JP2016050296A - Fluorochrome-coupled coelenterazine - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide techniques of making the light emission wavelength of a Renilla luciferase (Rluc)-coelenterazine light-emitting system longer by modifying coelenterazine.SOLUTION: A fluorochrome-coupled coelenterazine is represented by the general formula [I], where A is a fluorochrome moiety including a fluorochrome.SELECTED DRAWING: Figure 3

Description

  The present invention relates to coelenterazine to which a fluorescent dye is bound.

  In recent years, optical in vivo imaging has attracted attention as a technique widely used for elucidating diseases by visualizing life phenomena in the living body with light. Imaging using light is relatively inexpensive compared with other imaging methods such as MRI, and has advantages such as high sensitivity and non-invasiveness. As light used for imaging, a region of 600 nm to 900 nm called near infrared light is suitable. This is because the absorption coefficient of hemoglobin or water, which is a molecule in the living body, is small in the near-infrared region, and the influence of autofluorescent substances in the living body is small. This is because small and highly sensitive measurement can be performed.

  There are fluorescence, chemiluminescence, and bioluminescence as types of light used in in vivo imaging. Among these, bioluminescence has an advantage that a quantum yield is relatively high without using an excitation light source. The most useful in bioluminescence is the firefly luciferin-luciferase reaction. The emission of firefly luciferin has the highest quantum yield of 0.41 among bioluminescent species, and emits light at a relatively long wavelength of 560 nm, so it is often used for in vivo imaging. However, since firefly luciferin does not occur unless cofactors such as magnesium ion and ATP are present, this point is an adverse effect when applied to in vivo imaging.

  On the other hand, coelenterazine, which is a bioluminescent substrate derived from Renilla and Owan jellyfish, is also known. Unlike firefly luciferin, coelenterazine has been reported to emit light even in the absence of magnesium ions and ATP. However, since coelenterazine has a maximum emission wavelength peak of about 480 nm, in order to apply it to in vivo imaging, it is required to extend the emission wavelength to the near infrared region.

  Coelenterazine-v in which the structure of coelenterazine is modified in order to shift the emission wavelength of coelenterazine to the longer wavelength side is known (Non-Patent Document 1). In coelenterazine-v, the emission wavelength shifts to the 40 nm long wavelength side, but stability is difficult, and it is difficult to use for in vivo imaging.

  On the other hand, the structure of Renilla luciferase (Rluc), an enzyme using coelenterazine as a substrate, has been studied for its structure, and Rluc8 (Non-patent Document) has been improved in vivo stability by substituting 8 amino acids in its amino acid sequence. 2) was developed, and its active site was also clarified (Non-patent Document 3). Furthermore, Rluc8.6, which further improved in vivo stability by replacing 6 amino acid sequences in Rluc8, was also developed (Non-patent Document 3). Patent Document 4). Furthermore, since the active site of Rluc has been clarified, the region that does not affect the enzyme activity has also been clarified. Therefore, BRET (Bioluminescence Resonance Energy Transfer: Bioluminescence Resonance Energy Transfer) is bound by binding a fluorescent dye to Rluc and its variants. ) And FRET are also used to increase the emission wavelength (Non-Patent Document 5).

Galina. A. et al., Anal. Bioanal. Chem., 2010, 398, 1809-1817 Loening A. M., et. Al., Protein Eng. Des. Sel., 2006, 19, 391 Loening A. M., et. Al., J. Mol. Biol., 2007, 374, 1017 Loening A. M., et. Al., Nat. Methods, 2007, 4 (8), 641 Chen F. Q. et. Al., J. Chem. Soc. PERKIN TRANS. 1, 1992, 1607

  An object of the present invention is to provide a technique for extending the wavelength of light emitted by the Rluc-coelenterazine light-emitting system by modifying coelenterazine.

