**1. Introduction**

Fluorescent organic molecules with strong intramolecular hydrogen bonds (intraHBs) connected by proton donating and accepting groups have gained considerable attention

**Citation:** Kerdpol, K.; Daengngern, R.; Sattayanon, C.; Namuangruk, S.; Rungrotmongkol, T.; Wolschann, P.; Kungwan, N.; Hannongbua, S. Effect of Water Microsolvation on the Excited-State Proton Transfer of 3-Hydroxyflavone Enclosed in γ-Cyclodextrin. *Molecules* **2021**, *26*, 843. https://doi.org/10.3390/ molecules26040843

Academic Editors: Marina Isidori, Margherita Lavorgna and Rosa Iacovino Received: 10 January 2021 Accepted: 2 February 2021 Published: 5 February 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

in recent years owing to their unique fluorescent emission in term of large Stokes shift without self-absorption [1]. Their unique properties harnessing from the excited state intramolecular proton transfer (ESIntraPT) could be typically described by the characteristic four-level photocycle. Initially, the molecule in an enol form (E) in the ground (S0) state absorbing light in the shorter wavelength region results in the photoexcitation process from the S<sup>0</sup> state into the excited (S1) state. The intraHB of E is strengthened in the S<sup>1</sup> state because of the charge redistribution upon photoexcitation, leading to the transfer of proton from the donor (D: −NH2, −OH) to the acceptor (A: C=O, −N=), which changes the enol form (E\*) to the keto form (K\*) in the S<sup>1</sup> state. After that, the K\* emits the fluorescence in the remarkably longer wavelength than the absorption and relaxes to the S<sup>0</sup> state, resulting in the notably large Stokes shift (the difference between positions of absorption and emission peaks). Then the K changes to the E through the back proton transfer (BPT) process spontaneously in the S<sup>0</sup> state due to the PT barrierless and high exothermic reaction. Generally, their photophysical properties can be easily modulated using many strategies [2,3] such as introducing electron-donating and withdrawing substituents, the heteroatom substitution, and the π-conjugation into the main core structure, to give the desirable absorption and emission spectra as well as large Stokes shift. The ESIntraPT molecules with such tunable photophysical properties including derivatives of salicylates [4–6], salicylideneanilines [7–9], flavones [10–13], benzazoles [14–18], and chalcones [19–21] have been reported and widely used in various applications ranging from chemical sensing to light-emitting diodes [22–27].

Among various ESIntraPT molecules, 3-hydroxyflavone (3HF), which consists of a chromone ring (C-ring) and a phenyl ring (P-ring), is one of the best-known molecular systems. 3HF exhibits ESIntraPT and gives dual fluorescence corresponding to its E\* and K\* forms with a large Stokes shift and photostability [28–30]. Thus, 3HF has been used as a prototype for the ESIntraPT processes and as sensitive fluorescence probes for discovering binding sites in various bio-relevant targets such as DNA, protein, and biomembranes [31–33]. The photophysical properties and ESPT processes of 3HF in organic solvents have been extensively studied [34–42]. In non-polar solvent, only the K\* emission peak of 3HF in toluene was observed at 530 nm with large Stokes shift [33] because the ESIntraPT process effectively occurs, giving only K\* form. However, in a protic solvent, the dual emission peaks from E\* and K\* of 3HF in methanol were observed at 409 and 528 nm, respectively [33], because the IntraHB of 3HF is disrupted and the intermolecular hydrogen bonds (interHBs) between 3HF and protic solvents is formed depending on the nature of solvents and the arrangement of protic solvent around 3HF. This favorable formation of interHBs could reduce the formation of K\*, resulting in the low quantum yield in protic solvents or aqueous solution [40–44].

