Water-Soluble Single-Benzene Chromophores: Excited State Dynamics and Fluorescence Detection

Two water-soluble single-benzene-based chromophores, 2,5-di(azetidine-1-yl)-tereph- thalic acid (DAPA) and its disodium carboxylate (DAP-Na), were conveniently obtained. Both chromophores preserved moderate quantum yields in a wide range of polar and protonic solvents. Spectroscopic studies demonstrated that DAPA exhibited red luminescence as well as large Stokes shift (>200 nm) in aqueous solutions. Femtosecond transient absorption spectra illustrated quadrupolar DAPA usually involved the formation of an intramolecular charge transfer state. Its Frank–Condon state could be rapidly relaxed to a slight symmetry-breaking state upon light excitation following the solvent relaxation, then the slight charge separation may occur and the charge localization became partially asymmetrical in polar environments. Density functional theory (DFT) calculation results were supported well with the experimental measurements. Unique pH-dependent fluorescent properties endows the two chromophores with rapid, highly selective, and sensitive responses to the amino acids in aqueous media. In detail, DAPA served as a fluorescence turn-on probe with a detection limit (DL) of 0.50 μM for Arg and with that of 0.41 μM for Lys. In contrast, DAP-Na featured bright green luminescence and showed fluorescence turn-off responses to Asp and Glu with the DLs of 0.12 μM and 0.16 μM, respectively. Meanwhile, these two simple-structure probes exhibited strong anti-interference ability towards other natural amino acids and realized visual identification of specific analytes. The present work helps to understand the photophysic–structure relationship of these kinds of compounds and render their fluorescent detection applications.


Introduction
Amino acids are the fundamental building blocks of biological macromolecular proteins and play pivotal roles in many physiological processes and behaviors [1][2][3]. According to the structural characteristics of side groups, 20 common natural amino acids generally fall into four categories, i.e., hydrophobic, polar, and acidic as well as basic ones. Aspartic acid (Asp) and glutamic acid (Glu) are typical acidic amino acids, and are involved in many physiological processes, such as learning, memory, movement disorders, and other brain functions [4][5][6]. Arginine (Arg), lysine (Lys), and histidine (His) are well-known basic amino acids and play crucial roles in many biological processes such as cell division, healing of wounds, release of hormones, the immune system, and metabolism, etc. [7][8][9]. For various amino acids, maintaining an appropriate level in biological systems is of great importance and any serious alterations may cause related diseases. For example, high concentrations of Asp may cause motor neuron disease known as Lou-Gehrig' s disease [10], while excessive Lys in urine and plasma could even lead to cystinuria or hyperlysinemia [11,12]. Therefore, the development of versatile sensor systems for discriminating and quantifying of various amino acids becomes more important for human health and medical diagnosis of diseases. Scheme 1. Efficient single-benzene chromophores reported in previous literatu present work (b); their emission maxima and fluorescence quantum yields in DM given.

Photophysical Properties of DAPA and DAP-Na
UV−vis absorption and fluorescence emission spectra of water-solubl DAP-Na were firstly investigated in water and the results are shown in F absorption maximum of DAPA was located at 365 nm, while that of D blue-shifted and centered at 338 nm. The emission peak of DAPA was loca and exhibited an obviously bathochromic shift with respect to that of DAP-The obtained bathochromic shift phenomena are probably due to intramolecular charge transfer property [49]. Typically, these two single-b chromophores feature extraordinarily large Stokes shifts of up to 226 nm i special characteristic and almost zero overlap between their excitation a spectra ( Figure S1) commonly benefit fluorescent sensing applications. interest, DAP-Na displayed intensively green fluorescence with a quantum up to 0.42 in aqueous media. However, DAPA exhibited relatively weak with f around 0.08.

