**3.1 DCM derivatives as metal sensors**

Valeur and Bourson designed a DCM derivative, **DCM1**, which contains a receptor macrocycle (monoaza-15-crown-5) unit that is covalently attached to the electron-donating substituent (N,N-dimethylaniline unit) [47]. It was found that the resulting fluorosensor **DCM1** has almost identical photophysical properties to that of DCM. However, upon complexation with alkaline earth metal cations such as Li, Na, Mg, and Ca, the absorption spectra of **DCM1** undergo either hypsochromic shift or hypochromic shift. Similarly, with addition of alkaline metal cations, a substantial decrease in the fluorescence emission intensity and quantum yield was also observed. It is interesting to note that the fluorescence

**15**

**Figure 5.**

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

emission is slightly blueshifted and corresponding fluorescence lifetime is almost unchanged. It is well established that the ICT from the electron donor to the electron acceptor can be diminished if the electron-donating character of the donor moiety is reduced. In the **DCM1** fluorosensor, the nitrogen atom belongs to the crown, and therefore, upon complexation with cations, the donating ability is reduced and thus hinders the ICT character which more or less depends upon on the nature of the cation. From the observed sensing changes, it is understood that the charge transfer of the cation plays a key role and the reduction of

*Molecular structures of DCM and its derivatives as optical sensors for various analytes.*

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

**Figure 4.** *Schematic diagram of OFF–ON fluorogenic sensing mechanism [45, 46].*

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

**Figure 5.** *Molecular structures of DCM and its derivatives as optical sensors for various analytes.*

emission is slightly blueshifted and corresponding fluorescence lifetime is almost unchanged. It is well established that the ICT from the electron donor to the electron acceptor can be diminished if the electron-donating character of the donor moiety is reduced. In the **DCM1** fluorosensor, the nitrogen atom belongs to the crown, and therefore, upon complexation with cations, the donating ability is reduced and thus hinders the ICT character which more or less depends upon on the nature of the cation. From the observed sensing changes, it is understood that the charge transfer of the cation plays a key role and the reduction of

*Photophysics, Photochemical and Substitution Reactions - Recent Advances*

of off–on fluorogenic sensors is shown in **Figure 4**.

**3.1 DCM derivatives as metal sensors**

have two units: a signaling unit typically a fluorophore and a receptor (recognition unit) which are covalently connected with a π-spacer for rendering the recognition event to the fluorophore that ultimately changes fluorescence signal. A group of fluorogenic sensors which has either weak fluorescence or no fluorescence (off state) by nature and that becomes fluorescent (on state) upon the receptor recognizes the analyte/guest molecule, and this type of fluorogenic sensors are called as off–on sensors. Similarly, on–off sensors can also be designed, where a sensor initially exhibits fluorescence (on state) and after the recognition event, the sensor becomes nonfluorescent/weakly fluorescent (off state). A schematic representation

As discussed in the previous section, the DCM molecule and its derivatives are having unique advantages in terms of their photophysical properties such as red light emission, high quantum yield, and highly tunable fluorescence that is sensitive not only by solvent polarity but also structure modification. Unlike visible light fluorogenic sensors, red and NIR fluorogenic sensors (600–950 nm) have received considerable interest due to minimum fluorescence background, less light scattering, and less photodamage and are having certain advantages in bioimaging applications of live cells. Therefore, in recent years, there is a consistent growth of the colorimetric and fluorogenic sensors based on DCM and its analogues (**Figure 5**) for

sensing cations, anions, and neutral species, which are summarized below.

Valeur and Bourson designed a DCM derivative, **DCM1**, which contains a receptor macrocycle (monoaza-15-crown-5) unit that is covalently attached to the electron-donating substituent (N,N-dimethylaniline unit) [47]. It was found that the resulting fluorosensor **DCM1** has almost identical photophysical properties to that of DCM. However, upon complexation with alkaline earth metal cations such as Li, Na, Mg, and Ca, the absorption spectra of **DCM1** undergo either hypsochromic shift or hypochromic shift. Similarly, with addition of alkaline metal cations, a substantial decrease in the fluorescence emission intensity and quantum yield was also observed. It is interesting to note that the fluorescence

**14**

**Figure 4.**

*Schematic diagram of OFF–ON fluorogenic sensing mechanism [45, 46].*

charge-transfer efficiency from a nonemissive locally excited state to an emissive relaxed intramolecular charge-transfer state (RICT).

