) and 380 nm (PSS 380) followed by pH-reset of the systems (KOH and AcOH) and irradiating again. All variation points (irradiation and deprotonation/protonation) were performed successively on the same sample using about 2 mg of **2a** in 0.5 mL of deuterated methanol. The "reset" of the samples (KOH/AcOH) was carried out by adding to the mixture 40 µL of 0.4 M KOH solution in methanol-*d*₄ and, after few minutes, an addition of about 10 µL of glacial acetic acid. All points were monitored by ¹H NMR spectroscopy and the isomer percentage in solution was obtained by evaluating the integrals of the diagnostic signal.</figcaption></figure>
The behavior in anionic form is in line with a lower *E/Z* interconversion energy barriers in *S*₀ of the newly photoswitches **2a,b** with respect to that of compound **1** and corroborate the predictions of the computational studies. The low energy barrier enables the non-radiative mediated conversion of the *Z* isomers in the more stable *E* isomers at room temperature in base environment working as a reset button.

**2.5 Time resolved spectroscopy.**

¹H NMR spectroscopy studies clearly demonstrate that the irradiation of compounds **2a,b** in their neutral forms produces an interconversion between two thermally stable diastereomeric forms. On the other hand, similar experiments suggest that the irradiation of *E*-**2a,b** in their anionic forms does not result in the formation of the *Z* diastereomers in appreciable amounts. This phenomenon can be explained by formulating two different hypotheses: 1) the *E*-diastereomers of sulfonated compounds in the anionic form are not capable of forming the *Z* isomers; 2) the *E*-diastereoisomers in the anionic form are capable of interconverting into the corresponding *Z*-diastereomers, which, however, are thermally unstable (initial hypothesis).

In order to evaluate experimentally which of the two hypotheses is correct, we investigate the photoisomerization dynamics of **2a** and **2b** in the anionic form by means of Transient Absorption Spectroscopy (TAS). TAS spectra were recorded in methanol solution with KOH in excess, using an experimental set-up already described and used on several occasions.<sup>23,28,30,54</sup>

<figure><img src="image_17.png" /></figure>

**Figure 7.** TA data (Δ*A*, coded in false color, bottom panels) recorded for (A) **2a** and (B) **2b** in methanol solution with excess of KOH upon excitation with a 350nm, 60 fs, pump pulse, as a function of pump-probe time delay (in ps) and probe wavelength (nm). The top panels display the stationary differential spectra Δ*A* observed at time delays > 0.5 ns.
Figure 7 displays an overview of the femtosecond transient absorption (TA) data recorded for the anionic compounds **2a** and **2b** dissolved in MeOH with excess KOH upon 350 nm excitation. In both cases we observe two signatures of the S₁ excited state at very early times, which are stimulated emission (SE - negative signal coded in blue color in the 420-600 nm range) and excited state absorption (ESA – positive signal coded in red color at λ<sub>probe</sub> < 420 nm). In **2b** (Figure 7-B, bottom panel), the initial ESA appears to redshift, possibly due to the overlapping red-shifting SE (the so-called dynamic Stokes shift). Global analysis of the **2b** dataset with a multiexponential decay fitting function (see details in Figure S4) reveals a biexponential decay of the SE and ESA signals, with a 220 fs time constant dominating the decay of SE and of the blue side of the ESA, and a 1.0 ps time constant accounting for the decay of the residual, red-shifted SE and further decay of the ESA. Such a biexponential decay was already observed - with shorter timescales of 120 fs and 360 fs - in the parent compound **1**, where the first timescale was attributed to S₁ dynamics/relaxation while the second would characterize the S₁ lifetime and S₀ photoproduct formation time. The further time evolution in the **2b** dataset occurs on the 9 ps timescale tentatively attributed to vibrational relaxation in the ground state S₀. The final TA spectrum (Figure 7-B, top panel) displays a residual ground state bleach (at ~385 nm) and weakly positive photoproduct absorption (at ~430nm) as expected for the signature of the *E* to *Z* photoisomerization, in line with the above prediction of a redshift for the *Z* isomer absorption spectrum in gas phase.

