**2. Luciferin analogs of firefly luciferase**

Many researchers have synthesized luciferin analogs, and different substrates reacted with luciferases exhibit different luminescence activities [22–24]. Most luciferin analogs are formed by modifying the benzothiazole moiety of **1**. Analogs of **1** were first synthesized by White *et al*. in 1966. They showed that aminoluciferin (**2**, **Figure 1**), in which the hydroxyl group of benzothiazole is replaced with an amino group, can function as a substrate of Fluc and emit red bioluminescence [25].

## **2.1 Development of luciferin analogs based on LH2**

As mentioned above, many luciferin analogs are prepared by modifying the benzothiazole moiety of **1** [22–24]. For instance, *N*-cycloaminoluciferins (**2a–f**, **Figure 2A**) are prepared by cyclizing the NH2 of **2**. These analogs were reported by two independent groups, who synthesized them by different routes [26, 27] (**Figure 2B**–**C**). When reacted with Fluc, **2a–f** show longer wavelengths than **1**, probably reflecting the electron donation effect of cycloamine substitutes. Comparing the bioluminescence activities and emission wavelengths of analogs **2e** and **2f** on Fluc and Fluc mutant luciferase R218K, it was found that **2e**/Fluc and **2f**/R218K produced light at 604 and 614 nm, respectively, whereas **2e**/R218K and **2f**/Fluc produced no light [26]. The interaction between the active site of luciferase and the substrate is very critical, indicating that the structures of both reactants play essential controlling roles in luminescence activity.

Miller *et al*. synthesized CycLuc1 (**7a**, **Figure 3**) by fusing *N*-cycloalikylation of **2** with benzothiazole [28]. Analog **7a** exhibited a longer luminescence wavelength on Fluc (599 nm) than **1** on Fluc, and was emitted more intensely than **1** in a *Photuris pennsylvanica* firefly luciferase mutant (Ultra-Glo). The BLI of **7a** detects the signals from deep organs such as brains and lungs [21, 29]. Li *et al*. synthesized CybLuc (**7b**, **Figure 3**) by substituting the hydroxy group of **2** with a cycloamino group. Analog **7b** produced light at 603 nm and its BLI detected the signals from mouse brain [30].

Iwano *et al*. developed luciferin analogs **8a–g** (**Figure 4A**) by substituting the benzothiazole moiety of **1** with a simple benzene ring and extended π-conjugations [31]. Olefins were extended by the Wittig reaction from **10c–d** and **12e–f** as starting *Near-Infrared Luciferin Analogs for* In Vivo *Optical Imaging DOI: http://dx.doi.org/10.5772/intechopen.96760*

#### **Figure 2.**

*Structures and synthetic routes of luciferin analogs 2a–f. (A) N-cycloaminoluciferin analogs 2a–f, (B) the synthetic route reported by Miller et al. [28], and (C) the synthetic route reported by Hirano et al. [29].*

#### **Figure 3.** *Structures of CycLuc1 (7a) and CybLuc (7b).*

materials. In this synthesis, hydrolysis was stepwise followed by condensation with *d*-Cys(STrt)-OMe, thiazoline cyclization, and methyl ester deprotection (**Figure 4B**). The obtained analogs **8a–f** produced luminescent colors over a wide range (blue to red) [31]. Among these, AkaLumine (**8e**), which produces light at 675 nm, is a leading compound for NIR luciferin analogs, as described in Section 2.2. Later, analog **8e** was used as a reagent for BLI. In the same paper, 3-hydroxyl analog **8g** (**Figure 4A**) was also synthesized, but this analog produced no light [31]. Therefore, the position of the OH substituent is critical in the firefly bioluminescence reaction.

In contrast, the thiazoline site is rarely modified. Conley *et al*. synthesized a seleno-aminoluciferin analog **13a** (**Figure 5A**) in which the S of the thiazoline ring of **2** was replaced with Se [32], and Ioka *et al*. synthesized O- or C- substituted analogs **13b–c** (**Figure 5A**) [33]. Analog **13a**, which produced light at 600 nm, was synthesized by the cyclization reaction of selenocysteine (**Figure 5B**) [32]. Analog **13b** was obtained by synthesizing an amide **16b** synthesizing an amide from *d*-serine, cyclizing it with diethylaminosulfur trifluoride (DAST), and hydrolyzing it with Amano lipase (**Figure 5C**). Analog **13c** was prepared by coupling with bromothiazole **19** and pyrrolidione carboxylate **21** to form glutamate-linked benzothiazole **16c**, and cyclizing **16c** with trifluoroacetic acid (TFA) (**Figure 5D**). Interestingly, **13c** produces light at 547 nm, whereas **13b** is non-bioluminescence [33] but shows chemiluminescent ability. This result indicates that the thiazoline of **1** is an essential moiety for recognizing the activity site in luciferase.