Photophysical Properties of DAPA and DAP-Na
UV−vis absorption and fluorescence emission spectra of water-soluble DAPA and DAP-Na were firstly investigated in water and the results are shown in Figure 1a. The absorption maximum of DAPA was located at 365 nm, while that of DAP-Na was blue-shifted and centered at 338 nm. The emission peak of DAPA was located at 591 nm and exhibited an obviously bathochromic shift with respect to that of DAP-Na (535 nm). The obtained bathochromic shift phenomena are probably due to the stronger intramolecular charge transfer property [49]. Typically, these two single-benzene-based chromophores feature extraordinarily large Stokes shifts of up to 226 nm in water. This special characteristic and almost zero overlap between their excitation and emission spectra ( Figure S1) commonly benefit fluorescent sensing applications. Of particular interest, DAP-Na displayed intensively green fluorescence with a quantum yield (Φ f ) of up to 0.42 in aqueous media. However, DAPA exhibited relatively weak red emission with Φ f around 0.08.

Photophysical Properties of DAPA and DAP-Na
UV−vis absorption and fluorescence emission spectra of water-soluble DAPA and DAP-Na were firstly investigated in water and the results are shown in Figure 1a. The absorption maximum of DAPA was located at 365 nm, while that of DAP-Na was blue-shifted and centered at 338 nm. The emission peak of DAPA was located at 591 nm and exhibited an obviously bathochromic shift with respect to that of DAP-Na (535 nm). The obtained bathochromic shift phenomena are probably due to the stronger intramolecular charge transfer property [49]. Typically, these two single-benzene-based chromophores feature extraordinarily large Stokes shifts of up to 226 nm in water. This special characteristic and almost zero overlap between their excitation and emission spectra ( Figure S1) commonly benefit fluorescent sensing applications. Of particular interest, DAP-Na displayed intensively green fluorescence with a quantum yield (f) of up to 0.42 in aqueous media. However, DAPA exhibited relatively weak red emission with f around 0.08. Photophysical properties of these two single-benzene-based chromophores were investigated in highly polar and protonic solvents including DMF, DMSO, EtOH, and MeOH. DAPA showed obviously solvent-dependent properties and was characterized by single absorption maxima around 457 nm in DMF and 470 nm in DMSO, respectively Photophysical properties of these two single-benzene-based chromophores were investigated in highly polar and protonic solvents including DMF, DMSO, EtOH, and MeOH. DAPA showed obviously solvent-dependent properties and was characterized by single absorption maxima around 457 nm in DMF and 470 nm in DMSO, respectively (Figure 1b). In EtOH and MeOH, its absorption spectra at longer wavelengths were characterized by two bands located at 415-550 and 305-415 nm. Based on our understanding, the dicarboxylic acid of DAPA may be partially ionized, resulting in two balanced components (ionized state and carboxylic acid state) in the abovementioned solutions. If further considering the polarity of MeOH is larger than that of EtOH, the proportion of the component of ionized state (377 nm) in MeOH was slightly higher than that of carboxylic acid state (456 nm) as shown in Figure 1b. However, in water, the carboxy on one side was completely ionized, and thus DAPA was characterized by single absorption maxima around 365 nm. However, the absorption maxima of DAP-Na were insensitive to solvent properties ( Figure S2a (Table 1). [a] Absolute fluorescence quantum yields determined with a calibrated integrating sphere system (errors < 3%).

Theoretical Calculations
Time-dependent DFT calculations of DAPA and DAP-Na were performed in water at the CAM-B3LYP/6-31G(d,p) level to investigate their structural characteristics and photophysical properties (Figure 2a). The molecular orbital diagrams of DAPA and DPA-Na in both ground and excited states demonstrated HOMOs that were mainly localized over the benzene ring and the azetidines. However, LUMOs moved to the electron-withdrawing carbonyl moiety, indicating an effective push-pull system was established for these two single-benzene frameworks (Figure 2b,c). It is noteworthy that the absorptive energy gap of DAP-Na (6.48 eV) between the ground HOMO (−5.90 eV) and LUMO (0.58 eV) was larger than that of DAPA (5.94 eV). The similar trend of emissive energy gaps explicated the bathochromic shift phenomena from DAP-Na to DAPA. Moreover, their theoretical maxima absorption and emission bands agreed well with the experimental results, supporting our understanding about their photophysics.

