*3.4.1 Biothiols*

*Photophysics, Photochemical and Substitution Reactions - Recent Advances*

substances, thereby allowing selective detection of dopamine.

*3.3.3 Hydrogen peroxide (H2O2)*

*3.3.4 Hydrazine (N2H4)*

observed between the dopamine concentration and the fluorescence intensity. That means the observed fluorescence enhancement which is observed after addition of dopamine serves as an indicator to monitor dopamine content in a given sample. Besides, the fluorosensor does show any fluorescence response against other foreign

Zhang et al. have synthesized a new NIR and colorimetric fluorescent molecular probe, **DCBP7**, by covalently attaching dicyanomethylene-4*H*-benzopyran and phenylboronic acid for rapid detection of H2O2 [67]. The boronic acid functional group is attached primarily to have NIR fluorescence off–on switching. The sensing of H2O2 was successfully demonstrated by UV–visible absorption and fluorescence measurements. **DCBP7** exhibit a structured absorption band at 450 nm, and its solution appears pale yellow in color. However, with gradual addition of H2O2 (>20 equiv.), apparently, the absorption at 450 nm decreases, and a new absorption band starts evolving at 560 nm. Due to the large redshift (110 nm) of the absorption, the color of the solution (yellow) changed to purple, and colorimetric detection of H2O2 is visible even to the naked eye. The fluorescence measurements were also carried out to confirm the H2O2 sensing behavior of the probe molecule. The free probe molecule is non-fluorescent primarily because of phenylboronic group. However, after adding H2O2, the boronic acid group gets cleaved and generate a phenolate ion which is evident from the new fluorescent emission band at 670 nm. The Stokes shift (110 nm) of the phenolate band in the NIR region has been exploited further for detecting H2O2 and imaging live cells. Unlike the most conventional fluorescent probes, the developed **DCBP7** has been shown to have unique advantages such as deeper tissue penetration ability, lower background autofluorescence, and less damage to biological samples which ultimately allowed to in vivo studies of live cells.

Hydrazine is used as a common precursor in synthetic chemistry of many polymers, pharmaceutical intermediates, hydrazine fuel cells in power generation sector, and materials science [68, 69]. It is often used in rocket propulsion systems as an important propellant for its flammable and detonable characteristics. Moreover, hydrazine serves as an important metal corrosion inhibitor because of its strong reducing properties; hydrazine scavenges oxygen in water boilers that are used for feed and heating systems. However, hydrazine and its aqueous solutions are highly toxic to all living organisms when inhaled or in contact. It has been shown that hydrazine is mutagenic and carcinogenic which causes serious damage to the human central nervous system, kidneys, liver, and lungs [70]. Therefore, it is of great interest and importance to develop a reliable method for hydrazine detection with selectivity and sensitivity. With a view to develop efficient DCM-based NIR fluorophore for selective detection of hydrazine, a phenyl ring baring *O*-acetyl moiety was introduced onto the into dicyanomethylene-4*H*-benzopyran backbone and synthesized DCBP8 [71]. The absorption and fluorescence properties of DCBP8 were measured in PBS solution (pH = 7.4) containing 50% of ethanol. DCBP8 has absorption in between 300 and 450 nm region with a maximum at 434 nm. After treatment of DCBP8 with N2H4, gradually new absorption peaks started appearing at 551 nm at the expense of 434 nm absorption band. The absorption maximum shifted from 434 nm to 551 nm which indicates the efficiency of DCBP8 for colorimetric detection of N2H4 when absorption intensity ratio (A551/A434) and concentration of N2H4 ranging from 0 to 40 μM are plotted against each other, there is a

