Fluorescence Lifetimes of NIR-Emitting Molecules with Excited-State Intramolecular Proton Transfer

Molecular probes based on the excited-state intramolecular proton-transfer (ESIPT) mechanism have emerged to be attractive candidates for various applications. Although the steady-state fluorescence mechanisms of these ESIPT-based probes have been reported extensively, less information is available about the fluorescence lifetime characteristics of newly developed NIR-emitting dyes. In this study, four NIR-emitting ESIPT dyes with different cyanine terminal groups were investigated to evaluate their fluorescence lifetime characteristics in a polar aprotic solvent such as CH2Cl2. By using the time-correlated single-photon counting (TCSPC) method, these ESIPT-based dyes revealed a two-component exponential decay (τ1 and τ2) in about 2–4 nanoseconds (ns). These two components could be related to the excited keto tautomers. With the aid of model compounds (5 and 6) and low-temperature fluorescence spectroscopy (at −189 ℃), this study identified the intramolecular charge transfer (ICT) as one of the major factors that influenced the τ values. The results of this study also revealed that both fluorescence lifetimes and fractional contributions of each component were significantly affected by the probe structures.


Introduction
As one of the intrinsic properties of a fluorophore, fluorescence lifetime describes the characteristic time that a molecule remains in the excited state before returning to its ground state. Fluorescence lifetime is not dependent on the excitation methods (such as the wavelength of excitation, one or multiphoton excitation). Additionally, this photophysical property is basically not affected by the fluorescence intensity or fluorophore concentration. Since the excited state is a highly energetic state, its decay to its ground state is affected by both internal (e.g., fluorophore structure) and external conditions (e.g., solvent polarity [1] and the presence of fluorescence quencher). Due to the reasons mentioned above, fluorescence lifetime becomes a separate but complementary method to fluorescence intensity-based measurement [2].
Recently, excited-state intramolecular proton transfer (ESIPT) has emerged to be an attractive mechanism for the design of molecular probes, due to their unique fluorescent properties that include large Stokes shifts and dual emissions (arising from the enol and keto tautomers). The most common ESIPT fluorophores are derivatives of 2-(2 -hydroxyphenyl)benzoxazole (HBO) and 2-(2 -hydroxyphenyl) benzothiazole (HBT) (Figure 1). In previous studies, great efforts have been made to utilize the enol and keto emission, as well as their ratio, for a variety of chemical sensor applications [3]. There are also significant efforts to tune the ESIPT emission toward the near-infrared (NIR) region for improved imaging applications [4]. Most of the reported studies are relying on fluorescence intensity-based measurement, by using analyte-induced changes in fluorescence wavelengths (or color) and quantum yields.
Significant interests exist in studying the decay process of the excited ESIPT fluorophores by using time-resolved spectroscopic methods [5]. For example, HBO has been Significant interests exist in studying the decay process of the excited ESIPT fluoro-45 phores by using time-resolved spectroscopic methods [5]. For example, HBO has been 46 reported to exhibit a short lifetime (  20 ps) and a long lifetime (  295 ps) in hexane, 47 with the long lifetime species being associated with its keto tautomer [6]. Additionally, the 48 fluorescence of HBT is reported to exhibit one lifetime that is highly dependent on the 49 solvent polarity, showing   12-17 ps in CH3CN and   100 ps in cyclohexane [7]. By 50 using ultrafast infrared spectroscopy to monitor the process, the observed lifetime is iden-51 tified to be associated with the keto tautomer [7]. As new ESIPT-based fluorescent probes 52 have been developed, it is important to continue the evaluation of the fluorescence life-53 time characteristics of these new materials and to learn how the structural variation could 54 affect the lifetime parameters. After 58 deactivation of the keto excited state (K*), a ground state retro proton transfer occurs to regenerate 59 the enol form. In the illustration, the substituent "Y" represents an organic group, such as a pyri-60 dinium group in probe 1. 61 In an effort to tune the emission toward a longer wavelength, we have reported 62 probes 1-4 [8], [9], [10]. For example, probe 1 has been shown to be a useful fluorescent 63 dye for imaging intracellular mitochondria, cellular membranes and neuromast organs on 64 zebrafish [9], [11]. As a consequence of effective proton transfer, the dye gives only keto 65 emission with a large Stokes shift. This is in sharp contrast to simple HBT, whose emission 66 from enol/keto tautomers is quite sensitive to solvents [1]. As a unique structural feature, 67 dyes 1-4 incorporate a styryl group with a positively charged cyanine segment as indi-68 cated by the "cyanine unit" in Scheme 1, which enhances the intramolecular charge trans-69 fer (ICT) interaction in their excited states. A recent study investigated the coupling of 70 ESIPT and ICT of 3 by using ultrafast transient absorption spectroscopy and quantum 71 chemical calculations, showing an ultrafast proton transfer that is associated with solva-72 tion (~1.5 picoseconds) and conformation relaxation (~13 picoseconds) [12]. As a conse-73 quence of only keto emission, these dyes exhibit a clear "transparent window" between 74 their absorption and emission, such as dye 1 which reveals a transparent window between 75 500-580 nm (Supporting Information, Figure S11-S13). This is in sharp contrast to classical 76 organic fluorescent dyes, such as fluorescein, whose fluorescence spectra always have 77 some spectral overlap with their absorption. The lack of emission from the enol tautomer 78 indicated that the proton transfer happens effectively in the excited states. A fundamental 79 question is why the keto emission occurs nearly exclusively in these compounds. In order 80 After deactivation of the keto excited state (K*), a ground state retro proton transfer occurs to regenerate the enol form. In the illustration, the substituent "Y" represents an organic group, such as a pyridinium group in probe 1.