  The inventors of the present application considered that it may be possible to cause BRET by binding a fluorescent dye to coelenterazine, thereby making the emission wavelength longer than the emission wavelength of coelenterazine. As described above, an example in which a fluorescent dye is bound to the enzyme Rluc has been reported so far, but an example in which a fluorescent dye is bound to coelenterazine has not been reported. However, as is well known, since the substrate specificity of an enzyme is generally very high, it is expected that if a large structure such as a fluorescent dye is bound to coelenterazine, an enzyme reaction may not occur due to the high substrate specificity. In fact, when a fluorescent dye was bound to the hydroxyl group at the 2-position of coelenterazine, the enzyme reaction itself did not occur as expected. However, without giving up, this time, when a fluorescent dye was bound to the hydroxyl group at the 6-position of coelenterazine, the enzyme reaction did not occur with the enzyme Rluc. Surprisingly, Rluc8, which is a variant of Rluc described above, When Rluc8.6 was used, the enzyme reaction occurred to a sufficient extent, and it was found that the emission wavelength could be increased by BRET, and the present invention was completed.

  That is, the present invention provides a fluorescent dye-bound coelenterazine represented by the following general formula [I].

(In the formula [I], A represents a fluorescent dye part including a fluorescent dye).

  The present invention provides a novel Rluc substrate in which the emission wavelength of the Rluc-coelenterazine-based luminescence reaction is shifted to the longer wavelength side. Rluc is suitable as an enzyme label in in vivo imaging due to its low molecular weight, and also has the advantage that it emits light even in the absence of magnesium ions and ATP. The present invention further increases the emission wavelength. Since it is wavelengthized, the present invention is expected to contribute greatly to in vivo imaging.

The chemiluminescence spectrum of the coelenterazine-FITC conjugate (CTZ-6-FITC) synthesized in the following example is shown. It is a figure which compares the measurement result of the bioluminescence intensity of CTZ-6-FITC synthesize | combined with the following Example with the measurement result of CTZ-6-azide. It is a figure which shows the bioluminescence spectrum in the enzyme reaction using the enzyme Rluc or the enzyme Rluc8.6 of CTZ-6-FITC and CTZ-6-azide which were synthesize | combined in the following Example.

  As described above, the fluorescent dye-bound coelenterazine of the present invention has a structure represented by the above general formula [I]. In the general formula [I], A is a fluorescent dye part including a fluorescent dye, and preferably a fluorescent dye part including a fluorescent dye having an absorption wavelength peak in the range of 300 to 600 nm. The fluorescent dye-coupled coelenterazine of the present invention is produced by resonance transfer of fluorescence energy generated in the coelenterazine moiety by an enzymatic reaction with Rluc8 or Rluc8.6 (a recombinant plasmid encoding these can be obtained from Stanford University) to the fluorescent dye. By emitting light (BRET), the emission wavelength is longer than the emission peak wavelength (478 nm) of coelenterazine. As the fluorescent dye that causes such BRET, a fluorescent dye having an absorption wavelength peak in the range of 300 to 600 nm is preferable. Examples of the fluorescent dye having an absorption wavelength peak in the range of 300 to 600 nm include, but are not limited to, the following.

Fluorescein fluorescent dyes (fluorescein isothiocyanate (FITC), fluorescein, carboxynaphthofluorescein, etc.)

Fluorescein isothiocyanate (FITC)

Fluorescein

Carboxynaphthofluorescein

Alexa Fluor fluorescent dye (Alexa Fluor 480 (trade name) and Alexa Fluor 568 (trade name), etc.)

Alexa Fluor 480 (trade name)

Alexa Fluor 568 (trade name)

ATTO fluorescent dyes (ATTO 590 etc.)

ATTO 590

BODIPY fluorescent dye (BODIPY TR-X (trade name), etc.)

BODIPY TR-X

Coumarin fluorescent dyes (Coumarin 1 (trade name) and Coumarin 6 (trade name), etc.)

Coumarin 1 (product name)

Coumarin 6 (product name)

Cy-based fluorescent dye (Cy3, etc.)

Cy3

DY fluorescent dye (DY-405 (trade name), etc.)

HiLyte Fluor fluorescent dye (HiLyte Fluor 555 Labeling Kit-NH2 (trade name), etc.)

IRDye (trade name) -based fluorescent dye (IRDye 650 Alkyne (trade name) and IRDye 750 Carboxylate (trade name), etc.)

IRDye 650 Alkyne (trade name)

Nile Brue

Nile Red

PromoFluor fluorescent dye (PF-405 (trade name), etc.)