A red fluorosensor (**DCBP1**) was designed by replacing N,N-dimethylamine of **DCM** with bis(2-pyridylmethyl) amine (DPA) moiety and benzopyran backbone [48]. Screening of various alkaline and transition metals reveals that the **DCBP1** has more binding affinity towards Cu2+ ions than that of any other cations. The binding affinity is evident not only from the absorption spectrum but also colorimetric response where light pink color of free **DCBP1** solution changes to yellow color after coordinating with copper ions (**DCBP1-Cu2+**), which is visible even to the naked eye. Free **DCBP1** shows a characteristic emission band around 650 nm (with fluorescence high quantum efficiency, ϕ*DCBP1* = 0.40) which is redshifted 55 nm as compared to fluorescence emission of **DCM** (λem = 595 nm) due to insertion of a conjugated benzene unit onto the dicyanopyran backbone. Fluorescence behavior of **DCBP1** in presence of various metal ions was studied in a mixture of ethanol-water (60:40, v/v), and it is observed that only the addition of Cu2+ to **DCBP1** causes a significant decrease in fluorescence intensity. Surprisingly, when pyrophosphate (PPi) anion is added to the in situ generated **DCBP1** meatal complex **(DCBP1-Cu2+)**, the absorption at 505 nm increases with a isosbestic point at 447 nm, and the color of the solution also changes from pale yellow to pink (original color of **DCBP1**). Similarly, fluorescence emission of the **DCBP1-Cu2+** is turned on, and fluorescence emission intensity at 650 nm is also enhanced. From the fluorescence measurements, it is observed that **DCBP1** forms a 1:1 complex with pyrophosphate (PPi) anion, and association constant (Ka) is estimated to be very high at 4.6 × 105 M. Further, an investigation of a series of other anions reveals that the **DCBP1** probe molecule is highly selective and sensitive only towards PPi anion. The observed colorimetric response and on–off fluorescence response of DCBP1 were attributed to the inhibition of the ICT because of decreased the electron-donating ability of the amino group upon binding with Cu2+ ion. On the other hand, turn-on fluorescence is due to electrostatic interaction between PPi and **DCBP1-Cu2+**. Since, the two oxygen atoms of PPi somewhat strongly coordinated with the copper, and the nitrogen-copper bond gets weakened which restores the ICT; thereby fluorescence emission is enhanced. Therefore, the **DCBP1** molecule is demonstrated as both fluorescence on–off and off–on sensor when it is binding with Cu2+ ions and PPi, respectively.

In general, most of the fluorosensors exhibit on–off sensing behavior in solution phase because quenching of fluorescence emission is quite easy. However, developing off–on fluorosensor with processible technology is relatively a tedious and challenging task. Such fluorescence off–on sensors can be tailored to meet the specific needs via rational design approaches and have been paid much attention in recent years due to growing demand of various chemical and biological species detection by exploiting energy transduction principles such as radiant, electrical, mechanical, and thermal processes [49, 50]. Tian and co-workers have extended their previous research work [48] and developed a polymeric **DCM2** sensor based on a hydrophilic copolymer bearing the **DCM** moiety in the form of a fluorescent film which senses Cu2+ and PPi anion works based on off–on fluorescence mechanism (**Figure 6**) [51]. The sensor **DCM2** is decorated with a hydrophilic copolymer, poly(2-hydroxyethyl methacrylate) (PHEMA), that exhibits high hydrophilicity but insoluble in water. The hydrophilic chain segment was chosen mainly to improve the permeability of ions into the polymer backbone, and the **DCM** fluorophore is also grafted into the polymer backbone as metal ion-sensing units. The copolymer **DCM2** and the corresponding metal complex, **DCM2–Cu2+**, exhibit turn-off fluorescence for the selective targeting of Cu2+ (**Figure 6**). However, interestingly, upon adding PPi anion, the fluorescence of the copolymer is turned on with high sensitivity both in solution and in thin film over other anions such as AMP, ADP, ATP, and phosphate (Pi). Furthermore, the low-cost

