Comparison of Reactive Sites in 2(1H)-Quinolone Derivatives for the Detection of Biologically Important Sulfur Compounds

Novel fluorescent probes based on 2(1H)-quinolone skeleton containing a malonate group (Q1–Q3) were synthesized and proposed for biothiols detection. Their chemical reactivity toward thiols was compared to the reactivity of derivative having a dicyanovinyl group (Q4) as a reactive site. The detailed photophysical properties of these compounds were assessed through the determination of absorption and fluorescence spectra, fluorescence quantum yield, and fluorescence lifetime. In the presence of biothiols, an increase in the fluorescence intensity of compounds Q1–Q3 and a hypsochromic shift in their emission bands were observed. In contrast, the compound with the dicyanovinyl group (Q4) in the presence of biothiols and cyanide ion showed the quenching of fluorescence, while a fluorescence “turn on” effect was observed toward reactive sulfur species.


Introduction
In recent years, the interest in research on the biological role of sulfur species has increased significantly due to the importance of thiol compounds in both physiological and pathological processes [1][2][3][4]. Thiols that play an essential role in the biochemical processes of the cells include low-molecular compounds such as L-cysteine (L-Cys), L-glutathione (L-GSH), and N-acetyl-L-cysteine (L-ACC). The presence of a sulfhydryl group in thiol compounds affects their antioxidative properties as well as their high reactivity and affinity for heavy metals [5][6][7].
Peroxidative damage associated with the industrialization of the environment has been increasingly implicated as a cause of the rising incidence of cardiovascular disease, cancer, and neurodegenerative disorders [8,9]. For this reason, particular attention has been paid to the involvement of individual thiols in the detoxification processes of reactive oxygen species and electrophilic xenobiotics [10]. Changes in the levels of individual thiols in human plasma are observed in various pathological states. The change in the concentration and redox status of one of the thiols significantly affects the concentrations and the redox status of the remaining thiols. This can influence the activity of enzymes and the biological functions of the receptors, structural proteins, and antioxidant defenses. Therefore, it is crucial to determine the thiol redox status in physiological processes.
L-Cys belongs to amino acids containing the sulfhydryl group, which can be an active site in proteins and enzymes, for example, thiol proteinases. Despite its physiological neurotoxicity, L-Cys is necessary [11,12]. In cells, L-Cys is present at a very low level, while in the extracellular space, it is present mainly in the form of L-cystine [13]. Compared to the thiol tripeptide L-GSH, L-Cys is a less effective antioxidant and a much more dangerous pro-oxidant. The possibility of complex binding of metal ions confirms the antioxidant activity of L-Cys [14].
L-GSH is one of the most important antioxidants in the human body, which is responsible for oxidative homeostasis in cells [15][16][17]. The presence of L-GSH is necessary in cell division, in the regulation of intracellular metabolism and apoptosis [18,19]. Many factors affect L-GSH reduction. The most common are stress, incorrect diet, environmental factors, infections, genetic defects, and metabolic disorders. Low L-GSH level leads to various pathological states [20].
L-ACC, the N-acetylated precursor of L-Cys, is an endogenous sulfuric amino acid that occurs naturally in the human body. L-ACC has strong antioxidant properties [21]. As a result, L-ACC is a popular compound with mucolytic properties, commonly used in treating upper respiratory illnesses and other disorders [22,23]. The presence of sulfhydryl groups makes this compound an excellent candidate for L-GSH reduction and it is capable of binding transition metal ions and heavy metal ions. In addition, L-ACC also exhibits the ability to directly scavenge free radicals, e.g., hydroxyl radicals (•OH) [24,25].
The current literature is overflowing with fluorescent probes for detecting biothiols that work via different reaction mechanisms. Among the most important mechanisms are the Michael addition, cyclization with aldehyde, and other reactions [26][27][28][29]. The introduction of appropriate electron-withdrawing groups to the chromophore system changes the properties of the fluorescent probe. The presence of a dicyanovinyl moiety in the structure of the probe causes a redshift in the emission band and increases the value of Stokes shift. A number of fluorophores containing a dicyanovinyl moiety have been described in the literature as fluorescent probes for the detection of various nucleophiles, including biothiols [30,31], reactive sulfur species (RSS) [32], and cyanide anion [33]. Another example of an electron-withdrawing group is the malonate moiety, which can quench the fluorescence of fluorophore. It is a more selective moiety because it is only applicable as the recognition site for the detection of biothiols [34][35][36][37].
The 2(1H)-quinolone fluorophore was selected because of its desirable photophysical properties [38]. In addition, quinolones are important compounds due to their wide range of biological activities [39], and they can be easily modified via alkylation at the amide nitrogen, making them suitable as fluorescent probes [40][41][42].
