JP6095208B2 - Method for producing cyclized compound, and method for luminescence of solution containing cyclized compound - Google Patents

Description

  The present invention relates to a method for producing a cyclized compound and a light emitting method for a solution containing the cyclized compound.

Firefly luminescence, known as bioluminescence, emits light when firefly luciferase acts on firefly luciferin in the presence of ATP and magnesium ions. Since this quantum yield is extremely high, this light emitting system has been conventionally applied to ATP trace measurement and the like (for example, see Non-Patent Document 1).
In recent years, peptides have been synthesized using a compound in which firefly luciferin is bound to the ε-amino group of lysine and then firefly luciferase is allowed to act on the peptide to emit light (for example, Patent Document 1). Reference), a method for detecting C-reactive protein by causing C-reactive protein to act on a complex of a luciferin peptide compound and an antibody having a peptide bond to firefly luciferin and then causing firefly luciferase to further act to emit light is reported. (For example, see Patent Document 2).

JP 2010-30918 A JP2011-37791A

Chemical Dictionary 1st edition, pages 748 and 1360, 1994, published by Tokyo Chemical Co., Ltd.

  An object of this invention is to provide the manufacturing method of a cyclization compound, and the light-emission method of the solution containing a cyclization compound.

The manufacturing method of the cyclization compound which concerns on this invention includes the process of manufacturing the cyclization compound as described in following Chemical formula (II) by making an acid contact the compound as described in the following general formula (I).
(Wherein R ¹ , R ² and R ³ are each independently a substituent which becomes H by contact with H or the acid, and R ⁴ is OH or OH by contact with the acid. Which is a substituent).

  The acid is preferably a Bronsted acid or a Lewis acid. For example, hydrochloric acid, acetic acid, monofluoroacetic acid, difluoroacetic acid, trifluoroacetic acid, toluenesulfonic acid, sulfuric acid, nitric acid, phosphoric acid, perchloric acid, zinc bromide , Tin chloride, aluminum chloride, lithium bromide, lithium chloride, borane trifluoride, borane trichloride, borane tribromide, titanium tetrachloride, copper trifluoromethanesulfonate, silver trifluoromethanesulfonate, ytterbium trifluoromethanesulfonate, It may be one or more acids selected from the group consisting of tin trifluoromethanesulfonate, iodine, and bromine.

Moreover, the compound described in the chemical formula (I) may be produced from a protein or peptide containing an amino acid described in the following general formula (III) as a constituent amino acid.

The light emitting method according to the present invention includes a step of producing the cyclized compound represented by the chemical formula (II) by any one of the methods described above, and a step of bringing the cyclized compound into contact with luciferase.
The luciferase is preferably firefly luciferase.

  According to the present invention, it is possible to provide a method for producing a cyclized compound and a light emitting method for a solution containing the cyclized compound.

It is a figure which shows the light emission wavelength based on one Embodiment of this invention.

  Hereinafter, embodiments of the present invention completed based on the above knowledge will be described in detail with reference to examples. The objects, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description of the present specification, and those skilled in the art can easily reproduce the present invention from the description of the present specification. it can. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention and are shown for illustration or explanation, and the present invention is not limited to them. It is not limited. It will be apparent to those skilled in the art that various modifications and variations can be made based on the description of the present specification within the spirit and scope of the present invention disclosed herein.

The manufacturing method of the cyclization compound which concerns on this invention includes the process of manufacturing the cyclization compound as described in following Chemical formula (II) by making an acid contact the compound as described in following General formula (I).
(Wherein R ¹ , R ² and R ³ are each independently a substituent which becomes H by contact with H or the acid, and R ⁴ is OH or OH by contact with the acid. Which is a substituent).

The type of acid used in this production method is not particularly limited, and those skilled in the art will consider the properties of the substituents R ¹ , R ² , R ³ and R ⁴ of the compound described in the general formula (I). However, it can be appropriately selected appropriately.
For example, the acid may be a Bronsted acid or a Lewis acid. Examples of Bronsted acids include hydrochloric acid, acetic acid, monofluoroacetic acid, difluoroacetic acid, trifluoroacetic acid, toluenesulfonic acid, sulfuric acid, nitric acid, phosphoric acid, and perchloric acid, and should be hydrochloric acid or trifluoroacetic acid. Although preferable, it is not limited to these. Examples of Lewis acids include zinc bromide, tin chloride, aluminum chloride, lithium bromide, lithium chloride, borane trifluoride, borane trichloride, borane tribromide, titanium tetrachloride, copper trifluoromethanesulfonate, trifluoro Examples include, but are not limited to, silver methanesulfonate, ytterbium trifluoromethanesulfonate, tin trifluoromethanesulfonate, iodine, and bromine.

The substituents R ¹ , R ^2, and R ³ in the general formula (I) are not particularly limited as long as they are H or a substituent that becomes H by contact with the acid. The substituent R ⁴ is not particularly limited as long as it is OH or a substituent that becomes OH by contact with the acid.
In the case where R ² and / or R ³ is a substituent that becomes H by contact with an acid, when an acid is brought into contact with the compound represented by the general formula (I), first, R ² in the general formula (I) And a compound in which at least one of R ³ is H is generated, and the primary or secondary amino group thus generated undergoes an intramolecular nucleophilic attack on the carbonyl group at the α-position of the benzothiazole ring. The 3,4-dihydro-2H-pyrrole ring in the cyclized compound described in (II) is formed. When R ¹ is a substituent that becomes H by contact with an acid, it may be H before, at the same time as, or after this 3,4-dihydro-2H-pyrrole ring is formed. In addition, when R ⁴ is a substituent that becomes OH upon contact with an acid, it may become OH before, at the same time as, or after this 3,4-dihydro-2H-pyrrole ring is formed. good.