**20**

There is quest for developing molecular probes for rapid, selective, and sensitive detection of the highly toxic thiophenols which are of great importance in both environmental and biological science. James and co-workers have developed a novel near-infrared (NIR) and colorimetric fluorescent molecular probe, **DCBP9**, based on a dicyanomethylene-4H-pyran chromophore for selective detection of glutathione in living cells [72]. The molecular probe **DCBP9** was synthesized by Michael's addition of 2-(2-(4-hydroxystyryl)-4H- chromen-4-ylidene) malononitrile and 2,4-dinitrobenzene-1-sulphonyl chloride (DNBS) in the presence of pyridine at room temperature. Molecular probe has an intense absorption centred at 414 nm in a DMSO-PBS buffer solution; upon the addition of glutathione (GSH), the color of the solution turned to pink from slight yellow and clearly visible to the naked eyes. In addition, a new absorption band emerged at 560 nm with an isosbestic point at 446 nm which is assigned to the specific O–S cleavage, and the generation of phenolate ion with a distinct 146 nm redshift in absorbance is observed. Since the phenolate group is a much stronger electron donor than the sulphonate group, the ICT efficiency is significantly enhanced by the interaction of **DCBP9** with GSH and thus shifts the absorption to a longer wavelength region. Subsequent fluorescence experiments showed that the molecular probe alone is nonemissive (turn-off) in absence of GSH. However, when the probe is excited at 560 nm in presence of GSH, turn-on fluorescence and the intensity at 690 nm were dramatically enhanced. The turn-on fluorescence is due to the release of electron-withdrawing DNBS moiety via a GSH-induced O–S bond cleavage and produces phenolate ion, which possesses a strong ICT character and induces a turn-on NIR fluorescence. Having known chemical properties of thiophenols that are able to cleave sulphonamide selectively and efficiently under mild conditions, a dicyanomethylene-benzopyran-based NIR fluorescent probe **DCBP10** is designed for detection of thiophenols [73]. Upon adding thiophenols to the **DCBP10** solution, the DNBS moiety is cleaved and forms amine (–NH2) functional group at the phenyl ring. Since the amine is an electrondonating group, the ICT of the fluorophore is restored, and as a result, absorption and fluorescence emission properties of the probe were changed. This probe features remarkable large Stokes shift and shows a rapid, highly selective, and sensitive detection process for thiophenols with significant NIR turn-on fluorescent response. Therefore, **DCBP10** was successfully demonstrated as a potential NIR fluorescent probe that can be mitigated not only for quantitative detection of thiophenol in real water samples but also fluorescence imaging of thiophenol in living cells [73].

Slightly similar molecular structure **DCBP11** is redesigned by Li et al. that consists of dicyanomethylene-benzopyran scaffold and 2,4-dinitrophenyl (DNP) connected by ether linkage for probing for thiophenols [74]. It was demonstrated that **DCBP11** shows both colorimetric and rapid turn-on fluorescence sensing process for thiophenols with high selectivity and better sensitivity (DL = 70 nm). Moreover, it should be noted that a dual colorimetric and selective NIR fluorescence sensing phenomenon is also visual to the 'naked eye' without the need of advanced

instrumentation. In addition, quantitative detection of thiophenol in real water samples and fluorescent imaging of thiophenol in living cells and zebrafish were successfully demonstrated which suggests that this probe has a great potential for in vitro and in vivo applications. Another NIR probe, which contains a conjugated dicyanomethylene-benzopyran moiety as the NIR fluorophore **DCBP12** and an acrylate moiety as a receptor, is found to be promising for biothiols: cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) detection. **DCBP12** itself almost non-fluorescent due to alkene-induced quenching of photoinduced electrontransfer (PET) process; however, it becomes fluorescent upon sensing biothiols. It has been proven that the NIR fluorescence enhancement of **DCBP12** is due to Cys sensing originating from cleavage of acryloyl group of **DCBP12** that simultaneously releases a NIR fluorescent DCM-OH. A feasible sensing mechanism was proposed to understand the sensing process of Cys. The reaction of Cys with **DCBP12** involves two steps. Initially, a Michael addition reaction of the thiol functional group of Cys with the acryloyl group has taken place and then followed by a spontaneous intramolecular cyclization to release the NIR fluorescent, phenolate ion (**DCMO−)**. Further, the detection limit for Cys was estimated to be 81 nm. In addition, imaging of biothiols by **DCBP12** was also successfully demonstrated in living cells which indicates that this probe is suitable for imaging biological samples [75]. Therefore, **DCBP12** shows rapid response and high-selectivity and high-sensitivity biothiols particularly for Cys and Hcy, accompanied by distinct color changes seen by the naked eye and significant NIR turn-on fluorescence responses.