In an effort to tune the emission toward a longer wavelength, we have reported probes 1-4 [8][9][10]. For example, probe 1 has been shown to be a useful fluorescent dye for imaging intracellular mitochondria, cellular membranes and neuromast organs on zebrafish [9,11]. As a consequence of effective proton transfer, the dye gives only keto emission with a large Stokes shift. This is in sharp contrast to simple HBT, whose emission from enol/keto tautomers is quite sensitive to solvents [1]. As a unique structural feature, dyes 1-4 incorporate a styryl group with a positively charged cyanine segment as indicated by the "cyanine unit" in Scheme 1, which enhances the intramolecular charge transfer (ICT) interaction in their excited states. A recent study investigated the coupling of ESIPT and ICT of 3 by using ultrafast transient absorption spectroscopy and quantum chemical calculations, showing an ultrafast proton transfer that is associated with solvation (~1.5 picoseconds) and conformation relaxation (~13 picoseconds) [12]. As a consequence of only keto emission, these dyes exhibit a clear "transparent window" between their absorption and emission, such as dye 1 which reveals a transparent window between 500-580 nm (Supporting Information, Figures S11-S13). This is in sharp contrast to classical organic fluorescent dyes, such as fluorescein, whose fluorescence spectra always have some spectral overlap with their absorption. The lack of emission from the enol tautomer indicated that the proton transfer happens effectively in the excited states. A fundamental question is why the keto emission occurs nearly exclusively in these compounds. In order to shed some light on this fundamental process, we decide to evaluate/understand the fluorescence lifetime characteristics of compounds 1-4, which have not been investigated in previous studies.
to shed some light on this fundamental process, we decide to evaluate/understand the 81 fluorescence lifetime characteristics of compounds 1-4, which have not been investigated 82 in previous studies.  Compounds 1-5 were synthesized by using procedures as described in our previous 88 reports. In order to aid the study, model compound 6 was synthesized by reacting 3 with 89 excess methyl iodide in the presence of a base at room temperature. All products are char-90 acterized by 1 H-NMR, 13 C-NMR and MS spectra (ESI Figure S1-S10). Different from 3, the 91 hydroxy group in 6 is protected, which eliminates the proton transfer later. 1 H NMR spec-92 trum of 6 revealed only one resonance Ar-OCH3 signal at ~3.93 ppm, whose integration 93 matched well with the signals from the -CH2CH3 group (ESI Figure S8). A large coupling 94 constant (J =15 Hz) was observed from the vinyl protons in both spectra of 3 and 6 ( Figure 95 2), showing the trans-CH=CH linkage. Since only trans-CH=CH was detected in both com-96 pounds, the reaction sequence from 3 to 6 did not have any impact on the stereochemistry 97 of the vinyl bond linkage.

Synthesis
Compounds 1-5 were synthesized by using procedures as described in our previous reports. In order to aid the study, model compound 6 was synthesized by reacting 3 with excess methyl iodide in the presence of a base at room temperature. All products are characterized by 1 H-NMR, 13 C-NMR and MS spectra (ESI Figures S1-S10). Different from 3, the hydroxy group in 6 is protected, which eliminates the proton transfer later. 1 H NMR spectrum of 6 revealed only one resonance Ar-OCH 3 signal at~3.93 ppm, whose integration matched well with the signals from the -CH 2 CH 3 group (ESI Figure S8). A large coupling constant (J = 15 Hz) was observed from the vinyl protons in both spectra of 3 and 6 ( Figure 2), showing the trans-CH=CH linkage. Since only trans-CH=CH was detected in both compounds, the reaction sequence from 3 to 6 did not have any impact on the stereochemistry of the vinyl bond linkage.
to shed some light on this fundamental process, we decide to evaluate/understand the 81 fluorescence lifetime characteristics of compounds 1-4, which have not been investigated 82 in previous studies.  Compounds 1-5 were synthesized by using procedures as described in our previous 88 reports. In order to aid the study, model compound 6 was synthesized by reacting 3 with 89 excess methyl iodide in the presence of a base at room temperature. All products are char-90 acterized by 1 H-NMR, 13 C-NMR and MS spectra (ESI Figure S1-S10). Different from 3, the 91 hydroxy group in 6 is protected, which eliminates the proton transfer later. 1 H NMR spec-92 trum of 6 revealed only one resonance Ar-OCH3 signal at ~3.93 ppm, whose integration 93 matched well with the signals from the -CH2CH3 group (ESI Figure S8). A large coupling 94 constant (J =15 Hz) was observed from the vinyl protons in both spectra of 3 and 6 ( Figure 95 2), showing the trans-CH=CH linkage. Since only trans-CH=CH was detected in both com-96 pounds, the reaction sequence from 3 to 6 did not have any impact on the stereochemistry 97 of the vinyl bond linkage.

Spectroscopic Studies
The photophysical properties are listed in Table 1 (ESI Figures S11-S28). It was reported that both compound 3 and its effective chromophore 5 exhibited nearly identical absorption (e.g.,~447 in CH 2 Cl 2 ) ( Figure 3). However, the quantum yield of 3 was significantly higher than 5, due to the coupling with the ESIPT unit in the former [8]. The structural analogues, such as 1 & 4, also revealed similar appealing properties (i.e., high φ fl and large Stokes shifts of these ESIPT cyanine dyes) [9]. The role of the ESIPT unit in enhancing the fluorescence was further demonstrated by the synthesis of model compound 6, whose φ fl was much lower than its parent compound 3. Clearly, the intramolecular hydrogen bonding played an important role in maintaining the high fluorescence of this class of NIR-emitting dyes, partly due to increased molecular rigidity via hydrogen bonding. The photophysical properties are listed in Table 1 (ESI Figure S11-S28). It was re-103 ported that both compound 3 and its effective chromophore 5 exhibited nearly identical 104 absorption (e.g., ~447 in CH2Cl2) ( Figure 3). However, the quantum yield of 3 was signifi-105 cantly higher than 5, due to the coupling with the ESIPT unit in the former [8]. The struc-106 tural analogues, such as 1&4, also revealed similar appealing properties (i.e., high fl and 107 large Stokes shifts of these ESIPT cyanine dyes) [9]. The role of the ESIPT unit in enhancing 108 the fluorescence was further demonstrated by the synthesis of model compound 6, whose 109 fl was much lower than its parent compound 3. Clearly, the intramolecular hydrogen 110 bonding played an important role in maintaining the high fluorescence of this class of 111 NIR-emitting dyes, partly due to increased molecular rigidity via hydrogen bonding.   It should be noted that the absorption of 6 (max 399 nm) was blue-shifted from 5 by 118 about 37-48 nm. It appeared that the introduced methyl-protecting group on the phenol 119 perturbed the alignment of the lone pair on the oxygen atom (with the -bond on the 120 aromatic ring), resulting in a hypochromic shift. The observed hypochromic shift could 121 be attributed to the steric interaction of the methoxy group. In the ground state, the methyl 122 group would be forced to rotate away from the plane of the connected phenyl ring, due 123 to the steric interaction with the adjacent aromatic group (6 in Scheme 2). As a conse-124 quence, the lone pair electron on the oxygen atom (with the sp 2 hybridization) would be 125 away significantly from the desirable parallel position to the -orbitals of the phenyl ring. 126 This could prevent effective electron delocalization from the methoxy group to the phenyl 127 It should be noted that the absorption of 6 (λ max ≈ 399 nm) was blue-shifted from 5 by about 37-48 nm. It appeared that the introduced methyl-protecting group on the phenol perturbed the alignment of the lone pair on the oxygen atom (with the π-bond on the aromatic ring), resulting in a hypochromic shift. The observed hypochromic shift could be attributed to the steric interaction of the methoxy group. In the ground state, the methyl group would be forced to rotate away from the plane of the connected phenyl ring, due to the steric interaction with the adjacent aromatic group (6 in Scheme 2). As a consequence, the lone pair electron on the oxygen atom (with the sp 2 hybridization) would be away significantly from the desirable parallel position to the π-orbitals of the phenyl ring. This could prevent effective electron delocalization from the methoxy group to the phenyl ring, thereby leading to the hypochromic shift in UV-vis absorption. Free rotation of the methoxy group, relative to the connected aromatic ring, appeared to be a major factor that led to a large decrease in the fluorescence quantum yield of 6 (by a factor of~100 in comparison with that of 3).