In our previous studies, we have synthesized, characterized, and used the derivatives of 2(1H)-quinolone with dicyanovinyl group as fluorescent sensors for thiols [30,31] and cyanide anion [33]. In the presence of the studied analyte, these compounds exhibit fluorescence quenching (on-off) as the detection signal.
Herein, we synthesized and characterized the fluorescent probes based on a 2(1H)quinolone skeleton with two different reactive moieties. Their reactivity toward biothiols was compared. These compounds contain a malonate moiety (Q1-Q3) and a dicyanovinyl group (Q4) acting as an acceptor. The structures of the studied compounds are shown in Scheme 1. L-GSH is one of the most important antioxidants in the human body, which is responsible for oxidative homeostasis in cells [15][16][17]. The presence of L-GSH is necessary in cell division, in the regulation of intracellular metabolism and apoptosis [18,19]. Many factors affect L-GSH reduction. The most common are stress, incorrect diet, environmental factors, infections, genetic defects, and metabolic disorders. Low L-GSH level leads to various pathological states [20].
L-ACC, the N-acetylated precursor of L-Cys, is an endogenous sulfuric amino acid that occurs naturally in the human body. L-ACC has strong antioxidant properties [21]. As a result, L-ACC is a popular compound with mucolytic properties, commonly used in treating upper respiratory illnesses and other disorders [22,23]. The presence of sulfhydryl groups makes this compound an excellent candidate for L-GSH reduction and it is capable of binding transition metal ions and heavy metal ions. In addition, L-ACC also exhibits the ability to directly scavenge free radicals, e.g., hydroxyl radicals (•OH) [24,25].
The current literature is overflowing with fluorescent probes for detecting biothiols that work via different reaction mechanisms. Among the most important mechanisms are the Michael addition, cyclization with aldehyde, and other reactions [26][27][28][29]. The introduction of appropriate electron-withdrawing groups to the chromophore system changes the properties of the fluorescent probe. The presence of a dicyanovinyl moiety in the structure of the probe causes a redshift in the emission band and increases the value of Stokes shift. A number of fluorophores containing a dicyanovinyl moiety have been described in the literature as fluorescent probes for the detection of various nucleophiles, including biothiols [30,31], reactive sulfur species (RSS) [32], and cyanide anion [33]. Another example of an electron-withdrawing group is the malonate moiety, which can quench the fluorescence of fluorophore. It is a more selective moiety because it is only applicable as the recognition site for the detection of biothiols [34][35][36][37].
The 2(1H)-quinolone fluorophore was selected because of its desirable photophysical properties [38]. In addition, quinolones are important compounds due to their wide range of biological activities [39], and they can be easily modified via alkylation at the amide nitrogen, making them suitable as fluorescent probes [40][41][42].
In our previous studies, we have synthesized, characterized, and used the derivatives of 2(1H)-quinolone with dicyanovinyl group as fluorescent sensors for thiols [30,31] and cyanide anion [33]. In the presence of the studied analyte, these compounds exhibit fluorescence quenching (on-off) as the detection signal.
Herein, we synthesized and characterized the fluorescent probes based on a 2(1H)quinolone skeleton with two different reactive moieties. Their reactivity toward biothiols was compared. These compounds contain a malonate moiety (Q1-Q3) and a dicyanovinyl group (Q4) acting as an acceptor. The structures of the studied compounds are shown in Scheme 1. Scheme 1. The chemical structure of studied compounds Q1-Q4.

Synthesis and Characterization of Q1-Q3
The novel derivatives of 2(1H)-quinolone, Q1-Q3, were obtained as results of the presented syntheses in Scheme 2. The appropriate three formyl derivatives of 2(1H)-quinolone (1-3) were earlier described in the literature [31,32]. They were condensed with diethyl malonate (DEM) in the presence of piperidine as a catalyst in anhydrous ethanol, yielding the compounds Q1-Q3. The chemical structures of Q1-Q3 were confirmed via proton nuclear magnetic resonance ( 1 H NMR) and electrospray ionization mass Scheme 1. The chemical structure of studied compounds Q1-Q4.

Synthesis and Characterization of Q1-Q3
The novel derivatives of 2(1H)-quinolone, Q1-Q3, were obtained as results of the presented syntheses in Scheme 2. The appropriate three formyl derivatives of 2(1H)-quinolone (1-3) were earlier described in the literature [31,32]. They were condensed with diethyl malonate (DEM) in the presence of piperidine as a catalyst in anhydrous ethanol, yielding the compounds Q1-Q3. The chemical structures of Q1-Q3 were confirmed via proton nuclear magnetic resonance ( 1 H NMR) and electrospray ionization mass spectrometry (ESI MS) (Figures S1-S6). The obtained data agreed with the established structure of the molecules. The photophysical and spectroscopic properties of the novel compounds Q1-Q3 were determined based on the recorded absorption and emission spectra. Detailed charac-teristics of the obtained compounds Q1-Q3 are included in Table 1. Moreover, electronic absorption and emission spectra of Q1-Q3 compounds are provided in Figure 1.