Those skilled in the art can appropriately design such substituents R ¹ , R ² , R ³ and R ⁴ that become H by contact with an acid.
As the substituent R ¹ , for example, an aryl group, an aryl group, an unsubstituted alkyl group, a BOM group, a MOM group, a MEM, such as a Me group, a tert-Bu group, a Bn group, a PMB group, and an Allyl group. A substituent which forms an acetal together with the hydroxyl group of the compound described in formula (I), such as a group, a THP group and an EE group, an acyl group such as an Ac group, a Bz group and a Piv group, and TIPS Group, TES group, TMS group, TBDPS group and TBS group may be alkylsilyl groups, and TIPS group, TES group, TMS group, TBDPS group and TBS group are preferable alkylsilyl groups, Although it is more preferable that it is group, it is not limited to these.
The substituents R ² and R ³ may be the same or different, for example, a Moc group (methoxycarbonyl group), an Eoc group (ethoxycarbonyl group), a Boc group, an Alloc group, an Fmoc group, a Troc group, and A carbamate group such as a Cbz group, a Me group, an Et group, a tert-Bu group, a Bn group, a PMB group, and an Allyl group, such as an aryl or allyl substituted or unsubstituted alkyl group, and an Ac group; It may be an acyl group such as a Bz group or a Piv group, and may be a carbamate group such as a Moc group (methoxycarbonyl group), an Eoc group (ethoxycarbonyl group), a Boc group, an Alloc group, an Fmoc group, a Troc group, or a Cbz group. Although it is preferable that it is a Boc group, it is not limited to this. One of R ² and R ³ is preferably H, and in this case, the other is more preferably a Boc group, but the present invention is not limited to this.
R ⁴ is substituted with aryl or allyl such as methoxy group, ethoxy group, iso-propoxy group, tert-butoxy group, benzyloxy group, p-methoxybenzyloxy group and 2-propenyloxy group. Or an unsubstituted alkyloxy group, and preferably a tert-butoxy group, but is not limited thereto.

The method of bringing the acid into contact with the compound described in the general formula (I) is not particularly limited, and if necessary, the acid is added to the compound after being dispersed in an appropriate solvent, followed by stirring, shaking and standing. It can contact by the well-known method of these.
Further, the temperature and time for bringing the acid into contact with the compound represented by the general formula (I) are not particularly limited, and those skilled in the art will understand the compound represented by the general formula (I) and the ring represented by the chemical formula (II). The temperature can be appropriately set while taking into account the stability of the chemical compound and the degree of progress of the reaction. For example, the temperature may be -78 ° C to 130 ° C, and -20 ° C to 100 ° C. It is preferable that it is 0 degreeC-80 degreeC, and it is further more preferable that it is room temperature-70 degreeC. Further, the time may be 5 minutes to 1 week, preferably 10 minutes to 3 days, and more preferably 30 minutes to 1 day.

  The produced cyclized compound represented by the chemical formula (II) can emit light by the action of a luciferase such as firefly luciferase in the presence of ATP and magnesium ions. Thus, similar to firefly luciferin, It can be applied to various uses including micro measurement. In addition, the cyclized compound represented by the chemical formula (II) is extremely stable with respect to oxygen, light, acid, heat, and the like as compared with firefly luciferin, and further, among the artificially prepared firefly luciferin analogs. Then, strong emission intensity can be generated.

On the other hand, since the compound described in the general formula (I) does not emit light even when luciferase is allowed to act on it, the compound described in the general formula (I) is contacted with an acid at a stage where light emission is necessary, and the chemical formula (II) ) Can be switched to a state capable of emitting light. This function can be used, for example, for monitoring pH conditions and detecting Lewis acidic metals.
For example, an industrial product such as a measurement kit that utilizes luminescence contains a compound represented by the general formula (I) and an acid, and the chemical formula is obtained by contacting the compound with an acid at a stage where luminescence is required. It can convert into the cyclized compound as described in (II), and can be set as a luminescent substrate.

Moreover, since the compound described in the general formula (I) has an α-amino acid structure, a protein or a peptide can be produced using this compound. That is, a protein or peptide containing the amino acid described in the following general formula (III) as a constituent amino acid can be produced. In addition, the substituent R ¹ of the amino acid described in General Formula (III) is the same as the substituent R ¹ of the compound described in General Formula (I).

  The constituent amino acid other than the amino acid described in the general formula (III) contained in the protein or peptide may be an L-amino acid or a D-amino acid, but is preferably an L-amino acid. . In the present specification, those having a molecular weight of 5000 or more are referred to as proteins, and those having a molecular weight of less than 5000 are referred to as peptides.

By decomposing a protein or peptide containing the amino acid described in the general formula (III) as a constituent amino acid using an enzyme or an acid / alkali catalyst, the compound described in the general formula (I) can be liberated. . The liberated compound represented by the general formula (I) can generate a cyclized compound represented by the chemical formula (II) by contacting with an acid, and can further emit light by the action of luciferase. The protein or peptide containing the amino acid described in III) as a constituent amino acid can be used as a labeled protein or peptide having a luminescence function.
A person skilled in the art can appropriately select an enzyme for hydrolyzing the protein or peptide using an enzyme, and examples thereof include enzymes belonging to the EC 3.4 group. May be a protease such as, but not limited to, pepsin, subtilisin, trypsin, chymotrypsin, xin, furin, cathepsin D, papain, and thermolysin.
In addition, when chemically hydrolyzing, either an acid or a base may be used as a catalyst, but the compound represented by the general formula (I) which has been liberated is subsequently contacted with an acid to obtain a chemical formula (II). In view of the formation of the cyclized compound described in 1., hydrolysis with an acid is preferable. Here, the acid may be a Bronsted acid or a Lewis acid, but is preferably a Bronsted acid. Examples of Bronsted acids include hydrochloric acid, acetic acid, monofluoroacetic acid, difluoroacetic acid, trifluoroacetic acid, toluenesulfonic acid, sulfuric acid, nitric acid, phosphoric acid, and perchloric acid, and should be hydrochloric acid or trifluoroacetic acid. Although preferable, it is not limited to these.

When producing a protein or peptide containing the amino acid described in the general formula (III) as a constituent amino acid, the compound described in the general formula (I) has two reactive sites of an amino group and a carboxyl group. It can be contained not only at the end of the protein or peptide but also at any location, and can be contained at multiple locations as required. Thus, any location within the protein or peptide can be labeled. Furthermore, when it is made to contain in several places in protein or a peptide, emitted light intensity can be improved.
In addition, when hydrolyzing with an enzyme, it is specifically cleaved by an amino acid depending on the enzyme used. Therefore, in that case, it is necessary to design the surrounding amino acid sequence in consideration of the specificity of the enzyme so that the amino acid described in the general formula (III) can be cut out.