112
ring, thereby leading to the hypochromic shift in UV-vis absorption. Free rotation of the 128 methoxy group, relative to the connected aromatic ring, appeared to be a major factor that 129 led to a large decrease in the fluorescence quantum yield of 6 (by a factor of ~100 in com-130 parison with that of 3). 131 132 Scheme 2. Schematic illustration of molecular geometry in ground state of 6, and structural change 133 from the locally excited state of 6 to the excited species 6a and 6b; intramolecular charge transfer 134 (ICT). 135 Interestingly, compound 6 gave two emission peaks, with one at ~587 nm and the 136 other at ~660 nm ( Figure 3). The emission at ~587 nm matched the emission peak of 5, 137 which could be attributed to the effective chromophore (or cyanine subunit). It was as-138 sumed that the methyl group could adopt a coplanar position with the phenyl ring in the 139 excited state as shown in 6a, in which the lone pair orbital on the oxygen is aligned parallel 140 to the -orbitals for extended conjugation/interaction. Nearly identical emission between 141 6 (em  587 nm) and 5 (em  580 nm) supports the assumption, as the lone pair orbital on 142 the phenol of 5 is expected to be parallel with the -orbitals on the phenyl ring. The second 143 emission from 6 (em  660 nm) could be attributed to the excited species 6b, which has 144 lower energy than 6a after undergoing the intramolecular charge transfer (ICT). In order to explore the ICT nature of the fluorophores, we decided to acquire the 147 fluorescence spectra at low temperatures. In the previous studies on cyanine 1 & 4 [4], [9] 148 the molecular motion and bond changes associated with the ICT process are shown to be 149 frozen at the low temperature, while the ESIPT process could still operate. Thus, the meth-150 anol solution (10 μM) of model compounds 5 and 6 in quartz tubes was quickly cooled by 151 immersing the sample into liquid nitrogen in a quartz Dewar. The fluorescence spectra at 152 low temperature (−189 °C) were significantly blue-shifted from that at room temperature 153 20 °C (Figure 4), as the ICT interaction was removed in the excited states. The emission of 154 5 was blue shifted from 581 nm to 531 nm when the temperature was decreased to −189 155 °C. At room temperature, the solution of 6 exhibited two emission peaks in methanol (em 156  568 & 700 nm, Figure 4), similar to that in CH2Cl2 ( Figure 3). Interestingly, only one 157 emission peak was observed at −189 °C from 6. This observation pointed to the low energy 158 emission peak (em  700 nm) from 6 was largely associated with ICT. In other words, the 159 ICT interaction could play a more important role in 6. The charge transfer accounts for the 160 degrees of the redshift of the emission spectra from different extents of ICT nature [13]. 161 Since the ICT was disabled when the sample was frozen, the excited 6 could not generate 162 Scheme 2. Schematic illustration of molecular geometry in ground state of 6, and structural change from the locally excited state of 6 to the excited species 6a and 6b; intramolecular charge transfer (ICT).
Interestingly, compound 6 gave two emission peaks, with one at~587 nm and the other at~660 nm (Figure 3). The emission at~587 nm matched the emission peak of 5, which could be attributed to the effective chromophore (or cyanine subunit). It was assumed that the methyl group could adopt a coplanar position with the phenyl ring in the excited state as shown in 6a, in which the lone pair orbital on the oxygen is aligned parallel to the π-orbitals for extended conjugation/interaction. Nearly identical emission between 6 (λ em ≈ 587 nm) and 5 (λ em ≈ 580 nm) supports the assumption, as the lone pair orbital on the phenol of 5 is expected to be parallel with the π-orbitals on the phenyl ring. The second emission from 6 (λ em ≈ 660 nm) could be attributed to the excited species 6b, which has lower energy than 6a after undergoing the intramolecular charge transfer (ICT).

Low-Temperature Fluorescence
In order to explore the ICT nature of the fluorophores, we decided to acquire the fluorescence spectra at low temperatures. In the previous studies on cyanine 1 & 4 [4], [9] the molecular motion and bond changes associated with the ICT process are shown to be frozen at the low temperature, while the ESIPT process could still operate. Thus, the methanol solution (10 µM) of model compounds 5 and 6 in quartz tubes was quickly cooled by immersing the sample into liquid nitrogen in a quartz Dewar. The fluorescence spectra at low temperature (−189 • C) were significantly blue-shifted from that at room temperature 20 • C (Figure 4), as the ICT interaction was removed in the excited states. The emission of 5 was blue shifted from 581 nm to 531 nm when the temperature was decreased to −189 • C. At room temperature, the solution of 6 exhibited two emission peaks in methanol (λ em ≈ 568 & 700 nm, Figure 4), similar to that in CH 2 Cl 2 ( Figure 3). Interestingly, only one emission peak was observed at −189 • C from 6. This observation pointed to the low energy emission peak (λ em ≈ 700 nm) from 6 was largely associated with ICT. In other words, the ICT interaction could play a more important role in 6. The charge transfer accounts for the degrees of the redshift of the emission spectra from different extents of ICT nature [13]. Since the ICT was disabled when the sample was frozen, the excited 6 could not generate 6a & 6b, and the emission could only occur from 6. The emission of 6 at~700 nm at room temperature was not associated with the aggregation, as aggregation content would increase with decreasing temperature [14]. In summary, the low-temperature fluorescence supported the proposed fluorescence process in Scheme 2.
6a & 6b, and the emission could only occur from 6. The emission of 6 at ~700 nm at room 163 temperature was not associated with the aggregation, as aggregation content would in-164 crease with decreasing temperature [14]. In summary, the low-temperature fluorescence 165 supported the proposed fluorescence process in Scheme 2. Our initial study was carried out in CH2Cl2 (a polar aprotic solvent), in order to avoid 181 the dissociation of phenolic protons while providing good solubility. The steady-state flu-182 orescence of compound 1 in CH2Cl2 at room temperature is shown in Figure 5. On the 183 basis of a large Stokes shift, the emission peak at em  685 nm was attributed to the keto 184 emission. The absence of the absorption and emission signals between 500 and 580 nm led 185 to an optically transparent window, suggesting that the enol emission was either absent 186 or at a very low concentration.

