Screening the Optimal Probe by Expounding the ESIPT Mechanism and Photophysical Properties in Bis-HBX with Multimodal Substitutions

DFT and TD-DFT were used in this article to investigate the effects of different substitutions at multiple sites on the photophysical mechanism of bis-HBX in the gas phase. Four different substitution modes were selected, denoted as A1 (X=Me, Y=S), A2 (X=OMe, Y=S), B1 (X=Me, Y=NH), and C1 (X=Me, Y=O). The geometric parameters proved that the IHBs enhanced after photoexcitation, which was conducive to promote the ESIPT process. Combining the analysis of the PECs, it was revealed that the bis-HBX molecule underwent the ESIPT process, and the ease of the ESIPT process was in the order of A1 > A2> B1 > C1. In particular, the TICT process in A1 and B1 promoted the occurrence of the ESIPT process. In addition, the IC process was identified, particularly in C1. Meanwhile, the calculation of fluorescence lifetime and fluorescence rate further confirmed that A1 was the most effective fluorescent probe molecule. This theoretical research provides an innovative theoretical reference for regulating ESIPT reactions and optimizing fluorescent probe molecules.


Introduction
The excited-state intramolecular proton transfer (ESIPT) process is a photo-induced enol-keto tautomerization process [1][2][3] accompanied by strong intramolecular hydrogen bonds (IHBs) [4,5], which allows proton H from specific amino or hydroxyl atoms to transfer to carbonyl oxygen or azo nitrogen atoms after photoexcitation.As shown in Scheme 1, the integral ESIPT reaction is essentially a four-level photo-cycle process.Firstly, the enol form in the ground state is photoexcited to the Franck-Condon point of the S 1 state, generating the enol* form simultaneously.Secondly, the enol* form undergoes the ESIPT process to produce a tautomer keto (keto*) form.Thirdly, the keto* form relaxes back to the ground-state keto structure via releasing the fluorescence or non-radiative transition.Eventually, the keto form is converted to the initial thermodynamically stable enol form achieved by the reverse proton transfer (RPT) process.Generally speaking, the ESIPT system tends to exhibit a large Stokes shift and dual fluorescence [6][7][8].In the 1950s, Weller first observed the double fluorescence phenomenon during an experimental study on the dynamic characteristics of methyl salicylate and attributed it to the ESIPT reaction [9].Since then, the mechanism of the ESIPT reaction has attracted the attention of extensive researchers, most of whom have studied it experimentally and theoretically [10][11][12][13].2-(2′-hydroxyphenyl) benzazole (HBX) derivatives with ESIPT properties are typically characterized by high yield and low synthesis complexity [14].This distinctive nature makes them widely used in laser dyes [15], chemical sensors [16,17], molecular switches [18], light-emitting diode devices [19], and fluorescent probes for use in biological systems [20,21].Previous reports have shown that modifying the molecular structure can effectively regulate the ESIPT process of HBX derivatives.In 2017, Manojai et al. [22] investigated the effect of heteroatom substitution (X=NH, O, and S) on the photophysical properties of HBX derivatives.They proved that NH substitution increased the probability of the ESIPT process in HBT, resulting in redshift emission fluorescence.In 2020, yang et al. [23] probed the effects of -NH2 group substitutions at different positions on the ESIPT mechanism.They revealed that the para-substitution of the -NH2 group promoted the occurrence of the ESIPT process.In 2022, Zhang et al. [24] designed four molecules (BPN, BPNS, BPS, and BPSN) to explore the effects of different electron groups on the ESIPT process in depth.They believed that introducing electron-withdrawing groups accelerates the ESIPT process, while the electron-donating groups do the opposite.It should not be ignored that in previous studies on HBX derivatives, researchers mainly focused on the effects of substitutions on the mono-HBX [22][23][24][25].Recently, Lee et al. [26] designed and synthesized bis 2-(2′-hydroxyphenyl) benzazole (bis-HBX) fluorescent probe molecules based on two HBX moieties.The bis-HBX had extra proton-binding sites and was more effective for the detection of alkaline pH than mono-HBX [26,27].However, the internal mechanism of the effects of different substitutions at multiple sites on the fluorescence properties of bis-HBX was still deficient, and the fluorescence probe molecules with the strongest luminous efficiency were screened, which merits further attention.Therefore, a thorough theoretical investigation was urgently needed to better understand the ESIPT process and the effects of different substitutions at multiple sites on the fluorescence properties of bis-HBX.
In this study, we simultaneously adjusted two key parameters, namely the extension group of central phenol 2-(2 ′ -hydroxyphenyl) benzazole (HBX) derivatives with ESIPT properties are typically characterized by high yield and low synthesis complexity [14].This distinctive nature makes them widely used in laser dyes [15], chemical sensors [16,17], molecular switches [18], light-emitting diode devices [19], and fluorescent probes for use in biological systems [20,21].Previous reports have shown that modifying the molecular structure can effectively regulate the ESIPT process of HBX derivatives.In 2017, Manojai et al. [22] investigated the effect of heteroatom substitution (X=NH, O, and S) on the photophysical properties of HBX derivatives.They proved that NH substitution increased the probability of the ESIPT process in HBT, resulting in redshift emission fluorescence.In 2020, yang et al. [23] probed the effects of -NH 2 group substitutions at different positions on the ESIPT mechanism.They revealed that the para-substitution of the -NH 2 group promoted the occurrence of the ESIPT process.In 2022, Zhang et al. [24] designed four molecules (BPN, BPNS, BPS, and BPSN) to explore the effects of different electron groups on the ESIPT process in depth.They believed that introducing electron-withdrawing groups accelerates the ESIPT process, while the electron-donating groups do the opposite.It should not be ignored that in previous studies on HBX derivatives, researchers mainly focused on the effects of substitutions on the mono-HBX [22][23][24][25].Recently, Lee et al. [26] designed and synthesized bis 2-(2 ′ -hydroxyphenyl) benzazole (bis-HBX) fluorescent probe molecules based on two HBX moieties.The bis-HBX had extra proton-binding sites and was more effective for the detection of alkaline pH than mono-HBX [26,27].However, the internal mechanism of the effects of different substitutions at multiple sites on the fluorescence properties of bis-HBX was still deficient, and the fluorescence probe molecules with the strongest luminous efficiency were screened, which merits further attention.Therefore, a thorough theoretical investigation was urgently needed to better understand the ESIPT process and the effects of different substitutions at multiple sites on the fluorescence properties of bis-HBX.
In this study, we simultaneously adjusted two key parameters, namely the extension group of central phenol (X: Me or OMe) and the properties of heteroatoms (Y: S, O, N) (as shown in Scheme 2).Using DFT and TD-DFT methods, the effects of different substitutions at multiple sites on the photophysical mechanism and the ESIPT process in bis-HBX were rationally researched.Our research not only provides a new idea and method for improving the efficiency of the ESIPT process but also has an important reference value for designing and optimizing fluorescent probe molecules with high fluorescence efficiency.
were rationally researched.Our research not only provides a new idea and method for improving the efficiency of the ESIPT process but also has an important reference value for designing and optimizing fluorescent probe molecules with high fluorescence efficiency.