Rhodamine fluorescent dye (Rhodamine B, Rhodamine 101, etc.)

Rhodamine B

Rhodamine 101

SYTO fluorescent dye (SYTO 9 Stain (trade name), etc.)

Chlorophyll-based fluorescent dye

A in the general formula [I] is a fluorescent dye part including the fluorescent dye as described above. The fluorescent dye part may consist of only the fluorescent dye, or may include a linker that binds the fluorescent dye to coelenterazine. The linker binds the fluorescent dye and coelenterazine, so the structure is not particularly limited. Any structure that can bind the fluorescent dye and coelenterazine and does not adversely affect BRET is limited. is not. Preferable examples include alkylene groups and those having functional group residues used for bonding with fluorescent dyes at the ends of the alkylene groups. In the following example, the isothiocyanate group of FITC and the hydroxyl group at the 6-position of coelenterazine are bonded via the structure of —NH—CH ₂ —CH ₂ —O— (the rightmost —O— is coelenterazine). The residue of the 6-position hydroxyl group) can be considered as a linker. The size of the linker is not particularly limited, but in order for BRET to occur, the distance between the fluorescent dye and coelenterazine is desirably about 1 nm to 10 nm, so the number of atoms in the main chain of the linker is desirably about 3 to 5. (In the case of —NH—CH ₂ —CH ₂ —O—, the number of atoms in the main chain is 4).

  Since coelenterazine and its production method are also known, and the fluorescent dye and its production method are known, the fluorescent dye-bound coelenterazine of the present invention can be chemically synthesized according to common sense of organic chemistry. The synthesis method of what is FITC is described in detail.

  Since the enzyme Rluc and its modified Rluc8 and Rluc8.6 are used as enzyme labels as is well known, the fluorescent dye-conjugated coelenterazine of the present invention is used as an in vivo imaging as a substrate for Rluc8 and Rluc8.6 in exactly the same manner as coelenterazine. Can be used.

  Hereinafter, the present invention will be specifically described based on examples. However, the present invention is not limited to the following examples.

Example 1 Synthesis of FITC-Coelenterazine A total synthesis scheme of model molecules in which coelenterazine (CTZ) and FITC are combined is shown below. First, coelenteramine modified with an azide group was synthesized, and this was reacted with acetal-protected pyruvic aldehyde to synthesize coelenterazine modified with an azide group. Thereafter, the azide group was converted to an amine group by a hydroreduction reaction, which was combined with the isothiocyanate portion of FITC. As shown below, all syntheses in 12 steps were successful with a total yield of 0.58%. Details of each step will be described.

  Reagents were purchased from Wako Pure Chemical, Kanto Chemical, Tokyo Kasei, or Sigma-Aldrich and used as they were without purification. NMR used ECA500 of JEOL, and tetramethylsilane (TMS, 0 ppm) as an internal standard.

  Under a nitrogen atmosphere, Compound (1) (2.0 g, 21.0 mmol, 1.0 eq.) And THF (25 ml) were placed in a 200 ml three-necked flask and stirred. Next, N-bromosuccinimide (8.2 g, 46.1 mmol, 2.2 eq.) Dissolved in THF (25 ml) was added dropwise at 0 ° C. and stirred at room temperature for 20 minutes.

After completion of the reaction, impurities were filtered through celite. The filtrate was extracted with ethyl acetate, washed with H ₂ O and sat.NH ₄ Cl aq., Dried over Na ₂ SO ₄ and concentrated. Part of the resulting residue was separated and purified by silica gel column chromatography (eluent: n-hexane: ethyl acetate = 7: 3) and concentrated to obtain the target compound (2) (3.16 g, yield: 60%). Obtained as a white solid.

・ TLC (silica gel): R _f = 0.53 (eluent: n-hexane / ethyl acetate = 7/3 (v / v))
・¹ H-NMR (500 MHz, CDCl ₃ , TMS, rt)
δ = 8.04 (s, 1H), 5.05 (s, 2H)

ZnCl ₂ (1.62 g, 11.9 mmol, 3.0 eq.) Was put into a 100 ml three-necked eggplant flask (A), and vacuum-dried in advance. The flask was then charged with Et ₂ O (26.7 ml) and stirred.

