WO2010106896A1 - 波長が制御されたルシフェラーゼの発光基質および製造方法 - Google Patents

波長が制御されたルシフェラーゼの発光基質および製造方法 Download PDF

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WO2010106896A1
WO2010106896A1 PCT/JP2010/053177 JP2010053177W WO2010106896A1 WO 2010106896 A1 WO2010106896 A1 WO 2010106896A1 JP 2010053177 W JP2010053177 W JP 2010053177W WO 2010106896 A1 WO2010106896 A1 WO 2010106896A1
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compound
luciferin
emission wavelength
group
mmol
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French (fr)
Japanese (ja)
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昌次郎 牧
哲 小島
治樹 丹羽
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University of Electro Communications NUC
Campus Create Co Ltd
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Campus Create Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • C07D277/08—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
    • C07D277/12—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/60—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings condensed with carbocyclic rings or ring systems
    • C07D277/62—Benzothiazoles
    • C07D277/64—Benzothiazoles with only hydrocarbon or substituted hydrocarbon radicals attached in position 2
    • C07D277/66—Benzothiazoles with only hydrocarbon or substituted hydrocarbon radicals attached in position 2 with aromatic rings or ring systems directly attached in position 2
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
    • C09K2211/1037—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom with sulfur

Definitions

  • the present invention relates to a method for controlling the emission wavelength of luciferin and luciferin analogs.
  • the present invention also relates to a method for producing a luminescent substrate for luciferase having a controlled wavelength.
  • 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.
  • luciferin which is a luminescent substrate, emits light through a chemical reaction by the action of a luminescent enzyme luciferase.
  • 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.
  • AMP adenylylated
  • ATP adenosine triphosphate
  • Mg 2+ divalent magnesium ion
  • the luminous efficiency is very high, and the molecular mechanism of bioluminescence is elucidated.
  • the objects for which visualization is desired are expanding, and signs for various materials are desired. Because of these circumstances, a wide variety of luminescent materials using a firefly bioluminescent system are sold by many companies.
  • the luminous efficiency is very high, and the molecular mechanism of bioluminescence is elucidated. Because of these circumstances, a wide variety of luminescent materials using a firefly bioluminescent system are sold by many companies. 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.
  • luminescent labeling materials using firefly bioluminescent systems such as kit products are not inexpensive.
  • the luminescent substrate is luciferin.
  • 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.
  • multicolor luminescence 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 light emission, it is desirable to prepare a labeling material having light emission over a wavelength of about 450 nm or less to about 650 nm or more as a label.
  • Non-patent document 1 Chroma-Luc: approx. 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).
  • SLG green luminescence luciferase
  • SLO orange luminescence luciferase
  • SLR red luminescence luciferase
  • the present inventors also disclose luciferin-like compounds. These compounds are compounds having the same skeleton as luciferin.
  • the present inventors made a group of analogs of a luminescent substrate having a similar structure and analyzed the emission wavelength.
  • the emission wavelength is shifted by about 100 nm to the longer wavelength side, and the double bond portion is converted into a benzene ring, thereby generating an emission wavelength.
  • an index for changing a practical emission wavelength could be established.
  • the present invention relates to a method for producing a substrate of luciferase having a desired emission wavelength, and in order to shift the emission wavelength, an —OH group or an —NR 1 R 2 group (wherein R 1 And R 2 are each independently H or C 1-4 alkyl) in order to produce a compound in which one is substituted with the other and / or to shift the emission wavelength in luciferin or luciferin analogs
  • the present invention is the above production method, wherein in order to shift the emission wavelength, the formula I: A compound in which the —OH group of luciferin is substituted with a —NR 1 R 2 group (wherein R 1 and R 2 are each independently H or C 1-4 alkyl) or the general formula II: Wherein R 1 , R 2 and R 3 are each independently H or C 1-4 alkyl, X and Y are each independently C, N, S or O, and n is 0, 1, 2 or 3) or general formula III: Wherein R 1 , R 2 and R 3 are each independently H or C 1-4 alkyl, X and Y are each independently C, N, S or O, and n is -NR 1 R 2 group of 0, 1, 2 or 3 (wherein R 1 and R 2 are each independently H or C 1-4 alkyl) is substituted with an —OH group A step of producing a compound, a step of producing a compound in which the number n of double bonds of the general formula II or III is changed in order to shift the
  • the present invention provides luciferin analogs of general formula II: Where R 1 , R 2 and R 3 are each independently H or C 1-4 alkyl; X and Y are each independently C, N, S or O; n is 0, 1, 2 or 3.