One of the strategies to enhance the K\* emission intensity of 3HF in an aqueous solution is disrupting the interHBs of 3HF-water cage-like network using cyclodextrin (CD) [45–47]. The reduction of polarity and restricted environment inside CD's cavity is essential for many aspects of photophysical phenomena by inclusion complexes between 3HF and CD [45–47]. CDs are the cyclic oligosaccharides consisting of the crucial α-Dglucose unit, which exhibit the conical shape and the α-D-glucose units of 6, 7, and 8 are represented as α-, β-, and γ-cyclodextrins (α-, β-, and γ-CDs), respectively [48]. S. Das and N. Chattopadhyay experimentally studied the fluorescence anisotropy of the inclusion complexes of 3HF in α-, β-, and γ-CDs compared to 3HF in the aqueous medium. The fluorescence anisotropy of these probes decreased following the order α-, β-, and γ-CD, which is attributed to the disruption of the 3HF-water network in an aqueous medium [46]. It can be stated that the micro-environment of 3HF derivatives was able to alter and prevent the self-aggregation effectively by using CDs, especially γ-CD. From the investigation on ESPT processes of 3HF in β-, and γ-CDs [45,47], the intensity of K\* of 3HF/γ-CD inclusion complex is higher than that of 3HF/β-CD inclusion complex. Consequently, the encapsulation in CDs should be a possible method to tune the fluorescence emission of 3HF derivatives and other hydrophobic compounds [45–47,49–52].

From the studies of the multi-spectroscopic approaches and molecular docking of the encapsulation of 3HF in different small-ring CDs, the 3HF/γ-CD complex showed the strongest interaction, and it provided a higher fluorescent yield than that in water medium [45–47]. Understanding the role of CD in enhancement of the fluorescent yield at atomic level might help us to be able to adjust the fluorescent wavelength to fit for fluorescence probes for bio-labeling in aqueous medium. However, to the best of our knowledge, the detailed information of ESPT reaction in the S<sup>0</sup> and S<sup>1</sup> states of 3HF in γ-CD at atomic level has not been reported. In this work, we aimed to systematically investigate the effect of a water molecule on the photophysical properties and ESPT reactions of an isolated 3HF and its encapsulation. All-atom molecular dynamics (MD) simulations for 300 ns were performed to study the structure and dynamics properties of the two possible 3HF/γ-CD complexes. The detailed information of each complex both in the S<sup>0</sup> and S<sup>1</sup> states was then investigated using density functional theory (DFT) and time-dependent DFT (TD-DFT) methods. The important distances and simulated infrared (IR) vibrational spectra from the optimized structures as well as the topology analysis were used to describe the hydrogen-bonded strength. The frontier molecular orbitals (MOs) were analyzed to provide the charge distribution of the complex. The simulated absorption and emission spectra were calculated and compared with the experimental data. Moreover, the energies of E and K forms of 3HF in each system at the S<sup>0</sup> and S<sup>1</sup> states were discussed to explain why the fluorescent yield of K\* in aqueous medium increases when encapsulating 3HF into γ-CD. From the studies of the multi-spectroscopic approaches and molecular docking of the encapsulation of 3HF in different small-ring CDs, the 3HF/γ-CD complex showed the strongest interaction, and it provided a higher fluorescent yield than that in water medium [45–47]. Understanding the role of CD in enhancement of the fluorescent yield at atomic level might help us to be able to adjust the fluorescent wavelength to fit for fluorescence probes for bio-labeling in aqueous medium. However, to the best of our knowledge, the detailed information of ESPT reaction in the S0 and S1 states of 3HF in γ-CD at atomic level has not been reported. In this work, we aimed to systematically investigate the effect of a water molecule on the photophysical properties and ESPT reactions of an isolated 3HF and its encapsulation. All-atom molecular dynamics (MD) simulations for 300 ns were performed to study the structure and dynamics properties of the two possible 3HF/γ-CD complexes. The detailed information of each complex both in the S0 and S1 states was then investigated using density functional theory (DFT) and time-dependent DFT (TD-DFT) methods. The important distances and simulated infrared (IR) vibrational spectra from the optimized structures as well as the topology analysis were used to describe the hydrogen-bonded strength. The frontier molecular orbitals (MOs) were analyzed to provide the charge distribution of the complex. The simulated absorption and emission spectra were calculated and compared with the experimental data. Moreover, the energies of E and K forms of 3HF in each system at the S0 and S1 states were discussed to explain why the fluorescent yield of K\* in aqueous medium increases when encapsulating 3HF into γ-CD.

complex is higher than that of 3HF/β-CD inclusion complex. Consequently, the encapsulation in CDs should be a possible method to tune the fluorescence emission of 3HF de-

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rivatives and other hydrophobic compounds [45–47,49–52].