Transient Absorption Spectroscopy
To obtain a full understanding of the excited state dynamics, femtosecond transient absorption (fs-TA) measurements of DAPA were carried out in DMF, DMSO, EtOH, and MeOH upon an excitation at fs-420 nm. fs-TA spectra recorded at less than 0.5 ps delay time were characterized by a positive excited state absorption (ESA) band in the 470-850 nm region and accompanied by three peaks at 495, 610, and 790 nm (Figures 3a,c and S3a-c). As mentioned in previous reports, highly symmetric push-pull chromophores, such as DAPA, could be recognized as quadrupolar molecules and the solvent dependence of their fluorescence is usually related to the formation of an intramolecular charge transfer (ICT) state [51][52][53][54][55]. In our opinion, the Frank-Condon S1 state of DAPA could be rapidly relaxed to a slight symmetry-breaking state upon light excitation following the solvent relaxation, and then the slight charge separation may occur and the charge localization become partially asymmetrical in polar media [56,57]. To provide an overview of excited-state dynamics and charge transfer properties of the fluorophores, the decay curves in fs-TA spectra and multi-exponential fitting kinetic traces probed at different wavelengths were compared as shown in Figures 3b and S4a,b. The corresponding lifetimes of transient states are summarized in Table S1. Based on these spectral traces and time constants, a corresponding sequential model for global fitting was proposed as shown in Figure 3d. The first decay lifetime may reflect the time-constant of solvent relaxation (SR < 1 ps). The second one is associated with the formation of the charge separation state and symmetry-breaking state (CS~5 ps), implying the ICT localizing on the partial donor (azetdiine)--acceptor (carboxy) branch. The falling processes reflected the formation of the charge recombination process and the quenching of the excited state absorption (ESA) process (Table S1). Comparatively, the fs-TA spectra of parent compound 5 (diethyl 2,5-di(azetidine-1-yl)terephthalate) in DMSO displayed similarly spectral characteristics and photoinduced excited-state dynamics ( Figure S5). These results remarkably revealed that the ICT state can also be formed for these symmetric push-pull single-benzene chromophores based on the view of excited-state dynamics.

Transient Absorption Spectroscopy
To obtain a full understanding of the excited state dynamics, femtosecond transient absorption (fs-TA) measurements of DAPA were carried out in DMF, DMSO, EtOH, and MeOH upon an excitation at fs-420 nm. fs-TA spectra recorded at less than 0.5 ps delay time were characterized by a positive excited state absorption (ESA) band in the 470-850 nm region and accompanied by three peaks at 495, 610, and 790 nm (Figures 3a,c and S3a-c). As mentioned in previous reports, highly symmetric push-pull chromophores, such as DAPA, could be recognized as quadrupolar molecules and the solvent dependence of their fluorescence is usually related to the formation of an intramolecular charge transfer (ICT) state [51][52][53][54][55]. In our opinion, the Frank-Condon S1 state of DAPA could be rapidly relaxed to a slight symmetry-breaking state upon light excitation following the solvent relaxation, and then the slight charge separation may occur and the charge localization become partially asymmetrical in polar media [56,57]. To provide an overview of excitedstate dynamics and charge transfer properties of the fluorophores, the decay curves in fs-TA spectra and multi-exponential fitting kinetic traces probed at different wavelengths were compared as shown in Figures 3b and S4a,b. The corresponding lifetimes of transient states are summarized in Table S1. Based on these spectral traces and time constants, a corresponding sequential model for global fitting was proposed as shown in Figure 3d. The first decay lifetime may reflect the time-constant of solvent relaxation (τ SR < 1 ps). The second one is associated with the formation of the charge separation state and symmetrybreaking state (τ CS~5 ps), implying the ICT localizing on the partial donor (azetdiine)π-acceptor (carboxy) branch. The falling processes reflected the formation of the charge recombination process and the quenching of the excited state absorption (ESA) process (Table S1). Comparatively, the fs-TA spectra of parent compound 5 (diethyl 2,5-di(azetidine-1-yl)terephthalate) in DMSO displayed similarly spectral characteristics and photoinduced excited-state dynamics ( Figure S5). These results remarkably revealed that the ICT state can also be formed for these symmetric push-pull single-benzene chromophores based on the view of excited-state dynamics.