Molecules 2023, 28, x FOR PEER REVIEW 3 of 15 spectrometry (ESI MS) ( Figures S1-S6). The obtained data agreed with the established structure of the molecules. The photophysical and spectroscopic properties of the novel compounds Q1-Q3 were determined based on the recorded absorption and emission spectra. Detailed characteristics of the obtained compounds Q1-Q3 are included in Table  1. Moreover, electronic absorption and emission spectra of Q1-Q3 compounds are provided in Figure 1.   Compounds Q1-Q3 exhibit two absorption bands located in the ultraviolet and visible ranges. Their absorption maximum (λabs) is located in the range of 366-402 nm, while the emission maximum (λem) is in the range of 460-600 nm. The presence of the methoxy substituent at position 6 in the quinolone skeleton significantly affects the spectroscopic properties of Q3. This compound has a huge shift in λem and λabs toward longer   Figures S1-S6). The obtained data agreed with the established structure of the molecules. The photophysical and spectroscopic properties of the novel compounds Q1-Q3 were determined based on the recorded absorption and emission spectra. Detailed characteristics of the obtained compounds Q1-Q3 are included in Table  1. Moreover, electronic absorption and emission spectra of Q1-Q3 compounds are provided in Figure 1.   Compounds Q1-Q3 exhibit two absorption bands located in the ultraviolet and visible ranges. Their absorption maximum (λabs) is located in the range of 366-402 nm, while the emission maximum (λem) is in the range of 460-600 nm. The presence of the methoxy substituent at position 6 in the quinolone skeleton significantly affects the spectroscopic properties of Q3. This compound has a huge shift in λem and λabs toward longer Compounds Q1-Q3 exhibit two absorption bands located in the ultraviolet and visible ranges. Their absorption maximum (λ abs ) is located in the range of 366-402 nm, while the emission maximum (λ em ) is in the range of 460-600 nm. The presence of the methoxy substituent at position 6 in the quinolone skeleton significantly affects the spectroscopic properties of Q3. This compound has a huge shift in λ em and λ abs toward longer wavelengths compared to the unsubstituted derivative (Q1). Moreover, a small bathochromic effect is also observed for the methoxy derivative (Q2). Q2 possesses the highest molar extinction coefficient value compared with other derivatives (Q1 and Q3). Otherwise, the derivative Q3 is characterized by a Stokes shift above 100 nm, suggesting that the ground and excited state geometry is different. In comparison, the values below 100 nm indicate that the geometry of these two states are similar (Q1 and Q2). Additionally, the fluorescence quantum yield (Φ em ) and the lifetime of the singlet excited state (τ) of compound Q3 are also higher than Φ em and τ of derivatives Q1 and Q2, whereas the introduction of a dicyanovinyl group into the quinolone structure (Q4) causes an even larger bathochromic shift (λ em and λ abs ) and the highest values for Φ em and τ.
Our earlier paper [30,31] has shown that the 2(1H)-quinolone derivatives with a dicyanovinyl moiety are unstable in an aqueous medium and probably undergo hydrolysis. To compare the stability of Q3 with Q4, their absorption and emission spectra were recorded in a 0.1 M phosphate buffer with CH 3 CN (20%, v/v, pH = 7.4) over 36 min. As shown in Figure 2, compound Q3 is stable under these conditions, in contrast to the derivative Q4. It is evident that a compound with a malonate moiety (Q3) will be more suitable for potential use as a sensor for detecting biothiols.
Molecules 2023, 28, x FOR PEER REVIEW 4 of 15 wavelengths compared to the unsubstituted derivative (Q1). Moreover, a small bathochromic effect is also observed for the methoxy derivative (Q2). Q2 possesses the highest molar extinction coefficient value compared with other derivatives (Q1 and Q3). Otherwise, the derivative Q3 is characterized by a Stokes shift above 100 nm, suggesting that the ground and excited state geometry is different. In comparison, the values below 100 nm indicate that the geometry of these two states are similar (Q1 and Q2). Additionally, the fluorescence quantum yield (Φem) and the lifetime of the singlet excited state (τ) of compound Q3 are also higher than Φem and τ of derivatives Q1 and Q2, whereas the introduction of a dicyanovinyl group into the quinolone structure (Q4) causes an even larger bathochromic shift (λem and λabs) and the highest values for Φem and τ.
