Development of near‐infrared firefly luciferin analogue reacted with wild‐type and mutant luciferases

Abstract Interestingly, only the D‐form of firefly luciferin produces light by luciferin–luciferase (L–L) reaction. Certain firefly luciferin analogues with modified structures maintain bioluminescence (BL) activity; however, all L‐form luciferin analogues show no BL activity. To this date, our group has developed luciferin analogues with moderate BL activity that produce light of various wavelengths. For in vivo bioluminescence imaging, one of the important factors for detection sensitivity is tissue permeability of the number of photons emitted by L–L reaction, and the wavelengths of light in the near‐infrared (NIR) range (700–900 nm) are most appropriate for the purpose. Some NIR luciferin analogues by us had performance for in vivo experiments to make it possible to detect photons from deep target tissues in mice with high sensitivity, whereas only a few of them can produce NIR light by the L–L reactions with wild‐type luciferase and/or mutant luciferase. Based on the structure–activity relationships, we designed and synthesized here a luciferin analogue with the 5‐allyl‐6‐dimethylamino‐2‐naphthylethenyl moiety. This analogue exhibited NIR BL emissions with wild‐type luciferase (λ max = 705 nm) and mutant luciferase AlaLuc (λ max = 655 nm).


| INTRODUCTION
Firefly bioluminescence (BL) showed light emission caused by the reaction of firefly luciferin (1, Figure 1) catalyzed with firefly luciferase in the presence of Mg 2+ , in Nobuo Kitada and Ryohei Saito contributed equally to this work.
[This article is part of the Special Issue: In honor and memory of Prof. Koji Nakanishi. See the first articles for this special issue previously published in Volumes 31:12, 32:3, 32:4, 32:5, and 32:6. More special articles will be found in this issue as well as in those to come.] which 1 is first adenylated with ATP followed by the oxidative reaction with O 2 to generate oxyluciferin with a yellow-green light (λ max = 560 nm). 1,2 This reaction is termed as the luciferin-luciferase (L-L) reaction. Firefly luciferin 1 and luciferase are biosynthesized in the body of firefly, and 1 has a chiral center at C3 with the same stereochemistry as unnatural D-cysteine (D-form). Interestingly, despite the fact that the L-form of firefly luciferin has significantly low BL activity of L-L reaction 1,3,4 ; however, we reported that the L-form of firefly luciferin is able to produce light by conversion to D-form 1 through the luciferyl-CoA under the action of luciferase. 5 The L-L reaction is applied to optical imaging techniques in the fundamental research fields of medical and biological sciences. [6][7][8] One of the solutions to improve optical in vivo imaging technique is an increase in the permeability of light from deep site of biological tissue. Because the permeability of near-infrared (NIR) light is higher than that of visible light (450-600 nm) for biological tissue, 9,10 researchers have been engaged in developing luciferin analogues 11,12 and mutant luciferases 13 producing NIR light by the L-L reactions. These luciferin analogues and mutant luciferases successfully enabled high-resolution optical in vivo imaging compared with the use of the wild-type luciferin 1 and luciferase. Our group developed luciferin analogues producing light with various wavelengths, [14][15][16][17] and some of the analogues were tested for in vivo experiments. Then, we confirmed that the analogues enabled to detect light emission from the deep target tissue of mice with high sensitively. [18][19][20] In addition, Aka-BLI, which is the combination of a NIR luciferin analogue, TokeOni (2, Figure 1) with a mutant luciferase, Akaluc, produced NIR light and made it possible to detect the BL emission from the brain in a marmoset. 21 Although, there are a number of luciferin analogues, only limited analogues can produce NIR light (over 700 nm) reacted with wild-type luciferase. To design a new luciferin analogue, we have evaluated a structure-BL activity relationship of our luciferin analogues for the wavelength of L-L reaction with wild-type luciferase ( Figure 2). 14,17 One conclusion of the evaluations lead us to design analogue 3 based on the data of 2, 4, and 5. The BL emission maximum (λ BL ) of 2 with the dimethylamino group is 35-nm red shifted from that of 4 with the hydroxyl group, although 2 and 4 have the common phenyl-1,3-butadiene structure. 14 When 4 and 5, both of which contain the hydroxyl group, are compared, the λ BL of 5 is 50-nm red shifted from that of 4. 17 Hence, we designed 3 to have the 5-allyl-2-naphthylethenyl moiety and a dimethylamino group at C6. The structure-BL activity relationship predicts that the λ BL value of 3 will be 725 nm. In this report, we prepared 3 and investigated its BL activity with Photinus pyralis (Ppy) luciferase and Akaluc, 17 comparing its properties to those of 1, 2, and 5.