  As described above, the protein or peptide containing the amino acid described in the general formula (III) as a constituent amino acid does not become a luminescent substrate only by containing or liberating the compound described in the general formula (I). Since it becomes a luminescent substrate only after conversion to the cyclized compound described in (1), it is possible to adjust the timing of the luminescent substrate by contacting with an acid at a stage where luminescence is necessary.

The light-emitting method according to the present invention includes a step of bringing luciferase into contact with a solution containing the cyclized compound represented by the chemical formula (II) produced by the above-described method. By this light emitting method, the cyclized compound represented by the chemical formula (II) can emit light having a maximum emission wavelength of 547 nm. In addition, in this luminescent method, light having a higher intensity can be generated than when other firefly luciferin analogs are used.
The type of luciferase is not particularly limited as long as the cyclized compound described in the chemical formula (II) can emit light, but is preferably a luciferase that can use firefly luciferin as a substrate, and more preferably firefly luciferase. The luciferase may be naturally derived or recombinant.
A person skilled in the art can refer to the conditions for contacting luciferase, including the type of solvent for dissolving the cyclized compound represented by chemical formula (II), while referring to the conditions for causing firefly luciferin to emit light with firefly luciferase. It can be set appropriately as appropriate. For example, the cyclized compound represented by the chemical formula (II) may be caused to emit light by contacting luciferase at room temperature in the presence of ATP and magnesium ions in a pH 6-8 potassium phosphate buffer. It is not limited to this.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, the scope of the present invention is not limited to the following Example.

[Example 1] Preparation of substrate (1) tert-butyl (R) -2- (tert-butoxycarbonylamino) -5- (6-hydroxybenzo [d] thiazol-2-yl) -5-oxopentanoate Preparation of (Precursor 1) (1-1) Preparation of 2-bromo-6-methoxybenzo [d] thiazole

After adding dried copper (II) bromide (2.70 g, 11.6 mmol) and isobutyl nitrite (2 mL, 15.0 mmol) to acetonitrile (150 mL) at room temperature under an argon atmosphere, 6-methoxy A solution of benzo [d] thiazol-2-amine (1.80 g, 9.99 mmol) in acetonitrile (50 mL) was also added at room temperature. The resulting mixture was heated to 65 ° C. and stirred for 4 hours under an argon atmosphere while maintaining this temperature.
The reaction mixture was allowed to cool to room temperature, 2 M hydrochloric acid (30 mL) was added, and the mixture was extracted with chloroform (3 × 150 mL). The combined organic layers were dried over anhydrous sodium sulfate, and then the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography (silica gel 200 g; hexane: ethyl acetate (2: 1)) to give 2-bromo-6-methoxybenzo [d] thiazole (2.32 g, yield). 95%) was obtained as a reddish brown solid. The measured physical properties of the product are as follows.

2-Bromo-6-methoxybenzo [d] thiazole
IR (film) 1603, 1221 cm ^-1 .
¹ H NMR (400 MHz, CDCl ₃ ) δ 7.85 (1 H, d, J = 8.8 Hz), 7.24 (1 H, d, J = 2.4 Hz), 7.06 (1 H, dd, J = 2.4, 8.8 Hz ), 3.87 (3 H, s).
¹³ C NMR (100 MHz, CDCl ₃ ) δ 158.1, 146.8, 138.6, 135.5, 123.3, 115.8, 103.6, 55.9.
ESI-HRMS calcd for C ₈ H ₆ ⁷⁹ BrNOS 242.9353 (M + H) ⁺ , found, m / z 243.9459.

(1-2) Preparation of 2-bromobenzo [d] thiazol-6-ol

2-Bromo-6-methoxybenzo [d] thiazole (555 mg, 2.28 mmol) was dissolved in methylene chloride (10 mL) at room temperature under an argon atmosphere. To the resulting solution was added 1 M boron tribromide methylene chloride solution (4 mL) at room temperature. The resulting mixture was stirred at room temperature for 7 hours under an argon atmosphere.
The reaction mixture was added to ice water and extracted with chloroform (3 × 30 mL). The combined organic layers were dried over anhydrous sodium sulfate, and then the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography (silica gel 80 g; hexane: ethyl acetate (3: 1)) to give 2-bromobenzo [d] thiazol-6-ol (521 mg, yield 99). %) Was obtained as a brown solid. The measured physical properties of the product are as follows.

2-Bromobenzo [d] thiazol-6-ol
IR (film) 3118, 1598 cm ^-1 .
¹ H NMR (400 MHz, CD ₃ OD) δ 7.72 (1 H, d, J = 8.8 Hz), 7.26 (1 H, d, J = 2.4 Hz), 6.97 (1 H, dd, J = 2.4, 8.8 Hz).
¹³ C NMR (100 MHz, CD ₃ OD) δ 156.5, 145.6, 138.5, 134.8, 122.5, 116.0, 105.6.
ESI-HRMS calcd for C ₇ H ₄ ⁷⁹ BrNOS 228.9197 (M + H) ⁺ , found, m / z 229.9303.

(1-3) Preparation of 2-bromo-6- (tert-butyldimethylsilyloxy) benzo [d] thiazole

2-Bromobenzo [d] thiazol-6-ol (794 mg, 3.45 mmol) was dissolved in N, N-dimethylformamide (42 mL) at room temperature under an argon atmosphere, and subsequently tert-butyldimethylchlorosilane ( 1.75 g, 11.6 mmol) and imidazole (1.41 g, 20.7 mmol) were added at room temperature. The resulting mixture was stirred at room temperature under an argon atmosphere for 24 hours.
A saturated aqueous ammonium chloride solution (50 mL) was added to the reaction mixture, and the mixture was extracted with chloroform (3 × 30 mL). The combined organic layers were dried over anhydrous sodium sulfate, and then the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography (silica gel 80 g; hexane: ethyl acetate (3: 1)) to give 2-bromo-6- (tert-butyldimethylsilyloxy) benzo [d]. Thiazole (1.13 g, 95% yield) was obtained as a brown liquid. The measured physical properties of the product are as follows.