  • the present invention also provides luciferin analogs of general formula III: Wherein R 1 , R 2 and R 3 are each independently H or C 1-4 alkyl, X and Y are each independently C, N, S or O, and n is 0 , 1, 2 or 3.
  • the present invention also provides the above compound, wherein the —NR 1 R 2 group is an —OH group.
  • the present invention provides a luminescence detection kit comprising the compound produced by the above production method or the compound described above.
  • the present invention also provides a luminescence detection kit comprising a compound produced by the above production method and a plurality of compound groups selected from the compounds described above.
  • the present invention provides a luminescent agent using the compound produced by the above production method or the compound described above.
  • the present invention provides a luciferin analog having a desired emission wavelength.
  • a systematic conversion index that almost covers the visible light range is established.
  • the figure which shows the light emission wavelength of the luciferin analog group of this invention The figure which shows the structure which converts the light emission wavelength and light emission wavelength of the luciferin analog group of this invention.
  • the present invention relates to a method for producing a substrate of luciferase having a desired emission wavelength, and in order to shift the emission wavelength, an —OH group or an —NR 1 R 2 group (wherein R 1 And R 2 are each independently H or C 1-4 alkyl) in order to produce a compound in which one is substituted with the other and / or to shift the emission wavelength in luciferin or luciferin analogs Producing a compound in which the number of double bonds is increased or decreased, and / or producing a compound in which one of the double bond moiety or the benzene ring is substituted with the other in order to shift the emission wavelength.
  • a method of including is provided.
  • luciferin refers to the following formula I: Firefly luciferin having the structure
  • the luciferin derivative includes a non-natural compound capable of emitting light through a chemical reaction through the action of the luminescent enzyme luciferase.
  • Luciferin derivatives include compounds having a different skeleton from naturally occurring luciferins.
  • Luciferin derivatives include, for example, the following general formula II: It has the following structure.
  • R 1 , R 2 and R 3 can each independently be H or C 1-4 alkyl. Such lower alkyls as substituents are considered unlikely to affect activity.
  • R 1 and R 2 are methyl.
  • R 3 is H.
  • the —NR 1 R 2 group can be an —OH group.
  • C 1-4 alkyl refers to a saturated linear or branched alkyl group containing 1 to 4 carbon atoms, such as methyl, ethyl, n-propyl, iso-propyl, n-butyl. , Iso-butyl, sec-butyl and tert-butyl.
  • C 1 -C 3 alkyl refers to a saturated linear or branched alkyl group containing from 1 to 3 carbon atoms (eg, methyl, ethyl or iso-propyl).
  • R 3 may be C 1-4 alkyl.
  • Patent Document 2 shows that a luciferin-like compound in which the portion corresponding to the R 3 portion of the compound of the present invention is AMP can serve as a substrate for a firefly bioluminescence system.
  • X and Y can each independently be C, N, S or O.
  • the heteroatoms of X and Y may be C, N, S or O.
  • the various luciferin-like compounds described in the above-mentioned Patent Document 2 by the present inventors include luciferin-like compounds in which the corresponding parts of the compound of the present invention are various heteroatoms as a substrate for a firefly bioluminescence system. The results obtained are shown.
  • n the double bond unit (vinylene unit) represented as “n” can be changed to a desired length.
  • n is 0, 1, 2 or 3.
  • luciferin derivatives include, for example, the following general formula III: It has the following structure.
  • R 1 , R 2 and R 3 can each independently be H or C 1-4 alkyl. Such lower alkyls as substituents are considered unlikely to affect activity.
  • R 1 and R 2 are methyl.
  • R 3 is H.
  • the —NR 1 R 2 group can be an —OH group.
  • R 3 may be C 1-4 alkyl.
  • Patent Document 2 shows that a luciferin-like compound in which the portion corresponding to the R 3 portion of the compound of the present invention is AMP can serve as a substrate for a firefly bioluminescence system.
  • X and Y can each independently be C, N, S or O.
  • the heteroatoms of X and Y may be C, N, S or O.
  • the various luciferin-like compounds described in the above-mentioned Patent Document 2 by the present inventors include luciferin-like compounds in which the corresponding parts of the compound of the present invention are various heteroatoms as a substrate for a firefly bioluminescence system. The results obtained are shown.