| General
Commercially available reagents and solvents were used without further purification. For bioluminescence measurements, TokeOni (2) was provided by Kurogane Kasei Co., Ltd. and recombinant Ppy luciferase (QuantiLum ® recombinant luciferase, E1701, Promega) was used. Wako Silica gel 70 F254 thin-layer chromatography plates were used for analytical thin-layer chromatography, and Kanto Chemical Silica gel 60 N (spherical, neutral) was used for column chromatography. For preparative flash chromatography, an automated system (Smart Flash EPCLC AI-580S, Yamazen Corp., Japan) was used with universal columns of silica gel. Melting points were measured with a Yanaco MP-500P. IR spectra were obtained with a Nicolet 6700 spectrometer with an attenuated total reflection attachment. 1 H and 13 C nuclear magnetic resonance (NMR) spectra were recorded on a JEOL ECA-500 instrument (500 MHz for 1 H and 126 MHz for 13 C). High-resolution electrospray ionization mass spectra were obtained with a JEOL JMS-T100LC mass spectrometer. The optical purities of NIR analogue 3 was analyzed by high-performance liquid chromatography (HPLC; Agilent 1100 series) using a Daicel chiral column (Daicel Chemical Industries, OD-RH, 5 μm, 4.6 × 150 mm, flow rate 0.5 ml/min). Bioluminescence spectra were measured with an ATTO AB-1850 spectrophotometer. Bioluminescence intensities were monitored using an ATTO AB-2270 luminometer. Density functional theory (DFT) calculations were performed with the Gaussian 09 program (Rev. D.01). 22 DFT included the B3LYP function with the 6-31 + G(d) basis set. [23][24][25] Molecular graphics were prepared with GaussView, Version 5. 26 2.2 | Synthesis of NIR analogue 3

| Dimethylamine 3c
To a solution of bromoamine 3b (2.39 g, 8.54 mmol) in tetrahydrofuran (30 ml), sodium cyanoborohydride (2.63 g, 41.9 mmol) and formaldehyde (35% in H 2 O, 15 ml, 195 mmol) were added, and the mixture was stirred in an ice bath. The mixture was slowly added to acetic acid (4 ml, 70 mmol) and stirred for 14 h. To the reaction mixture, saturated NaHCO 3 aqueous solution (100 ml) was added to quench the reaction. Further, the reaction mixture was diluted with water and extracted with ethyl acetate (3 × 100 ml). The combined organic layers were dried over Na 2 SO 4 , filtered, and the solvents was removed under reduced pressure. The obtained residue was purified by silica gel column chromatography (hexane only to hexane/ethyl acetate = 3/1) to yield dimethylamine 3c (912 mg, 2.96 mmol, 35%) as a white solid: 1

| Allyl alcohol 3e
A solution of allyl dimethylamine 3d (2.15 mg, 7.99 mmol) in dry toluene (30 ml) under Ar at 0 C was slowly added 1.0-M diisobutylaluminium hydride (DIBAL-H) in toluene (16.0 ml, 16 mmol), and the mixture was stirred for 1 h at r.t. Then to the reaction mixture was added 1-M hydrochloric acid (10 ml). The mixed solution was extracted with ethyl acetate (3 × 100 ml). The combined organic layers were dried over Na 2 SO 4 , filtered, and the solvent was removed under reduced pressure. The obtained residue was purified by silica gel column chromatography (hexane/ethyl acetate = 3/1) to yield alcohol 3e (1.65 mg, 6.83 mmol, 85%) as a colorless oil: 1 13

