JP2013184909A - Luminous substrate of luciferase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel luminous substrate in a firefly bioluminescence system.SOLUTION: Compounds of formula (I) and their salts are provided. In the formula, R, Rand Rto Rare each independently H, HN-, HS- or Calkyl; Ris OH or NRR; Rand Rare each independently H or Calkyl; Ris H or Calkyl; X and Y are C, N, S or O; and n is 0, 1, 2 or 3.

Description

  The present invention relates to a compound that can be used as a luminescent substrate in a firefly bioluminescence system. More specifically, the present invention relates to a compound capable of providing a luminescent substrate material having a long emission wavelength in a firefly bioluminescence system.

<About firefly luciferin>
In recent years, visualization of biological events and phenomena has been emphasized, and expansion of materials for visualization has been desired. Along with this, diversification is also required in the labeling technology. In particular, labeling techniques for molecular imaging have developed significantly in conjunction with advances in diagnostic and testing equipment. For example, labeling techniques for applying to advanced technologies such as personalized medicine for cancer and heart disease have been energetically studied. In addition, with the advancement of measurement technology, the demand for more sensitive and high-performance instruments and labeling materials is rapidly increasing.

  Among the visualization techniques, the firefly bioluminescence system is said to have a very high luminous efficiency and to convert energy into light most efficiently. The interpretation of the molecular mechanism of bioluminescence is also progressing.

In this firefly bioluminescence, it is known that luciferin, which is a luminescent substrate, emits light through a chemical reaction by the action of a luminescent enzyme luciferase. In this reaction, the luminescent substrate is adenylylated (AMP) in the presence of adenosine triphosphate (ATP) and divalent magnesium ion (Mg ²⁺ ) in the luminescent enzyme, and the adenylyl derivative is the active substrate. Be guided to. This is then oxygenated to the peroxide anion and converted to a high energy peroxide, dioxetanone. Unstable dioxetanone releases protons and carbon dioxide while decomposing, and enters an excited singlet state. The light emission from this dianion-type excited singlet state is yellowish green, which is considered to be firefly light emission. The product after luminescence is called oxyluciferin.

  As mentioned above, firefly bioluminescence has very high luminous efficiency, and the elucidation of the molecular mechanism of bioluminescence is progressing, so a wide variety of luminescent materials using firefly bioluminescence systems are sold by many companies. Yes. However, since the development of firefly bioluminescence-related luminescent materials is progressing mainly in the medical biochemical field, research and development from the protein (enzyme) side is generally active, but low molecular weight compounds (substrates) There is very little approach from the side. In particular, there is almost no research on the relationship between structure and activity, such as skeleton transformation of luminescent substrates.

  Furthermore, although a luminescent enzyme can be supplied inexpensively by recombinant technology, a luminescent labeling material using a firefly bioluminescent system supplied as a kit product or the like is not inexpensive. This is due to the fact that the luminescent substrate is luciferin. At present, D-form luciferin, which is a natural luminescent substrate, is synthesized from D-cysteine, which is an unnatural amino acid, but D-cysteine is very expensive.

<Needs and Status of Long Wavelength Luminescence Using Bioluminescence System>
In order to observe multi-phenomena, multi-color emission is also required in detection systems using labels. For this reason, it is desirable that the wavelength range of the labeling material that can be used in the detection system is wider. Further, for use in deep in vivo labeling, a red light emitting labeling material is desired from the viewpoint that long wavelength light has better light transmittance than short wavelength light. For example, for research using multicolor emission, it is desirable to prepare a labeling material that emits light at a wavelength of about 450 nm to 650 nm or more, particularly at 680 nm or more, as a label.

  Currently available substrates as luminescent substrates for firefly bioluminescent systems include substrates with several emission wavelengths. The wavelengths at the shortest and longest ends of these substrates include coelenterazine blue (about 480 nm) and firefly red (about 613 nm). In addition, a longer wavelength red luminescent material (about 630 nm) using a railway insect luminescent enzyme has recently been commercially available. However, since long wavelength light has better light transmission, there is potential demand for further expansion of not only these emission wavelengths but also the longest emission wavelength.

Examples of existing red and blue light emitting products using bioluminescent systems are shown below:
1． Promega Corporation: Chroma-Luc: About 613nm (Non-patent Document 1)
This system uses a mutant of clicked beetle (click beetle) and a natural firefly luminescent substrate.
2． Toyobo Co., Ltd .: MultiReporter Assay System-Tripluc: About 630nm (Non-patent document 2)
This system is a system using a railworm red luminescent enzyme and a natural firefly luminescent substrate. The luminescent color is changed using luciferase genes of green luminescence luciferase (SLG, maximum emission wavelength 550 nm), orange luminescence luciferase (SLO, 580 nm) and red luminescence luciferase (SLR, 630 nm). This utilizes luminescent enzymes that give different luminescent colors.
3． The University of Tokyo: Aminoluciferin: About 610nm (Patent Document 1)
This discloses a luciferin derivative.
Four. Promega Corporation: Chroma-Luc: About 480nm (Non-patent Document 3)
This system is a system using coelenterazine and Renilla luciferase.
Five. Atoh Corporation: Sea firefly bioluminescence approximately 460nm (Non-patent Document 4)
This system uses a coelenterazine-based substrate and Cypridina luciferase.