Our earlier paper [30,31] has shown that the 2(1H)-quinolone derivatives with a dicyanovinyl moiety are unstable in an aqueous medium and probably undergo hydrolysis. To compare the stability of Q3 with Q4, their absorption and emission spectra were recorded in a 0.1 M phosphate buffer with CH3CN (20%, v/v, pH = 7.4) over 36 min. As shown in Figure 2, compound Q3 is stable under these conditions, in contrast to the derivative Q4. It is evident that a compound with a malonate moiety (Q3) will be more suitable for potential use as a sensor for detecting biothiols. Because of the observed difference in fluorescence of compounds with a malonate moiety (Q1-Q3) and the compound containing dicyanovinyl moiety (Q4), further investigations were carried out to explain the quenching mechanism acting in these compounds. The fluorescence decay profile of compounds Q1-Q3 and Q4 are presented in Figure S7 in acetonitrile with an excitation at 376.2 nm. Quantum yield of fluorescence (Φem) and fluorescence lifetime (τ) are determined by the ratio between the rate constants of radiative (kr) and non-radiative (knr) intramolecular decay processes [43,44]. From the presented data in Table S1, it is clear that the increase in fluorescence lifetime is due to a Because of the observed difference in fluorescence of compounds with a malonate moiety (Q1-Q3) and the compound containing dicyanovinyl moiety (Q4), further investigations were carried out to explain the quenching mechanism acting in these compounds. The fluorescence decay profile of compounds Q1-Q3 and Q4 are presented in Figure S7 in acetonitrile with an excitation at 376.2 nm. Quantum yield of fluorescence (Φ em ) and fluorescence lifetime (τ) are determined by the ratio between the rate constants of radiative (k r ) and non-radiative (k nr ) intramolecular decay processes [43,44]. From the presented data in Table S1, it is clear that the increase in fluorescence lifetime is due to a decrease in non-radiative decay. The k r /k nr = 0.1025 obtained for compound Q4 compared to the k r /k nr in the range 0.0019-0.0245 for compounds Q1-Q3, indicates that a radiative pathway, which is responsible for fluorescence, is more favorable in Q4. Malonate moiety in the structure of 2(1H)-quinolone (Q1-Q3) causes the ICT process and a non-radiative pathway to dominate.

Absorption and Fluorescence Responses of Q1-Q3 toward the Thiols
The reactivity of Q1-Q3 derivatives toward biothiols (L-Cys, L-GSH, and L-ACC), non-thiol amino acids (L-Glu, Gly, and L-Lys), and representative anions, including reactive sulfur species (HSO 3 − , SH − , and S 2− ), and reactive oxygen species (H 2 O 2 ) were tested and the results are shown in Figure 3. The measurements were carried out in a 0.1 M phosphate buffer with CH 3 CN (20%, v/v, and pH = 7.4). The absorption spectra recorded in the presence of various analytes for Q1-Q3 derivatives are presented in Figure 3A-C, and emission spectra are presented in Figure 3D-F, respectively. For compounds Q1-Q3, in the presence of biothiols (L-Cys, L-ACC, and L-GSH), the disappearance of the absorption band in the visible spectrum and the formation of a new band in the near ultraviolet range were observed. The absorption spectra for Q1-Q3 derivatives in the presence of L-Cys and L-GSH over time (0-30 min.) are presented in Figure S8. The location of the maximum of a new absorption band for compounds Q1-Q3 in the presence of biothiols depends on their chemical structure. For compounds Q1 and Q2, the absorption maximum is located at 330 nm, and for Q3 at 354 nm. In the presence of non-thiol amino acids (L-Glu, Gly, L-Lys) and the other analytes, absorption spectra of Q1-Q3 did not display any significant changes. As shown in Figure 3D-F, fluorescence intensity at wavelengths 380, 370, and 415 nm increased in the presence of biothiols for Q1, Q2, and Q3, respectively. The highest increase in fluorescence intensity was recorded for L-Cys, L-GSH, and L-ACC. Moreover, a slight increase in fluorescence was also seen for HSO 3 − and S 2− .  As shown in our earlier publications [30,33], the fluorescence of Q4 was complete quenched in the presence of L-Cys or cyanide anion. Similar changes in the absorptio spectra for all compounds (Q1-Q4) toward biothiols were observed. Here, to study th reactivity of the Q4 probe in more detail, the absorption and emission spectra of Q4 in th As shown in our earlier publications [30,33], the fluorescence of Q4 was completely quenched in the presence of L-Cys or cyanide anion. Similar changes in the absorption spectra for all compounds (Q1-Q4) toward biothiols were observed. Here, to study the reactivity of the Q4 probe in more detail, the absorption and emission spectra of Q4 in the presence of reactive sulfur species (HSO 3 − , SO 3 2− , SH − , and S 2− ) and reactive oxygen species (H 2 O 2 ) were also