2-Bromo-6- (tert-butyldimethylsilyloxy) benzo [d] thiazole
IR (film) 1597, 1270 cm ^-1 .
¹ H NMR (400 MHz, CDCl ₃ ) δ 7.79 (1 H, d, J = 8.8 Hz), 7.19 (1 H, d, J = 2.4 Hz), 6.94 (1 H, dd, J = 2.4, 8.8 Hz ), 0.97 (9 H, s), 0.20 (6 H, s).
¹³ C NMR (100 MHz, CDCl ₃ ) δ 154.0, 147.3, 138.4, 135.9, 123.2, 120.3, 111.1, 25.7, 18.2, -4.4.
ESI-HRMS calcd for C ₁₃ H ₁₈ ⁷⁹ BrNOSSi 343.0062 (M + H) ⁺ , found, m / z 344.0168.

(1-4) Preparation of di-tert-butyl (R) -5-oxopyrrolidine-1,2-dicarboxylate (1-4-1) of tert-butyl (R) -5-oxopyrrolidine-2-carboxylate Preparation

(R) -5-oxopyrrolidine-2-carboxylic acid (3.21 g, 24.9 mmol) was dissolved in tert-butyl acetate (100 mL) at room temperature under an argon atmosphere, and then 60% aqueous perchloric acid solution was added. (1.5 mL) was added at room temperature. The resulting mixture was stirred at room temperature under an argon atmosphere for 24 hours.
Saturated aqueous sodium hydrogen carbonate solution (50 mL) was added to the reaction mixture, and the mixture was extracted with chloroform (3 × 30 mL). The combined organic layers were dried over anhydrous sodium sulfate, and then the solvent was distilled off under reduced pressure to give tert-butyl (R) -5-oxopyrrolidine-2-carboxylate (4.62 g, yield quant.) Obtained as a white solid. The measured physical properties of the product are as follows.

(R) -5-oxopyrrolidine-2-carboxylate tert-butyl
IR (film) 3454, 1737 cm ^-1 .
¹ H NMR (400 MHz, CDCl ₃ ) δ 5.97 (1 H, br), 4.16 (1 H, m), 2.38 (3 H, m), 2.11 (1 H, m), 1.48 (9 H, s) .
¹³ C NMR (100 MHz, CDCl ₃ ) δ 177.8, 171.1, 82.5, 56.1, 29.4, 28.1, 24.9.
ESI-HRMS calcd for C ₉ H ₁₅ NO ₃ 185.1052 (M + H) ⁺ , found, m / z 186.1113.
[α] ²⁵ _D -1.11 (c 1.00, CHCl ₃ ).

Preparation of (1-4-2) (R) -5-oxopyrrolidine-1,2-dicarboxylate

(R) -5-oxopyrrolidine-2-carboxylate tert-butyl (367 mg, 1.98 mmol) and di-tert-butyl dicarbonate (0.55 mL, 24.6 mmol) were added to tetrahydrofuran ( After dissolving in 10 mL), N, N-dimethyl-4-aminopyridine (58.4 mg, 0.478 mmol) was added at room temperature. The resulting mixture was stirred at room temperature for 2 hours under an argon atmosphere.
Pure water (25 mL) was added to the reaction mixture, followed by extraction with ethyl acetate (3 × 50 mL). The combined organic layers were dried over anhydrous sodium sulfate, and then the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography (silica gel 300 g; hexane: ethyl acetate (1: 1)) to give (R) -5-oxopyrrolidine-1,2-dicarboxylic acid ditert- Butyl (591 mg, yield quant.) Was obtained as a yellow liquid. The measured physical properties of the product are as follows.

(R) -5-oxopyrrolidine-1,2-dicarboxylate ditert-butyl
IR (film) 1739,1717 cm ^-1 .
¹ H NMR (400 MHz, CDCl ₃ ) δ 4.38 (1 H, dd, J = 2.4, 9.3 Hz), 2.48 (1 H, m), 2.35 (1 H, m), 2.20 (1 H, m), 1.90 (1 H, m), 1.41 (9 H, s), 1.39 (9 H, s).
¹³ C NMR (100 MHz, CDCl ₃ ) δ 173.7, 170.4, 149.3, 83.2, 82.2, 59.6, 31.1, 27.9, 27.8, 21.6.
ESI-HRMS calcd for C ₁₄ H ₂₃ NO ₅ 285.1576 (M + H) ⁺ , found, m / z 286.1650.
[α] ²⁵ _D +33.6 (c 1.00, CHCl ₃ ).

(1-5) (R) -2- (tert-butoxycarbonylamino) -5- (6- (tert-butyldimethylsilyloxy) benzo [d] thiazol-2-yl) -5-oxopentanoic acid tert- Preparation of butyl

Solution A was prepared by dissolving 2-bromo-6- (tert-butyldimethylsilyloxy) benzo [d] thiazole (528 mg, 1.53 mmol) in tetrahydrofuran (15.3 mL) at room temperature under argon atmosphere. did. Alternatively, (R) -5-oxopyrrolidine-1,2-dicarboxylate ditert-butyl (558 mg, 1.96 mmol) was dissolved in tetrahydrofuran (10 mL) at room temperature under an argon atmosphere to obtain a solution B. Was prepared.
To the solution A thus prepared, a 2.7 M n-butyllithium hexane solution (0.63 mL) was added at −78 ° C. under an argon atmosphere, followed by stirring for 10 minutes. Furthermore, Solution B was added to the obtained mixture at −78 ° C. under an argon atmosphere, and the mixture was stirred for 2 hours while maintaining −78 ° C.
The reaction mixture was returned to room temperature, saturated aqueous ammonium chloride solution (50 mL) was added, and the mixture was extracted with chloroform (3 × 30 mL). The combined organic layers were dried over anhydrous sodium sulfate, and then the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography (silica gel 200 g; hexane: ethyl acetate (5: 1)) and then gel permeation chromatography to give (R) -2- (tert-butoxycarbonyl Amino) -5- (6- (tert-butyldimethylsilyloxy) benzo [d] thiazol-2-yl) -5-oxopentanoic acid tert-butyl (360 mg, 43% yield) was obtained as a yellow liquid. It was. The measured physical properties of the product are as follows.