  The present inventors have also disclosed luciferin-like compounds in Patent Document 2 and Patent Document 3. These compounds are compounds having the same skeleton as luciferin.

  Furthermore, the present inventors have disclosed in Patent Document 4 that luciferin-like compounds having a skeleton different from luciferin have various emission wavelengths. Also in these luciferin-like compounds, it is desired to further shift the emission wavelength to the longer wavelength side.

  Furthermore, the present inventors have disclosed a compound having a firefly luciferin-like structure in Patent Document 5 that emits light at an emission wavelength different from that of natural firefly luciferin. One of these compounds is currently sold as ACALUMINE ™.

Japanese Unexamined Patent Publication No. 2007-091695 International Publication WO2007 / 116687 JP 2006-219381 A JP 2010-215795 A JP 2009-184932 A

Promega Corporation General Catalog 2008-9 12.6 Upload vol. 79, 2005 p 1-10, Toyobo Biochemicals for Lifescience 2006/2007 p4-67. Promega Corporation General Catalog 2008-9 12.14 Ato Corporation General Catalog 2008-2009 p 247

  In view of the above situation, an object of the present invention is to provide a novel luminescent substrate having a long emission wavelength in a firefly bioluminescence system.

  As a result of intensive studies to achieve the above object, the present inventors have newly found that a compound having a novel skeleton has a long emission wavelength in a firefly bioluminescence system.

  The present invention provides a compound having the following general formula I or a salt thereof:

Where
R ₁ , R ₂ and R _{4 to} R ₉ are each independently H, H ₂ N-, HS- or C _1-4 alkyl;
R ₃ is OH or NR ₁₁ R ₁₂ where R ₁₁ and R ₁₂ are each independently H or C _1-4 alkyl;
R ₁₀ is H or C _1-4 alkyl,
X and Y are each independently C, N, S or O;
n is independently 0, 1, 2 or 3.

  The present invention provides a compound having the following general formula II or a salt thereof:

Where
R ₃ is OH or NR ₁₁ R ₁₂ where R ₁₁ and R ₁₂ are each independently H or C _1-4 alkyl;
R ₁₀ is H or C _1-4 alkyl,
X and Y are each independently C, N, S or O.

In addition, the present invention provides a compound or a salt thereof, in the above general formulas I and II, wherein R ₃ is N (CH ₃ ) ₂ .

In the general formula I, R ₁ , R ₂ and R _{4 to} R ₉ are H, R ₁₀ is H, X is N, and Y is S. , N is 0 and provides a compound of the following formula (a) or a salt thereof:

  The present invention also provides a luciferase luminescent substrate comprising the above compound or a salt thereof.

  The present invention also provides a luminescence detection kit comprising the above compound or a salt thereof.

  According to the present invention, it is possible to provide a novel luminescent substrate having a long luminescence wavelength in a firefly bioluminescence system.

FIG. 3 is a graph showing relative light emission intensity when Glowell G2 emission intensity is 100 in an example of the compound according to the present invention.

It is a figure which shows the relative light emission intensity when the light emission intensity of a firefly luciferin is set to 100 in one Example of the compound based on this invention.

FIG. 4 is a graph showing the relative light emission intensity of the 180-second integrated value when the light emission intensity of Glowell G2 is set to 100 in an example of the compound according to the present invention.

FIG. 3 is a graph showing the relative light emission intensity of the 180-second integrated value when the light emission intensity of firefly luciferin is 100, in one example of the compound according to the present invention.

It is a figure which shows the emission spectrum of one Example of the compound based on this invention.

  The present invention provides a compound having the following general formula I or a salt thereof:

Where
R ₁ , R ₂ and R _{4 to} R ₉ are each independently H, H ₂ N-, HS- or C _1-4 alkyl;
R ₃ is OH or NR ₁₁ R ₁₂ where R ₁₁ and R ₁₂ are each independently H or C _1-4 alkyl;
R ₁₀ is H or C _1-4 alkyl,
X and Y are each independently C, N, S or O;
n is independently 0, 1, 2 or 3.

As used herein, the term “C _1-4 alkyl” refers to a saturated linear or branched alkyl group containing from 1 to 4 carbon atoms, such as methyl, ethyl, n-propyl, iso-propyl, n-butyl. , Iso-butyl, sec-butyl and tert-butyl.

As mentioned above, it will be readily apparent to one skilled in the art that R ₁ , R ₂ and R ₄ -R ₉ may be C _1-4 alkyl. Also, those skilled in the art will readily realize that R ₁ , R ₂ and R _{4 to} R ₉ may be H ₂ N- and HS-.