Fluorescence Lifetime
When the fluorescence decay involves multicomponents [15], the fluorescence signal decay I(t) is assumed to be the sum of individual single exponential decay from each component: In the above equation, τ i are the individual lifetimes of each component, α i represent the amplitudes of the components at t = 0, and n is the number of decay times. In a mixture of fluorophores, the decay times τ i may be assigned to each excited species. The relative contributions of each component i are calculated from the pre-exponential values α i weighted by the lifetime τ i and given as a percentage, with Our initial study was carried out in CH 2 Cl 2 (a polar aprotic solvent), in order to avoid the dissociation of phenolic protons while providing good solubility. The steady-state fluorescence of compound 1 in CH 2 Cl 2 at room temperature is shown in Figure 5. On the basis of a large Stokes shift, the emission peak at λ em ≈ 685 nm was attributed to the keto emission. The absence of the absorption and emission signals between 500 and 580 nm led to an optically transparent window, suggesting that the enol emission was either absent or at a very low concentration. 6a & 6b, and the emission could only occur from 6. The emission of 6 at ~700 nm at room 163 temperature was not associated with the aggregation, as aggregation content would in-164 crease with decreasing temperature [14]. In summary, the low-temperature fluorescence 165 supported the proposed fluorescence process in Scheme 2. Our initial study was carried out in CH2Cl2 (a polar aprotic solvent), in order to avoid 181 the dissociation of phenolic protons while providing good solubility. The steady-state flu-182 orescence of compound 1 in CH2Cl2 at room temperature is shown in Figure 5. On the 183 basis of a large Stokes shift, the emission peak at em  685 nm was attributed to the keto 184 emission. The absence of the absorption and emission signals between 500 and 580 nm led 185 to an optically transparent window, suggesting that the enol emission was either absent 186 or at a very low concentration. Interestingly, fluorescence lifetime measurement of 1 revealed a two-exponential decay, with τ 1 = 1.05 ns (5.8%) and τ 2 = 3.38 ns (94.2%), based on the best curve fitting of reduced chi-square (χ 2 r = 1.02) shown in Figure 6. Since only one emission peak was observed in the steady-state fluorescence, the major component (τ 2 = 3.38 ns) was attributed to the emission from keto tautomer. It should be noted that the minor component (τ 1 = 1.05 ns) might not be associated with the locally excited (LE) state of enol tautomer, as the fast rate of ESIPT (e.g., in less than 200 picoseconds) [16] makes it unlikely to have the lifetime in the nanosecond range. The assumption was consistent with the steady-state fluorescence, as no enol emission was observed ( Figure 5). Interestingly, fluorescence lifetime measurement of 1 revealed a two-exponential de-192 cay, with 1 = 1.05 ns (5.8%) and 2 = 3.38 ns (94.2%), based on the best curve fitting of 193 reduced chi-square ( r 2 = 1.02) shown in Figure 6. Since only one emission peak was ob-194 served in the steady-state fluorescence, the major component (2 = 3.38 ns) was attributed 195 to the emission from keto tautomer. It should be noted that the minor component (1 = 1.05 196 ns) might not be associated with the locally excited (LE) state of enol tautomer, as the fast 197 rate of ESIPT (e.g., in less than 200 picoseconds) [16] makes it unlikely to have the lifetime 198 in the nanosecond range. The assumption was consistent with the steady-state fluores-199 cence, as no enol emission was observed ( Figure 5). It has been shown that pyridinium-containing styryl dyes typically exhibit a rela-206 tively short fluorescence lifetime. For example, 2-(4-(dimethylamino)styryl)-1-methylpyr-207 idinium iodide (DASPMI) in chloroform shows a three-exponential decay with 1 = 34 and 208 2 = 79 picoseconds, which are attributed to two different excited states, i.e., LE and ICT 209 states [17]. In a polar solvent such as MeOH, DASPMI exhibits a three-exponential decay 210 function, in which an additional component with a shorter lifetime (e.g.,   1 ps) is as-211 sumed to be caused by solvation [18]. In a recent study by Spalletti and coworkers, [16] 212 the pyridinium-containing dyes are further examined by ultrafast transient absorption, 213 revealing a three-exponential decay in CH2Cl2 (Table 2) Table 2. Fluorescence lifetime of two pyridinium-containing styryl dyes in CH2Cl2 (from reference 220 [19], Copyright © 2014, American Chemical Society). It has been shown that pyridinium-containing styryl dyes typically exhibit a relatively short fluorescence lifetime. For example, 2-(4-(dimethylamino)styryl)-1-methylpyridinium iodide (DASPMI) in chloroform shows a three-exponential decay with τ 1 = 34 and τ 2 = 79 picoseconds, which are attributed to two different excited states, i.e., LE and ICT states [17]. In a polar solvent such as MeOH, DASPMI exhibits a three-exponential decay function, in which an additional component with a shorter lifetime (e.g., τ ≈ 1 ps) is assumed to be caused by solvation [18]. In a recent study by Spalletti and coworkers, [16] the pyridinium-containing dyes are further examined by ultrafast transient absorption, revealing a three-exponential decay in CH 2 Cl 2 ( Table 2), which includes LE, solvation and ICT processes. It should be noticed that the lifetimes of these pyridinium-containing styryl dyes are on the time scale of picoseconds. In sharp contrast to what is generally reported in the literature, the lifetime of 1 revealed only two components with significantly longer lifetimes (on the scale of nanoseconds) for LE and ICT, showing the significant impact of intramolecular hydrogen bonding on the lifetime parameter. Table 2. Fluorescence lifetime of two pyridinium-containing styryl dyes in CH 2 Cl 2 (from reference [19], Copyright © 2022, American Chemical Society). Examination of compound 3 under the same condition also revealed similar fluores-225 cence lifetime characteristics in CH2Cl2, exhibiting a two-exponential decay with 1 = 2.04 226 ns (16%) and 2 = 2.75 ns (84%) ( Table 3). In order to shed some light on the observed 227 fluorescence decay, model compound 5 was used in the study, which was synthesized as 228 described in a previous report [8]. As shown in Scheme 1, the conjugation length of cya-229 nine fragment in 3 can be approximated by compound 5, as they exhibit nearly identical 230 absorption max (447 nm for 3 and 449 nm for 5 in CH2Cl2) [8]. Upon excitation with a 405 231 nm laser, however, the fluorescence lifetime measurement of 5 revealed a three-exponen-232 tial decay (Table 3). With a chi-square value  r 2 1.0, the fitting gave two major compo-233 nents 1 = 0.10 and 2 = 0.24 ns (>98%) and a minor component 3  1.77 ns. The minor 234 component (3) could be attributed to the ICT state since the intramolecular charge transfer 235 (ICT) state typically had a significantly longer lifetime in styryl dyes (see Table 2). It 236 should be noted that a dual fluorescence lifetime was expected from the excited state of a 237 donor-acceptor stilbene [20]. One of the two components with shorter lifetimes (1 = 0.10 238 and 2 = 0.24 ns) could be associated with the LE state. 239 1.17 *Data obtained with a 550 nm long path filter, by using the same conditions as for 1-5; **data ob-241 tained with a 650 nm long path filter, and τ ̅ represents the average lifetime, ̅ = 1 1 + 2 2 + 3 3 . 242