The TA data for compound **2a** display a qualitative difference as compared to **2b**. While the SE signature and decay look similar, the decay of the early ESA reveals the residual ground state bleach at 380nm and is followed by the rise of another transient absorption centered at 415nm, hereafter called ESAₓ. Notably, the global multiexponential fit of the entire dataset fails, since it systematically results in the presence of two nearly identical time constants associated with very large (i.e. unphysical), nearly opposite amplitudes. Only when fitting the data starting from time delays larger than 0.4 or 0.6 ps can we obtain a good fit (see details in Figure S5) revealing that the decay of ESAₓ occurs on the 2 ps timescale (±15% error bar), together with the decay of a very weak SE signal tail and with the ground state bleach recovery. Most of the SE signal actually decays earlier with a 320 fs timescale (see Figure S5-A). We therefore tentatively propose that ESAₓ be the signature of a (nearly) “dark” conformation of the S₁ state characterized by a very weak SE, which would decay to S₀ (GSB recovery) on the 2 ps timescale. Further spectral relaxation is again observed on the 10 ps timescale and assigned to S₀ vibrational relaxation (as in **2b**). The final TA spectrum for **2a** (Figure 7-A, top panel) resembles that of **2b**, and also suggests the formation of the *Z* isomer.
<figure><img src="image_18.png" /></figure>

**Figure 8.** Comparison of the ESA ($\lambda_{probe} = 400$ nm, top panel) and SE (bottom panel) kinetic traces recorded for **2a**, **2b** (together with the global fit in green), and **E-1**. The kinetic traces are extracted from the dataset displayed in figure 7 for **2a** and **2b** (SE monitored at $\lambda_{probe} = 460$ nm) and from the dataset displayed in figure 8 of ref 28 for **E-1** (SE monitored at $\lambda_{probe} = 475$ nm).

Figure 8 provides a qualitative comparison of the decay kinetics observed for the SE and ESA signals in compounds **E-1**, **2a** and **2b**. Qualitatively, the kinetics traces of **E-1** and **2b** are similar, except that the S<sub>1</sub> lifetime is shorter in **E-1**. For compound **2a**, while the SE signal decays on an intermediate timescale, the early ESA (Figure 8 top panel) decays as fast as in **E-1**, but is followed by the rise of the ESA<sub>x</sub>, which is longer-lived. We note, that the ESA<sub>x</sub> being assigned to a slightly longer-lived (2ps) population in a “dark” region of the S<sub>1</sub> state may correspond the “S<sub>1</sub>-trap” structure located in the calculations above. The weak SE assigned to this species is consistent with the small calculated energy gap (20 kcal/mol) and oscillator strength (f = 0.001).

While ¹H NMR spectroscopy (minute timescale) of sulfonated compounds **2a,b** in anionic form reveals an ineffective photoconversion of the *E*-isomer into the *Z*-isomer, the final TAS spectra observed in the Figure 7 (few 100ps timescale) and the predicted red-shift of the *Z* isomer absorption spectrum with respect to *E* strongly support that *E*-to-*Z* isomerization occurs on the picosecond timescale with a non-vanishing quantum yield. This observation corroborates our hypothesis that the failure to accumulate the anionic form of the *Z* isomer in solution is due to its thermal return to the *E* isomer on a minute timescale.

**3. Conclusions**

The set-reset function in molecular systems is an important step to construct memory elements in sequential logic process. Molecular photoswitch can represent an on-off switch
controllable by light. However, the insertion of a third control element, the “reset button”, could
represent an added value to the classical binary system.

Starting from our biomimetic photoswitch **1**,²⁸ we designed the insertion of a methane
sulfonic functional group on the pyrrolidinone nitrogen atom (compound **2a**), potentially capable of
significantly altering the electronic distribution within the structure. The synthesis of **2a**, and of its
analogue **2b** bearing a tosyl group, confirmed the initial hypothesis giving us the opportunity to
observe a light-induced alternation between the two thermal stable *E/Z* isomer in neutral condition
(phenolic form). Moreover, the deprotonation of the phenol group provided molecular systems in
which the *E* isomers can be photoconverted into the corresponding *Z* isomers in ultrafast timescale
(picoseconds), but these latter, due to the low thermal interconversion barrier, promptly evolves into
the starting *E* molecules. By re-establishing the initial protonation conditions, the two molecules
also re-established the photochemical properties, producing a "rewritable" double-controlled
systems. These molecules represent a prototype, which, when appropriately conjugated to biological
or synthetic structure, due to the versatility of the sulfonation reaction and the high stability of the
sulfonamide group in acid and basic environment, can confer "light and pH" modular properties.