As mentioned above, those skilled in the art will readily realize that R ₃ may be OH. For example, the above-mentioned Patent Document 4 by the present inventors shows that a luciferin-like compound in which the —OH group of luciferin is substituted with —N (CH ₃ ) ₂ group can be a substrate of a firefly bioluminescence system. ing. The compounds of the present invention are shown to have activity when R ₃ is a —N (CH ₃ ) ₂ group in the examples. Therefore, even if R ₃ is OH, it is unlikely to affect the activity.

As described above, when R ₃ is NR ₁₁ R ₁₂ , it can be easily conceived by those skilled in the art that R ₁₁ and R ₁₂ may be H or C _1-4 alkyl. Will.

As noted above, one skilled in the art can readily appreciate that R ₁₀ may be C _1-4 alkyl. For example, the above-mentioned Patent Document 2 by the present inventors shows a result that a luciferin-like compound in which a carboxyl group bonded to a heterocycle of luciferin is AMPed can be a substrate of a firefly bioluminescence system. Therefore, also in the compound of the present invention, it is considered that such a lower alkyl which substitutes a carboxyl group bonded to a heterocycle similar to the luciferin heterocycle is less likely to affect the activity.

  In the above general formula I, X and Y can each independently be C, N, S or O. One skilled in the art will readily realize that the heteroatoms of X and Y may be C, N, S or O. For example, the various luciferin-like compounds described in Patent Document 2 described above by the present inventors include luciferin-like compounds in which the portions corresponding to the compounds of the present invention have various heteroatoms as the substrate of the firefly bioluminescence system. The results obtained are shown.

  As described above, a person skilled in the art can easily conceive that the olefin chain unit represented as “n” in the general formula I can be changed to a desired length. Further, two n in the general formula I may be the same or different.

  In one embodiment, the present invention provides a compound having the following general formula II or a salt thereof:

Where
R ₃ is OH or NR ₁₁ R ₁₂ where R ₁₁ and R ₁₂ are each independently H or C _1-4 alkyl;
R ₁₀ is H or C _1-4 alkyl,
X and Y are each independently C, N, S or O.

In one embodiment, the present invention provides a compound having the following general formula III, wherein R ₃ is N (CH ₃ ) ₂ in general formula I:

In another embodiment, the invention relates to a compound of general formula II wherein R ₃ is N (CH ₃ ) ₂ , R ₁₀ is H, X is N, Y is S and n is Provides a compound of formula (a) which is 0:

  The compound represented by the above formula (a) has a light emission activity in a long wavelength region and has a broad emission spectrum. This compound emits light at a light emission wavelength of approximately 670 nm in a firefly bioluminescence system, and its emission spectrum has a broad base extending to a region exceeding 700 nm. That is, it can be said that the compound represented by the above formula (a) has a broad emission spectrum and has a light emitting ability on the longer wavelength side than existing materials. Therefore, the compound of the present invention is also useful as a lead compound for obtaining a light emitting substrate material having a light emission wavelength longer than that of an existing material.

  The present invention includes salts of the aforementioned compounds. A “salt” is envisioned only in the compounds of the present invention where any portion of the compound can form a base.

  The expression "salt" includes hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, phosphorous acid, nitrous acid, citric acid, formic acid, acetic acid, oxalic acid, maleic acid, lactic acid , Tartaric acid, fumaric acid, benzoic acid, mandelic acid, cinnamic acid, pamoic acid, stearic acid, glutamic acid, aspartic acid, methanesulfonic acid, ethanedisulfonic acid, p-toluenesulfonic acid, salicylic acid, succinic acid, trifluoroacetic acid and Any salt with an inorganic or organic acid that is non-toxic to living organisms, or where the nature of the compound of formula I is acidic, an alkali or alkaline earth base such as hydroxylated Also included are salts with inorganic bases such as sodium, potassium hydroxide, calcium hydroxide and the like.

  The compounds of the invention can be prepared, for example, according to the procedures described in the following examples. More detailed procedures are described in the examples below, but compounds of general formula I can be prepared, for example, according to the following procedures.

  First, a commercially available benzoic acid ester having an arbitrary substituent is used as a boronic acid derivative, and is reacted with a styrene-type compound derived from a commercially available benzaldehyde substituted body having an arbitrary substituent according to the following reaction formula, Compound 1 in which two benzene rings having a substituent are connected by a single bond or an olefin chain is obtained.

  Next, according to the following reaction formula, compound 1 is reduced with DIBAL-H and subsequently oxidized (swan oxidation or the like) to give an aldehyde to obtain compound 2.

  Next, the compound 2 is reacted with a wittig reagent to increase carbon and introduce a double bond. Similarly, double bonds can be increased to an arbitrary number by repeating reduction / oxidation and carbon increase. This step provides compound 3.

  Next, according to the following reaction formula, compound 3 is reacted with sodium hydroxide in an 80% aqueous ethanol solvent to be hydrolyzed to give a carboxylic acid to obtain compound 4.