Pyridinium-Based Stilbenes a Lifetime (ps) Transient
The fluorescence lifetime measurement of 6 also revealed a three-exponential decay 243 in CH2Cl2, in agreement with 5. Interestingly, multiexponential decay analysis of 6 re-244 vealed one minor component (1  0.11 ns; fractional intensity ~5.3%) and two major com-245 ponents (2  1.41 ns; 3  2.61 ns). The observed distribution from 6 was in contrast to that 246 from 5, whose distribution revealed two major components with shorter lifetimes (i.e., 1 247 and 2 less than 0.24 ns). Among the emissive species of 6, the component with 3  2.67 ns 248 was likely to be associated with the excited 6 (CT) that was the major emissive species. 249 The assumption was consistent with the observed increase in its fractional intensity when 250 Examination of compound 3 under the same condition also revealed similar fluores-225 cence lifetime characteristics in CH2Cl2, exhibiting a two-exponential decay with 1 = 2.04 226 ns (16%) and 2 = 2.75 ns (84%) ( Table 3). In order to shed some light on the observed 227 fluorescence decay, model compound 5 was used in the study, which was synthesized as 228 described in a previous report [8]. As shown in Scheme 1, the conjugation length of cya-229 nine fragment in 3 can be approximated by compound 5, as they exhibit nearly identical 230 absorption max (447 nm for 3 and 449 nm for 5 in CH2Cl2) [8]. Upon excitation with a 405 231 nm laser, however, the fluorescence lifetime measurement of 5 revealed a three-exponen-232 tial decay (Table 3). With a chi-square value  r 2 1.0, the fitting gave two major compo-233 nents 1 = 0.10 and 2 = 0.24 ns (>98%) and a minor component 3  1.77 ns. The minor 234 component (3) could be attributed to the ICT state since the intramolecular charge transfer 235 (ICT) state typically had a significantly longer lifetime in styryl dyes (see Table 2). It 236 should be noted that a dual fluorescence lifetime was expected from the excited state of a 237 donor-acceptor stilbene [20]. One of the two components with shorter lifetimes (1 = 0.10 238 and 2 = 0.24 ns) could be associated with the LE state. 239 1.17 *Data obtained with a 550 nm long path filter, by using the same conditions as for 1-5; **data ob-241 tained with a 650 nm long path filter, and τ ̅ represents the average lifetime, ̅ = 1 1 + 2 2 + 3 3 . 242 The fluorescence lifetime measurement of 6 also revealed a three-exponential decay 243 in CH2Cl2, in agreement with 5. Interestingly, multiexponential decay analysis of 6 re-244 vealed one minor component (1  0.11 ns; fractional intensity ~5.3%) and two major com-245 ponents (2  1.41 ns; 3  2.61 ns). The observed distribution from 6 was in contrast to that 246 from 5, whose distribution revealed two major components with shorter lifetimes (i.e., 1 247 and 2 less than 0.24 ns). Among the emissive species of 6, the component with 3  2.67 ns 248 was likely to be associated with the excited 6 (CT) that was the major emissive species. 249 The assumption was consistent with the observed increase in its fractional intensity when 250 Examination of compound 3 under the same condition also revealed similar fluorescence lifetime characteristics in CH 2 Cl 2 , exhibiting a two-exponential decay with τ 1 = 2.04 ns (16%) and τ 2 = 2.75 ns (84%) ( Table 3). In order to shed some light on the observed fluorescence decay, model compound 5 was used in the study, which was synthesized as described in a previous report [8]. As shown in Scheme 1, the conjugation length of cyanine fragment in 3 can be approximated by compound 5, as they exhibit nearly identical absorption λ max (447 nm for 3 and 449 nm for 5 in CH 2 Cl 2 ) [8]. Upon excitation with a 405 nm laser, however, the fluorescence lifetime measurement of 5 revealed a three-exponential decay (Table 3). With a chi-square value χ 2 r ≈1.0, the fitting gave two major components τ 1 = 0.10 and τ 2 = 0.24 ns (>98%) and a minor component τ 3 ≈ 1.77 ns. The minor component (τ 3 ) could be attributed to the ICT state since the intramolecular charge transfer (ICT) state typically had a significantly longer lifetime in styryl dyes (see Table 2). It should be noted that a dual fluorescence lifetime was expected from the excited state of a donor-acceptor stilbene [20]. One of the two components with shorter lifetimes (τ 1 = 0.10 and τ 2 = 0.24 ns) could be associated with the LE state. * Data obtained with a 550 nm long path filter, by using the same conditions as for 1-5; ** data obtained with a 650 nm long path filter, and τ represents the average lifetime, τ = α 1 τ 1 + α 2 τ 2 + α 3 τ 3 .
The fluorescence lifetime measurement of 6 also revealed a three-exponential decay in CH 2 Cl 2 , in agreement with 5. Interestingly, multiexponential decay analysis of 6 revealed one minor component (τ 1 ≈ 0.11 ns; fractional intensity~5.3%) and two major components (τ 2 ≈ 1.41 ns; τ 3 ≈ 2.61 ns). The observed distribution from 6 was in contrast to that from 5, whose distribution revealed two major components with shorter lifetimes (i.e., τ 1 and τ 2 less than 0.24 ns). Among the emissive species of 6, the component with τ 3 ≈ 2.67 ns was likely to be associated with the excited 6 (CT) that was the major emissive species.
The assumption was consistent with the observed increase in its fractional intensity when the long path optical filter wavelength was increased from 550 nm to 650 nm, which allowed only the photons to be detected above 550 nm or 650 nm. The components with τ 1 ≈ 0.1 ps and τ 2 ≈ 1.45 ns could be attributed to the excited 6 (LE) and 6a, respectively (Scheme 3). In summary, excited 3 exhibited a two-exponential decay, while its model compounds 5 and 6 showed a three-exponential decay. In other words, the results suggested that ESIPT functional group could play an important role in simplifying the fluorescence decay processes.
