Fluorescent Azobenzene-Containing Compounds: From Structure to Mechanism

: The reversible photoisomerization of azobenzenes has been extensively studied to con-struct systems with optical responsiveness; however, this process limits the luminescence of these compounds. Recently, there have been many efforts to design and synthesize ﬂuorescent azobenzene compounds, such as inhibition of electron transfer, inducing aggregation, and metal-enhancement, which make the materials ideal for application in ﬂuorescence probes, light-emitting devices, molecular detection, etc. Herein, we review the recently reported progress in the development of various ﬂuorescent azobenzenes and summarize the possible mechanism of their ﬂuorescence emission. The potential applications of these materials are also discussed. Finally, in order to guide research in this ﬁeld, the existing problems and future development prospects are discussed.

The photoisomerization of azobenzene molecules in the excited state is almost instantaneous, but its radiation deactivation is slow [22], which hinders the fluorescence of azobenzene derivatives [23], thus limiting further applications in multifunctional fluorescent probes, chemical sensors, and light-emitting devices. However, the rate of cis-trans isomerization depends on the chemical architecture of the system. Therefore, suppressing the photoisomerization or reducing the efficiency of other non-radiative transitions could enhance the fluorescence of these compounds.
Kunitake and Simomure reported the first fluorescent azobenzene-containing amphiphiles with aqueous-bilayer aggregates in 1987 [24]. Although many azobenzenebased chromophores that exhibit luminescence have been developed through exquisite molecular design, finding new luminous azoderivatives is still challenging. Thus, this field has at-1.
Inhibition of the electron transfer; 3.
Other mechanism.
In this review, we discuss the different mechanisms for producing fluorescent azobenzene derivatives with respect to molecular design. We discuss the range of azobenzene molecules linked with electron acceptors that feature a "push-pull" structure as well as non-planar structures with improved rigidity that suppress PET, ICT, and intramolecular motion. Moreover, some azobenzene aggregates exhibit unique optical properties induced by photoexcitation or selective solvents. In particular, when metal nanoparticles are combined with azo derivatives, enhanced fluorescence can be achieved. The azobenzene compounds exhibiting the strongest fluorescence utilize B-N coordination. In addition, the design of fluorescent azobenzene provides a new insight in the design of smart fluorescence switches, fluorescent materials, and novel fluorescent probes. Here, we discuss their structures, characteristics, and the possible mechanisms involved.
In particular, the sufficient lifetime of cis-azobenzenes, varying with different substituents at the ortho or para position, has been found to be one of the crucial factors favoring fluorescence enhancement (Figure 2c). Han et al. designed and synthesized a series of simple azobenzene derivatives 1 featuring substituents with various electronic properties to study the effects of substituents on the isomerization of azobenzene [50], as shown in Figure 2a. The substitution of electron-withdrawing groups on azobenzenes 1 indeed slowed the isomerization rate but obstructed the azobenzenes from aggregating (Figure 2b). On the other hand, azobenzenes 1 with more electron-donating substituents seemed to form aggregates more easily (Figure 2b), resulting in higher fluorescence quantum yield and obvious red shift of the π-π* transition [56,57]. Although the existence of strong electrondonating substituents leads to a significant increase in the photoisomerization rate, it tips the balance toward fluorescence quenching. If the azobenzene molecules are substituted by electron-donating and electron-withdrawing groups (e.g., -NH 2 and -NO 2 ), respectively, the asymmetric "push-pull" electronic structure will favor higher isomerization rate and shorter lifetime of the cis-isomer [58], which greatly thwarts the fluorescence. Rau and Shen designed a series of azobenzene derivatives 2a-2j containing steric hindrance [53], and found that the azobenzene derivatives 2f-2g (Figure 3), bearing substituents with larger steric hindrance at ortho and para positions, had the lowest E→Z efficiency and showed almost no isomerization transition. The bulky ortho-substituents, such as isopropyl [51], t-isopropyl [52] and decyloxy [54] groups, often sterically restrict the molecular movement required for isomerization, and hence fluorescence emission occurs. Slow thermal back isomerization and green fluorescence have been observed from one steric azobenzene derivative (3) featuring decyloxy groups due to the large ortho substituents [54] (Figure 4a,b). Thereafter, utilizing a series of derivatives of azobenzenes (4) substituted with electron-donating or electron-accepting groups resulted in appreciable change in fluorescence color from blue to yellow (see Figure 4c) [55]. The blue shift of its emission wavelength occurs with the electron-withdrawing ability of the substituent group becoming stronger. The emission wavelength of 4 was regulated by the electronic properties of the substituents, but its emission may be ascribed to the substituents with steric hindrance at the ortho and para-positions of the azobenzene molecules [55].