Next, compound 4 and commercially available cysteine protector 5 are converted into 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium chloride according to the following reaction formula. Reaction with (DMT-MM) to give an amide compound gives compound 6.

  Next, by reacting triphenylphosphine oxide with trifluoromethanesulfonic anhydride in an argon atmosphere according to the following reaction formula, compound 6 is derived into a cyclized product to obtain compound 7.

  Next, compound 8 is obtained by deprotecting the tBu group of compound 7 with trifluoroacetic acid (TFA) in methanol according to the following reaction formula.

One skilled in the art will understand that the corresponding compounds can be synthesized by procedures similar to the synthetic procedures described above, starting from starting materials in which the substituents of the compounds of general formula I are replaced with the desired substituents. Will. Those skilled in the art will also appreciate that the substituents of the compound can be substituted with the desired substituents in the appropriate steps of the synthetic procedure. Also, in the last step, a compound having the corresponding R ₁₀ could be obtained by substituting the carboxylic acid moiety with the desired ester.

The compound of the present invention emits light by being oxidized by a luminescent beetle luciferase when added to a system in which luminescent beetle luciferase, ATP and Mg ²⁺ are present. The compound of the present invention can be used alone as a luminescent substrate, but may be used in combination with other luminescent substrates as necessary. The compound of the present invention or a salt thereof can also be provided as a kit (luminescence detection kit) together with ATP and Mg ²⁺ . The kit can also contain other luminescent substrates and solutions prepared at an appropriate pH. Furthermore, the luminescent substrate composition prepared by adjusting the compound of the present invention to an appropriate pH together with ATP and Mg ²⁺ can be provided as a luminescence detection kit for the compound of the present invention or a salt thereof.

  Since the firefly bioluminescence system is an aqueous system, a hydrophilic organic compound may be present. For example, tetrafluoroacetic acid, acetic acid, formic acid and the like may be present. When the compound of the present invention is applied to a luminescent system, it is preferably used at a concentration of a luminescent substrate of 1 μM or more, for example, 5 μM or more in order to obtain a suitable luminescence intensity. The pH of the light emitting system is assumed to be 4 to 10, preferably 6 to 8, but is not particularly limited. If necessary, buffering agents such as potassium phosphate, Tris-HCl, glycine and HEPES can be used to stabilize the pH.

  In addition, the compound of the present invention can be made to emit light by various oxidases in the firefly luminescence beetle luciferase luminescence system. Luciferase has been isolated from various organisms such as North American firefly (Photinus pyralis) and Railroad worm, any of which can be used. Oxidases that can be used include, for example, light beetle luciferase, Iriomote luciferase and flavin-containing monooxygenase.

It is known that bioluminescence using the compound of the present invention as a luminescent substrate is enhanced in the presence of coenzyme A (CoA), pyrophosphate or Mg ion (Mg ²⁺ ) in the luminescent system. Therefore, these can be used as a luminescence enhancer of the luminescent beetle luciferase luminescence system. It is known that the luminescence enhancement effect of these compounds is remarkable when the concentration of CoA, pyrophosphate or Mg ²⁺ in the luminescence system is 5 μM or more, respectively, and is enhanced as the concentration increases.

In order to use the firefly bioluminescence system for measurement / detection, it is important to stabilize the luminescence so as to prevent inactivation of the enzyme and exhibit a plateau luminescence behavior. For example, Mg ²⁺ is effective for stabilizing luminescence in a firefly bioluminescence system. When Mg ²⁺ is present in the light emitting system, the light emission behavior changes so that attenuation after rising is suppressed. In particular, when pyrophosphate and Mg ²⁺ coexist in the luminescent system, the luminescence behavior changes greatly. That is, the stabilization of luminescence becomes extremely remarkable, and the luminescence behavior when a large excess of pyrophosphate and Mg ²⁺ coexists with the luminescent substrate rises rapidly and is maintained to form a plateau state. In the case of Mg ²⁺ alone, when the Mg ²⁺ concentration of the luminescent system is 0.5 mM or more, the luminescence stabilization effect is significant, and is enhanced as the concentration of Mg ²⁺ increases. In order to achieve a plateau emission behavior, magnesium pyrophosphate can be present, for example, at a concentration of 10 μM or more, preferably 100 μM or more. Further, the ratio of pyrophosphoric acid to Mg ²⁺ need not be an equivalent ratio. Although magnesium pyrophosphate has low water solubility, pyrophosphoric acid and Mg ²⁺ can be separately supplied by using this. These can be supplied to the luminescent system in free form and in salt form. Magnesium salts that can be used include inorganic acid salts such as magnesium sulfate and magnesium chloride, and organic acid salts such as magnesium acetate. Pyrophosphates include salts with alkali metals such as sodium and potassium, salts with alkaline earth metals such as magnesium and calcium, salts with iron and the like. These may be included in the light emitting system in an aqueous solution state. In consideration of the effect on the enzyme, the pH of the luminescent system is preferably 2-10.