**17**

**Figure 6.**

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

hydrophilic copolymer film of **DCM2–Cu2+** on a quartz plate shows a very rapid response towards PPi anion with turn-on orange-red fluorescence due to high permeability of its side chains. Recently, a new **DCM**-based NIR fluorescent probe (E)-4- (2-(4-(dicyanomethylene)-4H-chromen-2-yl)vinyl) phenyl picolinate (**DCBP2**) was designed and synthesized for Cu2+ ions with improved performance [52]. As shown in **Chart 5**, the sensor molecule **DCBP2** consists of **DCBP-OH** as a fluorophore and electron-withdrawing 2-pyridinecarbonyl group as the receptor for Cu2+ ions. The reaction between **DCM-OH** and 2-pyridinecarbonyl gives rise to picolinoyl ester of the **DCM**. Since 2-pyridinecarbonyl group is covalently anchored to the fluorophore, the ICT is blocked, and no fluorescence can be observed. At this stage when Cu2+ is added to the probe, the copper ions coordinate with nitrogen and oxygen atoms of 2-pyridinecarbonyl group fluorescence emission quenches. However, upon hydrolysing **DCBP2** with water, Cu2+ releases from coordination, and subsequently a phenolate ion (**DCBPO−)** is produced, which is a better electron-donating group and thus restores its ICT property which ultimately leads to a dramatic increase in fluorescence intensity at 676 nm. The sensing behavior of the probe **DCBP2** towards Cu2+ ions can also be conveniently followed by naked eye inspection and measuring absorption under mild conditions. The color of the solution appears yellow in absence of copper ions, which, however, changes to pink color upon adding copper ions and clearly visible to the naked eye. The free **DCBP2** gives absorption 558 nm upon, and binding with copper ions, the absorption shifts 415 nm (blueshift). Furthermore, this probe was

*Fluorescence on–off and off–on mechanism of DCBP1 (above) DCM2 copolymer (below).*

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

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

hydrophilic copolymer film of **DCM2–Cu2+** on a quartz plate shows a very rapid response towards PPi anion with turn-on orange-red fluorescence due to high permeability of its side chains. Recently, a new **DCM**-based NIR fluorescent probe (E)-4- (2-(4-(dicyanomethylene)-4H-chromen-2-yl)vinyl) phenyl picolinate (**DCBP2**) was designed and synthesized for Cu2+ ions with improved performance [52]. As shown in **Chart 5**, the sensor molecule **DCBP2** consists of **DCBP-OH** as a fluorophore and electron-withdrawing 2-pyridinecarbonyl group as the receptor for Cu2+ ions. The reaction between **DCM-OH** and 2-pyridinecarbonyl gives rise to picolinoyl ester of the **DCM**. Since 2-pyridinecarbonyl group is covalently anchored to the fluorophore, the ICT is blocked, and no fluorescence can be observed. At this stage when Cu2+ is added to the probe, the copper ions coordinate with nitrogen and oxygen atoms of 2-pyridinecarbonyl group fluorescence emission quenches. However, upon hydrolysing **DCBP2** with water, Cu2+ releases from coordination, and subsequently a phenolate ion (**DCBPO−)** is produced, which is a better electron-donating group and thus restores its ICT property which ultimately leads to a dramatic increase in fluorescence intensity at 676 nm. The sensing behavior of the probe **DCBP2** towards Cu2+ ions can also be conveniently followed by naked eye inspection and measuring absorption under mild conditions. The color of the solution appears yellow in absence of copper ions, which, however, changes to pink color upon adding copper ions and clearly visible to the naked eye. The free **DCBP2** gives absorption 558 nm upon, and binding with copper ions, the absorption shifts 415 nm (blueshift). Furthermore, this probe was