Recently, a red-emitting fluorescent probe **DCM4** was developed for selective detection of cysteine (Cys) over glutathione (GSH) and homocysteine (Hcy) by incorporating acryloyl group as the recognition unit into the 2-(2-(4-hydroxystyryl)-6-methyl-4H-pyran-4-ylidene) malononitrile (P-OH) fluorophore [76]. Selective detection of Cys is very important because, among biothiols, Cys is considered as the most significant biothiols of living organisms and plays a crucial role in multiple physiological processes that include mitochondrial protein turnover, protein biosynthesis, detoxification administration, and metabolism regulation [77]. Further, because of crucial physiological and pathological significance of Cys in biological systems, it is essential to develop a rapid and promising analytical tool for selective detection of Cys so as to unravel hidden physiological processes of Cys and understand the specific pathogenesis of Cys-related diseases. Basically, the probe design is almost similar with **DCBP12** used for thiol detection and **DCBP3** used for palladium detection. The **DCM4** molecule is almost non-fluorescent due to acryloyl group that blocks ICT and promotes non-radiative processes. Upon the addition of Cys, **DCM4** undergoes Michael addition of Cys and the acryloyl group to afford a transient intermediate, followed by the intramolecular cyclization to give highly fluorescent oxide anion. Therefore, accordingly by monitoring fluorescence intensity variations before and after the addition, the Cys can be detected. The fluorosensor **DCM4** has certain advantages. Firstly, probe **DCM4** has good selectivity for Cys over Hcy and GSH. Secondly, the probe senses Cys in the solution and responds in a short time (4 min) towards Cys. Thirdly, this probe exhibits high signal-to-noise ratio (~147-fold) and ultralow detection limit (41.696 nm). Thus, the **DCM4** was successfully demonstrated as off–on fluorosensor to monitor the Cys level in living cells with low cytotoxicity.

### *3.4.2 Selenocysteine*

Selenocysteine (Sec) is a cysteine (Cys) analogue which consists of selenol group in place of the thiol group in Cys and considered as a major form of biological selenium and known as the 21st proteinogenic amino acid that is specifically

**23**

*Photophysical Properties of 4-(Dicyanomethylene)-2-Methyl-6-(4-Dimethylaminostyryl)-4*H*…*

incorporated into selenoproteins (SePs). More than 50 human proteins are known to contain Sec [78]. Therefore, detection of Sec in physiological conditions is very important. In order to achieve NIR turn-on fluorescent detection of Sec selectively, the molecule **DCBP11** was designed which was originally used for thiol detection [74]. **DCBP11** senses the presence of Sec and shows colorimetric and NIR turn-on fluorescence response upon cleavage of ether bond and subsequent formation of **DCM-OH** [79]. Similar to many other DCM-based ICT molecules, **DCBP11** also shows a remarkable large Stokes shift at 146 nm. Besides, **DCBP11** is highly sensitive to Sec and exhibits a very small detection limit of 62 nm over a wide linear range (0.2–80 μM) of selenocysteines which allows quantitative estimation of the Sec. Moreover, it was further demonstrated that this NIR fluorescent probe can be employed to image both exogenous and endogenous Sec in living cells, indicating

A pH-sensitive fluorescent chemosensor, **DCBP-OH**, was designed based on dicyanomethylene-4H-benzopyran scaffold by employing D-π-A architecture [80]. At neutral pH, the **DCBP-OH** shows absorption at 450 nm which is attributed to the typical ICT band of DCM chromophore and very weak fluorescent (~ 574 nm). Interestingly, as the pH of the solution increases (from 7.15 to 11.00), the weak fluorescence emission band at 574 nm decreases, and simultaneously a new band at 692 nm started increasing. The evolution of new fluorescence band at 692 nm is assigned to the increase of the ICT process from the oxygen anion of phenolate group. That means the strong change in fluorescence intensity is a clear indication for determining pH of any solution from 7 to 11. Acid dissociation constant pKa value is calculated to be 7.21. Moreover, it was found that the fluorescence signal ratio (I692/I574) is found to be ratiometric induced by a large Stokes shift of about 118 nm. Furthermore, from the absorption and fluorescence measurements, it was proved that the pH response of **DCBP-OH** is reversible which makes **DCBP-OH** a simple naked-eye sensitive NIR fluorescent chemosensor for pH measurement. In a very recent work, the **DCBP-OH** probe has been slightly modified with triphenylphosphate and shown as NIR sensor for lysozyme detection in urine sample [81].