Next, THF (5.0 ml) was placed in a 50 ml two-necked eggplant flask that had been degassed in advance, and Benzylmagnesium Chloride 2.0M THF solution (4.37 ml, 8.70 mmol, 2.2 eq.) Was added dropwise thereto. This solution was added dropwise to the three-necked eggplant flask (A). Next, the compound (2) (1.0 g, 3.95 mmol, 1 eq.) Was placed in a 100 ml two-necked eggplant flask and vacuum-dried. Thereto was added THF (7 ml) and stirred, and the solution was added to the three-necked eggplant flask (A). Finally, (Ph ₃ P) ₂ PdCl ₂ (about 1 cup of micro spatula) was added and stirred at room temperature for 17 hours. Vacuum degassing was performed three times before and after the Pd catalyst was added.

After completion of the reaction, impurities were filtered through celite. The filtrate was extracted with ethyl acetate, washed with H ₂ O and sat. NaHCO ₃ aq., Dried over Na ₂ SO ₄ and concentrated. The obtained residue was separated and purified by silica gel flash column chromatography (eluent: n-hexane: ethyl acetate = 9: 1 → 4: 1) to give the target compound (3) (0.617 g, 60% yield) as yellow Obtained as a liquid.

・ TLC (silica gel): R _f = 0.25 (eluent: n-hexane / ethyl acetate = 4/1 v / v)
・₁ H-NMR (300 MHz, CDCl ₃ , TMS, rt)
δ = 8.04 (s, 1H), 7.21-7.34 (m, 5H), 4.37 (s, 2H), 4.09 (s, 2H)

Under a nitrogen atmosphere, put compound (4) (1.0 g, 4.5 mmol, 1.0 eq.), K ₂ CO ₃ (0.82 g, 5.9 mmol, 1.3 eq.), And acetone (20 ml) in a 200 ml two-necked flask. Stir. Next, 1,2-dibromoethane (4.1 g, 22.7 mmol, 22.7 eq.), KI (0.02 g, 0.1 mmol, 0.02 eq.) Dissolved in acetone (20 ml) was added to the reaction system, and 70 Stir at 16 ° C. for 16 hours.

After completion of the reaction, the mixture was extracted with CH ₂ Cl ₂ , washed with H ₂ O and sat. NaCl aq., Dried over Na ₂ SO ₄ and concentrated. The obtained residue was separated and purified by silica gel flash column chromatography (eluent: chloroform) to obtain the target compound (3) (0.446 g, yield 20%) as a white solid.

・ TLC (silica gel): R _f = 0.85 (eluent: chloroform 100%)
・₁ H-NMR (300 MHz, CDCl ₃ , TMS, rt)
δ = 7.68 (d, J = 9.0 Hz, 2H), 6.92 (d, J = 8.4 Hz, 2H), 4.32 (t, J = 5.7 Hz, 2H), 3.70 (t, J = 6.0 Hz, 2H), 1.33 (s, 12H)

In a nitrogen atmosphere, put compound (5) (350 mg, 1.07 mmol, 1.0 eq.), DMF (10 ml), NaN ₃ (83 mg, 1.28 mmol, 1.2 eq.) In a 50 ml two-necked flask at 100 ° C The mixture was stirred for 1 hour.

After completion of the reaction, the mixture was extracted with toluene, washed with H ₂ O and sat.NaCl aq., Dried over Na ₂ SO ₄ and concentrated. Then, the target compound (6) (299 mg, yield 97%) was obtained as a white solid.

・ TLC (silica gel): R _f = 0.63 (eluent: chloroform 100%)
・₁ H-NMR (300 MHz, CD ₃ OD, TMS, rt)
δ = 7.68 (d, J = 9.0 Hz, 2H), 6.93 (d, J = 8.4 Hz, 2H), 4.18 (t, J = 4.5 Hz, 5H), 3.59 (t, J = 4.8 Hz, 2H), 1.33 (s, 12H)

Compound (3) (0.660 g, 2.50 mmol, 1.0 eq.), Compound (6) (1.0 g, 3.46 mmol, 1.6 eq.), Ethanol (5 ml), Toluene (15) in a 100 ml three-necked flask under nitrogen atmosphere ml), and 1 M Na ₂ CO ₃ aq. (7 ml) were added and stirred. Finally, Pd (Ph ₃ P) ₄ (about 1 cup of micro spatula) was added and stirred at 100 ° C. for 20 hours. Vacuum degassing was performed three times before and after the Pd catalyst was added.