Geometric Parameters of Bis-HBX
All the geometric configurations of different states of bis-HBX (see Figures 1 and S1) were optimized completely by the B3LYP-D3/6-31G+(d,p) method without any restrictions on bond lengths or bond angles.In the excited state, we labeled the enol* structure as the S1 state and the keto* structure as the S1′ state.Some of the significant geometric parameters are displayed in Table 1, including bond lengths and bond angles.

Geometric Parameters of Bis-HBX
All the geometric configurations of different states of bis-HBX (see Figure 1 and Figure S1) were optimized completely by the B3LYP-D3/6-31G+(d,p) method without any restrictions on bond lengths or bond angles.In the excited state, we labeled the enol* structure as the S 1 state and the keto* structure as the S 1 ′ state.Some of the significant geometric parameters are displayed in Table 1, including bond lengths and bond angles.The intramolecular hydrogen bonding (IHB) parameters of bis-HBX from the S 0 to the S 1 state followed the similar varying tendency recorded in Table 1.We chose A 1 as a representative for ease of expounding and fully discussing the changing trend in IHB intensity upon photoexcitation.The bond length (O 1 -H 1 ) of the A 1 molecule was elongated from 0.994 Å (S 0 ) to 1.058 Å (S 1 ), and N 1 -H 1 was decreased from 1.720 Å (S 0 ) to 1.501 Å (S 1 ).Furthermore, the bond angle δ (O 1 -H 1 -N 1 ) was increased from 147.14 • to 153.08 • .It has been validated that the IHB (O 1 -H 1 . ..N 1 ) intensity is enhanced in the S 1 state, which is conducive to the ESIPT process [28][29][30].In addition, in the S 1 ′ state, we obtained tautomer configuration, and a new IHB (N 1 -H 1 . ..O 1 ) was formed with a bond length of 1.036 Å.The bis-HBX molecule underwent the ESIPT process, which was proved by the above-calculated results.The intramolecular hydrogen bonding (IHB) parameters of bis-HBX from the S0 to the S1 state followed the similar varying tendency recorded in Table 1.We chose A1 as a representative for ease of expounding and fully discussing the changing trend in IHB intensity upon photoexcitation.The bond length (O1-H1) of the A1 molecule was elongated from 0.994 Å (S0) to 1.058 Å (S1), and N1-H1 was decreased from 1.720 Å (S0) to 1.501 Å (S1).Furthermore, the bond angle δ (O1-H1-N1) was increased from 147.14° to 153.08°.It has been validated that the IHB (O1-H1...N1) intensity is enhanced in the S1 state, which is conducive to the ESIPT process [28][29][30].In addition, in the S1′ state, we obtained tautomer configuration, and a new IHB (N1-H1…O1) was formed with a bond length of 1.036 Å.The bis-HBX molecule underwent the ESIPT process, which was proved by the above-calculated results.