Inhibition the PET and ICT
Photoinduced electron transfer (PET) occurs between the electron acceptor (which behaves as a fluorophore) and the electron donor (which acts as the receptor unit) in the photoexcited state, which quenches the fluorescence. In a PET fluorescent molecular probe ( Figure 5), once the receptor unit binds to the guest, the PET is inhibited or even completely blocked, and the fluorophore emits fluorescence. The cis-isomers of 5 and 6 have a non-planar structure in the excited state, which limits the effective conjugation between -N=N-and the π electrons of the fluorophore, thus inhibiting electron transfer, albeit without binding to the guest. As shown in Figure 6b, the fluorescence of 5 increases significantly under UV irradiation [59].
Smitha and Asha synthesized a phenylphenol-bearing azobenzene 7 and its corresponding side-chain polymer 8 ( Figure 7a) which exhibits the electron transfer from the N=N to the group of fluorophore, to a certain extent [60]. The unusually intense fluorescence spectra of the small molecule 7 showed that the aggregation peak at 535 nm decreased with continuous UV irradiation, while the emission intensity of the molecularly dissolved species at 390 nm increased, as shown in Figure 7b. Interestingly, the dilute solution of 8 also exhibited enhanced fluorescence under irradiation (Figure 7c), which can be attributed to the inhibition of PET from the cis-isomer with non-planar geometry.  Furthermore, conjugated fluorophores having a strong "push-pull" electron effect mode providing a channel for electron transfer from the electron donor to the electron acceptor upon photoexcitation, which is known as intramolecular charge transfer (ICT), in a manner similar to PET. Mariyappan et al. designed and synthesized 9 and 10 ( Figure 8), which when appended with Zn 2+ , could be used in fluorescent chemosensors [61]. A significant increase in fluorescence intensity was observed, due to the synergy effects of chelation-enhanced fluorescence (CHEF) and ICT. After adding Zn 2+ , the molecular structure of 9 and 10 becomes more planar and rigid, as well as inhibiting the intramolecular charge transfer from azo to hydroxyl groups.

Fluorescence from Aggregation
Extensive studies have revealed that reasonable fluorescence can be found in aggregates, such as micelles [62][63][64][65][66][67][68][69][70], vesicles [71][72][73], etc., of azobenzene derivatives in a manner similar to aggregation-induced emission (AIE), proposed by Tang et al. [74]. Herein, we compared the fluorescence of the AIE phenomenon to that of the aggregates of azobenzene compounds in detail, so as to understand the unusual fluorescence behavior of azobenzene derivatives with different methods of isomerization inhibition.