  The compounds of the present invention may be used as substrates in chemiluminescence. Chemiluminescence occurs when the compound of the present invention is oxidized to produce a peroxide, and the decomposition product of the peroxide becomes a luminescent species in an excited state. Oxidation can proceed by air oxidation using, for example, potassium t-butoxy in DMSO. In the case of chemiluminescence, luminescence with a shorter wavelength than that in the firefly bioluminescence system is assumed.

  The compounds of the present invention can be used as luminescent labels in biological measurements / detections. For example, it can be used to label amino acids, polypeptides, proteins, nucleic acids and the like. Means for conjugating the compounds of the present invention to these materials are well known to those of skill in the art. For example, the compounds of the present invention can be coupled to the carboxyl and amino groups of the target substance using methods well known to those skilled in the art.

  In addition, the compound of the present invention can be used for measurement / detection utilizing detection of luminescent beetle luciferase activity by luminescence of a luminescent substrate. For example, a compound of the present invention is reacted under conditions suitable for reaction with a luminescent beetle luciferase as described above. The luminescence from the compound is then detected. For example, by administering the compound of the present invention to a cell or animal into which a luciferase gene has been introduced, the expression of a target gene or protein in vivo can be measured / detected.

  In the following examples, the present invention is specifically described, but the present invention is not limited to these ranges.

1) Instrument analysis and measurement equipment
pH measurement: Measured using pH test paper UNIV manufactured by Toyo Roshi Kaisha, Ltd. Moreover, it measured using pH / ION METER F-23 by Horiba as a pH meter.

¹ H nuclear magnetic resonance spectrum ( ¹ H NMR): Measured using a Lambda-270 type device (270 MHz) manufactured by JEOL Ltd. or an ECA500 type device (500 MHz) manufactured by JEOL Ltd. “ ¹ H NMR (measurement frequency, measurement solvent): δ chemical shift value (number of hydrogen, multiplicity, spin coupling constant)”. The chemical shift value (δ) is expressed in pp using tetramethylsilane (δ = 0) as an internal standard. Multiplicity is displayed as s (single line), d (double line), t (triple line), q (quadruple line), m (multiple line or complex overlapping signals) It was written. The spin coupling constant (J) is described in Hz.

¹³ C nuclear magnetic resonance spectrum ( ¹³ C NMR): Measured using a Lambda-270 type device (67.8 MHz) manufactured by JEOL Ltd. or an ECA500 type device (125 MHz) manufactured by JEOL Ltd. “ ¹³ C NMR (measurement frequency, measurement solvent): δ chemical shift value (multiplicity)” is described. The chemical shift value (δ) is expressed in ppm with tetramethylsilane (δ = 0) as an internal standard. The multiplicity is indicated by s (single line), d (double line), t (triple line), and q (quadruple line).

  Mass spectrum (MS): Measured by electron impact method (EI, ionization energy: 70 eV) using a JMS-600H mass spectrometer manufactured by JEOL Ltd. It measured by the electron spray ionization method (ESI) using the JEOL company JMS-T100LC type TOF mass spectrometer AccuTOF. The apparatus was set to a solvent removal gas of 250 ° C., an orifice 1 temperature of 80 ° C., a needle voltage of 2000 V, a ring lens voltage of 10 V, an orifice 1 voltage of 85 V, and an orifice 2 voltage of 5 V. Sample feeding was performed by the infusion method, and the flow rate was 10 μl / min. It was described as “MS (measurement method) m / z mass number (relative intensity)”.

Specific rotation: measured using a DIP-1000 polarimeter manufactured by JASCO Corporation. A sodium lamp was used as the light source, and a cylindrical glass cell (Φ10 × 100 mm) was used as the cell. The measured values are uncorrected, and the data is the average of 5 measurements. Each of D-form and L-form is described as “D or L: [α] measured ^temperature value (concentration, measurement solvent)”.

2) Chromatography Analytical thin layer chromatography (TLC):. E Merck Co. TLC plates, using silica gel 60F ₂₅₄ (Art.5715) thickness 0.25 mm. The detection of the compound on TLC was carried out by UV irradiation (254 nm or 365 nm) and immersion in a color former, followed by heating to cause color development. As the color former, p-anisaldehyde (9.3 ml) and acetic acid (3.8 ml) dissolved in ethanol (340 ml) and concentrated sulfuric acid (12.5 ml) were added.

Preparative thin layer chromatography (PTLC): E. Merck TLC plate, silica gel 60F ₂₅₄ (Art.5744) 0.5 mm thick, or E. Merck silica gel 60GF for thin layer chromatography ₂₅₄ (Art. 7730) was prepared on a 20 cm × 20 cm glass plate adjusted to a thickness of 1.75 mm.

Silica gel column chromatography: silica gel 60F ₂₅₄ (Art. 7734) manufactured by E. Merck was used.