pH-Dependent Fluorescence
As shown in Figure S11, in the NMR spectrum for DAPA, there were two split peaks at 3.72 and 3.25 ppm assigned to the proton (-NCH2-) close to the N atom. Based on our understanding, this special splitting should be originated from the formed hydrogen bonding between the carboxyl-H and N atom, thus resulting in the asymmetry or twist of the azetidine ring. It hints that the azetidine ring is pH-responsive. Spectroscopic properties of DAPA and DAP-Na were also examined under the pH range of 4.0-8.6 ( Figure S6). In Britton-Robinson buffer at pH~4.0, DAPA showed weak emission at 591 nm. When the pH increased from 6.0 to 8.0, the fluorescence intensity enhanced significantly along with a blue-shifted emission peak. It is anticipated that, with the increase of alkalinity, the acidic carboxyl group on DAPA was easily deprotonated and converted into the corresponding carboxylate. Based on the pH titration results, the pKa value of DAPA was estimated to be 6.59. Concomitantly, the absorption maxima of DAPA showed a slight red shift above pH~8.0. DAP-Na also exhibited remarkable pH-dependent fluorescence emission properties. The fluorescence emission of DAP-Na was quenched dramatically as the pH decreased from 8.6 to 7.0 ( Figure S6). Particularly, the fluorescence intensity was quite low when pH < 6.0, concomitantly with a red-shifted emission maximum. The pH investigations clearly demonstrate that these two single-benzene chromophores can be used as superiorly base-and acid-responsive fluorescent probes in aqueous media.

pH-Dependent Fluorescence
As shown in Figure S11, in the NMR spectrum for DAPA, there were two split peaks at 3.72 and 3.25 ppm assigned to the proton (-NCH 2 -) close to the N atom. Based on our understanding, this special splitting should be originated from the formed hydrogen bonding between the carboxyl-H and N atom, thus resulting in the asymmetry or twist of the azetidine ring. It hints that the azetidine ring is pH-responsive. Spectroscopic properties of DAPA and DAP-Na were also examined under the pH range of 4.0-8.6 ( Figure S6). In Britton-Robinson buffer at pH~4.0, DAPA showed weak emission at 591 nm. When the pH increased from 6.0 to 8.0, the fluorescence intensity enhanced significantly along with a blue-shifted emission peak. It is anticipated that, with the increase of alkalinity, the acidic carboxyl group on DAPA was easily deprotonated and converted into the corresponding carboxylate. Based on the pH titration results, the pKa value of DAPA was estimated to be 6.59. Concomitantly, the absorption maxima of DAPA showed a slight red shift above pH~8.0. DAP-Na also exhibited remarkable pH-dependent fluorescence emission properties. The fluorescence emission of DAP-Na was quenched dramatically as the pH decreased from 8.6 to 7.0 ( Figure S6). Particularly, the fluorescence intensity was quite low when pH < 6.0, concomitantly with a red-shifted emission maximum. The pH investigations clearly demonstrate that these two single-benzene chromophores can be used as superiorly base-and acid-responsive fluorescent probes in aqueous media.