**Figure 4.** *Structures of 8a–g (A) and their synthetic routes 8a–f (B).*

#### **2.2 Structure–activity relationships for developing NIR luciferin analogs**

Based on these structure–activity relationships, additional luciferin analogs have been designed and synthesized for NIR light production. For example, Anderson *et al*. synthesized iLH2 (**22**, **Figure 6**) by inserting an olefin into the structure of **1**. Analog **22** produced NIR light at 706 nm [34]. However, the luciferase used at that time was a mutant (S284T), and the luminescence wavelength on Fluc was 670 nm. The same authors developed an *in vivo* dual-imaging technique that combines **1** and **22** with two different luciferases. This system can potentially observe new biological events by tracking two processes simultaneously [35]. Hall *et al*. synthesized NH2–NpLH2 (**23**, **Figure 6**) by extended conjugation of **2**. Analog **23** produced no light with Fluc, but its luminescence wavelength was extended to 743 nm by reaction with CBR2, a mutant luciferase of click beetles (*Pyrophorus plagiophthalamus*) [36]. All of these studies achieved long-wavelength emissions from mutant luciferases, but their luminescence activity is much lower than that of combinations of **1** and wild type *Ppy* luciferase.

*Near-Infrared Luciferin Analogs for* In Vivo *Optical Imaging DOI: http://dx.doi.org/10.5772/intechopen.96760*

**Figure 5.** *Structures of 13a–c (A) and their synthetic routes 13a (B), 13b (C) and 13c (D).*

#### **Figure 6.** *Structures of NIR luciferin analogs iLH2 (22) and NH2-NpLH2 (23).*

Meanwhile, Maki's group has developed a number of analogs based on the structure of **8e**, which are expected to produce NIR light. Miura *et al*. formed a mother skeleton by a coupling reaction, and thus synthesized biphenyl analogs **24a–c** (**Figure 7** and **8A**) [37]. Analog **24a** produced light at 675 nm, but the luminescence intensity was weak. Although its conjugation was more extended than in **8e**, the luminescence wavelength of **24a** did not change as that of **8e** (675 nm). This result suggests that the biphenyl moiety rotates and reduces the fluorescent intensity.

Kiyama *et al*. synthesized cyclic amino analogs of **8e** (**25a–d**, **Figure 7**) [38] from 4-fluorobenzaldehyde **32** as the starting material. They replaced the F group with various secondary amines, and conducted the Horner–Wadsworth–Emmons

**Figure 7.** *Structures of NIR luciferin analogs 24–29.*

reaction, condensation and cyclization to obtain the final compounds **25a–d** (**Figure 8B**). Despite containing an electron-donating amino group, **25a–d** produced luminescence at almost the same wavelengths (656–667 nm) as **8e** (668 nm). However, the luminescence intensity of **25a** was approximately four times stronger than that of **8e**. The fluorescence quantum yields of **8e** and these cyclic amino analogs **25a–d** were almost identical, suggesting that the luminescence intensity largely depends on the reactivity with luciferase.

The luminescent wavelength can be lengthened not only by extending the π-conjugations and introducing an electron donate substituent, but also by introducing an allyl group. Kitada *et al*. synthesized allyl analogs **26a–b** (**Figure 7**) by introducing allyl groups into **8c**, **8e** and naphthol analogs **27a–d** (**Figure 7**). The analogs were introduced by two routes: Pd-catalyzed Stille coupling (**Figure 8C**) and Claisen rearrangement (**Figure 8D**) [39]. Although these analogs delivered very low luminescence intensities, their wavelength shift was long (approximately 15–35 nm). As the allyl group itself does not affect the π-conjugations of the substrate structure, it was considered that induce fitting was occurred at the luciferase active site and stabilized the substrate metabolite to lower energy state conformation. To develop a long-wavelength, Kitada *et al*. synthesized NIR analog (**28** in **Figure 7**) by introduced both an electron-donating NMe2 and an allyl group. When reacted with Fluc, **28** produced NIR light at a sufficiently long-wavelength (705 nm), but the luminescence intensity was only 1.3% of that of **8e**. Although the allyl group extends the luminescent wavelength, it greatly reduces the luminescence intensity, which is a major disadvantage.

The aromatic ring site has also been targeted in the development of potential NIR emitters. Saito *et al*. synthesized three analogs **29a–c** (**Figure 7**) in which the *Near-Infrared Luciferin Analogs for* In Vivo *Optical Imaging DOI: http://dx.doi.org/10.5772/intechopen.96760*

**Figure 8.** *Synthetic routes of NIR luciferin analogs 24a–c (A), 25a–d (B), 26a–b (C) and 27a–d (D).*

aromatic ring of **8e** was replaced with an *N*-heteroaromatic ring [40]. Interestingly, the luminescence wavelengths of three analogs depended on the positions and numbers of their N atoms; **29a** produced red light at 645 nm, seMpai (**29b**) produced NIR light at 675 nm, and **29c** produced orange light at 625 nm. This result highlights the importance of interactions between the luciferase active site and the N atoms of the heterocycle. Although the luminescence wavelength of all three analogs were shorter than 700 nm, the wavelength was changed with a single atom, suggesting that interaction with the luciferase active site is an important part of molecular design.