3) Basic operation The reaction solution was cooled by immersing the reaction vessel in a dewar filled with refrigerant. Ice water was used at room temperature to 4 ° C, and liquid nitrogen-acetone was used as a refrigerant at 4 to -90 ° C. The extracted solution after the reaction was dried by washing with saturated brine and then adding anhydrous sodium sulfate or anhydrous magnesium sulfate. About what performed neutralization with the resin after reaction, the cation exchange resin Amberlite IR120B NA or the anion exchange resin Amberlite IRA400 OH AG made by Organo Corporation was used. The solution was concentrated under reduced pressure using a rotary evaporator under reduced pressure of an aspirator (20 to 30 mmHg). The trace amount of solvent was removed using a vacuum pump (about 1 mmHg) equipped with a trap cooled in a liquid nitrogen bath. All solvent mixing ratios were expressed in volume ratios.

4) Solvent Distilled water was distilled and ion-exchanged using a GS-200 type distilled water production apparatus manufactured by Advantech Toyo Corporation.

  Toluene, methanol, ethanol, isopropanol, dichloromethane, tetrahydrofuran, N, N-dimethylformamide, 2-butanone, dehydrated solvent for organic synthesis or special grade solvent manufactured by Kanto Chemical Co., Ltd., using molecular sieves (4A) Used.

The solvent for NMR measurement used the following as it was. CDCl ₃ : 99.7 ATOM% D, 0.03% TMS made by ISOTEC Inc., CD ₃ OD: 99.8 ATOM% D (˜0.7 ATOM% ¹³ C) made by ISOTEC Inc., 0.05% TMS.

[Example 1]
The following biphenyl type compound 8 was synthesized according to the following synthesis scheme.

  (Synthesis of biphenyl derivative 2)

Ethyl 4-iodobenzoate (250 μl, 1.5 mmol), tetrakis (triphenylphosphine) palladium (0) (100 mg, 5%), potassium carbonate (539.0 mg, 3.8 mmol) in N, N-dimethylformamide (6 ml) Dissolved and put under argon atmosphere. 4- (N, N-dimethylamino) phenylboronic acid (1) (315.0 mg, 1.9 mmol) was added, and the mixture was heated with stirring at 90 ° C. for 14 hours. Furthermore, tetrakis (triphenylphosphine) palladium (0) (27 mg, 2%), potassium carbonate (125.0 mg, 0.90 mmol), 4- (N, N-dimethylamino) phenylboronic acid (1) (97.0 mg, 0.58 mmol) was added and stirred for 24 hours. The reaction mixture was subjected to silica gel column chromatography [ethyl acetate] to remove insoluble matters, and then concentrated under reduced pressure. The obtained crude product was purified by silica gel column chromatography [hexane-ethyl acetate (4: 1) → (0: 1)] to give biphenyl derivative 2 (229.0 mg, 0.85 mmol, 56%) as a white solid. Obtained.
¹ H NMR (500MHz, CDCl ₃ )
δ1.40 (3H, t, J = 6.9Hz), 3.00 (6H, s), 4.38 (2H, q, J = 6.9Hz), 6.79 (2H, d, J = 9.2Hz), 7.55 (2H, d , J = 9.2Hz), 7.61 (2H, d, J = 8.0Hz), 8.05 (2H, d, J = 8.6Hz)

  (Synthesis of carboxylic acid 3)

Biphenyl derivative 2 (148.0 mg, 0.54 mmol) was dissolved in ethanol (6 ml), 1M aqueous sodium hydroxide solution (6 ml) was added, and the mixture was refluxed for 4 hours. The reaction mixture was neutralized with a saturated aqueous ammonium chloride solution (9 ml), filtered and washed with water. Carboxylic acid 3 (122.0 mg, 0.50 mmol, 92%) was obtained as a yellow solid.
¹ H NMR (500MHz, DMSO-D6)
δ2.91 (6H, s), 6.77 (2H, d, J = 6.9Hz), 7.54 (2H, d, J = 7.5Hz), 7.49 (2H, d, J = 7.5Hz), 7.89 (2H, d , J = 7.5Hz),

  (Synthesis of cysteine protector 5)

TFA (1.5 ml) was added and dissolved in D-cysteine methyl ester hydrochloride (4) (308 mg, 1.8 mmol) and triphenylmethanol (515 mg, 1.98 mmol). The mixture was stirred at room temperature for 2 hours, concentrated under reduced pressure, and neutralized with an anion exchange resin IRA400 OH AG. The resin was filtered and the resulting solution was concentrated under reduced pressure. The obtained residue was purified by silica gel chromatography [silica gel 30 g; hexane-ethyl acetate (1: 1)] to obtain cysteine-protected 5 (658 mg, 1.7 mmol, 97%) as a pale yellow oil.
¹ H NMR ¹ H NMR (270 MHz, CDCl ₃ )
δ2.47 (1 H, dd, J = 8.1, 13), 2.60 (1 H, dd, J = 5.1, 13), 3.21 (1 H, dd, J = 5.1, 8.1 Hz), 3.66 (3 H, s), 7.18-7.32 (9 H, complex), 7.40-7.54 (6 H, complex)