Sensitive and Discriminative Detection of Amino Acids
Next, to inspect the practical applications of these two highly pH-sensitive chromophores, the fluorescent sensing performances of DAPA and DAP-Na towards 20 natural amino acids with different pI values were examined (pI values of used amino acids: Arg, 10 [58,59]. As shown in Figure 4a, DAPA underwent a significant intensity increase along with~30 nm blue-shifted emission in the presence of basic amino acids (100 µM, Arg and Lys). Addition of other natural amino acids induced negligible responses, although His induced a little emission enhancement in comparison with that of Arg or Lys. The fluorescence enhancement of DAPA upon addition of different amino acids revealed high selectivity to Arg and Lys (Figure 4b). Moreover, a visual fluorescence color change of the aqueous solution from dark red to bright yellow upon addition of Arg or Lys was clearly identified under UV lamp (λ ex = 365 nm). As depicted in Figure S7a, the selectivity of DAP-Na for Asp and Glu detection was also evaluated. When adding 20 natural amino acids to aqueous DAP-Na, only Asp and Glu produced dramatical fluorescence quenching as well as a 20 nm red shift. While, basic amino acids (Arg, Lys, and His) induced a slight emission enhancement of DAP-Na and all the other amino acids caused few changes. The fluorescence variation of DAP-Na to various amino acids also revealed the strong identifying capability to Asp and Glu ( Figure S7b).