measured. The analysis of the absorption and emission spectra changes shows that RSS can interact with compound Q4. In their presence, a complete disappearance of the absorption band in the visible range of the spectrum and a formation of a new band with a maximum located in the near ultraviolet range were observed ( Figure 4A). An analogous effect was observed in the case of emission spectra. As can be seen in Figure 4B The dependence of changes in the fluorescence intensity of Q1-Q3 derivatives wit time in the presence of a 10-fold excess of L-Cys and L-GSH in a 0.1 M phosphate buffe with CH3CN (20%, v/v, pH = 7.4) were also carried out, and the results are presented i Figure 5. The results of kinetic study showed that the fluorescence emission of compound Q1-Q3 increased and reached a maximum after about 400 s. Thus, from the experimen conducted, it follows that the fluorescence response of the tested derivatives to biothio is obtained rapidly. The highest value of the rate of increase in the fluorescence signa intensity was observed for Q3 in the presence of L-Cys. Furthermore, competitive selectivity studies of three biothiols with some representa tive amino acids (Gly, L-Glu, and L-Lys) were carried out. As illustrated in Figure 6, all o the compounds, Q1-Q3, demonstrated an ability to selectively detect the thiol group i The dependence of changes in the fluorescence intensity of Q1-Q3 derivatives with time in the presence of a 10-fold excess of L-Cys and L-GSH in a 0.1 M phosphate buffer with CH 3 CN (20%, v/v, pH = 7.4) were also carried out, and the results are presented in Figure 5. The results of kinetic study showed that the fluorescence emission of compounds Q1-Q3 increased and reached a maximum after about 400 s. Thus, from the experiment conducted, it follows that the fluorescence response of the tested derivatives to biothiols is obtained rapidly. The highest value of the rate of increase in the fluorescence signal intensity was observed for Q3 in the presence of L-Cys. The dependence of changes in the fluorescence intensity of Q1-Q3 derivatives wit time in the presence of a 10-fold excess of L-Cys and L-GSH in a 0.1 M phosphate buffe with CH3CN (20%, v/v, pH = 7.4) were also carried out, and the results are presented i Figure 5. The results of kinetic study showed that the fluorescence emission of compound Q1-Q3 increased and reached a maximum after about 400 s. Thus, from the experimen conducted, it follows that the fluorescence response of the tested derivatives to biothiol is obtained rapidly. The highest value of the rate of increase in the fluorescence signa intensity was observed for Q3 in the presence of L-Cys. Furthermore, competitive selectivity studies of three biothiols with some representa tive amino acids (Gly, L-Glu, and L-Lys) were carried out. As illustrated in Figure 6, all o the compounds, Q1-Q3, demonstrated an ability to selectively detect the thiol group i the presence of other biologically relevant nucleophiles, such as the amino groups. Furthermore, competitive selectivity studies of three biothiols with some representative amino acids (Gly, L-Glu, and L-Lys) were carried out. As illustrated in Figure 6, all of the compounds, Q1-Q3, demonstrated an ability to selectively detect the thiol group in the presence of other biologically relevant nucleophiles, such as the amino groups. However, it is worth noting that the selectivity of Q2 and Q3 is sign probably due to the presence of an electron-donating group in the qui The pH value significantly influences the spectroscopic propert response of the chemosensors in the presence of analytes. Therefore, th the fluorescence intensity of compounds Q1-Q3 relative to L-Cys, L-G solutions of different pH (3.1-10.5) was studied (Figures 7A-C). In the ols, the compounds Q1-Q3 displayed minimal emission over all ana 508 nm, 468 nm, and 600 nm, respectively, indicating good stability of t the broad pH range. The fluorescence intensities of Q1-Q3 in the pr were pH dependent. Compounds Q1-Q3 at a pH 3.1-5.0 show neglig the presence of biothiols, while the highest fluorescence intensity after observed at pH 7.4. However, above this value, the fluorescence inte creased. This proves that the most active form of thiols is the nucleop anion). This result implies that derivatives Q1-Q3 can potentially det physiological conditions (at a pH of 7.4). For comparison, the impact rescence intensity of compound with a dicyanovinyl group, Q4, on the is presented in Figure 7D. The compound Q4, in contrast to the Q showed a decrease in the fluorescence intensity at a pH above 6.5. Add ence of pH value on the detection of HSO3 − via Q4 was investigated However, it is worth noting that the selectivity of Q2 and Q3 is significantly increased probably due to the presence of an electron-donating group in the quinolone structure.