  (Synthesis of amide 6)

Carboxylic acid 3 (15.0 mg, 0.062 mmol) and cysteine protector 5 (31.2 mg, 0.082 mmol) were dissolved in N, N-dimethylformamide (2 ml). To this solution was added 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium chloride (DMT-MM) (21.6 mg, 0.073 mmol) and the mixture was added. Stir at room temperature for 3 hours. Thereafter, the mixture was extracted with dichloromethane (20 ml × 3), the organic layers were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography [hexane-ethyl acetate (2: 1)] to obtain amide 6 (32.9 mg, 0.054 mmol, 87%)) as a transparent oil.
¹ H NMR (500 MHz, CDCl ₃ )
δ2.75 (2H, complex), 3.01 (6H, s), 3.75 (3H, s), 4.85 (1H, dd, J = 12.6, 4.6Hz), 6.81 (2H, d, J = 8.6Hz), 7.15 -7.34 (9H, complex), 7.35-7.42 (6H, complex), 7.55 (2H, d, J = 8.6Hz), 7.63 (2H, d, J = 8.0Hz), 7.78 (2H, d, J = 8.0 Hz)

  (Synthesis of cyclized product 7)

Amide 6 (14.0 mg, 0.023 mmol) and triphenylphosphine oxide (19.0 mg, 0.068 mmol) were dissolved in dichloromethane (1.5 ml) under an argon atmosphere, trifluoromethanesulfonic anhydride (50 μl, 0.29 mmol) was added, Stir for 2.5 hours. Thereafter, the mixture was extracted with dichloromethane (20 ml × 2), and the organic layer was washed with saturated brine (30 ml). Furthermore, the organic layers were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The obtained residue was purified by preparative thin layer chromatography [20 cm × 20 cm × 1.75 mm × 1 sheet; hexane-ethyl acetate (1: 1)], and cyclized product 7 (7.0 mg, 0.020 mmol, 86% ) Was obtained as a yellow solid.
¹ H NMR (500MHz, CDCl ₃ )
δ3.00 (6H, s), 3.61-3.73 (2H, complex), 3.83 (3H, s), 5.29 (1H, t, J = 9.2Hz), 6.79 (2H, d, J = 6.3Hz), 7.54 (2H, d, J = 5.9Hz), 7.59 (2H, d, J = 4.6Hz), 7.87 (2H, d, J = 4.6Hz)

  (Synthesis of biphenyl type compound 8)

Cyclized product 7 (7.7 mg, 0.023 mmol) was dissolved in THF: distilled water (2: 1, 2 ml). To this solution was added lithium hydroxide-hydrate (2.0 mg, 0.047 mmol) and stirred at room temperature for 30 minutes. Ethyl acetate (30 ml) was added to the reaction mixture, and the product was extracted with water (20 ml × 2). The aqueous layers were combined, acidified with 1M hydrochloric acid, and extracted with ethyl acetate (20 ml × 2). Thereafter, the extract was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain biphenyl type compound 8 (4.0 mg, crude) as a red solid.
¹ H NMR (500MHz, CDCl ₃ )
δ3.01 (6H, s), 3.73 (2H, m), 5.34 (1H, t, J = 9.2Hz), 6.80 (2H, d, J = 8.0Hz), 7.55 (2H, d, J = 8.6Hz) ), 7.62 (2H, d, J = 8.5Hz), 7.86 (2H, d, J = 8.0Hz)

[Example 2: Measurement of relative emission intensity and bioluminescence spectrum]
1) Measuring device (High-performance liquid chromatography (HPLC))
An Agilent 1100 series HPLC manufactured by Agilent Technologies was used. The breakdown of the equipment is degasser, quaternary pump, manual injector, column compartment, diode array detector, fluorescence detector, and ChemStation (PC software). The column used was CHIRALCELOD-RH (inner diameter 0.46 cm, length 15 cm) manufactured by Daicel Chemical Industries, Ltd.

(PH measurement)
This was performed using an F-23 type glass electrode type hydrogen ion concentration indicator manufactured by Horiba.

(Measurement of photon quantity)
Measurement was performed using a Luminescencer Octa AB-2270 manufactured by ATTO Corporation.

(Measurement of emission spectrum)
The measurement was performed using a weak emission fluorescence spectrum apparatus AB-1850 manufactured by ATTO Corporation. All measured spectra are spectra corrected for the characteristics of the detector.

2) Reagents Ultrapure water was collected from Milli-RX12α manufactured by Millipore.

  For methanol and t-butanol, special grade solvents manufactured by Kanto Chemical Co., Ltd. were used.

  For luciferase (derived from North American firefly Photinus pyralis), a recombinant type produced by Promega was used.

  ATP-Mg manufactured by Sigma was used.

  Phosphate buffer solution was prepared by dissolving dipotassium hydrogen phosphate dodecahydrate (special grade) and potassium dihydrogen phosphate dihydrate (special grade) manufactured by Wako Pure Chemical Industries, Ltd. in ultrapure water. Adjusted and used.