| Allyl aldehyde 3f
To a solution of alcohol 3e (1.46 mg, 6.07 mmol) in dichloromethane (50 ml), Dess-Martin periodinane (2.71 g, 6.39 mmol) and pyridine (1.0 ml, 12 mmol) were added, and the mixture was stirred for 5 h at r.t. The reaction mixture was diluted with water and extracted with chloroform (3 × 100 ml). The combined organic layer was dried over Na 2 SO 4 , filtered, and the solvent was removed under reduced pressure. The obtained residue was purified by silica gel column chromatography (hexane/ethyl acetate = 5/1) to yield allyl aldehyde 3f (477 mg, 2.00 mmol, 33%) as a yellow oil: 1

| Carboxylic acid 3h
A solution of allyl ethyl ester 3g (198 mg, 0.642 mmol) in 2-propanol (4 ml) was added 1-M NaOH aq. (2 ml), and the mixture was heated at reflux for 3 h. After cooling, the reaction mixture was neutralized by adding 1-M HCl aq. The mixed solution was extracted with ethyl acetate (3 × 100 ml). The combined organic layers were dried over Na 2 SO 4 , filtered, and the solvent was removed under reduced pressure to give carboxylic acid 3h (148 mg, 0.525 mmol, 82%) as a green-yellow solid: 1

| Thiazolidine ester 3j
To a solution of trifluoromethanesulfonic anhydride (Tf 2 O) (0.30 ml, 1.8 mmol) in dichloromethane (5 ml), a solution of amide 3i (540 mg, 0.843 mmol) in dichloromethane (5 ml) was added under Ar at 0 C, and the mixture was stirred for 10 min. Saturated NaHCO 3 aq. was added to the reaction mixture for neutralization. The product was extracted with chloroform (3 × 50 ml). The combined organic layer was dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure. The crude products were purified by silica gel column chromatography (hexane/ethyl acetate = 5/1) to yield thiazolidine ester 3j (122 mg, 0.322 mmol, 39%) as orange oil: 1 13

| Synthesis of luciferin analogue 3
Analogue 3 was prepared according the procedure as shown in Scheme 1. The synthesis of 3 was started from bromination of commercially available methyl ester 3a to obtain 3b. Dimethylation of 3b followed by allylation yielded 3d. Alcohol 3e was prepared from 3d via diisobutylaluminium hydride (DIBAL-H) reduction. Allylaldehyde 3f was prepared via oxidation of 3e, Finally, acid hydrolysis of 3j produced target analogue 3.
3.2 | Bioluminescence activity of analogues 3 and 5 BL activity and emission spectrum of 3 together with those of 1, 2, and 5 were investigated with wild-type recombinant Ppy luciferase and a mutant luciferase, Akaluc (Table 1 and Figure 3).
Before investigating BL properties, the D-and L-forms of 3 were separated by HPLC with a chiral octadecylsilane column, and their fractions were screened for BL measurements. The D-form of 3 showed sufficient luminescence with Ppy luciferase, whereas the L-form of 3 showed negligible luminescence similar to the background (Table S1). Similar to wild-type luciferin 1, NIR luciferin analogue produces light only in the D-form and not in the L-form. We used only D-form of 3 for the following experiments. The light intensity (Rel. Int.) obtained through the L-L reaction with Ppy luciferase during the initial 600 s for 3 was 1.3% as a relative value compared with that for 1 (Table 1), and the Rel. Int. value was similar to that of 5 (0.8%). The light intensity of 3 with Akaluc was weaker than that with Ppy luciferase and could not be determined relative intensity. These results indicate that 3 and 5 have weak BL activities compared with 2.
The λ BL value of 3 was recorded at 705 nm with Ppy luciferase (Table 1 and Figure 3), which red shifted from that of 5 (690 nm). The λ BL values of 3 and 5 are 135 and 120 nm longer than that of 1, respectively, and even 30 and 15 nm longer than that of 2, respectively. On the other hand, the emission spectra of 3 measured with Akaluc showed the λ BL value at 665 nm (Table 1 and Figure 3), which is red shifted by 15 nm compared with that of 2. Also, the λ BL value of 5 reacted with Akaluc was observed at 660 nm that is same as that of 2. To investigate the cause of the variation in λ BL values for 3 and 5, chemiluminescence reaction of the methyl esters of 1-3 and 5 were performed in DMSO containing t-BuOK under air. The chemiluminescence emission maxima (λ CL ) of 1-3 and 5 were observed at 595, 685, 685, and 620 nm, respectively (Table 1 and Figure S1). The λ CL value of 3 is same as that of 2, and the λ CL value of 5 is blue shifted by 65 nm compared with that of 2.
4 | DFT AND TIME-DEPENDENT DFT CALCULATIONS FOR OXY-2 , OXY-3 , AND OXY-5 To further evaluate the observed λ BL and λ CL values for 3, the electronic properties of the oxyluciferin form of 3 (oxy-3) together with that of the oxyluciferin form of 5 (oxy-5) were investigated using DFT and timedependent DFT (TD-DFT) calculations with the B3LYP/6-31 + G(d) method. Prior performing a search for the most stable optimized structures of oxy-3 and oxy-5, we found the most stable optimized structures of the luciferin forms 3 and 5. We then used the structures of 3 and 5 shown in Figure 4 as the basis for starting conformations of oxy-3 and oxy-5 for further calculations because the structures of 3 and oxy-3 have steric hindrance between the allyl and dimethylamino groups, and their dimethylamino groups are twisted and pyramidal. Next, we analyzed the electronic transition properties of the oxyluciferin forms (Table 2). In the case of oxy-5, the phenolate anion and its sodium salt model were calculated in the manner similar to the previous literature. 2 Table 2 summarizes vertical excitation energies (E ex ), excitation wavelengths (λ ex ), oscillator strengths (f ), and configurations of the allowed transitions to the excited singlet states with the lowest energies for oxy-3, oxy-5 (phenolate), and oxy-5(ONa) together with those for oxy-2. 16 The S 0 ! S 1 transitions of oxy-3 and oxy-5(phenolate) are π, π* transitions corresponding to the highest occupied molecular orbital (HOMO) ! lowest unoccupied Time-dependent density functional theory calculation data for oxy-2, oxy-3, and oxy-5