(R) -2- (tert-Butoxycarbonylamino) -5- (6- (tert-butyldimethylsilyloxy) benzo [d] thiazol-2-yl) -5-oxopentanoic acid tert-butyl
IR (film) 1717 cm ^-1 .
¹ H NMR (400 MHz, CDCl ₃ ) δ 7.99 (1 H, d, J = 9.2 Hz), 7.34 (1 H, d, J = 2.4 Hz), 7.07 (1 H, dd, J = 2.4, 9.2 Hz ), 5.18 (1 H, d, J = 8.0 Hz), 4.30 (1 H, d, J = 6.0 Hz), 3.31 (2 H, m), 2.30 (1 H, m), 2.11 (1 H, m ), 1.47 (9 H, s), 1.40 (9 H, s), 1.00 (9 H, s), 0.24 (6 H, s).
¹³ C NMR (100 MHz, CDCl ₃ ) δ 194.3, 171.5, 164.0, 156.1, 155.5, 148.6, 139.1, 126.2, 121.7, 111.9, 82.3, 79.9, 53.6, 34.5, 28.4, 28.1, 27.2, 25.7, 18.4,- 4.2.
ESI-HRMS calcd for C ₂₇ H ₄₃ N ₂ O ₆ SSi 550.2533 (M + H) ⁺ , found, m / z 551.2599.
[α] ²⁵ _D -7.16 (c 1.00, CHCl ₃ ).

Preparation of (1-6) (R) -2- (tert-butoxycarbonylamino) -5- (6-hydroxybenzo [d] thiazol-2-yl) -5-oxopentanoic acid tert-butyl

(R) -2- (tert-Butoxycarbonylamino) -5- (6- (tert-butyldimethylsilyloxy) benzo [d] thiazol-2-yl) -5-oxopentanoic acid tert-butyl (133 mg, 0.242 mmol) was dissolved in tetrahydrofuran (3 mL) at room temperature under an argon atmosphere. To the resulting solution, a mixed solution consisting of 1 M tetra-n-butylammonium fluoride in tetrahydrofuran (1 mL), acetic acid (0.057 mL), and pure water (0.018 mL) was added at room temperature under an argon atmosphere. 0.3 mL was added. The resulting mixture was stirred at room temperature for 1 hour under an argon atmosphere.
A saturated aqueous ammonium chloride solution (20 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (3 × 30 mL). The combined organic layers were dried over anhydrous sodium sulfate, and then the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography (silica gel 80 g; hexane: ethyl acetate (1: 1)) to give (R) -2- (tert-butoxycarbonylamino) -5- (6 Tert-Butyl-hydroxybenzo [d] thiazol-2-yl) -5-oxopentanoate (Precursor 1, 100 mg, 95% yield) was obtained as a yellow solid. The measured physical properties of the product are as follows.

(R) -2- (tert-Butoxycarbonylamino) -5- (6-hydroxybenzo [d] thiazol-2-yl) -5-oxopentanoic acid tert-butyl
IR (film) 3322, 1729,1700 cm ^-1 .
¹ H NMR (400 MHz, CDCl ₃ ) δ 7.89 (1 H, d, J = 9.0 Hz), 7.30 (1 H, d, J = 2.5 Hz), 7.09 (1 H, dd, J = 2.5, 9.0 Hz ), 5.35 (1 H, d, J = 8.0 Hz), 4.28 (1 H, d, J = 5.2 Hz), 3.29 (2 H, m), 2.28 (1 H, m), 2.10 (1 H, m ), 1.46 (9 H, s), 1.43 (9 H, s).
¹³ C NMR (100 MHz, CDCl ₃ ) δ 194.0, 171.6, 162.8, 157.1, 156.0, 147.7, 139.4, 126.4, 117.7, 106.8, 82.7, 80.9, 53.7, 34.5, 28.4, 28.1, 27.1.
ESI-HRMS calcd for C ₂₁ H ₂₈ N ₂ O ₆ S 436.1668 (M + H) ⁺ , found, m / z 437.1746.
[α] ²⁵ _D -17.2 (c 1.00, CHCl ₃ ).

(2) Preparation of 3-hydroxy-2- (6-hydroxybenzo [d] thiazole-2-carboxamido) propanoic acid (precursor 2) (2-1) 6-hydroxybenzo [d] thiazole-2-carbo Preparation of nitrile

To 6-methoxybenzo [d] thiazole-2-carbonitrile (766 mg, 4.03 mmol) was added pyridinium chloride (19.5 g) at room temperature under an argon atmosphere. The obtained mixture was heated to 200 ° C. under an argon atmosphere to melt pyridinium chloride, and then stirred for 1 hour while maintaining the temperature at 200 ° C.
The reaction mixture was allowed to cool to room temperature, 2 M hydrochloric acid (250 mL) was added, and the mixture was further extracted with ethyl acetate (3 × 100 mL). The combined organic layers were dried over anhydrous sodium sulfate, and then the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography (silica gel 85 g; hexane-ethyl acetate (1: 1)) to give 6-hydroxybenzo [d] thiazole-2-carbonitrile (710 mg, yield). 89%) was obtained as a white solid. The measured physical properties of the product are as follows.

6-Hydroxybenzo [d] thiazole-2-carbonitrile
mp 155-170 ° C decomp.
IR (film): 3217, 2228 cm ^-1 .
¹ H NMR (400 MHz, CD ₃ OD) δ 7.94 (1 H, d, J = 6.8 Hz), 7.36 (1 H, d, J = 2.4 Hz), 7.13 (1 H, dd, J = 6.8, 2.4 Hz).
¹³ C NMR (100 MHz, CD ₃ OD) δ 160.3, 147.3, 139.0, 133.9, 126.6, 119.6, 114.3, 107.0.
ESI-HRMS calcd for C ₈ H ₄ N ₂ OS 176.0044 (M + H) ⁺ , found, m / z 177.0100.