  • n the double bond unit (vinylene unit) represented as “n” can be changed to a desired length.
  • n is 0, 1, 2 or 3.
  • luciferin and luciferin analogs include salts thereof.
  • a “salt” is envisioned only in the compounds of the present invention where any portion of the compound can form a base.
  • 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
  • the method for producing a luciferase substrate having a desired emission wavelength produces a compound in which —OH group and —NR 1 R 2 group in luciferin or a luciferin analog are substituted in order to shift the emission wavelength.
  • “One of the —OH group or —NR 1 R 2 group is substituted with the other” means that in the case of a compound having an —OH group, the —OH group is substituted with the —NR 1 R 2 group, and — In the case of a compound having an NR 1 R 2 group, this means that the —NR 1 R 2 group is substituted with an —OH group.
  • the emission wavelength can be shifted to the longer wavelength side by about 20 to 30 nm or more.
  • the emission wavelength can be shifted to the short wavelength side by about 20 to 30 nm or more.
  • the emission wavelength is shifted from about 450 nm to about 430 nm of the luciferin derivative of formula IV.
  • the production method of the present invention includes a step of producing a compound in which the number of double bonds in luciferin or a luciferin analog is increased or decreased in order to shift the emission wavelength.
  • Increasing the number of double bonds refers to adding / inserting a double bond, that is, adding / inserting a vinylene unit in a carbon bond of luciferin or a luciferin analog. For example, in the compounds of general formulas II and III, this means increasing the number of n.
  • “Reducing the number of double bonds” means removing double bonds, ie, removing vinylene units, at the carbon bond of luciferin or a luciferin analog.
  • the number of n in the compounds of general formulas II and III, it means to reduce the number of n.
  • the number of double bonds in the luciferin analog, ie, the number of n can be increased or decreased depending on the desired emission wavelength, but is preferably between 0 and 3.
  • the emission wavelength can be shifted to the long wavelength side by about 100 nm or more.
  • the emission wavelength can be shifted to the short wavelength side by about 100 nm or more.
  • the emission wavelength is shifted from about 450 nm to about 560 nm of the luciferin derivative of formula IV.
  • luciferin or luciferin analogs can be produced using various methods well known to those skilled in the art. For example, it can be synthesized by a synthetic route as shown in the following examples.
  • the production method of the present invention includes a step of producing a compound in which a double bond portion and a benzene ring in luciferin or a luciferin analog are substituted in order to shift the emission wavelength.
  • the emission wavelength can be shifted to the long wavelength side by about 20 to 30 nm or more.
  • the emission wavelength can be shifted to the short wavelength side by about 20 to 30 nm or more.
  • a compound in which “one of the double bond moiety or the benzene ring is substituted with the other” means that the double bond in the carbon bond of the luciferin or luciferin analog forms a condensed ring together with the adjacent benzene ring.
  • Formula VII For example, Formula VII:
  • a derivative containing a naphthalene ring is formed. Further, in the case of a derivative in which one vinylene is bonded to the benzene ring of naphthol, such as the derivative of the above formula VIII, a derivative containing an anthracene ring is formed by substituting the vinylene moiety with a benzene ring.
  • the number of benzene rings in the condensed ring can be increased or decreased depending on the desired emission wavelength, but is preferably 0 to 3, more preferably 0 to 2.
  • a compound in which the double bond moiety and the benzene ring in luciferin or a luciferin analog are substituted can be produced using various methods well known to those skilled in the art. For example, it can be synthesized by a synthetic route as shown in the following examples.
  • the present invention also provides a luciferase derivative obtained by the above method for producing a luciferase substrate.
  • the compounds encompassed by the general formulas II and III can be produced by the production method of the present invention.
  • a compound in which the —NR 1 R 2 groups of the general formulas II and III are —OH groups can be produced by the production method of the present invention.
  • a compound in which the —NR 1 R 2 group of the luciferin derivative is an —OH group can be produced using various methods well known to those skilled in the art. For example, it can be synthesized by a synthetic route as shown in the following examples.
  • Example 7 describes a synthesis procedure of a dimethylaniline type compound in which R 1 and R 2 of the general formula II are each methyl, R 3 is H, and n is 2. Briefly, an aldehyde form such as commercially available 4-dimethylaminocinnamic aldehyde is used as a starting material, and reacted with carbethoxymethylenetriphenylphosphorane to obtain an ethyl ester form. Subsequently, this ethyl ester body is converted into a carboxyl body in an aqueous sodium hydroxide solution.