  Potassium t-butoxide used was made by Tokyo Chemical Industry Co., Ltd.

  For distilled water and acetonitrile, a solvent for high performance liquid chromatography manufactured by Kanto Chemical Co., Inc. was used.

  Trifluoroacetic acid (made by Wako Pure Chemical Industries, Ltd.) (special grade) was used.

  As comparative controls, firefly luciferin (following formula (b)) manufactured by Wako Pure Chemical Industries, Ltd. and Akalumine (trademark) (following formula (c)) manufactured by Wako Pure Chemical Industries, Ltd. were used.

  A Belwell Glowell G2 (yellow) was used as a standard light source.

3) Sample preparation (substrate solution)
The substrate was weighed and diluted with an electronic balance. As a solvent, phosphate buffer (50 mM, pH 6.0) was used for bioluminescence measurement, and t-butanol was used for chemiluminescence measurement.

(Enzyme solution)
The luciferase was diluted with Tris-HCl buffer (50 mM, pH 8.0) to 1 μg / μl, and divided into small portions. This was used as a stock solution, and the necessary amount was diluted each time. The stock solution was stored in a -80 ° C freezer.

(ATP-Mg solution)
ATP-Mg was diluted with ultrapure water.

4) Relative luminescence intensity Relative luminescence intensity of firefly luciferin, red luminescence and biphenyl type compound 8 of Example 1 was measured. In a 200 μL polystyrene tube, potassium phosphate buffer (0.5 M, pH 8.0, 20 μl), substrate solution (100 μM, 20 μl), enzyme solution (10 μg / ml, 20 μl), then ATP-Mg solution (200 μM, 40 μl) Were mixed to measure the emission intensity.

  The results are shown in FIGS. FIG. 1 shows the relative luminescence intensity in one example of the compound according to the present invention when the luminescence intensity of Glowell G2 is 100, and FIG. 3 shows the relative luminescence intensity of the 180-second integrated value at this time. FIG. 2 shows the relative luminescence intensity of one example of the compound according to the present invention when the luminescence intensity of firefly luciferin is 100, and FIG. 4 shows the relative luminescence intensity of the 180-second integrated value at this time.

5) Bioluminescence spectrum The bioluminescence spectra of firefly luciferin, red luminescence and biphenyl type compound 8 were measured. In a 200 μL polystyrene tube, potassium phosphate buffer (0.5 M, pH 8.0, 5 μl), substrate solution (100 μM, 5 μl), enzyme solution (1 mg / ml, 5 μl), then ATP-Mg solution (200 μM, 10 μl) Were mixed and the emission spectrum was measured. The exposure time for emission spectrum measurement was 60 seconds. However, firefly luciferin was performed in 5 seconds.

The results are shown in FIG. FIG. 5 is a graph showing an emission spectrum of one example of the compound according to the present invention. As shown in FIG. 5, firefly luciferin had an emission maximum wavelength of 565 nm, and Acalumine had an emission maximum wavelength of 673 nm. Biphenyl type compound 8 had an emission maximum wavelength of 670 nm. Therefore, biphenyl type compound 8 had a maximum emission wavelength on the longer wavelength side than natural firefly luciferin.

  Further, as shown in FIG. 5, biphenyl type compound 8 had a broad emission spectrum. The emission spectrum of the biphenyl type compound 8 extends to the region exceeding 700 nm. A biphenyl-type luminescent substrate such as this compound has never been found, and is a completely new skeleton as a luminescent substrate material. Therefore, this compound is also useful as a lead compound for obtaining a light emitting material having an emission wavelength on the longer wavelength side.

  Since the present invention can provide a novel luminescent substrate having a long emission wavelength in a firefly bioluminescence system, it can be suitably used as a luminescent material that is widely useful in the medical and life science fields.

Claims (6)

A compound having the following general formula I or a salt thereof:
Where
R ₁ , R ₂ and R _{4 to} R ₉ are each independently H, H ₂ N-, HS- or C _1-4 alkyl;
R ₃ is OH or NR ₁₁ R ₁₂ where R ₁₁ and R ₁₂ are each independently H or C _1-4 alkyl;
R ₁₀ is H or C _1-4 alkyl,
X and Y are each independently C, N, S or O;
n is independently 0, 1, 2 or 3. A compound having the following general formula II or a salt thereof:
Where
R ₃ is OH or NR ₁₁ R ₁₂ where R ₁₁ and R ₁₂ are each independently H or C _1-4 alkyl;
R ₁₀ is H or C _1-4 alkyl,
X and Y are each independently C, N, S or O. The compound or a salt thereof according to claim 1 or 2, wherein R ₃ is N (CH ₃ ) ₂ . A compound having the following formula or a salt thereof:
  A luminescent substrate for luciferase comprising the compound according to any one of claims 1 to 4 or a salt thereof.   A luminescence detection kit comprising the compound according to any one of claims 1 to 4 or a salt thereof.