Compound
Transition  16 molecular orbital (LUMO) configuration and the S 0 ! S 2 transition of oxy-5(ONa) is a π, π* transition corresponding to the HOMO ! LUMO + 1 configuration. Although the λ BL value of 3 with Ppy luciferase is red shifted from that of 2, the λ ex values of oxy-2 and oxy-3 are similar. Results indicate that λ BL values were mainly determined by the effect of the active site of Ppy luciferase to stabilize the excited states of oxy-2 and oxy-3.
Because the HOMO-LUMO transition of oxy-2 has charge-transfer character, the S 1 state is more highly polarized than the ground state. 20 The HOMO and LUMO of oxy-3 have primary electronic distributions at the (6-dimethylaminonaphtalenyl) and 2-ethenyl-1,-3-thiazolone moieties, respectively (Figure 4), indicating that the HOMO-LUMO transition of oxy-3 also has charge-transfer character. Thus, the S 1 state of oxy-3 also has polarized character. The environment surrounding the excited oxy-3 in Ppy luciferase will be more polar than that surrounding the excited oxy-2, resulting in the redshifted λ BL value of 3. The electronic distributions of the HOMO and LUMO of oxy-3 indicate that the allyl group has no contribution to the π electronic conjugation. The calculations showing that the λ ex values of oxy-5(phenolate) and oxy-5(ONa) are red shifted from that of oxy-3 are opposite to the λ BL data with Ppy luciferase and Akaluc. Although the oxido (O − ) group of oxy-5(phenolate) and oxy-5(ONa) has the potential to donate more electron density than that of the dimethylamiono group of oxy-3, the anionic property of the oxido group in the luciferase active site may be weakened.

| CONCLUSION
We synthesized luciferin analogue 3 and investigated their luminescence properties. The λ BL values for 3 upon reaction with Ppy luciferase and mutant luciferase Akaluc were 705 and 665 nm, respectively. Furthermore, the results of BL and TD-DFT calculations suggest that the allyl group of 3 induced the excited oxy-3 to be more stable in the active site of luciferase, thus increasing the λ BL value of 3 to over 700 nm. A λ BL value of over 700 nm is quite noteworthy; however, the intensity of 3 was very weak compared with those of 1 and 2. We should modify the new analogue design to produce a higher bioluminescence intensity for animal experiments.