After completion of the reaction, impurities were filtered through celite. The filtrate was extracted with ethyl acetate, washed with H ₂ O and sat. NaHCO ₃ aq., Dried over Na ₂ SO ₄ and concentrated. The resulting residue was separated and purified by flash column chromatography (eluent: chloroform: ethyl acetate = 19: 1 → 9: 1) to obtain the target compound (7) (0.590 g, yield 68%) as a white solid. It was.

・ TLC (silica gel): R _f = 0.24 (eluent: chloroform / ethyl acetate = 19/1 v / v)
・ 1H-NMR (300 MHz, CDCl ₃ , TMS, rt)
δ = 8.33 (s, 1H), 7.89 (d, J = 9 Hz, 2H), 7.19-7.38 (m, 5H), 7.01 (d, J = 8.7 Hz, 2H), 4.37 (s, 2H), 4.18 -4.22 (m, 4H), 3.63 (t, J = 4.8 Hz, 2H)

Under a nitrogen atmosphere, add compound (9) (10.0 g, 82.7 mmol, 1.0 eq.) And t-butyldimethylsilyl chloride (13.64 g, 90.5 mmol, 1.1 eq.) To a 1 L three-necked eggplant flask, CH ₂ Dissolved in Cl ₂ (400 mL) and cooled to 0 ° C. Triethylamine (14 mL, 99.2 mmol, 1.2 eq.) Was added thereto, and the mixture was slowly returned to room temperature and allowed to react overnight.

After completion of the reaction, the reaction solution was partially concentrated, extracted with CH ₂ Cl ₂ , washed with H ₂ O and sat.NaCl aq., Dried over Na ₂ SO ₄ and concentrated. The obtained liquid was separated and purified by silica gel column chromatography (n-hexane: ethyl acetate = 9: 1) and concentrated to obtain the target compound (10) (18.68 g, yield 96%) as a colorless liquid.

・ TLC (silica): R _f = 0.63 (n-hexane / ethyl acetate = 4/1)
・ 1H-NMR (300 MHz, CDCl ₃ , TMS, rt)
δ = 9.89 (s, 1H), 7.79 (d, J = 8.7 Hz, 2H), 6.95 (d, J = 8.4 Hz, 2H), 0.96 (s, J = 23.7 Hz, 9H), 0.25 (s, 6H )

  Under a nitrogen atmosphere, compound (10) (18.6 g, 78.7 mmol, 1.0 eq.) Was added to a 1 L three-necked eggplant flask and dissolved in 300 mL of methanol. To this, sodium borohydride (3.57 g, 94.3 mmol, 1.2 eq.) Was added and reacted at room temperature for 30 minutes.

After completion of the reaction, the reaction mixture was concentrated, extracted with CH ₂ Cl ₂ , washed with H ₂ O and sat.NaCl aq., Dried over Na ₂ SO ₄ , concentrated, and the target compound (13) (14.65 g , Yield 78%) was obtained as a colorless liquid. The obtained colorless liquid was used for the next reaction without further purification.

・ TLC (silica gel): R _f = 0.09 (eluent: n-hexane / ethyl acetate = 9/1 v / v)
・ 1H-NMR (300 MHz, CDCl ₃ , TMS, rt)
δ = 7.24 (d, J = 8.4 Hz, 2H), 6.83 (d, J = 8.4 Hz, 2H), 4.61 (s, 2H), 0.95 (s, 9H), 0.20 (s, 6H)

Under a nitrogen atmosphere, compound (13) (7.0 g, 29.3 mmol, 1.0 eq.) Was added to a 500 mL three-necked eggplant flask and dissolved in CH ₂ Cl ₂ (150 mL). This was cooled to 0 ° C., triethylamine (8.0 mL, 58.6 mmol, 2.0 eq.) Was added, and the mixture was stirred for 15 minutes. After returning this to room temperature, methanesulfonyl chloride (3.4 g, 44.6 mmol, 1.5 eq.) Dissolved in CH ₂ Cl ₂ (50 mL) was added and reacted for 3 hours.