Infrared (IR) Vibrational Spectra
Due to the IR vibrational spectra providing direct evidence for the formation and fracture of hydrogen bonds (HBs) during the ESIPT process [31], we simulated the IR vibrational spectra of four probes in different electronic states.
From the simulated IR spectra (Figure 2), we found that the stretching vibration frequencies of O 1 -H 1 in A 1 , A 2 , B 1 , and C 1 were separately redshifted from 3244 cm −1 , 3279 cm −1 , 3193 cm −1 , and 3322 cm −1 in the S 0 state to 2148 cm −1 , 2266 cm −1 , 2359 cm −1 , and 2654 cm −1 in the S 1 state, accompanied with redshifts of 1096 cm −1 , 1013 cm −1 , 798 cm −1 , and 668 cm −1 , respectively.The above data indicated that the redshift value followed the order: A 1 > A 2 > B 1 > C 1 , as we all know the weakening and strengthening of the IHB can be explained by the blue-and redshifts in the IR vibrational frequencies of the O-H bond [32][33][34], respectively, so we can infer that the ESIPT process was more prone to occur in A 1 .Meanwhile, in the S 1 ′ state, a new vibrational peak was observed, corresponding to the stretching vibration frequency of N 1 -H 1 , which was located around 3225 cm −1 (A 1 ), 3125 cm −1 (A 2 ), 3425 cm −1 (B 1 ), and 3374 cm −1 (C 1 ).The above discussion confirmed that the four probe molecules underwent the ESIPT process after photoexcitation.
underwent the ESIPT process with distortions in molecular configuration.

Infrared (IR) Vibrational Spectra
Due to the IR vibrational spectra providing direct evidence for the formation fracture of hydrogen bonds (HBs) during the ESIPT process [31], we simulated the I brational spectra of four probes in different electronic states.
From the simulated IR spectra (Figure 2), we found that the stretching vibratio quencies of O1-H1 in A1, A2, B1, and C1 were separately redshifted from 3244 cm −1 , cm −1 , 3193 cm −1 , and 3322 cm −1 in the S0 state to 2148 cm −1 , 2266 cm −1 , 2359 cm −1 , and cm −1 in the S1 state, accompanied with redshifts of 1096 cm −1 , 1013 cm −1 , 798 cm −1 , an cm −1 , respectively.The above data indicated that the redshift value followed the ord > A2 > B1 > C1, as we all know the weakening and strengthening of the IHB can be expla by the blue-and redshifts in the IR vibrational frequencies of the O-H bond [32-34 spectively, so we can infer that the ESIPT process was more prone to occur in A1.M while, in the S1′ state, a new vibrational peak was observed, corresponding to the str ing vibration frequency of N1-H1, which was located around 3225 cm −1 (A1), 3125 cm −1 3425 cm −1 (B1), and 3374 cm −1 (C1).The above discussion confirmed that the four p molecules underwent the ESIPT process after photoexcitation.1) [36,37]:

RDG Scatter Plot and Topological Analyses
The obtained electron density matrix equation was Equation (2): As clarified in Figures S2 and S3, the blue section of the color level corresponds to the HB effect, and the intensity of HB gradually increased with the deepening of the color.In addition, the spatial interactions and van der Waals forces were represented by the red and green areas, respectively.Through analyzing the RDG versus Sign(I 2 )r scatter plot, the relationship of IHB intensity in A 1 , A 2 , B 1 , and C 1 was clear-cut and unambiguous.As shown in Figure 3, with the substitution modes of C 1 , B 1 , A 2 , and A 1 , the Sign(I 2 )r values became more and more negative, unveiling that the strength of IHB in different surroundings obeys the order of A 1 > A 2 > B 1 > C 1 .In the S 0 state, the prong peaks of A 1 , A 2 , B 1 , and C 1 were located at −0.050, −0.049, −0.050, and −0.044, respectively.After photoexcitation, the prong peaks of A 1 , A 2 , B 1 , and C 1 shifted to the more negative region (−0.087,−0.081, −0.075, and −0.063, respectively).The interaction type was judged to be an IHB interaction, corresponding to O 1 -H 1 . ..N 1 .The IHB intensity of the four compounds was enhanced after photoexcitation, as indicated by the above-mentioned data, which is conducive to the ESIPT process.Moreover, comparison of the left-shifted peak positions in the four compounds revealed that the strength of IHB followed the order of A 1 > A 2 > B 1 > C 1 in the S 1 state.This was consistent with our previous analysis conclusion, revealing that different substitutions at multiple sites did affect the ESIPT process.
on RDG versus Sign(I2)r, the expression of RDG function was as shown in Equation [36,37]: The obtained electron density matrix equation was Equation (2): As clarified in Figures S2 and S3, the blue section of the color level corresponds to t HB effect, and the intensity of HB gradually increased with the deepening of the color.addition, the spatial interactions and van der Waals forces were represented by the r and green areas, respectively.Through analyzing the RDG versus Sign(I2)r scatter pl the relationship of IHB intensity in A1, A2, B1, and C1 was clear-cut and unambiguous.shown in Figure 3, with the substitution modes of C1, B1, A2, and A1, the Sign(I2)r valu became more and more negative, unveiling that the strength of IHB in different surroun ings obeys the order of A1 > A2 > B1 > C1.In the S0 state, the prong peaks of A1, A2, B1, a C1 were located at −0.050, −0.049, −0.050, and −0.044, respectively.After photoexcitatio the prong peaks of A1, A2, B1, and C1 shifted to the more negative region (−0.087,−0.0 −0.075, and −0.063, respectively).The interaction type was judged to be an IHB interactio corresponding to O1-H1...N1.The IHB intensity of the four compounds was enhanced af photoexcitation, as indicated by the above-mentioned data, which is conducive to t ESIPT process.Moreover, comparison of the left-shifted peak positions in the four co pounds revealed that the strength of IHB followed the order of A1 > A2 > B1 > C1 in the state.This was consistent with our previous analysis conclusion, revealing that differe substitutions at multiple sites did affect the ESIPT process.Topological analysis, first proposed by Bader [38], is also one of the most comm methods for measuring the strength of HBs [39].The obvious bond paths and bond criti points (BCPs) [40] in the IHB region are captured in Figure S4, and we have listed t relevant parameters in Table 2.As is known to all, ρ(r) and ν(r) can gauge the intensity IHBs with advantages, while the larger ρ(r) is and the more negative ν(r) is, the strong IHB intensity will be.It could clearly be seen that the IHBs of the four molecules were enhanced in the S1 state.Moreover, a horizontal comparison of the absolute values of  in bis-HBX showed that the HB values followed the order of HB (BCP2) > HB(BCP4 HB(BCP6) > HB(BCP8), indicating that the IHB intensity of A1 was the strongest of t four molecules in the S1 state.It is well known that the stronger the IHB intensity is, t Topological analysis, first proposed by Bader [38], is also one of the most common methods for measuring the strength of HBs [39].The obvious bond paths and bond critical points (BCPs) [40] in the IHB region are captured in Figure S4, and we have listed the relevant parameters in Table 2.As is known to all, ρ(r) and ν(r) can gauge the intensity of IHBs with advantages, while the larger ρ(r) is and the more negative ν(r) is, the stronger IHB intensity will be.It could clearly be seen that the IHBs of the four molecules were all enhanced in the S 1 state.Moreover, a horizontal comparison of the absolute values of E HB in bis-HBX showed that the E HB values followed the order of E HB (BCP2) > E HB (BCP4) > E HB (BCP6) > E HB (BCP8), indicating that the IHB intensity of A 1 was the strongest of the four molecules in the S 1 state.It is well known that the stronger the IHB intensity is, the more favorable the ESIPT process is.Therefore, through the analysis of the above methods, we believe that the stronger the IHBs interaction, the easier the ESIPT process.