*Photophysics, Photochemical and Substitution Reactions - Recent Advances*

relaxed intramolecular charge-transfer state (RICT).

is estimated to be very high at 4.6 × 105

Cu2+ ions and PPi, respectively.

charge-transfer efficiency from a nonemissive locally excited state to an emissive

A red fluorosensor (**DCBP1**) was designed by replacing N,N-dimethylamine of **DCM** with bis(2-pyridylmethyl) amine (DPA) moiety and benzopyran backbone [48]. Screening of various alkaline and transition metals reveals that the **DCBP1** has more binding affinity towards Cu2+ ions than that of any other cations. The binding affinity is evident not only from the absorption spectrum but also colorimetric response where light pink color of free **DCBP1** solution changes to yellow color after coordinating with copper ions (**DCBP1-Cu2+**), which is visible even to the naked eye. Free **DCBP1** shows a characteristic emission band around 650 nm (with fluorescence high quantum efficiency, ϕ*DCBP1* = 0.40) which is redshifted 55 nm as compared to fluorescence emission of **DCM** (λem = 595 nm) due to insertion of a conjugated benzene unit onto the dicyanopyran backbone. Fluorescence behavior of **DCBP1** in presence of various metal ions was studied in a mixture of ethanol-water (60:40, v/v), and it is observed that only the addition of Cu2+ to **DCBP1** causes a significant decrease in fluorescence intensity. Surprisingly, when pyrophosphate (PPi) anion is added to the in situ generated **DCBP1** meatal complex **(DCBP1-Cu2+)**, the absorption at 505 nm increases with a isosbestic point at 447 nm, and the color of the solution also changes from pale yellow to pink (original color of **DCBP1**). Similarly, fluorescence emission of the **DCBP1-Cu2+** is turned on, and fluorescence emission intensity at 650 nm is also enhanced. From the fluorescence measurements, it is observed that **DCBP1** forms a 1:1 complex with pyrophosphate (PPi) anion, and association constant (Ka)

other anions reveals that the **DCBP1** probe molecule is highly selective and sensitive only towards PPi anion. The observed colorimetric response and on–off fluorescence response of DCBP1 were attributed to the inhibition of the ICT because of decreased the electron-donating ability of the amino group upon binding with Cu2+ ion. On the other hand, turn-on fluorescence is due to electrostatic interaction between PPi and **DCBP1-Cu2+**. Since, the two oxygen atoms of PPi somewhat strongly coordinated with the copper, and the nitrogen-copper bond gets weakened which restores the ICT; thereby fluorescence emission is enhanced. Therefore, the **DCBP1** molecule is demonstrated as both fluorescence on–off and off–on sensor when it is binding with

In general, most of the fluorosensors exhibit on–off sensing behavior in solution phase because quenching of fluorescence emission is quite easy. However, developing off–on fluorosensor with processible technology is relatively a tedious and challenging task. Such fluorescence off–on sensors can be tailored to meet the specific needs via rational design approaches and have been paid much attention in recent years due to growing demand of various chemical and biological species detection by exploiting energy transduction principles such as radiant, electrical, mechanical, and thermal processes [49, 50]. Tian and co-workers have extended their previous research work [48] and developed a polymeric **DCM2** sensor based on a hydrophilic copolymer bearing the **DCM** moiety in the form of a fluorescent film which senses Cu2+ and PPi anion works based on off–on fluorescence mechanism (**Figure 6**) [51]. The sensor **DCM2** is decorated with a hydrophilic copolymer, poly(2-hydroxyethyl methacrylate) (PHEMA), that exhibits high hydrophilicity but insoluble in water. The hydrophilic chain segment was chosen mainly to improve the permeability of ions into the polymer backbone, and the **DCM** fluorophore is also grafted into the polymer backbone as metal ion-sensing units. The copolymer **DCM2** and the corresponding metal complex, **DCM2–Cu2+**, exhibit turn-off fluorescence for the selective targeting of Cu2+ (**Figure 6**). However, interestingly, upon adding PPi anion, the fluorescence of the copolymer is turned on with high sensitivity both in solution and in thin film over other anions such as AMP, ADP, ATP, and phosphate (Pi). Furthermore, the low-cost

M. Further, an investigation of a series of

**16**

successfully applied for the quantitative estimation of Cu2+ in various types of water samples and also demonstrated its utility in imaging living cells.