After completion of the reaction, the reaction solution was partially concentrated, extracted with CH ₂ Cl ₂ , washed with H ₂ O and sat.NaCl aq., Dried over Na ₂ SO ₄ and concentrated. The obtained liquid was separated and purified by silica gel column chromatography (n-hexane: ethyl acetate = 95: 5) and concentrated to obtain the target compound (12) (5.09 g, yield 68%) as a colorless liquid.

・ TLC (silica gel): R _f = 0.78 (eluent: n-hexane / ethyl acetate = 9/1 v / v)
・ 1H-NMR (500 MHz, CDCl ₃ , TMS, rt)
δ = 7.24 (d, J = 8.6 Hz, 2H), 6.81 (d, J = 8.6 Hz, 2H), 4.55 (s, 2H), 0.98 (s, 9H), 0.20 (s, 6H)

  Under an argon atmosphere, the compound (12) (4.0 g, 15.6 mmol, 1 eq.) Was placed in a 200 mL three-necked eggplant flask and vacuum dried. Then, THF (40 ml) was added and stirred. Next, magnesium (775 mg, 31.9 mmol, 2.0 eq.) Was added to a 200 mL three-necked eggplant flask (A), followed by vacuum drying. To this was added THF (10 mL). Then, the THF solution of compound (12) was put in a dropping funnel attached to (A). Then, 1,2-dibromoethane (1 mL, catalyst amount) was added to (A) and stirred while warming by hand to activate magnesium. Then, it was dripped over 10 minutes from the dropping funnel. After the dropwise addition, the reaction solution was heated at 50 ° C. for 1 hour and then returned to room temperature to obtain a brown solution compound. The obtained compound was used for the next reaction immediately without further purification.

  Under an argon atmosphere, 3.2 mL (15.6 mmol, 1 eq.) Of ethyl diethoxyacetate was added to a 200 mL two-necked eggplant flask, dissolved in 30 mL of THF, and cooled to −78 ° C. To this, the THF solution of the compound synthesized earlier was added dropwise over 20 minutes, and reacted for 2 hours.

After the reaction, water was added to stop the reaction, and the temperature was returned to room temperature. Extracted with ethyl acetate, washed with H ₂ O and sat. NaHCO ₃ aq., Dried over Na ₂ SO ₄ and concentrated. The obtained liquid was separated and purified by silica gel flash column chromatography (n-hexane: ethyl acetate = 95: 5) and concentrated to obtain the target compound (13) (2.98 mg, 54% yield) as a colorless liquid. It was.

-TLC (silica gel): R _f = 0.56 (eluent: n-hexane: ethyl acetate = 9/1 v / v)
・ 1H-NMR (300 MHz, CDCl ₃ , TMS, rt)
δ = 7.07 (d, J = 8.4 Hz, 2H), 6.78 (d, J = 8.4 Hz, 2H), 4.63 (s, 1H), 3.81 (s, 2H), 3.74-3.63 (m, 2H), 3.58 -3.48 (m, 2H), 1.24 (t, J = 6.9 Hz, 6H), 0.98 (s, 9H), 0.18 (s, 6H)

Compound (7) (500 mg, 1.44 mmol, 1 eq.), Compound (13) (814 g, 2.31 mmol, 1.6 eq.), EtOH (25 ml), water in a 30 ml two-necked eggplant flask under an argon stream (8 ml) and finally cooled to 0 ° C., charged with HCl (4 ml) under N ₂ and stirred at 80 ° C. overnight. Before adding HCl, vacuum deaeration was performed three times.

  After the reaction, the reaction mixture was concentrated, and the resulting solid was purified by silica gel column chromatography (ethyl acetate / methanol = 20/1) and concentrated to give the target compound (8) (287 mg, 40% yield). Was obtained as a yellow solid.