Fluorescence Attribution and Mechanism Analysis
We performed electron-hole analysis on bis-HBX molecules in the S 1 state, as shown in Figure 4. Furthermore, they were intuitively analyzed by utilizing the inter-fragment charge transfer (IFCT) method.During the emission process, from the right side to the central benzene ring direction, 0.776 electrons, 0.691 electrons, 0.675 electrons, and 0.188 electrons were transmitted, respectively.Moreover, the characteristics of hole and electron distribution indexes are included in Table 3.It was well known that for analogs, the lower the Sr index and the more positive the t index, the more complete the separation of electrons and holes.In the light of the t and Sr indexes, a significant separation of holes and electrons was implied through the S 0 →S 1 transition in A 1 and B 1 , which proved that A 1 and B 1 had significant ICT (intramolecular charge transfer) characteristics.Furthermore, combined with the configuration of bis-HBX, the dihedral angles δ (C 1 -C 2 -C 3 -N 2 ) of A 1 and B 1 had abnormal twists of 47 degrees and 24.69 degrees, respectively, during photoexcitation.Perspicuously, it can be confirmed that the typical twisted intramolecular charge transfer (TICT) [41,42] process existed in A 1 and B 1 .From the above analysis results, it can be seen that A 1 and B 1 first underwent the TICT process in the S 1 state, and then underwent the ESIPT process.Thus, we know that the TICT process in A 1 and B 1 facilitated the ESIPT process.
ods, we believe that the stronger the IHBs interaction, the easier the ESIPT process.