Acknowledgment

Mar. M. and J.L. thank Dr. Ilias Nikolinakos and Dr. Olivier Crégut for assistance with the TA data acquisition. We acknowledge support from the French ANR via the "PhotoMecha" project (ANR-20-CE29-0021-01), and from the Interdisciplinary Thematic Institute QMat as part of the ITI 2021-2028 program of the University of Strasbourg, CNRS and Inserm via the IdEx Unistra (ANR 10 IDEX 0002), SFRI STRAT'US (ANR 20 SFRI 0012), and Labex NIE (ANR-11-LABX-0058-NIE) projects of the French Investments for the Future Program. A.C., M.O., and M.P. acknowledge the MUR for the financial support under the project CN00000041-"National Center for Gene Therapy and Drugs based on RNA Technology"-CUP B63C2200061 0006 Mission 4 Component 2 (M4C2)-investment 1.4 [CN3] of the National Recovery and Resilience Plan (PNRR) funded by the European Union "Next Generation EU". M. P. acknowledge the University of Siena for the financial support of the project Chromo-GEnUP through the F-CUR2022 funding line (2265-2022-PM-CONRICMIUR_PC-FCUR2022_003).
**Supporting Information**

The Supporting Information is available free of charge at https://pubs.acs.org/doi/

Synthesis, additional irradiation experiments, TAS data, and computational details.

**4. References**

(1) Fan, L. Z.; Lin, M. Z. *Wiley Interdiscip. Rev. Dev. Biol.* **2015**, *4* (5), 545.

(2) Zhou, X. X.; Lin, M. Z. *Curr. Opin. Chem. Biol.* **2013**, *17* (4), 682.

(3) Shen, L.; Lu, Q.; Zhu, A.; Lv, X.; An, Z. *ACS Macro Lett.* **2017**, *6* (6), 625.

(4) Abendroth, J. M.; Bushuyev, O. S.; Weiss, P. S.; Barrett, C. J. *ACS Nano* **2015**, *9* (8), 7746.

(5) Sauer, M. *Proc. Natl. Acad. Sci. U. S. A.* **2005**, *102* (27), 9433.

(6) Feringa, B. L.; Koumura, N.; Van Delden, R. A.; Ter Wiel, M. K. J. *Appl. Phys. A Mater. Sci. Process.* **2002**, *75* (2), 301.

(7) Tahara, R.; Morozumi, T.; Nakamura, H.; Shimomura, M. *J. Phys. Chem. B* **1997**, *101* (97), 7736.

(8) Blanco-Lomas, M.; Samanta, S.; Campos, P. J.; Woolley, G. A.; Sampedro, D. *J. Am. Chem. Soc.* **2012**, *134*, 6960.

(9) Kienzler, M. a.; Reiner, A.; Trautman, E.; Yoo, S.; Trauner, D.; Isacoff, E. Y. *J. Am. Chem. Soc.* **2013**, *135*, 17683.

(10) Quandt, G.; Höfner, G.; Pabel, J.; Dine, J.; Eder, M.; Wanner, K. T. *J. Med. Chem.* **2014**, *57*, 6809.

(11) Velema, W. A.; Szymanski, W.; Feringa, B. L. *J. Am. Chem. Soc.* **2014**, *136* (6), 2178.

(12) García-López, V.; Chen, F.; Niewski, L. G.; Duret, G.; Aliyan, A.; Kolomeisky, A. B.; Robinson, J. T.; Wang, G.; Pal, R.; Tour, J. M. *Nature* **2017**, *548* (7669), 567.

(13) Ndong, M.; Bomble, L.; Sugny, D.; Justum, Y.; Desouter-Lecomte, M. *Phys. Rev. A - At. Mol. Opt. Phys.* **2007**, *76* (4), 1.

(14) Muramatsu, S.; Kinbara, K.; Taguchi, H.; Ishii, N.; Aida, T. *J. Am. Chem. Soc.* **2006**, *128* (11), 3764.

(15) Andréasson, J.; Pischel, U. *Chem. Soc. Rev.* **2015**, *44* (5), 1053.

(16) Kink, F.; Collado, M. P.; Wiedbrauk, S.; Mayer, P.; Dube, H. *Chem. - A Eur. J.* **2017**, *23* (26), 6237.

(17) Andréasson, J.; Pischel, U. *Coord. Chem. Rev.* **2021**, *429*, 213695.

(18) Zanirato, V.; Pollini, G. P.; De Risi, C.; Valente, F.; Melloni, A.; Fusi, S.; Barbetti, J.; Olivucci, M. *Tetrahedron* **2007**, *63*, 4975.