Molecules 2022, 27, x FOR PEER REVIEW 9 of 14 the long path optical filter wavelength was increased from 550 nm to 650 nm, which al-251 lowed only the photons to be detected above 550 nm or 650 nm. The components with 1 252  0.1 ps and 2  1.45 ns could be attributed to the excited 6 (LE) and 6a, respectively 253 (Scheme 3). In summary, excited 3 exhibited a two-exponential decay, while its model 254 compounds 5 and 6 showed a three-exponential decay. In other words, the results sug-255 gested that ESIPT functional group could play an important role in simplifying the fluo-256 rescence decay processes. Previous studies have shown that HBT typically gives only one lifetime (on a pico-261 second time scale) that is associated with its keto tautomer [7]. Since the proton transfer in 262 HBT has essentially no barrier and can occur at a very fast rate [5], it was unlikely to have 263 the lifetime of the enol tautomer of 3 on a nanosecond scale. This pointed to the possibility 264 that the singlet keto tautomer and its ICT species were potential lifetime components. The 265 assumption was further supported by the observation of a multiexponential decay from 6 266 that cannot undergo ESIPT. On the basis of the above reasoning, the observed two lifetime 267 components from 3 was likely to be associated with species that appeared after the ESIPT 268 event. One possible species was the singlet keto tautomer, which was followed by another 269 species after undergoing ICT, as shown in Scheme 4. The proposed decay involved the 270 assumption that the deactivation of the excited states was basically determined by the 271 styryl-cyanine fragment and ICT process. The assumption was consistent with the known 272 literature examples of pyridinium-containing styryl dyes [19], whose lifetime components 273 included LE and ICT (Table 2). Previous studies have shown that HBT typically gives only one lifetime (on a picosecond time scale) that is associated with its keto tautomer [7]. Since the proton transfer in HBT has essentially no barrier and can occur at a very fast rate [5], it was unlikely to have the lifetime of the enol tautomer of 3 on a nanosecond scale. This pointed to the possibility that the singlet keto tautomer and its ICT species were potential lifetime components. The assumption was further supported by the observation of a multiexponential decay from 6 that cannot undergo ESIPT. On the basis of the above reasoning, the observed two lifetime components from 3 was likely to be associated with species that appeared after the ESIPT event. One possible species was the singlet keto tautomer, which was followed by another species after undergoing ICT, as shown in Scheme 4. The proposed decay involved the assumption that the deactivation of the excited states was basically determined by the styryl-cyanine fragment and ICT process.
The assumption was consistent with the known literature examples of pyridinium-containing styryl dyes [19], whose lifetime components included LE and ICT (Table 2). s 2022, 27, x FOR PEER REVIEW 9 of 14 the long path optical filter wavelength was increased from 550 nm to 650 nm, which al-251 lowed only the photons to be detected above 550 nm or 650 nm. The components with 1 252  0.1 ps and 2  1.45 ns could be attributed to the excited 6 (LE) and 6a, respectively 253 (Scheme 3). In summary, excited 3 exhibited a two-exponential decay, while its model 254 compounds 5 and 6 showed a three-exponential decay. In other words, the results sug-255 gested that ESIPT functional group could play an important role in simplifying the fluo-256 rescence decay processes. Previous studies have shown that HBT typically gives only one lifetime (on a pico-261 second time scale) that is associated with its keto tautomer [7]. Since the proton transfer in 262 HBT has essentially no barrier and can occur at a very fast rate [5], it was unlikely to have 263 the lifetime of the enol tautomer of 3 on a nanosecond scale. This pointed to the possibility 264 that the singlet keto tautomer and its ICT species were potential lifetime components. The 265 assumption was further supported by the observation of a multiexponential decay from 6 266 that cannot undergo ESIPT. On the basis of the above reasoning, the observed two lifetime 267 components from 3 was likely to be associated with species that appeared after the ESIPT 268 event. One possible species was the singlet keto tautomer, which was followed by another 269 species after undergoing ICT, as shown in Scheme 4. The proposed decay involved the 270 assumption that the deactivation of the excited states was basically determined by the 271 styryl-cyanine fragment and ICT process. The assumption was consistent with the known 272 literature examples of pyridinium-containing styryl dyes [19], whose lifetime components 273 included LE and ICT (Table 2). It should be noticed that all four compounds containing an ESIPT group (i.e., 1-4) revealed a two-exponential decay (Table 3). For compounds 1-3, their solution in CH 2 Cl 2 gave a predominant excited component that involved the ICT process (with a fractional intensity as high as 94%). However, both components in the decay were present in significant fractional intensities (e.g., f 1 ≈ 54% and f 2 ≈ 46%) in CH 2 Cl 2 for compound 4. Observation of significant fractional contribution from both components indicated that none of them could be associated with the enol tautomer since the steady-state fluorescence of 4 observed only keto emission [10]. The large variation in the fractional contribution could be related to the extent of ICT interaction, which deserves further investigation.

Materials and Methods
All starting materials and the essential solvents were purchased from Sigma-Aldrich, TGI, Ark Pharma, Fischer Scientific, Alfa-Asaer and Across Organics and directly used without further purification. Starting materials 2-(Benzo[d]thiazol-2-yl)-4-methylphenol (8) and 2-(benzo[d]oxazol-2-yl)-4-methylphenol (10) were synthesized by using literature procedures. All deuterated solvents were purchased from Cambridge Isotopes and used as received. All NMR data were recorded on Varian 300 and 500 MHz instruments with all spectra referenced to deuterated solvents. HRMS data were acquired on an ESI-TOF MS system (Waters, Milford, MA). UV−vis studies were carried out in Hewlett-Packard-8453 diode array-based spectrophotometer at 25 • C. Fluorescence spectral analysis was conducted by using a HORIBA Fluoromax-4 spectrofluorometer.
Fluorescence lifetime was measured by using a time-correlated single-photon counting (TC-SPC) method, on a Horiba DeltaPro lifetime system, which is capable of measuring a lifetime range of 30 ps-1 s. The instrument is equipped with a picosecond photon detection module comprising a fast, cooled, photomultiplier with 230-850nm response. All measurements were performed by exciting the sample solutions with a Horiba DeltaDiode TM DD-405 Laser (peak wavelength at 405 nm +/−10 nm).