  • an aldehyde form such as commercially available 4-dimethylaminocinnamic aldehyde is used as a starting material, and reacted with carbethoxymethylenetriphenylphosphorane to obtain an ethyl ester form.
  • this ethyl ester body is converted into a carboxyl body in an aqueous sodium hydroxide solution.
  • a D-cysteine-S-trityl compound is reacted in hydrogen chloride and 1,4-dioxane solution to prepare a methyl ester form.
  • this methyl ester form is reacted with a carboxyl form to form an amide form.
  • this amide form is cyclized with triphenylphosphine oxide and trifluoromethanesulfonic anhydride to form a heterocyclic ring to obtain a thiazoline form.
  • the methyl ester portion of the thiazoline is converted to a desired substituent to obtain the desired compound.
  • Example 3 describes a synthetic procedure for a compound in which the —NR 1 R 2 group of general formula III is an —OH group, R 3 is H, and n is 1. More detailed procedures are described in the examples below. One skilled in the art would be able to synthesize the desired compound using synthetic procedures similar to those described in the examples below.
  • the compound produced by the production method of the present invention is oxidized by the luminescent beetle luciferase when added to a system containing luminescent beetle luciferase, ATP and Mg 2+ to emit light.
  • a compound can be used alone as a luminescent substrate, but may be used in combination with other luminescent substrates as necessary.
  • Such compounds can also be provided as a kit with ATP and Mg 2+ .
  • the kit can also contain other luminescent substrates and solutions prepared at an appropriate pH.
  • such a compound can be provided as a kit with a luminescent substrate composition prepared by adjusting the compound of the present invention to an appropriate pH together with ATP and Mg 2+ .
  • the compound produced by the production method of the present invention can be used as various luminescent agents.
  • the compound manufactured by the manufacturing method of this invention can be utilized as a sensor.
  • a hydrophilic organic compound may be present.
  • tetrafluoroacetic acid, acetic acid, formic acid and the like may be present.
  • 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 ⁇ M or more, for example, 5 ⁇ M or more in order to obtain a suitable luminescence intensity.
  • the pH of the luminescent 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.
  • the compound of the present invention can be made to emit light by various oxidases in the luminescent beetle luciferase luminescence system.
  • Luciferase has been isolated from various organisms such as North American firefly (Photinus pyralis) and railroad worm (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.
  • Mg ions are effective for stabilizing luminescence in a firefly bioluminescence system.
  • the light-emission behavior changes so that attenuation after rising is suppressed.
  • the luminescence behavior changes greatly.
  • the stabilization of luminescence becomes extremely remarkable, and the luminescence behavior when a large excess of pyrophosphate and Mg ions coexist with the luminescent substrate rises rapidly and is maintained to form a plateau state.
  • Mg ion alone, when the Mg ion concentration of the light emitting system is 0.5 mM or more, the light emission stabilizing effect is remarkable, and is enhanced as the Mg ion concentration increases.
  • magnesium pyrophosphate can be present, for example, at a concentration of 10 ⁇ M or more, preferably 100 ⁇ M or more.
  • the ratio of pyrophosphoric acid and Mg ion does not need to be an equivalent ratio.
  • magnesium pyrophosphate Although magnesium pyrophosphate has low water solubility, it can be used to supply pyrophosphate and Mg ions separately. These can be supplied to the luminescent system in free form and in salt form.
  • Mg 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.
  • the pH of the luminescent system is preferably 2-10.
  • the compound of the present invention may be used as a substrate for 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 compound of the present invention can be used as a luminescent label in biological measurement / detection.
  • 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.
  • 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.
  • 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.
  • 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.
  • the compound of the present invention can emit light at different emission wavelengths.
  • the compound produced by the production method of the present invention realizes light emission in a wide range of emission wavelengths.
  • RGB light emission which is the three primary colors of light
  • firefly luciferin analogs can be realized with firefly luciferin analogs.
  • the blue wavelength shorter than 450 nm and the red wavelength longer than 650 nm, which correspond to both ends, are emission wavelength regions that could not be achieved by existing firefly bioluminescence systems.
  • light emission by the compound produced by the production method of the present invention can correspond to RGB which is the three primary colors of light. It is well known that any color tone can be obtained by combining the three primary colors of light. Therefore, by combining compounds with emission wavelengths corresponding to the three primary colors, light emission of infinite color tone can be obtained.