Fluorescence Attribution and Mechanism Analysis
We performed electron-hole analysis on bis-HBX molecules in the S1 state, as show in Figure 4. Furthermore, they were intuitively analyzed by utilizing the inter-fragm charge transfer (IFCT) method.During the emission process, from the right side to central benzene ring direction, 0.776 electrons, 0.691 electrons, 0.675 electrons, and 0.1 electrons were transmitted, respectively.Moreover, the characteristics of hole and electr distribution indexes are included in Table 3.It was well known that for analogs, the low the Sr index and the more positive the t index, the more complete the separation of el trons and holes.In the light of the t and Sr indexes, a significant separation of holes a electrons was implied through the S0→S1 transition in A1 and B1, which proved that A1 a B1 had significant ICT (intramolecular charge transfer) characteristics.Furthermore, co bined with the configuration of bis-HBX, the dihedral angles δ (C1-C2-C3-N2) of A1 and had abnormal twists of 47 degrees and 24.69 degrees, respectively, during photoexci tion.Perspicuously, it can be confirmed that the typical twisted intramolecular char transfer (TICT) [41,42] process existed in A1 and B1.From the above analysis results, it c be seen that A1 and B1 first underwent the TICT process in the S1 state, and then underw the ESIPT process.Thus, we know that the TICT process in A1 and B1 facilitated the ESI process.In order to gain a deeper understanding of the effects of different substitutions at multiple sites on photophysical properties in bis-HBX, we simulated the absorption and fluorescence spectra of bis-HBX (see Figure 5), which were obtained based on the optimized S 0 , S 1 , and S 1 ′ configurations.As shown in Table 4, the relevant photophysical parameters were recorded, and the absorption peak, oscillator strength, and corresponding orbital transition contributions were also included.The bis-HBX had double absorption peaks that could be distinctly observed from t data listed in Table S1, in which the calculated maximum absorption peak was very co sistent with the experimental value, proving the feasibility of our method [26].In additio as shown in Figure 5, the short-wavelength emission peaks of A2, A1, B1, and C1 were 4 nm, 463 nm, 432 nm, and 419 nm, showing a blueshift from A2 to C1, respectively.T proton transfer tautomers in A2, A1, B1, and C1 corresponding to the longer waveleng fluorescence peaks were 633 nm, 622 nm, 566 nm, and 541 nm, respectively.Obvious with the introduction of different substitution forms, the significant redshift was p duced in the longer wavelength emission peaks of A2, A1, B1, and C1, and the degree redshift followed the order of A1 > A2 > B1 > C1.However, only one short-wavelength fl orescence was observed in A1 in accord with the emission peak in the experiment; th The bis-HBX had double absorption peaks that could be distinctly observed from the data listed in Table S1, in which the calculated maximum absorption peak was very consistent with the experimental value, proving the feasibility of our method [26].In addition, as shown in Figure 5, the short-wavelength emission peaks of A 2 , A 1 , B 1 , and C 1 were 481 nm, 463 nm, 432 nm, and 419 nm, showing a blueshift from A 2 to C 1 , respectively.The proton transfer tautomers in A 2 , A 1 , B 1 , and C 1 corresponding to the longer wavelength fluorescence peaks were 633 nm, 622 nm, 566 nm, and 541 nm, respectively.Obviously, with the introduction of different substitution forms, the significant redshift was produced in the longer wavelength emission peaks of A 2 , A 1 , B 1 , and C 1 , and the degree of redshift followed the order of A 1 > A 2 > B 1 > C 1 .However, only one short-wavelength fluorescence was observed in A 1 in accord with the emission peak in the experiment; thus, we attributed it to the S 1 (TICT) state.Moreover, in order to study the fluorescence properties in bis-HBX more comprehensively, we used Equations ( 3) and (4) [36,37]: We calculated the fluorescence lifetime and rate in the bis-HBX and listed them in Table 5.In the formula, f 1 represents the oscillator strength and E delegates the wavenumber.It can be observed that A 1 exhibited the greatest fluorescence lifetime, which may be due to its lower oscillation intensity.Similarly, a larger fluorescence lifetime resulted in a lower fluorescence rate, with A 1 exhibiting the lowest fluorescence rate.All the above analyses indicated that A 1 was the fluorescence probe molecule with the strongest luminous efficiently in bis-HBX, and we were also able to confirm that different substitutions at multiple sites did affect the spectral characteristics of bis-HBX.
Finally, we conducted a detailed analysis of the excited-state decay process in bis-HBX.We have provided a diagrammatic sketch of the excitation and emission processes in bis-HBX (Figure 6) according to Table 4. Perspicuously, from the oscillator strength in Table 3, the excited states of A 1 , A 2 , and B 1 were separated from each other.It was interesting that the excited states of S 1 (0.4634) and S 5 (0.4930) in C 1 were coupled together and formed an intersection point where the internal conversion (IC) took place.The vertical transition S 1 →S 0 that occurred at the Franck-Condon region of A 1 , A 2 , B 1 , and C 1 was accompanied by shorter wavelength fluorescence measurements of 463 nm, 481 nm, 432 nm, and 419 nm, respectively.Subsequently, in the S 1 state, from the higher vibration level to the lowest vibration level, a vibration relaxation process occurred in which A 1 , A 2 , B 1 , and C 1 recovered from the tautomer structure to the S 0 state, while at 622 nm, 633 nm, 566 nm, and 541 nm, respectively, they emitted long-wavelength fluorescence.Finally, we conducted a detailed analysis of the excited-state decay process in bis-HBX.We have provided a diagrammatic sketch of the excitation and emission processes in bis-HBX (Figure 6) according to Table 4. Perspicuously, from the oscillator strength in Table 3, the excited states of A1, A2, and B1 were separated from each other.It was interesting that the excited states of S1 (0.4634) and S5 (0.4930) in C1 were coupled together and formed an intersection point where the internal conversion (IC) took place.The vertical transition S1→S0 that occurred at the Franck-Condon region of A1, A2, B1, and C1 was accompanied by shorter wavelength fluorescence measurements of 463 nm, 481 nm, 432 nm, and 419 nm, respectively.Subsequently, in the S1 state, from the higher vibration level to the lowest vibration level, a vibration relaxation process occurred in which A1, A2, B1, and C1 recovered from the tautomer structure to the S0 state, while at 622 nm, 633 nm, 566 nm, and 541 nm, respectively, they emitted long-wavelength fluorescence.

Frontier Molecular Orbitals and NBO Population
It is particularly acknowledged that frontier molecular orbitals (FMOs) are an effective means to reflect charge distribution and recombination [43].Since the first single transition of the target compound is mainly related to the highest occupied orbital (HOMO) and the lowest unoccupied orbital (LUMO), Figure 7 only renders these two orbitals.During the photoexcitation process, the electronic clouds on the O1 and N1 atoms were drastically reduced and increased, respectively.This result indicated that there was a strong binding ability between the N1 atom and the H1 proton, which provided the driving force for the ESIPT process.For the purpose of studying the changes in electron density distribution on O1 and N1 quantitatively, the NBO charge distributions of bis-HBX are recorded in Table 6.The negative charges on the O1 shrank from −0.693 a. u., −0.707 a. u., −0.693 a. u., and −0.696 a. u. in the ground state to −0.677 a. u., −0.685 a. u., −0.684 a. u., and −0.676