Fluorescence from Aggregated Azobenzenes without Photo Irradiation
Theoretically, molecules with planar configuration are able to improve the luminous efficiency of molecular fluorescence to a certain extent; however, these molecules are prone to aggregate in face-face stacking, resulting in exciplex formation, which further leads to fluorescence quenching. However, the molecular structures with appropriate steric hindrance and available rotating or vibratory units, such as 1-methyl-1,2,3,4,5-pentaphenylsilole ( Figure 9) [74], are the salient characteristics of AIEgens. Another factor reinforcing the AIE phenomenon is the restriction of intramolecular motion (including intramolecular rotation and intramolecular vibration) in the aggregates. In solution, non-radiative transition is the dominant deactivation process due to the rotational and vibrational motions in the unique AIE molecules, as shown in Figure 9. Upon addition of poor solvents, the solubility decreases, and aggregates start to appear, restricting intramolecular motions. At this point, radiative transition, the luminescence process, becomes the main mode of energy release from the excited state [75]. Following the AIE mechanism, the aggregates of luminous azobenzenes achieved typically rigid molecular structures, and radiative decay was activated because of the restriction of intramolecular motion [76]. Increased fluorescence was achieved with azo compound 11 (Figure 10), the earliest luminescent azobenzene reported, via bilayer aggregates with a head-to-tail orientation [24]. However, fluorescence was significantly lower when the bilayer structure was replaced by a parallel arrangement in C 8 AzoC 10 N + . In AIEgens, the intramolecular motion of dilute solutions and the steric hindrance or non-planar structures of the aggregated state generally prevent intermolecular π-π stacking. In sharp contrast, fluorescence from azobenzene aggregates is more complicated, and π-π stacking could exist in the aggregates [32,71]. Tsuda et al. demonstrated that dendrimer azobenzene 12 can self-assemble to form a large bilayer of vesicles stabilized by the π-π stacking of azobenzene moieties, leading to fluorescence, which was not observed in the previously reported AIEgens [71] (Figure 10). Motivated by the AIE phenomenon, the aggregation luminescence of azobenzenes has been extensively investigated [77][78][79][80]. Three fluorescent azo-phenol boron dipyrromethenes (BODIPYs), which have excellent fluorescent properties and are very amenable to modification [80][81][82][83], exhibited time-dependent AIE enhancement [77]. Nanoaggregates of 13b and 13c (Figure 11) initially demonstrated weak fluorescence after the addition of water, while favorable time-dependent fluorescence results were achieved. However, increasing the water content to >70% almost resulted in the elimination of the luminescence of 13a in a manner similar to aggregation-caused quenching. Han et al. discovered the luminescent structure of azo nanofiber 14 ( Figure 11) with multiple responses [78,79], and demonstrated AIE enhancement in polymer matrices (poly(methyl methacrylate) and poly(4-chlorostyrene)). These two examples demonstrate that the presence of ortho and para phenol groups may lead to the existence of an emissive tautomeric hydrazone form that restricts cis-trans isomerization [23,60,84].
As we have shown in our previous publications, a series of azopyridine-containing three-arm star salts nTBMB (15) [85] that exhibit a typical AIE effect upon addition of a poor solvent (Figure 11b,c), such as H 2 O, were developed. The photoisomerization of 15 was completely blocked because of the formation of aggregates due to the high polarity of the water molecules and H bonding between H 2 O and cationic azopyridinium moieties.
In addition to multiazobenzene-containing compounds, several other azobenzenecontaining polymers that exhibit AIE fluorescence ( Figure 12) have been reported. Zhu et al. fabricated the side-chain azoindole polymer (16), which exhibited strong fluorescence emission in a mixture of H 2 O and THF and when irradiated with UV in CH 2 Cl 2 [86]. Fluorescence upon water addition was attributed to AIE, while the emission induced by UV may be caused by the inhibition of the PET of cis-16. The block copolymer (17), which belongs to the AIE system, easily self-assembles into luminescent vesicles in water because of its amphiphilic properties, which limit intramolecular motion owing to the tight stacking of the azo groups in the vesicles [72,73]. Azobenzene-containing side-chain alternating amphiphilic copolymer 18 features acidity-enhanced AIE in micelles because of the special alternating amphiphilic topology [87].  Fluorescence of azobenzene polymers has attracted considerable research attention because such polymers are applied as potential drug delivery carriers and fluorescent probes for living systems and possess smart multiple-response mechanisms that allow them to out-perform macromolecules. The azobenzene-containing amphiphilic polymers 19-30 (Figures 13 and 14), bearing long-chain ester groups between the polymer main chain and the azobenzene part, formed micelles in the presence of selective solvents [62][63][64][65][66][67][68][69][70]88]. Such polymers exhibit luminescence possibly because the azobenzene groups are confined to the core region of the micelles. In contrast, 25 and 26 may produce fluorescence because of the presence of the poly(p-phenylenevinylene) main chain in the place of the azobenzene moiety [88]. Owing to the diverse plasticity of polymers, several multi-response fluorescent azobenzene polymers have been designed [69,70,72,73,89] (Figure 14). Azobenzene-containing monomer 27 and its homopolymer 28 exhibited fluorescence in response to UV radiation and pH changes in the mixtures of H 2 O and DMF [69,70]. In addition, multi-response fluorescence (due to pH and UV radiation changes) has also been reported in sphere micelles from another cyclic azobenzene-containing amphiphilic diblock copolymer 30 [89]. Fluorescence enhancement from micelle-like structure of block copolymer 29 has been observed. This was believed to be due to H bonding with azo carboxylic acid in response to changes in pH, UV radiation, and temperature [70]. The strong H bonding resulted in the formation of a tight micellar structure and considerable fluorescence under acidic conditions, while the response was adverse under basic conditions (Figure 14b). However, upon UV irradiation, the tighter stacking of the cis-isomer led to an increase in fluorescence under neutral and basic conditions (Figure 14c.). Moreover, as the temperature rose above lower critical solution temperature, 29 underwent a thermal-induced transition from coil to sphere, which limited the rotation of the carboxyl azobenzenes and, thus, enhanced the fluorescence emission intensity (Figure 14d).