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The pH value significantly influences the spectroscopic properties and the optical response of the chemosensors in the presence of analytes. Therefore, the impact of pH on the fluorescence intensity of compounds Q1-Q3 relative to L-Cys, L-GSH, and L-ACC in solutions of different pH (3.1-10.5) was studied ( Figure 7A-C). In the absence of biothiols, the compounds Q1-Q3 displayed minimal emission over all analyzed pH range at 508 nm, 468 nm, and 600 nm, respectively, indicating good stability of these derivatives in the broad pH range. The fluorescence intensities of Q1-Q3 in the presence of biothiols were pH dependent. Compounds Q1-Q3 at a pH 3.1-5.0 show negligible fluorescence in the presence of biothiols, while the highest fluorescence intensity after adding thiols was observed at pH 7.4. However, above this value, the fluorescence intensity gradually decreased. This proves that the most active form of thiols is the nucleophilic form (thiolate anion). This result implies that derivatives Q1-Q3 can potentially detect biothiols under physiological conditions (at a pH of 7.4). For comparison, the impact of pH on the fluorescence intensity of compound with a dicyanovinyl group, Q4, on the detection of L-Cys is presented in Figure 7D. The compound Q4, in contrast to the Q1-Q3 derivatives, showed a decrease in the fluorescence intensity at a pH above 6.5. Additionally, the influence of pH value on the detection of HSO 3 − via Q4 was investigated. As can be seen in Figure S9, an increase in the fluorescence intensity after adding HSO 3 − in the pH range from 2.6 to 7.5 is observed. This test showed that the Q4 derivative is most suitable for detecting this reactive sulfur form in a wide pH range. We propose that the reaction of compounds Q1-Q3 and sulfhydryl compounds proceeds via nucleophilic Michael addition to the α,β-unsaturated double bond. 2-Mercaptoethanol (ME) was selected as the sulfhydryl compound for the study confirming the proposed reaction mechanism (Scheme 3). The product of the reaction of ME with compound Q3 was confirmed via proton nuclear magnetic resonance ( 1 H NMR) spectroscopy. In the aromatic part of the spectrum, the signal corresponding to the double bond in the malonate moiety of compound Q3 disappeared, and two new signals at 2.74 and 4.72 ppm appeared in the aliphatic part ( Figure S10). Moreover, the product of the reaction of Q3 and ME was characterized using mass spectroscopy. For this purpose, the mass spectrum was recorded at a 100-fold excess ME ( Figure S11). The characteristic molecular ion peak at m/z = 446.1243 ([M + Na] + ) corresponding to the expected adduct of Q3-ME (calculated for C20H25NO7SNa m/z = 446.1249) was observed in the mass spectrum. The obtained result demonstrated that the thiol compound (ME) could react with the unsaturated double bond of the compound Q3 to yield product Q3-ME at a 100-fold excess ME. Additionally, the emission spectra of compounds Q1-Q3 were recorded in the presence of biothiols (L-Cys, L-GSH, and L-ACC) that were previously treated with a thiolblocking agent (NEM) ( Figure S12). The presence of NEM caused the blocking of the group thiol (−SH) in the biothiols [44][45][46]. As shown, biothiols treated with NEM did not We propose that the reaction of compounds Q1-Q3 and sulfhydryl compounds proceeds via nucleophilic Michael addition to the α,β-unsaturated double bond. 2-Mercaptoethanol (ME) was selected as the sulfhydryl compound for the study confirming the proposed reaction mechanism (Scheme 3). The product of the reaction of ME with compound Q3 was confirmed via proton nuclear magnetic resonance ( 1 H NMR) spectroscopy. In the aromatic part of the spectrum, the signal corresponding to the double bond in the malonate moiety of compound Q3 disappeared, and two new signals at 2.74 and 4.72 ppm appeared in the aliphatic part ( Figure S10). Moreover, the product of the reaction of Q3 and ME was characterized using mass spectroscopy. For this purpose, the mass spectrum was recorded at a 100-fold excess ME ( Figure S11). The characteristic molecular ion peak at m/z = 446.1243 ([M + Na] + ) corresponding to the expected adduct of Q3-ME (calculated for C 20 H 25 NO 7 SNa m/z = 446.1249) was observed in the mass spectrum. The obtained result demonstrated that the thiol compound (ME) could react with the unsaturated double bond of the compound Q3 to yield product Q3-ME at a 100-fold excess ME. We propose that the reaction of compounds Q1-Q3 and sulfhydryl compounds proceeds via nucleophilic Michael addition to the α,β-unsaturated double bond. 2-Mercaptoethanol (ME) was selected as the sulfhydryl compound for the study confirming the proposed reaction mechanism (Scheme 3). The product of the reaction of ME with compound Q3 was confirmed via proton nuclear magnetic resonance ( 1 H NMR) spectroscopy. In the aromatic part of the spectrum, the signal corresponding to the double bond in the malonate moiety of compound Q3 disappeared, and two new signals at 2.74 and 4.72 ppm appeared in the aliphatic part ( Figure S10). Moreover, the product of the reaction of Q3 and ME was characterized using mass spectroscopy. For this purpose, the mass spectrum was recorded at a 100-fold excess ME ( Figure S11). The characteristic molecular ion peak at m/z = 446.1243 ([M + Na] + ) corresponding to the expected adduct of Q3-ME (calculated for C20H25NO7SNa m/z = 446.1249) was observed in the mass spectrum. The obtained result demonstrated that the thiol compound (ME) could react with the unsaturated double bond of the compound Q3 to yield product Q3-ME at a 100-fold excess ME. Additionally, the emission spectra of compounds Q1-Q3 were recorded in the presence of biothiols (L-Cys, L-GSH, and L-ACC) that were previously treated with a thiolblocking agent (NEM) ( Figure S12). The presence of NEM caused the blocking of the group thiol (−SH) in the biothiols [44][45][46]. As shown, biothiols treated with NEM did not Additionally, the emission spectra of compounds Q1-Q3 were recorded in the presence of biothiols (L-Cys, L-GSH, and L-ACC) that were previously treated with a thiol-blocking agent (NEM) ( Figure S12). The presence of NEM caused the blocking of the group thiol (−SH) in the biothiols [44][45][46]. As shown, biothiols treated with NEM did not enhance the fluorescence of the studied compounds Q1-Q3. This suggests that the fluorescent response toward thiols results from the probes' reaction with the sulfhydryl group present in the amino acid structure.