(2-2) Preparation of methyl 6-hydroxybenzo [d] thiazole-2-carboxylate

To a methanol solution (50 mL) of 6-hydroxybenzo [d] thiazole-2-carbonitrile (633 mg, 3.59 mmol) was added potassium carbonate (496 mg, 3.59 mmol) at room temperature, and then the resulting mixture was obtained. Was stirred at room temperature for 24 hours.
To the reaction mixture was added 2 M hydrochloric acid (3 mL), and the mixture was extracted with ethyl acetate (3 × 60 mL). The combined organic layers were dried over anhydrous sodium sulfate, and then the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography (silica gel 100 g; hexane-ethyl acetate (1: 1)) to give methyl 6-hydroxybenzo [d] thiazole-2-carboxylate (751 mg, 94% yield) was obtained as a yellow solid. The measured physical properties of the product are as follows.

6-hydroxybenzo [d] methyl thiazole-2-carboxylate
mp 197-200 ° C.
IR (film): 3157, 1739 cm ^-1 .
¹ H NMR (400 MHz, CD ₃ OD) δ 7.95 (1 H, d, J = 8.9 Hz), 7.37 (1 H, d, J = 2.3 Hz), 7.11 (1 H, dd, J = 2.3, 8.9 Hz), 4.01 (3 H, s).
¹³ C NMR (100 MHz, CD ₃ OD) δ 163.6, 158.7, 155.9, 147.7, 139.3, 125.9, 118.3, 106.3.
ESI-HRMS calcd for C ₉ H ₇ NO ₃ S 209.0147 (M + H) ⁺ , found, m / z 210.0203.

(2-3) Preparation of methyl 6- (methoxymethoxy) benzo [d] thiazole-2-carboxylate

To a solution of methyl 6-hydroxybenzo [d] thiazole-2-carboxylate (284 mg, 1.35 mmol) in N, N-dimethylformamide (5 mL), sodium hydride (60 mg, 1.50 mmol) and chloromethyl methyl ether (510 μL, 6.75 mmol) was added at room temperature. The resulting mixture was stirred at room temperature for 30 minutes.
Pure water (10 mL) was added to the reaction mixture, followed by extraction with ethyl acetate (3 × 60 mL). The combined organic layers were dried over anhydrous sodium sulfate, and then the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography (silica gel 80 g; hexane-ethyl acetate (1: 1)) to give methyl 6- (methoxymethoxy) benzo [d] thiazole-2-carboxylate ( 207 mg, 61% yield) was obtained as a yellow liquid. The measured physical properties of the product are as follows.

Methyl 6- (methoxymethoxy) benzo [d] thiazole-2-carboxylate
IR (film): 1743, 1093 cm ^-1 .
¹ H NMR (CDCl ₃ ) δ 8.08 (1 H, d, J = 9.2 Hz), 7.57 (1 H, d, J = 2.5 Hz), 7.23 (1 H, dd, J = 2.5, 9.4 Hz), 5.23 (2 H, s), 4.03 (3 H, s), 3.48 (3 H, s).
¹³ C NMR (CDCl ₃ ) δ 161.2, 157.2, 155.9, 148.5, 138.5, 126.2, 118.7, 106.9, 94.8, 56.3, 53.6.
ESI-HRMS calcd for C ₁₁ H ₁₁ NO ₄ S 253.0409 (M + H) ⁺ , found, m / z 254.0486.

(2-4) Preparation of methyl 3-hydroxy-2- (6-hydroxybenzo [d] thiazole-2-carboxamido) propanoate

To a methanol solution (10 mL) of methyl 6- (methoxymethoxy) benzo [d] thiazole-2-carboxylate (232 mg, 0.91 mmol), triethylamine (1.8 mL, 12.8 mmol) and D-form or L-form serine methyl Hydrochloride (1.01 g, 6.39 mmol) was added at room temperature. The resulting mixture was stirred at room temperature for 3 days.
To the reaction mixture was added 2 M hydrochloric acid (3 mL), and the mixture was extracted with chloroform (3 × 60 mL). The combined organic layers were dried over anhydrous sodium sulfate, and then the solvent was distilled off under reduced pressure. Trifluoroacetic acid (3 mL) was added to the obtained residue, and the mixture was stirred at room temperature for 30 minutes. The mixture was concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography (silica gel 100 g; hexane-ethyl acetate (1: 1)) to give 3-hydroxy-2- (6-hydroxybenzo [ d] Methyl thiazole-2-carboxamido) propanoate (207 mg, 61% yield) was obtained as a white solid. The measured physical properties of the product are as follows.

3-hydroxy-2- (6-hydroxybenzo [d] thiazole-2-carboxamido) propanoic acid methyl ester
IR (film): 3373, 3262, 1749, 1653 cm ^-1 .
¹ H NMR (400 MHz, CD ₃ OD) δ 7.92 (1 H, d, J = 8.8 Hz), 7.34 (1 H, d, J = 2.4 Hz), 7.07 (1 H, dd, J = 2.4, 8.8 Hz), 4.74 (1 H, t, J = 4.0, 7.2 Hz), 4.05 (1 H, dd, J = 4.0, 7.2 Hz), 3.80 (3 H, s).
¹³ C NMR (100 MHz, CD ₃ OD) δ 171.8, 162.1, 159.8, 159.6, 147.8, 140.1, 126.2, 117.6, 62.8, 56.4, 53.1.
ESI-HRMS calcd for C ₁₂ H ₁₂ N ₂ O ₅ S 296.0467 (M + H) ⁺ , found, m / z 297.0569.
D: [α] ²⁵ _D -4.7 (c 1.00, CH ₃ OH).
L: [α] ²⁵ _D +6.3 (c 1.00, CH ₃ OH).