Light-Driven Self-Assembly and Fluorescence Enhancement
Not only can aggregation occur spontaneously, but some nonfluorescent azobenzene derivatives 1, 3, 4, 31-33 can self-assemble under photoirradiation into luminous spherical aggregates in their cis state ( Figure 2, Figure 4, and Figure 15) [50,54,55,90,91]. The curved cis-isomer has higher polarity and hydrophilicity than the rod-shaped transisomer; in contrast, the long alkyl chains at para position of the azobenzene impart inherent hydrophobicity, resulting in the strong fluorescence due to spontaneous formation of spherical organic aggregates. A long-lived crystalline aggregate of striking fluorescent azobenzene derivative 34 was fabricated by Han et al. [32]. Unlike the luminescence of the previously mentioned AIEgens, the aggregates lying in their strong π-π stacking between aromatic rings (Figure 16), as well as the van der Waals interaction between the long alkyl chains, significantly promoted the stacking of cis-34. Guo et al. observed similar light-driven fluorescence enhancement in 35, which is named AOB-t8 (Figure 17a) [92]. The cis-AOB-t8 solution exhibited 306-times higher fluorescence intensity than the trans-AOB-t8 solution (Figure 17b), because of the significant structural mutation from fiber-like H-aggregation in trans-AOB-t8 to layered J-aggregation in cis-AOB-t8 (Figure 17c,d). From small molecules to polymers, azobenzene-containing compounds can exhibit notable fluorescence behaviors either in a selective solvent or upon light irradiation.

Enhanced Fluorescence in Special Cases
Metal-enhanced fluorescence (MEF) refers to fluorescence enhancement of dyes in the vicinity of free or immobilized metal particles [93]. Our previous work observed that the fluorescence of azopyridine 36 (AzoPy) was enhanced upon addition of silver nanoparticles ( Figure 18) [94]. Furthermore, when Br 2 was inserted into the AzoPy molecule and Ag NPs, the MEF effect became more intense. Compared with Ag···AzoPy, Ag···Br-Br···AzoPy showed three-times greater fluorescence emission enhancement owing to the extended distance between the azopyridine molecule and the Ag NP surface due to the added Br 2 .
Studies have demonstrated that azobenzene-containing heteroatomic coordination has a similar locking effect. In a series of organometallic azobenzene Pd(II) complexes 44 ( Figure 20) [102][103][104][105], cyclopalladation increases the rigidity of the azo bonds, thus reducing the efficiency of photoisomerization and leading to fluorescence.   The lone pair electrons of the N atom in boron-substituted azobenzenes 45-47 can be used to form an N-B coordination bond, and the N-B action restricts the rotation of the N-C bond, thus inhibiting photoisomerization. In addition, the N-B interaction perturbs the orbital energy levels, leading to significant fluorescence during the relaxation process of S 1 -S 0 [106][107][108][109][110]. Kawashima et al. reported the most intensely flu-orescent boron-substituted azobenzene (45), taking advantage of the B-N interaction ( Figure 20) [57,[106][107][108][109]. The B-N-coordinated structure is extremely compact, imparting extraordinary rigidity to the molecule, which considerably inhibits any conformational change of the azobenzene that might prevent the photoisomerization process. Hence, 45 exhibits intense green fluorescence with significantly high quantum yield.

Conclusions and Outlook
This paper reviewed methods that achieve luminescence in azobenzene, including those involving orthoor para-substitution to equalize the rate of photoisomerization, inhibiting various possible non-radiative transitions (such as PET and ICT) and aggregation. It is possible to design a PET-type azobenzene molecule and then inhibit the PET moiety by inducing cis-isomerization through UV irradiation; the azobenzene molecules with a push-pull electronic structure are designed to match the metal ions that inhibit the ICT of azo molecules. These two mechanisms enable switchable fluorescence in azobenzene compounds. Studies have demonstrated that azobenzene exhibits AIE due to optical drive or in the presence of selective solvents. In particular, the azo molecules compounded with metal nanoparticles also exhibit enhanced fluorescence because of the MEF effect. Strong intermolecular forces such as those induced by H bonding, cyclic structures, metal coordination, and B atom coordination can be utilized to lock the azobenzene molecular structure, thus inhibiting its isomerization and causing fluorescence. Luminescence in azobenzene is not always caused by a single factor; it is often the result of multiple mechanisms. Although several cases of luminescence in azobenzene have been described herein, the luminescence efficiency in most of these examples is more limited compared to that of other existing organic fluorescent materials; consequently, improving the luminescence efficiency of azobenzene compounds is one of the main research goals of researchers in this field at present. This review highlights the close relationship between the molecular structures of azobenzenes and their fluorescence mechanisms to inspire research of fluorescent azobenzenes and to provide a detailed understanding of their remarkable luminescence capabilities.