The quantum chemical calculations were performed to understand the reasons for the enhancement or quenching of the fluorescence of compounds Q1-Q3 and Q4, which contain two different moieties as reaction sites for biothiols. In the DFT calculation (B3LYP/6-31+G (d,p)) [47], the structures of the derivatives Q3 and Q4 and their products of the reaction with 2-mercaptoethanol (Q3-ME and Q4-ME) were optimized. The energy gap (∆E) for compounds Q3 and Q4 between the HOMO (highest occupied molecular orbital) and the LUMO (lowest unoccupied molecular orbital) was found to be 3.52 and 3.23 eV, respectively. After the addition of 2-mercaptoethanol, the ∆E increased to 3.95 and 4.05 eV for compounds Q3-ME and Q4-ME, respectively. These differences are illustrated in Figure 8. The difference in the spectroscopic properties of the derivatives Q3 and Q4 is related to their energy gap values. Based on the obtained data, it can be unequivocally stated that the derivative with the malonate moiety (Q3) has a more significant energy gap. Therefore, according to the literature [48], it will have a lower fluorescence quantum yield. In contrast, the derivative with the dicyanovinyl moiety (Q4) has a lower energy gap and a higher quantum fluorescence yield. Moreover, comparing these compounds, it can be noticed that smaller the energy gap, the more significant the redshift in the emission. The small energy gap value characterizes the product of the reaction of Q3 with ME (Q3-ME) and an increase in fluorescence intensity was observed. In contrast, the product Q4-ME is characterized by a more significant energy gap and fluorescence quenching after the reaction of Q4 with thiols was observed. present in the amino acid structure.
The quantum chemical calculations were performed to understand the r the enhancement or quenching of the fluorescence of compounds Q1-Q3 and contain two different moieties as reaction sites for biothiols. In the DFT c (B3LYP/6-31+G (d,p)) [47], the structures of the derivatives Q3 and Q4 and thei of the reaction with 2-mercaptoethanol (Q3-ME and Q4-ME) were optimized. T gap (ΔE) for compounds Q3 and Q4 between the HOMO (highest occupied orbital) and the LUMO (lowest unoccupied molecular orbital) was found to b 3.23 eV, respectively. After the addition of 2-mercaptoethanol, the ΔE increas and 4.05 eV for compounds Q3-ME and Q4-ME, respectively. These difference trated in Figure 8. The difference in the spectroscopic properties of the derivativ Q4 is related to their energy gap values. Based on the obtained data, it can be cally stated that the derivative with the malonate moiety (Q3) has a more sign ergy gap. Therefore, according to the literature [48], it will have a lower fluoresce tum yield. In contrast, the derivative with the dicyanovinyl moiety (Q4) has a ergy gap and a higher quantum fluorescence yield. Moreover, comparing t pounds, it can be noticed that smaller the energy gap, the more significant the the emission. The small energy gap value characterizes the product of the reac with ME (Q3-ME) and an increase in fluorescence intensity was observed. In co product Q4-ME is characterized by a more significant energy gap and flu quenching after the reaction of Q4 with thiols was observed.