(2-5) Preparation of 3-hydroxy-2- (6-hydroxybenzo [d] thiazole-2-carboxamido) propanoic acid

To a solution of methyl D-form or L-form 3-hydroxy-2- (6-hydroxybenzo [d] thiazole-2-carboxyamido) propanoate (12.8 mg, 0.046 mmol) in methanol (1 mL), 25 mM hydroxylated Aqueous sodium solution (5 mL) was added at room temperature. The resulting mixture was stirred at room temperature for 25 minutes.
To the reaction mixture was added 2 M hydrochloric acid (0.1 mL), and the mixture was extracted with ethyl acetate (3 × 30 mL). The combined organic layers were dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to give 3-hydroxy-2- (6-hydroxybenzo [d] thiazole-2-carboxamido) propanoic acid (10.1 mg, yield). 100%) was obtained as a white solid. The measured physical properties of the product are as follows.

3-hydroxy-2- (6-hydroxybenzo [d] thiazole-2-carboxamido) propanoic acid
IR (film): 3388, 2942, 1729, 1652 cm ^-1 .
¹ H NMR (CD ₃ OD) δ 7.90 (1 H, d, J = 8.8 Hz), 7.32 (1 H, d, J = 2.4 Hz), 7.04 (1 H, dd, J = 8.8 2.4 Hz), 4.65 (1 H, t, J = 3.4 Hz), 4.05 (1 H, dd, J = 3.4, 11.7 Hz), 3.96 (1 H, dd, J = 3.4, 11.7 Hz).
¹³ C NMR (CD ₃ OD) δ 171.5, 160.6, 159.1, 157.4, 146.8, 138.7, 124.9, 117.1, 106.1, 61.5, 55.1.
ESI-HRMS calcd for C ₁₁ H ₁₀ N ₂ O ₅ S 282.0310 (M ⁺⁺ H), found, m / z 283.0379.
D: [α] ²⁵ _D -8.9 (c 1.00, CH ₃ OH).
L: [α] ²⁵ _D +10.2 (c 1.00, CH ₃ OH).

[Example 2] Cyclization reaction (1) tert-butyl (R) -2- (tert-butoxycarbonylamino) -5- (6-hydroxybenzo [d] thiazol-2-yl) -5-oxopentanoate Cyclization reaction from (1-1) acidic conditions

(R) -2- (tert-Butoxycarbonylamino) -5- (6-hydroxybenzo [d] thiazol-2-yl) -5-oxopentanoic acid tert-butyl (Precursor 1, 20.8 mg, 0.0476 mmol) Was dissolved in trifluoroacetic acid (1 mL) at room temperature under an argon atmosphere. The resulting mixture was stirred at room temperature under an argon atmosphere for 24 hours.
After the reaction mixture was concentrated under reduced pressure, the resulting residue was purified by preparative thin-layer reversed-phase silica gel column chromatography (20 cm x 20 cm x 1.75 mm x 1; pure water: methanol (1: 1)). (R) -5- (6-hydroxybenzo [d] thiazol-2-yl) -3,4-dihydro-2H-pyrrole-2-carboxylic acid (10.3 mg, 82% yield) Obtained as a brown solid. The measured physical properties of the product are as follows.

(R) -5- (6-Hydroxybenzo [d] thiazol-2-yl) -3,4-dihydro-2H-pyrrole-2-carboxylic acid
IR (film) 3088, 2924, 1677 cm ^-1 .
¹ H NMR (400 MHz, CD ₃ OD) δ 7.91 (1 H, d, J = 9.2 Hz), 7.35 (1 H, d, J = 2.5 Hz), 7.07 (1 H, dd, J = 2.5, 9.2 Hz), 4.96 (1 H, m), 2.51 (1 H, m), 2.29 (1 H, m).
¹³ C NMR (100 MHz, CD ₃ OD) δ 173.7, 173.4, 158.3, 157.8, 147.2, 138.1, 124.7, 116.9, 106.0, 73.8, 35.1, 26.1.
ESI-HRMS calcd for C ₁₂ H ₁₀ N ₂ O ₃ S 262.0412 (M + H) ⁺ , found, m / z 263.0490.
[α] ²⁵ _D -26.5 (c 1.00, CH ₃ OH).

(1-2) Basic conditions

(R) -2- (tert-Butoxycarbonylamino) -5- (6-hydroxybenzo [d] thiazol-2-yl) -5-oxopentanoic acid tert-butyl (Precursor 1, 20.8 mg, 0.0476 mmol) Was dissolved in 4 M hydrochloric acid in ethyl acetate (500 μL) at room temperature under an argon atmosphere. The resulting solution was stirred at room temperature for 24 hours under an argon atmosphere. As a result of TLC analysis (reverse phase (RP-18), water: methanol (1: 2)) of the reaction mixture, at this point, the deprotected product and the cyclized product ((R) -5-5) were obtained. (6-hydroxybenzo [d] thiazol-2-yl) -3,4-dihydro-2H-pyrrole-2-carboxylic acid) was produced (deprotected: near Rf value origin, UV irradiation positive, Ninhydrin coloration positive. Cyclized: Rf value = 0.7, UV irradiation positive, ninhydrin coloration negative).
The reaction mixture was concentrated under reduced pressure, and the obtained residue was dissolved in methanol (1.0 mL). To the resulting solution, 3 equivalents of any base of N, N-dimethyl-4-aminopyridine, triethylamine, or sodium hydroxide was added at room temperature, and the mixture was stirred at room temperature for 30 minutes. As a result, in both cases, the compound was decomposed in both the deprotected form and the cyclized form, and the cyclized product as the target product could not be obtained.

(2) Cyclization reaction from 3-hydroxy-2- (6-hydroxybenzo [d] thiazole-2-carboxamido) propanoic acid

(2-1) Trifluoroacetic acid
To a solution of D-form or L-form 3-hydroxy-2- (6-hydroxybenzo [d] thiazole-2-carboxyamido) propanoic acid (3.4 mg, 0.0081 mmol) in dichloromethane (2 ml) under an argon atmosphere, 0 After adding trifluoroacetic acid (10 μl) at 0 ° C., the resulting mixture was stirred at 0 ° C. for 30 min.
As a result of TLC analysis of the reaction mixture, the compound was decomposed, and the cyclized product, which was the target product, could not be obtained from either D-form or L-form precursor 2.