(19) Léonard, J.; Schapiro, I.; Briand, J.; Fusi, S.; Paccani, R. R.; Olivucci, M.; Haacke, S. *Chem. - A Eur. J.* **2012**, *18*, 15296.

(20) Dunkelberger, A. D.; Kieda, R. D.; Shin, J. Y.; Rossi Paccani, R.; Fusi, S.; Olivucci, M.; Fleming Crim, F. *J. Phys. Chem. A* **2012**, *116* (14), 3527.

(21) Lumento, F.; Zanirato, V.; Fusi, S.; Busi, E.; Latterini, L.; Elisei, F.; Sinicropi, A.; Andruniów, T.; Ferré, N.; Basosi, R.; Olivucci, M. *Angew. Chemie Int. Ed.* **2007**, *46* (3), 414.

(22) Sinicropi, A.; Martin, E.; Ryazantsev, M.; Helbing, J.; Briand, J.; Sharma, D.; Léonard, J.; Haacke, S.; Cannizzo, A.; Chergui, M.; Zanirato, V.; Fusi, S.; Santoro, F.; Basosi, R.; Ferré, N.; Olivucci, M. *Proc. Natl. Acad. Sci. U. S. A.* **2008**, *105* (46), 17642.

(23) Briand, J.; Braem, O.; Rehault, J.; Leonard, J.; Cannizzo, A.; Chergui, M.; Zanirato, V.; Olivucci, M.; Helbing, J.; Haacke, S. *Phys. Chem. Chem. Phys.* **2010**, *12* (13), 3178.
(24) Léonard, J.; Briand, J.; Fusi, S.; Zanirato, V.; Olivucci, M.; Haacke, S. *New J. Phys.* **2013**, *15*, 105022.

(25) Pagano, K.; Paolino, M.; Fusi, S.; Zanirato, V.; Trapella, C.; Giuliani, G.; Cappelli, A.; Zanzoni, S.; Molinari, H.; Ragona, L.; Olivucci, M. *J. Phys. Chem. Lett.* **2019**, *10* (9), 2235.

(26) Gueye, M.; Paolino, M.; Gindensperger, E.; Haacke, S.; Olivucci, M.; Léonard, J. *Faraday Discuss.* **2020**, *221*, 299.

(27) Gueye, M.; Manathunga, M.; Agathangelou, D.; Orozco, Y.; Paolino, M.; Fusi, S.; Haacke, S.; Olivucci, M.; Léonard, J. *Nat. Commun.* **2018**, *9* (1), 313.

(28) Paolino, M.; Gueye, M.; Pieri, E.; Manathunga, M.; Fusi, S.; Cappelli, A.; Latterini, L.; Pannacci, D.; Filatov, M.; Léonard, J.; Olivucci, M. *J. Am. Chem. Soc.* **2016**, *138* (31), 9807.

(29) Paolino, M.; Giovannini, T.; Manathunga, M.; Latterini, L.; Zampini, G.; Pierron, R.; Léonard, J.; Fusi, S.; Giorgi, G.; Giuliani, G.; Cappelli, A.; Cappelli, C.; Olivucci, M. *J. Phys. Chem. Lett.* **2021**, *12* (16), 3875.

(30) Filatov(Gulak), M.; Paolino, M.; Pierron, R.; Cappelli, A.; Giorgi, G.; Léonard, J.; Huix-Rotllant, M.; Ferré, N.; Yang, X.; Kaliakin, D.; Blanco-González, A.; Olivucci, M. *Nat. Commun.* **2022**, *13* (1), 6433.

(31) Ikonnikov, E.; Paolino, M.; Garcia-Alvarez, J. C.; Orozco-Gonzalez, Y.; Granados, C.; Röder, A.; Léonard, J.; Olivucci, M.; Haacke, S.; Kornilov, O.; Gozem, S. *J. Phys. Chem. Lett.* **2023**, *14* (26), 6061.

(32) Zimmer, M. *Chem. Rev.* **2002**, *102*, 759.

(33) Subach, F. V.; Verkhusha, V. V. *Chem. Rev.* **2012**, *112*, 4308.

(34) Paolino, M.; Saletti, M.; Reale, A.; Licciardi, M.; Varvarà, P.; Marquette, A.; Léonard, J.; Bonechi, C.; Donati, A.; Giorgi, G.; Giuliani, G.; Carlotti, B.; Ortica, F.; Latterini, L.; Gentile, M.; Paccagnini, E.; Olivucci, M.; Cappelli, A. *Chem. – A Eur. J.* **2022**, *28* (50), e202201477.