Sensitive and Discriminative Detection of Amino Acids
Next, to inspect the practical applications of these two highly pH-sensitive chromophores, the fluorescent sensing performances of DAPA and DAP-Na towards 20 natural amino acids with different pI values were examined (pI values of used amino acids: Arg, 10 [58,59]. As shown in Figure 4a, DAPA underwent a significant intensity increase along with ~30 nm blue-shifted emission in the presence of basic amino acids (100 M, Arg and Lys). Addition of other natural amino acids induced negligible responses, although His induced a little emission enhancement in comparison with that of Arg or Lys. The fluorescence enhancement of DAPA upon addition of different amino acids revealed high selectivity to Arg and Lys (Figure 4b). Moreover, a visual fluorescence color change of the aqueous solution from dark red to bright yellow upon addition of Arg or Lys was clearly identified under UV lamp (ex = 365 nm). As depicted in Figure S7a, the selectivity of DAP-Na for Asp and Glu detection was also evaluated. When adding 20 natural amino acids to aqueous DAP-Na, only Asp and Glu produced dramatical fluorescence quenching as well as a 20 nm red shift. While, basic amino acids (Arg, Lys, and His) induced a slight emission enhancement of DAP-Na and all the other amino acids caused few changes. The fluorescence variation of DAP-Na to various amino acids also revealed the strong identifying capability to Asp and Glu ( Figure S7b). Present single-benzene-based probes of DAPA and DAP-Na exhibited highly selective responses to basic (Arg and Lys) and acidic (Asp and Glu) amino acids, respectively. It is worthy to further appraise the anti-interference ability of the probes in   The competitive experiments demonstrated that most of these natural amino acids did not significantly induce the fluorescent sensing of DAPA towards Arg or Lys. Similarly, we also verified the co-presence of other natural amino acids did not obviously influence the fluorescent detection of DAP-Na towards Asp or Glu ( Figure S7c,d).
Sensitivity is another crucial factor for determining the practical usability of the fluorescent probes. Accordingly, the sensing performances of DAPA for Arg and Lys were systematically studied. Figures 5a and S8a demonstrated that the fluorescence dependence of DAPA on Arg and Lys ranged from 2 to 200 µM, respectively. As depicted, with incremental addition of Arg or Lys to aqueous DAPA solution, the fluorescence intensity was gradually enhanced (turn-on) and accompanied by the emission peak shift from 591 to 558 nm (∆λ = 33 nm). Figure S9a,b represent the corresponding plots of the fluorescence intensity rations (I/I 0 ) at 558 nm versus the concentrations of Arg and Lys. A relatively linear correlation was observed over a concentration range from 12 to 100 µM for both amino acids, and then the plot reached a plateau as the concentration of amino acids above 140 µM. To quantitatively evaluate the sensitivity of DAPA, the detection limits (DLs) were investigated using the standard IUPAC 3δ method [60]. The DL values for Arg and Lys were calculated to be 0.50 µM and 0.41 µM, respectively. In contrast, the fluorescence quenching (turn-off) responses of DAP-Na with respect to Asp and Glu were also carefully investigated (Figure 5b, Figures S8b and S9c,d). Related DL values were found to be 0.12 µM for Asp and 0.16 µM for Glu, respectively. Compared with recently-reported fluorescence probes, the structures of DAPA and DAP-Na are the simplest but display efficient sensing performances to different amino acids in aqueous media (Table S2).
the real-life applications. Therefore, a series of competitive experiments were conducted (Figures 4c,d and S7c,d). When Arg or Lys was added to the aqueous solution of DAPA with the co-existing interfering amino acids, 16 among 18 kinds of amino acids show no obvious effect. Asp and Glu show little interference because the released H + might weaken the basicity of Arg or Lys, thus reducing the positive response of DAPA to Arg or Lys. The competitive experiments demonstrated that most of these natural amino acids did not significantly induce the fluorescent sensing of DAPA towards Arg or Lys. Similarly, we also verified the co-presence of other natural amino acids did not obviously influence the fluorescent detection of DAP-Na towards Asp or Glu ( Figure S7c,d).
Sensitivity is another crucial factor for determining the practical usability of the fluorescent probes. Accordingly, the sensing performances of DAPA for Arg and Lys were systematically studied. Figures 5a and S8a demonstrated that the fluorescence dependence of DAPA on Arg and Lys ranged from 2 to 200 μM, respectively. As depicted, with incremental addition of Arg or Lys to aqueous DAPA solution, the fluorescence intensity was gradually enhanced (turn-on) and accompanied by the emission peak shift from 591 to 558 nm (Δ = 33 nm). Figure S9a,b represent the corresponding plots of the fluorescence intensity rations (I/I0) at 558 nm versus the concentrations of Arg and Lys. A relatively linear correlation was observed over a concentration range from 12 to 100 μM for both amino acids, and then the plot reached a plateau as the concentration of amino acids above 140 μM. To quantitatively evaluate the sensitivity of DAPA, the detection limits (DLs) were investigated using the standard IUPAC 3δ method [60]. The DL values for Arg and Lys were calculated to be 0.50 μM and 0.41 μM, respectively. In contrast, the fluorescence quenching (turn-off) responses of DAP-Na with respect to Asp and Glu were also carefully investigated (Figures 5b, S8b, and S9c,d). Related DL values were found to be 0.12 μM for Asp and 0.16 μM for