・ TLC (silica gel): R _f = 0.43 (eluent: ethyl acetate / methanol = 20/1, v / v)
¹ H-NMR (500 MHz, MeOD, TMS, rt)
δ = 7.55 (1H, s), 7.41 (2H, J = 7.5 Hz, d), 7.31 (2H, J = 7.0 Hz, t), 7.23 (5H, m), 7.04 (2H, J = 9.0 Hz, d ), 6.74 (2H, J = 8.5 Hz, d), 4.39 (2H, s), 4.22 (2H, J = 5.0 Hz, t), 4.11 (2H, s), 3.64 (2H, J = 4.5 Hz, t )

Under an argon stream, the compound (8) (19.8 mg, 0.04 mmol, 1.0 eq), CH ₂ Cl ₂ (3 mL), and MeOH (5 ml) were dissolved in a 30 ml two-necked eggplant flask and stirred. After degassing three times, 5% Pd / C (about 1 cup of micro spatula) was added and vacuum degassing was performed again. Then, after replacing with a hydrogen balloon, it was vacuum degassed again, filled with hydrogen, and stirred for 3 hours. After completion of the reaction, celite filtration was performed, and the filtrate was concentrated. The following reaction was performed without purification.

・ TLC (ODS-18): R _f = 0 (eluent: water / methanol = 1/1 v / v)
・ MALDI-TOF-MS (positive mode, CHCA): m / z = 467.08 [M + H] ⁺

  Under a nitrogen stream, the compound (14) crude, THF (3.0 ml), MeOH (5 ml), and fluorescein isothiocyanate (7.8 mg, 0.02 mmol) were dissolved in a 30 ml two-necked eggplant flask and stirred at 40 ° C. for 3 hours. Thereafter, the reaction solution is concentrated, and scraped and purified by reverse-phase column chromatography (eluent: water: methanol = 1: 4), followed by reverse-phase column chromatography (eluent: water: acetonitrile = 1: 2). I did it. Then, it refine | purified by reprecipitation using acetone and hexane. The purified target compound (15) was obtained as a red solid (2.12 mg, yield 6%).

・ TLC (ODS-18): R _f = 0.58 (eluent: water / MeOH = 1/4, v / v)
¹ H-NMR (500 MHz, MeOD, TMS, rt)
δ = 8.11 (1H, J = 2.0 Hz d), 7.71 (1H, J = 7.0 Hz, d), 7.59 (2H, s), 7.38 (2H, J = 7.5 Hz, d), 7.28 (2H, J = 7.0 Hz, t), 7.22 (1H, J = 7.5 Hz, t), 7.14 (5H, m), 6.68 (6H, m), 6.54 (2H, J = 8.5 Hz q), 4.40 (2H, s), 4.30 (2H, J = 5.5 Hz, t), 4.60 (4H, m)

Example 2 Luminescent properties The chemiluminescent properties of the 6-position conjugate of coelenterazine (CTZ) and FITC synthesized in Example 1 (CTZ-6-FITC) were examined.

1-1 Apparatus and measurement conditions Measurement was performed under the following apparatus conditions. Methanol, dimethyl sulfoxide (Kanto Chemical, for fluorescence analysis), and PBS buffer (pH 7.4, Nippon Gene) were used as measurement solvents. A fluorometer was used in the measurement of the chemiluminescence spectrum.

・ Fluorometer F-7000 (Model FL-3-11, Horiba Jobin Yvon Co., Kyoto, Japan)
Measurement wavelength: 350-650 nm
Wavelength feed width: 0.5 nm
Scanning speed: 1200 nm / min
Slit width (detection light): 25 nm
・ Measurement sample CTZ-6-FITC
·Measurement condition

    The sample was dissolved in methanol to give a 1 mM solution. 100 μL of the above methanol solution, 100 μL of PBS buffer or carbonate buffer (pH 9.6) was placed in a measurement container, and 2 mL of DMSO was added thereto and mixed, and measurement was performed immediately.

1-2 Chemiluminescence spectrum Figure 1 shows the chemiluminescence spectrum. The spectrum was normalized by setting the maximum emission intensity to 1.0.