(35) Barondeau, D. P.; Kassmann, C. J.; Tainer, J. A.; Getzoff, E. D. *Biochemistry* **2005**, *44* (6), 1960.

(36) Becke, A. D. *J. Chem. Phys.* **1993**, *98* (7), 5648.

(37) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Petersson, G. A.; Nakatsuji, H.; Li, X.; Caricato, M.; Marenich, A.; Bloino, J.; Janesko, B. G.; Gomperts, R.; Mennucci, B.; Hratchian, H. P.; Ortiz, J. V.; Izmaylov, A. F.; Sonnenberg, J. L.; Williams-Young, D.; Ding, F.; Lipparini, F.; Egidi, F.; Goings, J.; Peng, B.; Petrone, A.; Henderson, T.; Ranasinghe, D.; Zakrzewski, V. G.; Gao, J.; Rega, N.; Zheng, G.; Liang, W.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Throssell, K.; Montgomery, J. A. J.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Keith, T.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Millam, J. M.; Klene, M.; Adamo, C.; Cammi, R.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Farkas, O.; Foresman, J. B.; Fox, D. J. inc., Wallingford CT 2016,.

(38) Dreuw, A.; Head-Gordon, M. *Chem. Rev.* **2005**, *105* (11), 4009.

(39) Filatov(Gulak), M.; Paolino, M.; Kaliakin, D.; Olivucci, M.; Kraka, E.; Min, S. K. *Commun. Phys.* **2024**, *7* (1), 219.

(40) Conyard, J.; Heisler, I. A.; Chan, Y.; Bulman Page, P. C.; Meech, S. R.; Blancafort, L. *Chem. Sci.* **2018**, *9* (7), 1803.

(41) Schapiro, I.; Fusi, S.; Olivucci, M.; Andruniów, T.; Sasidharanpillai, S.; Loppnow, G. R. *J. Phys. Chem. B* **2014**, *118* (42), 12243.

(42) Filatov, M.; Olivucci, M. *J. Org. Chem.* **2014**, *79*, 3587.

(43) Lami, M.; Barneschi, L.; Saletti, M.; Olivucci, M.; Cappelli, A.; Paolino, M. *ChemPhotoChem* **2024**, *n/a* (n/a), e202400093.

(44) Migani, A.; Olivucci, M. In *Conical Intersections. Electronic Structure, Dynamics and Spectroscopy*;
Domcke, W., Yarkony, D. R., Köppel, H., Eds.; Advanced series in physical chemistry; World Scientific: Singapore, 2004; Vol. 15, pp 271–320.

(45) Filatov, M.; Paolino, M.; Min, S. K.; Kim, K. S. *J. Phys. Chem. Lett.* **2018**, *9* (17), 4995.

(46) Filatov, M.; Paolino, M.; Min, S. K.; Choi, C. H. *Chem. Commun.* **2019**, *55* (36), 5247.

(47) Baek, Y. S.; Lee, S.; Filatov, M.; Choi, C. H. *J. Phys. Chem. A* **2021**, *125* (9), 1994.

(48) Ruiz-Barragan, S.; Robb, M. A.; Blancafort, L. *J. Chem. Theory Comput.* **2013**, *9* (3), 1433.

(49) K. Nagayama, A. Kumar, K. W. and R. R. E. *J. Magn. Reson.* **1980**, *40*, 321.

(50) Jeener, J.; Meier, B. H.; Bachmann, P.; Ernst, R. R. *J. Chem. Phys.* **1979**, *71* (11), 4546.

(51) Bax, A.; Griffey, R. H.; Hawkins, B. L. *J. Magn. Reson.* **1983**, *55* (2), 301.

(52) Bax, A.; Summers, M. F. *J. Am. Chem. Soc.* **1986**, *108* (8), 2093.

(53) Weber, W.; Helms, V.; McCammon, J. a; Langhoff, P. W. *Proc. Natl. Acad. Sci. U. S. A.* **1999**, *96* (May), 6177.

(54) Skilitsi, A. I.; Agathangelou, D.; Shulov, I.; Conyard, J.; Haacke, S.; Mély, Y.; Klymchenko, A.; Léonard, J. *Phys. Chem. Chem. Phys.* **2018**, *20* (11), 7885.