**DCBP3** is designed based on the Pd(0)-catalyzed Tsuji-Trost allylic oxidative insertion reaction and dicyanomethylene benzopyran moiety [53]. Photophysical properties revealed that the probe **DCBP3** exhibits high sensitivity and selectivity towards the detection of both Pd(0) and Pd(II) under reducing conditions. The probe **DCBP3** shows a major absorption band with a maximum at 450 nm, and after treating with palladium, another new absorption peak started appearing around 560 nm. On the other hand, **DCBP3** displays no fluorescence at 700 nm when excited at 560 nm. However, upon the addition of palladium, the fluorescence emission peak at 700 nm increases gradually. Additionally, marked color changes were also noticed. All the photophysical properties have been explained based on palladium-triggered cleavage reaction that produced a free **DCBP-OH**. Moreover, the probe **DCBP3** is little affected with pH variation and has low cytotoxicity.

### **3.2 DCM derivatives as anion sensors**

As discussed in Section 3.1, the molecules **DCBP1** and **DCM2** form copper complexes (**DCBP1 Cu2+** and **DCBP1-Cu2+**), and their fluorescence emission quenches drastically [48, 51]. In situ generated **DCBP1 Cu2+** and **DCBP1-Cu2+** recognize PPi anion which can be tracked from spectrophotometrically and fluorescence measurements. Later, the molecule **DCBP1** was modified by decorating with a lithium iminodiacetate group in place of N-aryl group [54]. The synthesized NIR fluorophore, **DCBP4**, selectively binds with Cu2+ ions because of lithium iminodiacetate receptor and found to have very good solubility in aqueous water. The photophysical properties of metallated fluorophore (**DCBP4-Cu2+**) were found to be modified upon interacting selectively with pyrophosphate (PPi) anion. When PPi is gradually added to the solution of **DCBP4-Cu2+**, a new redshifted peak at 503 nm appeared and increased gradually with an isosbestic point at 450 nm. The absorption spectral changes are very much evident to the naked eye where the pale brown color of the **DCBP4-Cu2+** solution changes to red color. On the other hand, simultaneously turned on fluorescence and emission intensity in the NIR region (675 nm) are enhanced gradually and stabilized upon the addition of 15 equiv. of PPi. The fluorescence off–on switching and the colorimetric response of **DCBP4-Cu2+** are interpreted in terms of ICT variations upon sensing the receptor.

A near-infrared (NIR) fluorescent chemosensor, **DCBP5**, was developed on the basis of dicyanomethylene-4H-benzopyran derivative for detecting fluoride anions [55]. Chemodosimeter **DCBP5** was synthesized by the Knoevenagel condensation of 4-dicyanomethylene-2-methyl-4H-pyran and 4-(tert-butyldiphenylsilyloxy) benzaldehyde. With the addition of F<sup>−</sup> ions to the **DCBP5** sensor, absorption band cantered at 447 nm slowly decreases, and at lower F<sup>−</sup> concentration (<30 μM), a new absorption emerges at 454 nm gradually. When the F<sup>−</sup> concentration was further increased beyond 50 μM, the new absorption band at 454 nm decreases, and a concomitant increase of a new band at 645 nm was observed with an isosbestic point at 510 nm. The large redshift (190 nm) is also noticeable to the naked eye in which the initial pale yellow color of the **DCBP5** solution changes to blue color upon adding fluoride ions. It should also be noted that the sensing process is very fast, and within 30 s the sensing is noticeable to the naked eye. The observed isosbestic point of **DCBP5** sensor upon addition of the F<sup>−</sup> ions clearly indicates formation of a new species which is attributed to phenolate group generation due to Si–O cleavage. Similar supporting results were also observed from fluorescence measurements. The **DCBP5** molecule is non-fluorescent due to the presence of silyl group. However, the sensor **DCBP5** turn-on fluorescence with gradual addition of