1-3 Consideration of chemiluminescence characteristics of CTZ-6-FITC From Table 1, the maximum emission wavelength is as shown in Table 1. The emission peak of CTZ (document values) is also shown.

  From FIG. 1 and Table 1, the energy-transfer from the donor coelenterazine to the acceptor FITC was observed, and the chemiluminescence wavelength was successfully increased to 61 nm. This confirms that energy transfer occurred by binding FITC to coelenterazine. The emission peak derived from CTZ was 449 nm at pH 7.4 and 438 nm at pH 9.6. In general, CTZ is known to emit light in the form of Phenolate anion in the absence of a base. However, under neutral conditions, CTZ-6-FITC is more blue-shifted. This is probably due to the loss of the 6-position hydroxyl group of coelenterazine. Under basic conditions, CTZ takes the form of Pyrazine anion, which is also expected to blue-shift for the same reason.

2 Bioluminescence properties of CTZ-6-FITC
2-1 Equipment and measurement conditions The following equipment was used for measurement. A fluorescence spectrophotometer was used for the bioluminescence spectrum, and a luminescence plate reader was used for the bioluminescence intensity.

Fluorometer F-7000 (Hitachi Co. Ltd., Tokyo, Japan)
Measurement wavelength: 360-650 nm
Wavelength feed width: 0.2 nm
Scanning speed: 1200 nm / min
Luminescence plate reader: Lumat LB9507 (single tube type luminometer)
Measurement time: 1 sec

・ Measurement sample Substrate CTZ-6-azide
CTZ-6-FITC
Enzyme Rluc
Rluc8
Rluc8.6

CTZ-6-azide

  Using vectors expressing Rluc, Rluc8, and Rluc8.6 (source: Rluc is Promega, Rluc8 and Rluc8.6 are Dr. Sanjiv S. Gambhir, Stanford Univ.) Rluc8.6 was expressed, cells were lysed using Promega Renilla Luciferase Lysis Buffer, and the eluate was used as an enzyme solution.

Measurement conditions Bioluminescence measurement was performed according to the protocol of Promega's Renilla Luciferase Assay System (Cat. No. E2810). Substrates CTZ-6-FITC and CTZ-6-azide were dissolved in degassed methanol to 1 mM. This was diluted with Promega's Renilla Luciferase Lysis Buffer to 10 μM to obtain a substrate solution.

  Measurement was performed immediately after adding 20 μL of each of the cell solutions (Rluc, Rluc8, Rluc8.6) to 100 μL of the above substrate solution. All substrates and enzyme solutions were used immediately after preparation.

2-2 Enzyme recognition
The measurement result of CTZ-6-FITC is shown in FIG. The results shown in FIG. 2 indicate that CTZ-6-azide and CTZ-6-FITC were recognized by enzymes in Rluc8 and Rluc8.6. Table 2 compares the strength of natural CTZ with that of each. In the comparison of strength, the combination of CTZ-Rluc was set to 100%.

  From the results in Table 2, CTZ-6-azide and CTZ-6-FITC showed almost the same luminescence intensity as natural coelenterazine in the enzyme Rluc8.6. This suggests that Rluc8 and Rluc8.6 are recognized even when the dye is modified at the 6-position.

5-2-3 Bioluminescence spectrum
The bioluminescence of CTZ-6-FITC and CTZ is shown in FIG. From FIG. 3, the maximum bioluminescence wavelengths are as shown in Table 3.

  From the above results, CTZ-6-FITC succeeded in increasing the wavelength by about 40 nm compared to the natural one. It can also be seen that the CTZ-derived emission peak of CTZ-6-azide and CTZ-6-FITC is around 400 nm. This suggests the possibility of emitting light in the form of a neutral species by eliminating the 6-position hydroxyl group of natural coelenterazine.

Claims (3)

A fluorescent dye-bound coelenterazine represented by the following general formula [I].
(In the formula [I], A represents a fluorescent dye part including a fluorescent dye).   The fluorescent dye-bound coelenterazine according to claim 1, wherein the fluorescent dye is a fluorescent dye having an absorption wavelength peak in the range of 300 to 600 nm.   The fluorescent dye-bound coelenterazine according to claim 2, wherein the fluorescent dye is fluorescein isothiocyanate.