Kwak et al. developed different types of copolymers by decorating with the DCM moiety into a certain polymer chain which are sensible to external environment and useful to probe dye molecules [82]. The photophysical properties in solution, solid film, and aggregation revealed that ICT characteristics of the copolymers are modifying. More interestingly, it was observed that the fluorescent properties of DCM-type dyes within the polymers are significantly dependent upon the polarity of the polymer matrix. Three copolymers (P(St-*co*-**2**), P(MMA-*co*-**2**), and P(AN-*co*-**2**)) have shown quite unusual photophysical properties which are completely different from the corresponding DCM-type monomer. The copolymers show a blueshift in fluorescence emission relative to the monomer. The aggregates of copolymers prepared in polar medium (DMF) by adding methanol showed a significant blueshift in fluorescence emission, and aggregates prepared from non-polar medium (1,4-dioxan)/methanol exhibit a prominent redshift. Similarly, it was also observed that the fluorescence intensity of P(St-*co*-**2**) and P(MMA-*co*-**2**) decreased by aggregation while that of P(AN-*co*-**2**) increased. Such interesting solvatochromism and unusual aggregation behavior of the three copolymers were exploited further for selective sensing of volatile organic compounds (VOC).

*DOI: http://dx.doi.org/10.5772/intechopen.93149*

**3.5 DCM derivatives as pH sensor**

**3.6 DCM derivatives as polarity sensor**

that **DCBP11** has great potential for biological applications.

*Photophysical Properties of 4-(Dicyanomethylene)-2-Methyl-6-(4-Dimethylaminostyryl)-4*H*… DOI: http://dx.doi.org/10.5772/intechopen.93149*

incorporated into selenoproteins (SePs). More than 50 human proteins are known to contain Sec [78]. Therefore, detection of Sec in physiological conditions is very important. In order to achieve NIR turn-on fluorescent detection of Sec selectively, the molecule **DCBP11** was designed which was originally used for thiol detection [74]. **DCBP11** senses the presence of Sec and shows colorimetric and NIR turn-on fluorescence response upon cleavage of ether bond and subsequent formation of **DCM-OH** [79]. Similar to many other DCM-based ICT molecules, **DCBP11** also shows a remarkable large Stokes shift at 146 nm. Besides, **DCBP11** is highly sensitive to Sec and exhibits a very small detection limit of 62 nm over a wide linear range (0.2–80 μM) of selenocysteines which allows quantitative estimation of the Sec. Moreover, it was further demonstrated that this NIR fluorescent probe can be employed to image both exogenous and endogenous Sec in living cells, indicating that **DCBP11** has great potential for biological applications.

## **3.5 DCM derivatives as pH sensor**

*Photophysics, Photochemical and Substitution Reactions - Recent Advances*

naked eye and significant NIR turn-on fluorescence responses.

sor to monitor the Cys level in living cells with low cytotoxicity.

Selenocysteine (Sec) is a cysteine (Cys) analogue which consists of selenol group in place of the thiol group in Cys and considered as a major form of biological selenium and known as the 21st proteinogenic amino acid that is specifically

Recently, a red-emitting fluorescent probe **DCM4** was developed for selective detection of cysteine (Cys) over glutathione (GSH) and homocysteine (Hcy) by incorporating acryloyl group as the recognition unit into the

2-(2-(4-hydroxystyryl)-6-methyl-4H-pyran-4-ylidene) malononitrile (P-OH) fluorophore [76]. Selective detection of Cys is very important because, among biothiols, Cys is considered as the most significant biothiols of living organisms and plays a crucial role in multiple physiological processes that include mitochondrial protein turnover, protein biosynthesis, detoxification administration, and metabolism regulation [77]. Further, because of crucial physiological and pathological significance of Cys in biological systems, it is essential to develop a rapid and promising analytical tool for selective detection of Cys so as to unravel hidden physiological processes of Cys and understand the specific pathogenesis of Cys-related diseases. Basically, the probe design is almost similar with **DCBP12** used for thiol detection and **DCBP3** used for palladium detection. The **DCM4** molecule is almost non-fluorescent due to acryloyl group that blocks ICT and promotes non-radiative processes. Upon the addition of Cys, **DCM4** undergoes Michael addition of Cys and the acryloyl group to afford a transient intermediate, followed by the intramolecular cyclization to give highly fluorescent oxide anion. Therefore, accordingly by monitoring fluorescence intensity variations before and after the addition, the Cys can be detected. The fluorosensor **DCM4** has certain advantages. Firstly, probe **DCM4** has good selectivity for Cys over Hcy and GSH. Secondly, the probe senses Cys in the solution and responds in a short time (4 min) towards Cys. Thirdly, this probe exhibits high signal-to-noise ratio (~147-fold) and ultralow detection limit (41.696 nm). Thus, the **DCM4** was successfully demonstrated as off–on fluorosen-