Conclusions
In conclusion, the fluorescence lifetimes of compounds 1-4 have been determined by using time-correlated single-photon counting (TCSPC) method ( Figure 6; ESI Tables S1-S4, Figures S29-S39). A polar aprotic solvent, such as CH 2 Cl 2 , was used in the study, which provides good solubility while avoiding the phenolic proton dissociation that could lead to a new component to complicate the study. The experimental study revealed two lifetime components in the range of 1.0-3.6 nanoseconds (ns), depending on the structure of terminal cyanine segments. The identified lifetime components were mainly associated with the excited keto tautomers, as only keto emission was observed in the steady-state fluorescence spectra. For each compound, the lifetime difference (τ 2 − τ 1 ) in the two identified components also exhibited significant differences, ranging from 0.7 ns for 3 to 2.33 ns for 1, which is in agreement with the large structural difference between the probes.
The study was further carried out by comparison of 3 with its structural similar model styryl compounds 5-6, in which the intramolecular proton transfer is no longer possible. Evaluation of their fluorescence decay revealed three lifetime constants for 5-6 (ESI Tables S5-S8, Figures S40-S51), in contrast to two for 3. For example, the observed three lifetimes from 6 were τ 1 ≈ 0.1, τ 2 ≈ 1.4, and τ 3 ≈ 2.6 ns. Our study suggested that the component with the longest lifetime (τ 3 ≈ 2.6 ns) could be attributed to the ICT, while the component with the shortest lifetime to the locally excited 6 (LE) (Scheme 3). A molecular modeling study further confirmed that the methoxy group in the ground state of 6 was not coplanar with the attached aromatic ring (ESI Figure S52), which could be responsible for the additional lifetime component (τ 1 ≈ 0.1). Thus, intramolecular hydrogen bonding played an essential role in maintaining molecular co-planarity, which is desirable in the excited state (for ICT) and responsible for a simplified decay pathway.
The experimental finding showed that the excited state of ESIPT compounds 1-4 exhibited a two-exponential decay in a polar aprotic solvent, in contrast to the mono-exponential decay typically observed from HBT. The multiexponential decay of 1-4 was consistent with the known cyanine-containing styryl dyes (e.g., DASPMI) and model compounds 5-6. The results showed the large impact of the cyanine segment that contributed to the enhanced ICT interaction in the excited states. The ICT interaction in these ESIPT compounds had a significant impact on the fluorescence lifetime characteristics, including lifetime parameters (τ i ) and fractional intensities (f i ). These findings will provide useful data to guide the further development of this class of materials.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules28010125/s1. Figure S1. 1 H-NMR spectra of compound 9 in CDCl 3 . Figure S2. 1H-NMR spectra of compound 11 in CDCl 3 . Figure S3. 1H-NMR spectra of compound 1 in DMSO-d6. Figure S4. 1H-NMR spectra of compound 2 in DMSO-d 6 . Figure S5. 1H-NMR spectra of compound 3 in DMSO-d 6 . Figure S6. 1H-NMR spectra of compound 4 in DMSO-d 6 . Figure S7. 1H-NMR spectra of compound 5 in DMSO-d 6 . Figure S8. 1H-NMR spectra of compound 6 in DMSO-d 6 . Figure S9. 13 C-NMR spectra of compound 6 in DMSO-d 6 . Figure S10. ESI-MS spectra of compound 6. Figure S11. UV-vis absorption (solid line) and emission spectra (broken line) of compound 1 in DCM (10 µM). The excitation wavelength was 430 nm. Figure S12. UV-vis absorption (solid line) and emission spectra (broken line) of compound 1 in MeCN (10 µM). The excitation wavelength was 400 nm. Figure S13. UV-vis absorption (solid line) and emission spectra (broken line) of compound 1 in MeOH (10 µM). The excitation wavelength was 400 nm. Figure S14. UV-vis absorption (solid line) and emission spectra (broken line) of compound 2 in DCM (10 µM). The excitation wavelength was 410 nm. Figure S15. UV-vis absorption (solid line) and emission spectra (broken line) of compound 2 in MeCN (10 µM). The excitation wavelength was 390 nm. Figure S16. UV-vis absorption (solid line) and emission spectra (broken line) of compound 2 in MeOH (10 µM). The excitation wavelength was 400 nm. Figure S17. UV-vis absorption (solid line) and emission spectra (broken line) of compound 3 in DCM (10 µM). The excitation wavelength was 450 nm. Figure S18. UV-vis absorption (solid line) and emission spectra (broken line) of compound 3 in MeCN (10 µM). The excitation wavelength was 430 nm. Figure S19. UV-vis absorption (solid line) and emission spectra (broken line) of compound 3 in MeOH (10 µM). The excitation wavelength was 430 nm. Figure S20. UV-vis absorption (solid line) and emission spectra (broken line) of compound 4 in DCM (10 µM). The excitation wavelength was 440 nm. Figure S21. UV-vis absorption (solid line) and emission spectra (broken line) of compound 4 in MeCN (10 µM). The excitation wavelength was 420 nm. Figure S22. UV-vis absorption (solid line) and emission spectra (broken line) of compound 4 in MeOH (10 µM). The excitation wavelength was 420 nm. Figure S23. UV-vis absorption (solid line) and emission spectra (broken line) of compound 5 in DCM (10 µM). The excitation wavelength was 450 nm. Figure S24. UV-vis absorption (solid line) and emission spectra (broken line) of compound 5 in MeCN (10 µM). The excitation wavelength was 410 nm. Figure S25. UV-vis absorption (solid line) and emission spectra (broken line) of compound 5 in MeOH (10 µM). The excitation wavelength was 430 nm. Figure S26. UV-vis absorption (solid line) and emission spectra (broken line) of compound 6 in MeOH (10 µM). The excitation wavelength was 400 nm. Figure S27. UV-vis absorption (solid line) and emission spectra (broken line) of compound 6 in MeCN (10 µM). The excitation wavelength was 385 nm. Figure S28. UV-vis absorption (solid line) and emission spectra (broken line) of compound 6 in MeOH (10 µM). The excitation wavelength was 385 nm. Table S1. Fluorescence lifetime data of compound 1. Table S2. Fluorescence lifetime data of compound 2. Table S3. Fluorescence lifetime data of compound 3. Table S4. Fluorescence lifetime data of compound 4. Table S5. Fluorescence lifetime data of compound 5 (two-exponential). Table S6. Fluorescence lifetime data of compound 5 (three-exponential). Table S7. Fluorescence lifetime data of compound 6 (550 nm long path filter). Table S8. Fluorescence lifetime data of compound 6 (650 nm long path filter). Figure S29. The fluorescence lifetime of IRF (purple square) and decay for compound 1 in MeCN (green circle) and fitted curve of two exponential functions. The residuals are reported in the lower panel. With DD-405L (λ em = 406 nm) as light source and 650 nm long path filter. Figure S30. The fluorescence lifetime of IRF (purple square) and decay for compound 1 