Frontier Molecular Orbitals and NBO Population
It is particularly acknowledged that frontier molecular orbitals (FMOs) are an effective means to reflect charge distribution and recombination [43].Since the first single transition of the target compound is mainly related to the highest occupied orbital (HOMO) and the lowest unoccupied orbital (LUMO), Figure 7 only renders these two orbitals.During the photoexcitation process, the electronic clouds on the O 1 and N 1 atoms were drastically reduced and increased, respectively.This result indicated that there was a strong binding ability between the N 1 atom and the H 1 proton, which provided the driving force for the ESIPT process.For the purpose of studying the changes in electron density distribution on O 1 and N 1 quantitatively, the NBO charge distributions of bis-HBX are recorded in Table 6.The negative charges on the O 1 shrank from −0.693 a. u., −0.707 a. u., −0.693 a. u., and −0.696 a. u. in the ground state to −0.677 a. u., −0.685 a. u., −0.684 a. u., and −0.676 a. u. in the excited state, while that of the N 1 atom was enlarged during the photo-absorption process, which was confirmed by the analytical results of the FMOs.

Potential Energy Curves (PECs)
With the aim of uncovering the mechanism of the ESIPT process with different substitutions at multiple sites more intuitively, the potential energy curves (PECs) in bis-HBX were scanned via prolonging the O1-H1 bond length from 0.9 Å to 1.9 Å in increments of 0.1 Å based on the optimized structures in the gas phase [44].
As shown in Figure 8, the PECs of the four probe molecules showed an ascending trend in the S0 state.This demonstrates that it was difficult for the proton transfer (PT) process to occur.After photoexcitation, the energy barriers in the four probe molecules were diminished to 0.24 kcal/mol (A1), 0.55 kcal/mol (A2), 0.67 kcal/mol (B1), and 1.15 kcal/mol (C1), respectively.It is obvious that it was more possible for the four probe molecules to undergo the PT process in the S1 state.It is a remarkable fact that the order of energy barriers in bis-HBX followed the order of A1 < A2 < B1 < C1, which verified that A1 can occur the ESIPT process more easily.Although the B3LYP/6-31+G (d,p) can provide qualitative descriptions of the hypersurface, it may underestimate some of the energy  With the aim of uncovering the mechanism of the ESIPT process with different substitutions at multiple sites more intuitively, the potential energy curves (PECs) in bis-HBX were scanned via prolonging the O 1 -H 1 bond length from 0.9 Å to 1.9 Å in increments of 0.1 Å based on the optimized structures in the gas phase [44].
As shown in Figure 8, the PECs of the four probe molecules showed an ascending trend in the S 0 state.This demonstrates that it was difficult for the proton transfer (PT) process to occur.After photoexcitation, the energy barriers in the four probe molecules were diminished to 0.24 kcal/mol (A 1 ), 0.55 kcal/mol (A 2 ), 0.67 kcal/mol (B 1 ), and 1.15 kcal/mol (C 1 ), respectively.It is obvious that it was more possible for the four probe molecules to undergo the PT process in the S 1 state.It is a remarkable fact that the order of energy barriers in bis-HBX followed the order of A 1 < A 2 < B 1 < C 1 , which verified that A 1 can occur the ESIPT process more easily.Although the B3LYP/6-31+G (d,p) can provide qualitative descriptions of the hypersurface, it may underestimate some of the energy differences and energy barriers.We recalculated the PECs using Cam-B3LYP/TZVP [45,46] theory (see Figure 8; the blue font is the corrected energy barrier), taking the corrected results as the reference.It was very clear and concise that the energy barriers calculated using Cam-B3LYP/TZVP theory were consistent with the order of A 1 < A 2 < B 1 < C 1 .In addition, to further confirm the ESIPT process mechanism, we also performed transitionstate (TS) calculations and obtained the intrinsic reaction coordinate (IRC) to validate the reasonableness and accuracy.Meanwhile, we have listed the ESIPT barriers via the IRC paths for bis-HBX in Table 7, which is consistent with the simulation results of the PECs.We further confirmed that A 1 can cause the ESIPT process to occur more easily.To date, we have explained the effects of different substitutions at multiple sites in the ESIPT process of bis-HBX molecules and reasonably elucidated the whole kinetic process of the studied molecules.This provides important reference values for the application of fluorescent probe molecules.
reasonableness and accuracy.Meanwhile, we have listed the ESIPT barriers via the I paths for bis-HBX in Table 7, which is consistent with the simulation results of the PE We further confirmed that A1 can cause the ESIPT process to occur more easily.To da we have explained the effects of different substitutions at multiple sites in the ESIPT p cess of bis-HBX molecules and reasonably elucidated the whole kinetic process of studied molecules.This provides important reference values for the application of fl rescent probe molecules.