  • compounds with emission wavelengths corresponding to the three primary colors are used for measurement / detection utilizing the detection of luminescent beetle luciferase activity by the emission of the luminescent substrate, as a result, depending on the degree of emission of each of the three primary colors Light emission of different colors will be obtained. Therefore, it will be possible to simultaneously determine the degree of light emission of each of the three primary colors from the obtained light emission color tone. It would also be possible to select and measure and detect the desired wavelength using a filter or the like.
  • a luminescent substrate that emits light at a shorter wavelength (about 500 nm or less) than firefly luciferin can transfer energy to the green fluorescent protein (GFP) of the luminescent jellyfish. By such energy transfer, fluorescence (about 520 nm) is emitted from the green GFP.
  • GFP green fluorescent protein
  • a BRET (Bioluminescence Resonance Energy Transfer) type luminescence system using a GFP / luminescent beetle luciferase fusion protein can be constructed.
  • the BRET-type luminescence system allows bioimaging of various protein post-translational modifications and gene expression.
  • IR Infrared absorption spectrum
  • KBr Tablet method
  • CHCl 3 , CH 3 OH solution method
  • FT-730 Fourier transform infrared spectrophotometer manufactured by HORIBA, Ltd. The measured value was described in wave number (cm ⁇ 1 ). In addition, wide absorption was described as br.
  • 1 H nuclear magnetic resonance spectrum ( 1 H NMR): Measured using a Lambda-270 type apparatus (270 MHz) manufactured by JEOL Ltd. It was described as “ 1 H NMR (measurement frequency, measurement solvent): ⁇ chemical shift value (number of hydrogen, multiplicity, spin coupling constant)”.
  • the spin coupling constant (J) is described in Hz.
  • Mass spectrum 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 ⁇ l / min. It was described as “MS (measurement method) m / z mass number (relative intensity)”.
  • TLC Chromatography Analytical thin layer chromatography
  • Preparative thin layer chromatography 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.
  • Solvent Distilled water was distilled and ion-exchanged using a GS-200 type distilled water production apparatus manufactured by Advantech Toyo Corporation.
  • CDCl 3 99.7 ATOM% D, 0.03% TMS manufactured by ISOTEC Inc.
  • CD 3 OD 99.8 ATOM% D ( ⁇ 0.7 ATOM% 13 C) manufactured by ISOTEC Inc., 0.05% TMS.
  • Example 1 Synthesis of a dimethylaniline-type luminescent substrate
  • 6-Cyano-2-naphthol 49.6 mg, 0.292 mmol
  • D-cysteine hydrochloride monohydrate 140.5 mg, 0.797 mmol
  • dehydrated ethanol 5.0 mL
  • 1M aqueous sodium hydroxide 2.5 mL
  • the reaction mixture was allowed to cool, 1M hydrochloric acid (2 mL) was added, and the mixture was washed with distilled water to give analog 1 (43.4 mg, 54%) as a yellow solid.
  • 6-Cyano-2-naphthol 50.2mg, 0.297mmol
  • t-butyldimethylsilyl chloride 143mg, 0.95mmol
  • imidazole 160.7mg, 2.37mmol
  • DMF 0.5mL
  • Water 40 mL was added to the reaction mixture and extracted with ethyl acetate (3 ⁇ 60 mL).
  • the organic layer was dried over sodium sulfate and concentrated under reduced pressure.
  • the obtained residue was purified by column chromatography ⁇ silica gel 36 g; hexane-ethyl acetate (8: 1) ⁇ to obtain TBS protector 2 (69.9 mg, 83%) as a colorless oil.
  • TBS protector 2 99.3 mg, 0.35 mmol
  • 1M diisobutylaluminum 1M diisobutylaluminum (toluene solution)
  • dehydrated toluene (10 mL) under an argon atmosphere and stirred for 1 hour.
  • the reaction mixture was ice-cooled, acetone (10 mL), saturated aqueous Rochelle salt solution (20 mL), water (30 mL) were added, and the mixture was extracted with ethyl acetate (3 ⁇ 50 mL).
  • the organic layer was dried by adding sodium sulfate, and concentrated under reduced pressure.
  • the ester 4 (90.8 mg, 0.253 mmol) was dissolved in isopropyl alcohol (3 mL), 1M aqueous sodium hydroxide solution (5 mL) was added, and the mixture was stirred for 5 hours.