General
The reagents and solvents were commercial products from Sigma-Aldrich and were used directly without further purification. The course of the reactio purity of the obtained compounds were routinely checked using thin layer ch raphy (TLC) on silica gel 60 F254 plates (Merck, Germany). Plates were visuali UV light (λ = 254 nm or 365 nm). The crude probes were purified using colum tography on silica gel 60 (0.063-0.200 mm, Merck, Germany). Melting points wer on the Boeöthius melting point apparatus (type PHMK 05, Radebeul, Germany) uncorrected. Proton nuclear magnetic resonance ( 1 H NMR) spectra of the prev reported compounds were recorded on a Bruker Avance DPX 250 (Rh

General
The reagents and solvents were commercial products from Sigma-Aldrich (Poland) and were used directly without further purification. The course of the reaction and the purity of the obtained compounds were routinely checked using thin layer chromatography (TLC) on silica gel 60 F254 plates (Merck, Germany). Plates were visualized under UV light (λ = 254 nm or 365 nm). The crude probes were purified using column chromatography on silica gel 60 (0.063-0.200 mm, Merck, Germany). Melting points were checked on the Boeöthius melting point apparatus (type PHMK 05, Radebeul, Germany), and were uncorrected. Proton nuclear magnetic resonance ( 1 H NMR) spectra of the previously unreported compounds were recorded on a Bruker Avance DPX 250 (Rheinstetten, Germany) spectrometer in deuterated solvent (dimethyl sulfoxide, DMSO-d 6 ) with TMS as the internal standard. The chemical shifts (δ values) and coupling constants (J values) are expressed in parts per million (ppm) and hertz (Hz), respectively. NMR peak multiplicities are described as follows: s (singlet), d (doublet), ddd (doublet of doublets of doublets), t (triplet), q (quartet), m (multiplet), and brs (broad singlet). High-resolution mass spectrometry (HRMS) experiments were performed with a mass spectrometer equipped with an electrospray ionization (ESI) source operated in a positive ion mode and quadrupole-time-of-flight mass analyzer (Synapt G2-Si mass spectrometer, Waters). Data were identified as hydrogen ion adducts ([M + H] + ).
Fluorescence quantum yields were determined via a comparative method using fluorescein as a reference compound [49]. The fluorescence lifetimes of the compounds were obtained as previously described [32]. Moreover, the radiative decay rate constant k r and the non-radiative decay rate constant k nr for compounds Q1-Q4 were determined using the following Equations (1) and (2): where Φ em and τ are the fluorescence quantum yield and average fluorescence lifetime, respectively.

General Procedure Synthesis of Compound Q1-Q3
Diethyl malonate (DEM, 1.2 mmol) was put in the solution of appropriate 3-formyl-2(1H)-quinolone derivative (1 mmol) in absolute ethanol (10 mL), and then two drops of piperidine were added. The resulting mixture was stirred at 70 • C for 12 h under the inert atmosphere. The progress of the reaction was monitored via TLC (eluent: CH 3 CN:CH 2 Cl 2 , 1:1, v/v). After completion, the mixture was cooled to room temperature and filtered to yield a solid product Q1 (0.18 g; 58%; R f = 0.77) with a melting point of 168-170 • C, 1

Computational Methods
All the quantum chemical calculations were performed with the Gaussian 09 package [50,51]. Geometries of the compound Q3 and Q4 were optimized via Becke's LYP (B3LYP) exchange-correlation functional with 6-31G (d, p) basis set based on density functional theory (DFT). These computations were carried out in a gas phase. A harmonic frequency analysis characterized the optimized structures as local minima. The timedependent density functional theory (TD-DFT) at the B3LYP/6-31G ** level was used to calculate the electronic transition energies (HOMO and LUMO).

Conclusions
In summary, three compounds, Q1-Q3, based on a 2(1H)-quinolone skeleton with malonate moiety have been synthesized and characterized. These compounds exhibit a fluorescence response to the sulfhydryl group in biothiols (L-Cys, L-GSH, and L-ACC) in a 0.1 M phosphate buffer with CH 3 CN (20%, v/v, pH = 7.4). The results show that the reaction of Q3 with thiols proceeds via the nucleophilic addition of the sulfhydryl group to the malonate moiety. The responses of compounds containing a malonate group (Q1-Q3) and a dicyanovinyl group (Q4) were compared with the tested sulfhydryl compounds. Our studies on variously substituted 2(1H)-quinolones allow us to conclude that the applied reactive system influences the interaction of the analyte with the sensor. Based on the observed reactivity of the compounds Q1-Q4, we conclude that the compounds possessing a malonate group in the structure (Q1-Q3) are more useful for the detection of biothiols (L-Cys, L-GSH, and L-ACC) because these compounds distinguish biothiols among reactive sulfur forms as opposed to the compound possessing a dicyanovinyl group (Q4), which is less selective toward reactive sulfur species.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/molecules28165965/s1, Figures S1-S3 1H NMR spectra of Q1-Q3 in DMSO-d 6 ; Figures S4-S6 HRMS spectra of Q1-Q3; Figure S7 The fluorescence decay profiles of the compounds Q1-Q4 in acetonitrile with excitation at 376.2 nm. Lamp response was determined using Ludox. The fitting of the decay profiles to a three-exponential function. Residuals are shown versus time; Figure Table S1 The fluorescence lifetimes, quantum yields, and rate constant for radiative and non-radiative decay in acetonitrile. Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available in the article and supplementary material.