(2-2) Diethylamino sulfur trifluoride
D-form or L-form 3-hydroxy-2- (6-hydroxybenzo [d] thiazole-2-carboxyamido) propanoic acid (2.8 mg, 0.0067 mmol) in dichloromethane of 7 mM diethylaminosulfur trifluoride under argon atmosphere Solution (2 ml) was added at room temperature. The resulting mixture was heated to 90 degrees and stirred for 30 minutes while maintaining this temperature.
As a result of TLC analysis of the reaction mixture, the compound was decomposed, and the cyclized product, which was the target product, could not be obtained from either D-form or L-form precursor 2.

(2-3) Hydrochloric acid
To a dichloromethane solution (2 ml) of 3-hydroxy-2- (6-hydroxybenzo [d] thiazole-2-carboxyamido) propanoic acid (5.4 mg, 0.012 mmol) in D-form or L-form under argon atmosphere, 2 M hydrochloric acid aqueous solution (50 μL) was added at 0 ° C., and the mixture was stirred for 30 minutes while maintaining 0 ° C.
As a result of TLC analysis of the reaction mixture, the compound was decomposed, and the cyclized product, which was the target product, could not be obtained from either D-form or L-form precursor 2.

[Example 3] Measurement of emission intensity and wavelength (1) Preparation of solution In measuring emission intensity and wavelength, first, the following four solutions were prepared.
Solution 1: (R) -2- (tert-butoxycarbonylamino) -5- (6-hydroxybenzo [d] thiazol-2-yl) -5-oxopentane in 50 mM potassium phosphate buffer at pH 6 Acid tert-butyl (precursor 1), 3-hydroxy-2- (6-hydroxybenzo [d] thiazole-2-carboxamido) propanoic acid (precursor 2, using D-form), (R) -5- A substrate which is (6-hydroxybenzo [d] thiazol-2-yl) -3,4-dihydro-2H-pyrrole-2-carboxylic acid (cyclized) or firefly luciferin was dissolved at a concentration of 100 μM. Solution solution 2: Aqueous solution containing Mg and ATP at a concentration of 200 μM respectively 3: Solution of pH 8 500 mM potassium phosphate buffer solution 4: pH 8 50 mM phosphate containing glycerin at a concentration of 35% Firefly Lucif in potassium buffer Solution (from Photinus pyralis (firefly), Sigma-Aldrich Co., or Promega Corp. recombinant) hydrolase was dissolved at a concentration of 10 [mu] g / mL

(2) Measurement of luminous intensity 20 μL of solutions 1, 3 and 4 were weighed out and mixed uniformly, and then placed in a measuring instrument (Lumit sensor AB-2270-R manufactured by Atto Corporation). The mixed solution in the measuring instrument was made to emit light by adding 40 μL of Solution 2 in the dark, and the emission intensity was measured.
Table 1 shows the measurement results of the emission intensity of each of the other substrates when the emission intensity of firefly luciferin is 1.

  As Table 1 shows, the precursors 1 and 2 were not significantly different from the background in the emission intensity ratio, and no luminescence was observed, whereas the cyclized product was very high with respect to the background. A strong light was observed among the firefly luciferin derivatives.

(3) Measurement of emission wavelength The emission wavelength of each of the cyclized product and firefly luciferin in which the emission was observed in the above "(2) Measurement of emission intensity" was measured. The method is as follows.
Weigh 4.0 μL of solutions 1, 3 and 4 prepared in “(2) Measurement of luminescence intensity”, mix them uniformly, and then place them in a measuring instrument (Ate Co., Ltd., weak emission fluorescence spectrum measurement apparatus AB-1850). It was. The mixed solution in the measuring instrument was caused to emit light by adding 8.0 μL of Solution 2 in the dark, and the emission wavelength was measured.
The results are shown in FIG. The normalized intensity was determined by setting the intensity of the maximum emission wavelength to 1 for each of the cyclized product and firefly luciferin.

[Example 4] Measurement of stability (R) -5- (6-hydroxybenzo [d] thiazol-2-yl) -3,4-dihydro-2H-pyrrole-2-carboxylic acid (cyclized product) In order to compare the stability with firefly luciferin, each of these compounds (5.0 mg) was added at room temperature in the presence of air in the solvent (500 μL) and reagents (about 3 equivalents for each compound) shown in Table 2. The mixture was stirred for 1 hour, and it was measured whether the compound remained or decomposed after stirring.
The results are shown in Table 2. For example, when “100%” is described, it indicates that the compound was recovered 100% as it was without being decomposed or converted, and “decomposition” is described. In some cases, the original compound was not recovered at all due to decomposition or conversion of the compound.

  As a result, firefly luciferin is easily decomposed and unstable at any pH and solvent, whereas the cyclized product is completely in any of water and organic solvents in strong acid to neutral region. It was shown to be extremely stable without decomposition.

Claims (6)

A method for producing a cyclized compound represented by the following chemical formula (II) by contacting an acid with a compound represented by the following general formula (I)
(Wherein R ¹ , R ² and R ³ are each independently H or a substituent which becomes H upon contact with the acid, and R ⁴ is OH or the acid and Is a substituent which becomes OH by contact of The method according to claim 1 , wherein the acid is a Bronsted acid or a Lewis acid. The acid is hydrochloric acid, acetic acid, monofluoroacetic acid, difluoroacetic acid, trifluoroacetic acid, toluenesulfonic acid, sulfuric acid, nitric acid, phosphoric acid, perchloric acid, zinc bromide, tin chloride, aluminum chloride, lithium bromide, lithium chloride , Borane trifluoride, borane trichloride, borane tribromide, titanium tetrachloride, copper trifluoromethanesulfonate, silver trifluoromethanesulfonate, ytterbium trifluoromethanesulfonate, tin trifluoromethanesulfonate, iodine, and bromine The production method according to claim 1, wherein the production method is one or more acids selected from the group. 4. The compound according to any one of claims 1 to 3, wherein the compound represented by the chemical formula (I) is produced from a protein or peptide containing the amino acid represented by the following general formula (III) as a constituent amino acid. The manufacturing method as described.
A step of producing the cyclized compound represented by the chemical formula (II) by the method according to any one of claims 1 to 4 ,
Contacting the cyclized compound with luciferase;
A light emitting method comprising: 6. The luminescence method according to claim 5 , wherein the luciferase is a firefly luciferase.