#### **2. Results and Discussion 2. Results and Discussion**

#### *2.1. Possible Inclusion Complexes 2.1. Possible Inclusion Complexes*

From the docking study, 3HF could form two possible inclusion complexes with γ-CD through its chromone ring (C-ring, Form I) or phenyl ring (P-ring, Form II) insertion into the hydrophobic cavity as depicted in Figure 1B. Although a higher occurrence (65%) was found for Form I, the interaction energies of both forms were likely comparable (Form I: −21.42 kcal/mol and Form II: −20.78 kcal/mol). Additionally, the previous docking studies [46,47] suggested that Form II was more stable. Thus, in the present work the 3HF/γ-CD complexes in both forms were further studied by MD simulations and DFT calculations. From the docking study, 3HF could form two possible inclusion complexes with γ-CD through its chromone ring (C-ring, Form I) or phenyl ring (P-ring, Form II) insertion into the hydrophobic cavity as depicted in Figure 1B. Although a higher occurrence (65%) was found for Form I, the interaction energies of both forms were likely comparable (Form I: −21.42 kcal/mol and Form II: −20.78 kcal/mol). Additionally, the previous docking studies [46,47] suggested that Form II was more stable. Thus, in the present work the 3HF/γ-CD complexes in both forms were further studied by MD simulations and DFT calculations.

**Figure 1.** (**A**) Chemical structure of 3-Hydroxyflavone, 3HF. (**B**) Docked structures of the two possible 3HF/γ-CD complexes, Form I and Form II, where their percentage of occurrence and the lowest interaction energy retrieved from 100 independent docking runs are also given. **Figure 1.** (**A**) Chemical structure of 3-Hydroxyflavone, 3HF. (**B**) Docked structures of the two possible 3HF/γ-CD complexes, Form I and Form II, where their percentage of occurrence and the lowest interaction energy retrieved from 100 independent docking runs are also given.

#### *2.2. 3HF Mobility in γ-CD Cavity and Water Accessibility 2.2. 3HF Mobility in γ-CD Cavity and Water Accessibility*

To study the inclusion complexes in solution, the four different initial structures of Form I and Form II obtained from molecular docking and QM calculation were simulated by 300-ns MD simulations (MD1-MD4). All trajectories were analyzed and discussed as follows. The plots of RMSD and Rgyr of complex in Supplemental Figure S1 suggested that in Form I 3HF spontaneously released from the γ-CD cavity at ~68 ns, ~54 ns, and ~198 ns To study the inclusion complexes in solution, the four different initial structures of Form I and Form II obtained from molecular docking and QM calculation were simulated by 300-ns MD simulations (MD1-MD4). All trajectories were analyzed and discussed as follows. The plots of RMSD and Rgyr of complex in Supplemental Figure S1 suggested that in Form I 3HF spontaneously released from the γ-CD cavity at ~68 ns, ~54 ns, and ~198 ns for MD1, MD2 and MD3. Interestingly, it feasibly moved back to form a complexation with γ-CD, resulting in Form I (MD3 ~201 ns) and Form II (MD1 ~70 ns and MD2 ~57 ns) as considerably seen by the plot of distance between the center of mass (Cm) of each 3HF ring and the C<sup>m</sup> of the primary rim of the γ-CD in Figure 2, and the plot of distance between