Glu, respectively. Compared with recently-reported fluorescence probes, the structures of DAPA and DAP-Na are the simplest but display efficient sensing performances to different amino acids in aqueous media (Table S2). Aiming to better understand the sensing mechanism, 1 H NMR spectra were evaluated to study the interactions between the probes and the amino acids. As depicted in Figure 6, the signals of aromatic protons (Ha) in DAPA were assigned to 7.46 ppm. After adding Arg, the aromatic protons shifted up-field obviously and the chemical shifts changed up to 0.33 ppm. Such results suggest DAPA was easily converted into the species of carboxylate anion through the deprotonation of basic Arg, thus increasing the electron cloud density on the benzene ring of DAPA [61]. Upon addition of Asp into the Aiming to better understand the sensing mechanism, 1 H NMR spectra were evaluated to study the interactions between the probes and the amino acids. As depicted in Figure 6, the signals of aromatic protons (H a ) in DAPA were assigned to 7.46 ppm. After adding Arg, the aromatic protons shifted up-field obviously and the chemical shifts changed up to 0.33 ppm. Such results suggest DAPA was easily converted into the species of carboxylate anion through the deprotonation of basic Arg, thus increasing the electron cloud density on the benzene ring of DAPA [61]. Upon addition of Asp into the D 2 O solution of DAP-Na, all the protons assigned to DAP-Na shifted downfield ( Figure S10). Specifically, the downfield shift magnitude of the aromatic proton signal (H a ) was close to 1.12 ppm, and that of methylene (H b ) attached to the nitrogen atom was close to 0.53 ppm. Such extraordinary shifts suggest that the Lewis-basic nitrogen of azetidine is most probably protonated by Asp, explaining why DAP-Na exhibited dramatically quenching phenomena toward acidic amino acids [45].
D2O solution of DAP-Na, all the protons assigned to DAP-Na shifted downfield ( Figure  S10). Specifically, the downfield shift magnitude of the aromatic proton signal (Ha′) was close to 1.12 ppm, and that of methylene (Hb′) attached to the nitrogen atom was close to 0.53 ppm. Such extraordinary shifts suggest that the Lewis-basic nitrogen of azetidine is most probably protonated by Asp, explaining why DAP-Na exhibited dramatically quenching phenomena toward acidic amino acids [45].
NMR spectra of the synthesized compounds were recorded on a Bruker 600 MHz spectrometer and their high-resolution mass spectra were determined by a Bruker maxis UHR-TOF mass spectrometer. UV-vis absorption spectra were measured on a Hitachi U-3900 spectrophotometer. Fluorescence measurements were performed on a time-correlated single-photon-counting FLS920 fluorescence spectrometer from Edinburgh Instruments. Absolute fluorescence quantum yields (f) were measured on the Hamamatsu C9920-02G quantum efficiency measurer.
The femtosecond transient absorption (fs-TA) setup adopted in the present work was based on a PHAROS laser system from Light Conversion (1030 nm, ~200 fs, 200 μJ/pulse, and 100 kHz repetition rate), nonlinear frequency mixing techniques, and the Femto-TA100 spectrometer (Time-Tech Spectra) [62,63]. Briefly, the 1030 nm output pulse from the regenerative amplifier was split in two parts with an 80% beam splitter.
NMR spectra of the synthesized compounds were recorded on a Bruker 600 MHz spectrometer and their high-resolution mass spectra were determined by a Bruker maxis UHR-TOF mass spectrometer. UV-vis absorption spectra were measured on a Hitachi U-3900 spectrophotometer. Fluorescence measurements were performed on a time-correlated single-photon-counting FLS920 fluorescence spectrometer from Edinburgh Instruments. Absolute fluorescence quantum yields (Φ f ) were measured on the Hamamatsu C9920-02G quantum efficiency measurer.
The femtosecond transient absorption (fs-TA) setup adopted in the present work was based on a PHAROS laser system from Light Conversion (1030 nm,~200 fs, 200 µJ/pulse, and 100 kHz repetition rate), nonlinear frequency mixing techniques, and the Femto-TA100 spectrometer (Time-Tech Spectra) [62,63]. Briefly, the 1030 nm output pulse from the regenerative amplifier was split in two parts with an 80% beam splitter. The reflected part was used to pump an ORPHEUS Optical Parametric Amplifier (OPA) which generates a wavelength-tunable laser pulse from 300 nm to 15 µm. Here, 420 nm was used as the pump beam. The transmitted 1030 nm beam was split again into two parts. One part with less than 50% was attenuated with a neutral density filter and focused into a YAG window to generate a white light continuum from 500 to 1600 nm used for probe beam. The probe beam was focused with an Ag parabolic reflector onto the sample. After the sample, the probe beam was collimated and then focused into a fiber-coupled spectrometer with CMOS sensors and detected at a frequency of 10 kHz. The intensity of the pump pulse was controlled by a variable neutral-density filter wheel. The delay between the pump and probe pulses was controlled by a motorized delay stage. The pump pulses were chopped by a synchronized chopper at 5 kHz and the absorbance change was calculated with two adjacent probe pulses (pump-blocked and pump-unblocked).
Kinetic modeling: Kinetic modeling was carried out via target analysis on a composite data set of the fs-TA spectra in order to capture the complete dynamics. Using target analysis, the entire TA data set was fitted over the whole wavelength region and all the time delays with the application of a kinetic model. In this work, CarpetView (version 1.1.10) software was used for kinetic modelling of the transient absorption data (www.lightcon. com, accessed on 21 March 2022).