instrumentation. In addition, quantitative detection of thiophenol in real water samples and fluorescent imaging of thiophenol in living cells and zebrafish were successfully demonstrated which suggests that this probe has a great potential for in vitro and in vivo applications. Another NIR probe, which contains a conjugated dicyanomethylene-benzopyran moiety as the NIR fluorophore **DCBP12** and an acrylate moiety as a receptor, is found to be promising for biothiols: cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) detection. **DCBP12** itself almost non-fluorescent due to alkene-induced quenching of photoinduced electrontransfer (PET) process; however, it becomes fluorescent upon sensing biothiols. It has been proven that the NIR fluorescence enhancement of **DCBP12** is due to Cys sensing originating from cleavage of acryloyl group of **DCBP12** that simultaneously releases a NIR fluorescent DCM-OH. A feasible sensing mechanism was proposed to understand the sensing process of Cys. The reaction of Cys with **DCBP12** involves two steps. Initially, a Michael addition reaction of the thiol functional group of Cys with the acryloyl group has taken place and then followed by a spontaneous intramolecular cyclization to release the NIR fluorescent, phenolate ion (**DCMO−)**. Further, the detection limit for Cys was estimated to be 81 nm. In addition, imaging of biothiols by **DCBP12** was also successfully demonstrated in living cells which indicates that this probe is suitable for imaging biological samples [75]. Therefore, **DCBP12** shows rapid response and high-selectivity and high-sensitivity biothiols particularly for Cys and Hcy, accompanied by distinct color changes seen by the

**22**

*3.4.2 Selenocysteine*

A pH-sensitive fluorescent chemosensor, **DCBP-OH**, was designed based on dicyanomethylene-4H-benzopyran scaffold by employing D-π-A architecture [80]. At neutral pH, the **DCBP-OH** shows absorption at 450 nm which is attributed to the typical ICT band of DCM chromophore and very weak fluorescent (~ 574 nm). Interestingly, as the pH of the solution increases (from 7.15 to 11.00), the weak fluorescence emission band at 574 nm decreases, and simultaneously a new band at 692 nm started increasing. The evolution of new fluorescence band at 692 nm is assigned to the increase of the ICT process from the oxygen anion of phenolate group. That means the strong change in fluorescence intensity is a clear indication for determining pH of any solution from 7 to 11. Acid dissociation constant pKa value is calculated to be 7.21. Moreover, it was found that the fluorescence signal ratio (I692/I574) is found to be ratiometric induced by a large Stokes shift of about 118 nm. Furthermore, from the absorption and fluorescence measurements, it was proved that the pH response of **DCBP-OH** is reversible which makes **DCBP-OH** a simple naked-eye sensitive NIR fluorescent chemosensor for pH measurement. In a very recent work, the **DCBP-OH** probe has been slightly modified with triphenylphosphate and shown as NIR sensor for lysozyme detection in urine sample [81].

## **3.6 DCM derivatives as polarity sensor**

Kwak et al. developed different types of copolymers by decorating with the DCM moiety into a certain polymer chain which are sensible to external environment and useful to probe dye molecules [82]. The photophysical properties in solution, solid film, and aggregation revealed that ICT characteristics of the copolymers are modifying. More interestingly, it was observed that the fluorescent properties of DCM-type dyes within the polymers are significantly dependent upon the polarity of the polymer matrix. Three copolymers (P(St-*co*-**2**), P(MMA-*co*-**2**), and P(AN-*co*-**2**)) have shown quite unusual photophysical properties which are completely different from the corresponding DCM-type monomer. The copolymers show a blueshift in fluorescence emission relative to the monomer. The aggregates of copolymers prepared in polar medium (DMF) by adding methanol showed a significant blueshift in fluorescence emission, and aggregates prepared from non-polar medium (1,4-dioxan)/methanol exhibit a prominent redshift. Similarly, it was also observed that the fluorescence intensity of P(St-*co*-**2**) and P(MMA-*co*-**2**) decreased by aggregation while that of P(AN-*co*-**2**) increased. Such interesting solvatochromism and unusual aggregation behavior of the three copolymers were exploited further for selective sensing of volatile organic compounds (VOC).