Calculation Details
In the gas phase, the geometric structures of different electronic states of bis-HBX were fully optimized by DFT [46][47][48][49][50][51] and TD-DFT [52-55] based on B3LYP-D3/6-31+g (d, p) [47,48] levels.We chose six different functionals to calculate the absorption spectra of bis-HBX, and the results are listed in Table 8.The calculated values of B3LYP-D3 (295 nm and 368 nm) were compatible with the experiment values (290 nm and 363 nm) [26].Therefore, it was reasonable to use the B3LYP-D3 functional to evaluate the excited-state properties of the system.Based on the optimized geometric configurations, we calculated

Figure 1 .
Figure 1.Optimized geometry of bis-HBX in different states.

Figure 1 .
Figure 1.Optimized geometry of bis-HBX in different states.It is noteworthy that, in contrast to C 1 , the dihedral angle δ (C 1 -C 2 -C 3 -N 2 ) of A 1 , A 2, and B 1 existed a torsion between the central benzene ring and the right five-membered ring.The δ (C 1 -C 2 -C 3 -N 2 ) of A 1 , called the dihedral angle, enlarged from 47.02 • (S 0 ) to 13.29 • (S 1 ′ ), with a torsion of 60.31 • .Similarly, both the A 2 and B 1 molecules underwent torsions of 28.16 • and 24.69 • , respectively.All the alterations indicated that A 1 , A 2 , and B 1 underwent the ESIPT process with distortions in molecular configuration.

Figure 2 .
Figure 2. Simulated IR spectra of O1-H1 and N1-H1 stretching vibration frequencies of bis-H different electronic states in the gas phase.
Considering that using the scatter plot of RDG versus Sign(I2)r can effectively r IHB interactions in real space [35], and Lu et al. recently proposed the IRI isosurface b

Figure 2 .
Figure 2. Simulated IR spectra of O 1 -H 1 and N 1 -H 1 stretching vibration frequencies of bis-HBX in different electronic states in the gas phase.

2. 3 .
RDG Scatter Plot and Topological Analyses Considering that using the scatter plot of RDG versus Sign(I 2 )r can effectively reveal IHB interactions in real space [35], and Lu et al. recently proposed the IRI isosurface based on RDG versus Sign(I 2 )r, the expression of RDG function was as shown in Equation (

Figure 3 .
Figure 3. Sign(I 2 )r scatter plots versus reduced density gradient (RDG) in bis-HBX in the S 1 state.

Figure 4 .Table 3 .
Figure 4. Hole-electron analysis in the S1′ state of bis-HBX (the blue isosurface represents the dis bution of electrons, and the pink isosurface represents the distribution of holes).

Figure 4 .
Figure 4. Hole-electron analysis in the S 1 ′ state of bis-HBX (the blue isosurface represents the distribution of electrons, and the pink isosurface represents the distribution of holes).

Figure 6 .
Figure 6.Abridged sketch of the excitation process in bis-HBX.

Figure 6 .
Figure 6.Abridged sketch of the excitation process in bis-HBX.

Figure 7 .
Figure 7. Frontier molecular orbitals of bis-HBX in the gas phase.

Figure 7 .
Figure 7. Frontier molecular orbitals of bis-HBX in the gas phase.

Figure 8 .
Figure 8.The PECs of bis-HBX in different states.

Figure 8 .
Figure 8.The PECs of bis-HBX in different states.

Table 2 .
Calculated BCP parameters of bis-HBX.a: density of all electrons; b: potential energy density; c: Laplacian of electron density; d: Lagrangian kinetic energy; e: energy density; f: hydrogen bond energy E HB = V(r)/2.

Table 2 .
Calculated BCP parameters of bis-HBX.a: density of all electrons; b: potential energy d sity; c: Laplacian of electron density; d: Lagrangian kinetic energy; e: energy density; f: hydrog bond energy HB = V(r)/2.

Table 3 .
Indexes illustrating the distribution of holes and electrons in bis-HBX.Sr: the extent of hole-and-electron overlap; t: the separation of electrons and holes.

Table 6 .
NBO charge allotments (a.u.) of the O1 and N1 in bis-HBX in different states.

Table 6 .
NBO charge allotments (a.u.) of the O 1 and N 1 in bis-HBX in different states.