the C<sup>m</sup> of each 3HF ring and the C<sup>m</sup> of the secondary rim of the γ-CD in Supplementary Materials Figure S2. This is in contrast for Form II, in which the 3HF was well encapsulated inside the hydrophobic cavity in all MD1-MD4 systems throughout the simulation time (RMSD of 1.2–4.0 Å, and Rgyr of 6.0−7.0 Å). Moreover, it can be noticeable that the both rings of 3HF in Form I (MD3 and MD4) were fluctuated higher than those in Form II, suggesting that the complex in Form II was found to be more stable in aqueous solution; in other words, the P-ring insertion is the suitable binding mode of 3HF for encapsulation with γ-CD in consistent with the lower water accessibility to the encapsulated 3HF (*n(r)* of 2.0 ± 0.7 and 2.5 ± 1.2 at O1 and O2, respectively, in Figure 3). In Form I, the hydroxyl oxygen O1 of 3HF positioned closer to the wider rim of γ-CD had a significantly higher interaction with waters (4.4 ± 0.8), and in vice versa less waters (2.0 ± 0.2) can access to the carbonyl oxygen O2. No peak detected within ~2.8 Å of the O3 of 3HF, suggesting that this atom had a very weak hydration interaction as found in some flavonoids/CD complexes [53,54]. It is worth noting that such accessible water molecules at O1 and O2 sites may involve into proton transfer processes either blocking ESIntraPT or assisting ESInterPT in 3HF/γ-CD inclusion complex. To study the water assisted PT in 3HF/γ-CD either ground state or excited state, the model of 3HF/γ-CD with a water molecule placed between these two sites of 3HF was further investigated by DFT and TD-DFT calculations and discussed in the next sections. tween the Cm of each 3HF ring and the Cm of the secondary rim of the γ-CD in Supplementary Materials Figure S2. This is in contrast for Form II, in which the 3HF was well encapsulated inside the hydrophobic cavity in all MD1-MD4 systems throughout the simulation time (RMSD of 1.2–4.0 Å, and Rgyr of 6.0−7.0 Å). Moreover, it can be noticeable that the both rings of 3HF in Form I (MD3 and MD4) were fluctuated higher than those in Form II, suggesting that the complex in Form II was found to be more stable in aqueous solution; in other words, the P-ring insertion is the suitable binding mode of 3HF for encapsulation with γ-CD in consistent with the lower water accessibility to the encapsulated 3HF (*n(r)* of 2.0 ± 0.7 and 2.5 ± 1.2 at O1 and O2, respectively, in Figure 3). In Form I, the hydroxyl oxygen O1 of 3HF positioned closer to the wider rim of γ-CD had a significantly higher interaction with waters (4.4 ± 0.8), and in vice versa less waters (2.0 ± 0.2) can access to the carbonyl oxygen O2. No peak detected within ~2.8 Å of the O3 of 3HF, suggesting that this atom had a very weak hydration interaction as found in some flavonoids/CD complexes [53,54]. It is worth noting that such accessible water molecules at O1 and O2 sites may involve into proton transfer processes either blocking ESIntraPT or assisting ESInterPT in 3HF/γ-CD inclusion complex. To study the water assisted PT in 3HF/γ-CD either ground state or excited state, the model of 3HF/γ-CD with a water molecule placed between these two sites of 3HF was further investigated by DFT and TD-DFT calculations and discussed in the next sections.

for MD1, MD2 and MD3. Interestingly, it feasibly moved back to form a complexation with γ-CD, resulting in Form I (MD3 ~201 ns) and Form II (MD1 ~70 ns and MD2 ~57 ns) as considerably seen by the plot of distance between the center of mass (Cm) of each 3HF ring and the Cm of the primary rim of the γ-CD in Figure 2, and the plot of distance be-

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**Figure 2.** The plots of distance measured from the Cm of each 3HF ring to the Cm of the primary rim of γ-CD (all 7 O6 atoms) for the four MD simulations MD1-MD4 with different initial structures of complexes in Form I and Form II. **Figure 2.** The plots of distance measured from the C<sup>m</sup> of each 3HF ring to the C<sup>m</sup> of the primary rim of γ-CD (all 7 O6 atoms) for the four MD simulations MD1-MD4 with different initial structures of complexes in Form I and Form II.