in MeOH (green circle) and fitted curve of two exponential functions. The residuals are reported in the lower panel. With DD-405L (λ em = 406 nm) as light source and 650 nm long path filter. Figure S31. The fluorescence lifetime of IRF (purple square) and decay for compound 2 in DCM (green circle) and fitted curve of two exponential functions. The residuals are reported in the lower panel. With DD-405L (λ em = 406 nm) as light source and 650 nm long path filter. Figure S32. The fluorescence lifetime of IRF (purple square) and decay for compound 2 in MeCN (green circle) and fitted curve of two exponential functions. The residuals are reported in the lower panel. With DD-405L (λ em = 406 nm) as light source and 650 nm long path filter. Figure S33. The fluorescence lifetime of IRF (purple square) and decay for compound 2 in MeOH (green circle) and fitted curve of two exponential functions. The residuals are reported in the lower panel. With DD-405L (λ em = 406 nm) as light source and 650 nm long path filter. Figure S34. The fluorescence lifetime of IRF (purple square) and decay for compound 3 in DCM (green circle) and fitted curve of two exponential functions. The residuals are reported in the lower panel. With DD-405L (λ em = 406 nm) as light source and 650 nm long path filter. Figure S35. The fluorescence lifetime of IRF (purple square) and decay for compound 3 in MeCN (green circle) and fitted curve of two exponential functions. The residuals are reported in the lower panel. With DD-405L (λ em = 406 nm) as light source and 650 nm long path filter. Figure S36. The fluorescence lifetime of IRF (purple square) and decay for compound 3 in MeOH (green circle) and fitted curve of two exponential functions. The residuals are reported in the lower panel. With DD-405L (λ em = 406 nm) as light source and 650 nm long path filter. Figure S37. The fluorescence lifetime of IRF (purple square) and decay for compound 4 in DCM (green circle) and fitted curve of two exponential functions. The residuals are reported in the lower panel. With DD-405L (λ em = 406 nm) as light source and 650 nm long path filter. Figure S38. The fluorescence lifetime of IRF (purple square) and decay for compound 4 in MeCN (green circle) and fitted curve of two exponential functions. The residuals are reported in the lower panel. With DD-405L (λ em = 406 nm) as light source and 650 nm long path filter. Figure S39. The fluorescence lifetime of IRF (purple square) and decay for compound 4 in MeOH (green circle) and fitted curve of two exponential functions. The residuals are reported in the lower panel. With DD-405L (λ em = 406 nm) as light source and 650 nm long path filter. Figure S40. The fluorescence lifetime of IRF (purple square) and decay for compound 5 in DCM (green circle) and fitted curve of two exponential functions. The residuals are reported in the lower panel. With DD-405L (λ em = 406 nm) as light source and 550 nm long path filter. Figure S41. The fluorescence lifetime of IRF (purple square) and decay for compound 5 in MeCN (green circle) and fitted curve of two exponential functions. The residuals are reported in the lower panel. With DD-405L (λ em = 406 nm) as light source and 550 nm long path filter. Figure S42. The fluorescence lifetime of IRF (purple square) and decay for compound 5 in MeOH (green circle) and fitted curve of two exponential functions. The residuals are reported in the lower panel. With DD-405L (λ em = 406 nm) as light source and 550 nm long path filter. Figure S43. The fluorescence lifetime of IRF (purple square) and decay for compound 5 in DCM (green circle) and fitted curve of three exponential functions. The residuals are reported in the lower panel. With DD-405L (λ em = 406 nm) as light source and 550 nm long path filter. Figure S44. The fluorescence lifetime of IRF (purple square) and decay for compound 5 in MeCN (green circle) and fitted curve of three exponential functions. The residuals are reported in the lower panel. With DD-405L (λ em = 406 nm) as light source and 550 nm long path filter. Figure S45. The fluorescence lifetime of IRF (purple square) and decay for compound 5 in MeOH (green circle) and fitted curve of three exponential functions. The residuals are reported in the lower panel. With DD-405L (λ em = 406 nm) as light source and 550 nm long path filter. Figure S46. The fluorescence lifetime of IRF (purple square) and decay for compound 6 in DCM (green circle) and fitted curve of three exponential functions. The residuals are reported in the lower panel. With DD-405L (λ em = 406 nm) as light source and 550 nm long path filter. Figure S47. The fluorescence lifetime of IRF (purple square) and decay for compound 6 in MeCN (green circle) and fitted curve of three exponential functions. The residuals are reported in the lower panel. With DD-405L (λ em = 406 nm) as light source and 550 nm long path filter. Figure S48. The fluorescence lifetime of IRF (purple square) and decay for compound 6 in MeOH (green circle) and fitted curve of three exponential functions. The residuals are reported in the lower panel. With DD-405L (λ em = 406 nm) as light source and 550 nm long path filter. Figure S49. The fluorescence lifetime of IRF (purple square) and decay for compound 6 in DCM (green circle) and fitted curve of three exponential functions. The residuals are reported in the lower panel. With DD-405L (λ em = 406 nm) as light source and 650 nm long path filter. Figure S50. The fluorescence lifetime of IRF (purple square) and decay for compound 6 in MeCN (green circle) and fitted curve of three exponential functions. The residuals are reported in the lower panel. With DD-405L (λ em = 406 nm) as light source and 650 nm long path filter. Figure S51. The fluorescence lifetime of IRF (purple square) and decay for compound 6 in MeOH (green circle) and fitted curve of three exponential functions. The residuals are reported in the lower panel. With DD-405L (λ em = 406 nm) as light source and 650 nm long path filter. Figure S52. The simulated molecular geometry of compound 6 on the ground state via DFT method (a) and on the excited state via TD-DFT method (b) at B3LYP/6-31G (d, p) level. Figure S53. UV-vis absorption (solid line) and emission spectra (broken line) of compound 6 (10 µM) in Ethylene Glycol (MEG). The excitation wavelength was 385 nm. Figure S54. The fluorescence lifetime of IRF (purple square) and decay for compound 6 in MEG (green circle) and fitted curve of three exponential functions. The residuals are reported in the lower panel. With DD-405L (λ em = 406 nm) as light source and 550 nm long path filter. Figure S55. The fluorescence lifetime of IRF (purple square) and decay for compound 6 in MEG (green circle) and fitted curve of three exponential functions. The residuals are reported in the lower panel. With DD-405L (λ em = 406 nm) as light source and 650 nm long path filter.