**19**

*3.3.2 Dopamine*

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

F<sup>−</sup> ion which is evident from the fluorescence emission measurements in which a new fluorescent band started emerging in the NIR region (at 718 nm). Since the in situ generated phenolate group is a much stronger electron-donating group than the silyl group, the ICT efficiency restored after **DCBP5** interaction with that of F<sup>−</sup> ions. Further, the results also revealed that the **DCBP5** is not just an off–on fluorescent sensor, but it is also a ratiometric and colorimetric sensor which is the

Hydrogen sulphide (H2S) is involved as a signaling molecule in various physiological processes that include modulation of neuronal transmission, regulation of release of insulin, relaxation of the smooth muscle, and reduction of the metabolic rate [56, 57]. From the animal model study of critical illness, it was realized that the H2S donor protect from lethal hypoxia and reperfusion injury and exert antiinflammatory effects [58]. Physiological H2S concentration is estimated to vary from nano- to millimolar levels [59], and once this limit is crossed, the cells release H2S that can cause certain diseases, such as Alzheimer, Down syndrome, diabetes, and other diseases of mental deficiency [60]. Hence, a reliable in vivo study is essential to measure accurately H2S concentration thereby preventing deceases. A NIR probe, **DCBP6**, that comprises dicyanobenzopyran and 4-azidostyryl group as receptor was developed for selective detection of H2S [61]. The probe **DCBP6** selectively reacts with H2S and reduces the azido group (–N3) to amine (–NH2), and the corresponding molecule becomes highly fluorescent than the parent **DCBP6**. Upon H2S detection, the **DCBP6** probe solution changes which is visible to the naked eye and causes a large Stokes shift (>100 nm in different solvents). Besides, the reduced probe **DCBP6NH2** exhibit two-photon absorption (TPA) which is having more advantages than traditional one-photon absorption probes in fluorescence microscopy such as less phototoxicity, better three-dimensional spatial localization, deeper penetration depth, and lower self-absorption. Further, the probe molecule **DCBP6** was successfully used as fluorescent probe for monitoring H2S in living cells and tissues and in vivo in mice via fluorescence bioimaging investigations. More or less at the same time, Xu and coworkers have reported the same molecular probe for in vivo detection of H2S [62].

A catecholamine compound dopamine is known as a neurotransmitter that regulates a wide range of cognitive functions such as behavior, learning, motivation, and memory [63–65]. The dopamine content in the human brain is an important factor that can cause various diseases that include Parkinson's disease, and in fact it is used as a marker in the diagnosis of several conditions related to neurotransmitters. Therefore, there is a strong quest for developing efficient and rapid methods that can selectively determine and continuously sense the dopamine levels on a real-time basis. The DCM fluorosensor (**DCM3-Fe2+**) was developed for selective detection of dopamine based on on–off sensing mechanism [66]. The electron-donor part of DCM fluorophore is modified with a ligand, diethyliminodiacetic acid, such that it selectively complexes with iron(II) ions. In the absence of dopamine, the sensor molecule **DCM3-Fe2+ is** weakly fluorescent due to inhibition of ICT because of Fe2+ complexation with the donor moiety (off-state fluorescence). However, a much stronger fluorescence emission was observed upon gradual addition of dopamine owing to the release of Fe2+ from DCM complex. A good linear relationship was

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

ideal characteristic of any sensor.

*3.3.1 Hydrogen sulphide (H2S)*

**3.3 DCM derivatives for detection of neutral species**

F<sup>−</sup> ion which is evident from the fluorescence emission measurements in which a new fluorescent band started emerging in the NIR region (at 718 nm). Since the in situ generated phenolate group is a much stronger electron-donating group than the silyl group, the ICT efficiency restored after **DCBP5** interaction with that of F<sup>−</sup> ions. Further, the results also revealed that the **DCBP5** is not just an off–on fluorescent sensor, but it is also a ratiometric and colorimetric sensor which is the ideal characteristic of any sensor.