Synthesis of Compound DAP-Na
Diethyl 2,5-di(azetidin-1-yl) terephthalate (0.065 g, 0.20 mmol) was dissolved in THF (1 mL) and methanol (3 mL), followed by addition of 1 M NaOH (aq. 0.39 mL). The reaction mixture was heated up to 80 • C and stirred for 18 h. The reaction mixture was then cooled to room temperature and the precipitate was isolated by centrifugation, rinsed with CH 2 Cl 2 , and dried under reduced pressure to afford DAP-Na as a white solid (0.035 g, 56%). 1

Conclusions
In summary, we developed two compact single-benzene-based chromophores, DAPA and its disodium carboxylate DAP-Na, which displayed favorable properties including good water solubility, large Stokes shift, intense luminescence in most polar and protonic solvents, and highly pH-responsive characteristics. Fs-TA spectra illustrated that quadrupolar DAPA usually involved the formation of an ICT state. The Frank-Condon state could be rapidly relaxed to a slight symmetry-breaking state upon light excitation following the solvent relaxation, then the slight charge separation may occur and the charge localization become partially asymmetrical in polar environment. DFT theoretical calculation results support with the experimental measurements well. Given these advantages, these two fluorescent probes were capable of rapid, highly selective, and sensitive detection of amino acids in aqueous media. DAPA can be successfully used in sensing Arg and Lys via fluorescence turn-on response and displayed DL values of 0.50 µM for Arg and 0.41 µM for Lys, respectively. In contrast, DAP-Na demonstrated a remarkable fluorescence quenching response toward Asp and Glu and the DLs were calculated to be 0.12 and 0.16 µM, respectively. The present work provides two structurally simple probes that help to understand their photophysic-structure relationship and render their fluorescent detection applications.

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/molecules27175522/s1. Figure S1. Normalized fluorescence excitation and emission spectra of DAPA (red lines) and DAP-Na (green lines) in water solution (c = 20.0 µM). Figure S2. (a) UV−vis absorption and (b) normalized fluorescence emission spectra of DAP-Na in various solvents (λ ex = 350 nm; c = 20.0 µM). Figure S3. Femtosecond transient absorption spectra of DAPA in different solvents after excitation at fs-420 nm. Figure S4. Experimental decay curves of DAPA in transient absorption and their fitting kinetic traces probed at 610 nm and 790 nm in different solvents. Figure S5.   Figure S10. Partial 1 H NMR spectra of DAP-Na upon addition of Asp in D 2 O. Figure S11. 1 H NMR spectrum of DAPA in d 6 -DMSO. Figure S12. 13 C NMR spectrum of DAPA in d 6 -DMSO. Figure S13. 1 H NMR spectrum of DAP-Na in D 2 O. Figure S14. 13 C NMR spectrum of DAP-Na in D 2 O. Figure S15. ESI-HRMS spectrum for DAPA. Figure S16. ESI-HRMS spectrum for DAP-Na. Table S1. Time constants of multiple exponential fitting of femtosecond TA data of different system, with relative amplitudes given for DAPA and 5 in different solvents. Table S2. Performance comparison of DAPA and DAP-Na for detection of different amino acids with the recently reported probes [32,33,[64][65][66][67]. Detection limits of DAPA to basic amino acids (Arg and Lys) and DAP-Na to acidic amino acids (Asp and Glu) were determined based on the fluorescence titration.