  • the reaction mixture was neutralized by adding cation exchange resin IR-120BNa. The resin was filtered off, and the filtrate was concentrated under reduced pressure to obtain carboxylic acid 5 (54.9 mg, quant.) As a pale yellow solid.
  • D-cysteine-S-trityl compound (504 mg, 1.39 mmol) was dissolved in methanol (100 ml), and 4N hydrogen chloride solution in 1,4-dioxane (5.4 ml) was added. The mixture was stirred at room temperature for 17 days, and then neutralized with an anion exchange resin “IRA400 OH AG. The resin was filtered off and the resulting solution was concentrated under reduced pressure. The residue was purified by silica gel column chromatography ⁇ silica gel 33.6 g; hexane-ethyl acetate (1: 1) ⁇ to obtain methyl ester 6 (455 mg, 86%) as a pale yellow oil.
  • Carboxylic acid 5 (54.9 mg, 0.254 mmol), 1-ethyl-3- (3-dimethylaminopropyl) hydrochloride in an N, N-dimethylformamide solution (1 ml) of methyl ester 6 (145 mg, 0.381 mmol) in an argon atmosphere
  • Carbodiimide (145 mg, 0.762 mmol) and 4-dimethylaminopyridine (155 mg, 1.27 mmol) were added, and the mixture was stirred at room temperature for 4 hours.
  • Water 50 ml was added to the reaction mixture, and the mixture was extracted with ethyl acetate (3 ⁇ 50 ml). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure.
  • Triphenylphosphine oxide (91 mg, 0.327 mmol) and trifluoromethanesulfonic anhydride (100 ⁇ L, 0.546 mmol) were added to a solution of amide 7 (60.3 mg, 0.109 mmol) in dichloromethane (3 ml) under an argon atmosphere at room temperature for 1 hour. Stir. Water (50 ml) was added to the reaction mixture, and the mixture was extracted with chloroform (3 ⁇ 50 ml). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure.
  • the obtained residue was purified by preparative thin layer chromatography (20 cm ⁇ 20 cm ⁇ 0.5 mm ⁇ 2 sheets; hexane-ethyl acetate (1: 2) ⁇ to obtain thiazoline 8 (17.4 mg, 55%) as a yellow solid. Obtained.
  • Acetic anhydride (2.0 ml, 21 mmol) and 4-dimethylaminopyridine (1.13 g, 9.93 mmol) were added to a dichloromethane solution (40 ml) of p-hydroxycinnamic acid (502.2 mg, 3.06 mmol) and stirred at room temperature for 4 hours. .
  • Water (150 ml) was added to the reaction mixture and extracted with chloroform (3 ⁇ 150 ml). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure.
  • Triphenylphosphine oxide (33.2 mg, 0.119 mmol) and trifluoromethanesulfonic anhydride (50 ⁇ l, 0.297 mmol) were added to a dichloromethane solution (11 ml) of amide 12 (31.3 mg, 0.0553 mmol) under an argon atmosphere. Stir for hours. Water (50 ml) was added to the reaction mixture, and the mixture was extracted with chloroform (3 ⁇ 50 ml). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure.
  • Silyl derivative 15 (773 mg, 3.28 mmol) was dissolved in toluene (5 ml), carbethoxymethylenetriphenylphosphorane (1.24 g, 3.55 mmol) was added, and the mixture was heated to reflux for 3.5 hours. The reaction mixture was allowed to cool, water (50 ml) was added, and the mixture was extracted with ethyl acetate (3 ⁇ 50 ml). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography ⁇ silica gel 92 g; hexane: chloroform (1: 2) ⁇ to obtain ester 16 (895 mg, 89%) as a yellow oil.
  • Ethyl ester 16 (197 mg, 0.643 mmol) was dissolved in toluene (2 ml), a toluene solution of diisobutylaluminum hydride (3.0 ml, 3.0 mmol) was added, and the mixture was stirred at room temperature for 3 hours. Water (100 ml) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (3 ⁇ 10 ml). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure.
  • Alcohol 17 (62.8 mg, 0.239 mmol) was dissolved in a dichloromethane solution (1.5 ml), Dess Martin reagent (205 mg, 0.484 mmol) was added, and the mixture was stirred at room temperature for 1.5 hours.
  • Ethyl ester 19 (9.7 mg, 0.0292 mmol) was dissolved in isopropyl alcohol solution (1 ml), 1M sodium hydroxide aqueous solution (1 ml, 1 mmol) was added, and the mixture was stirred at 50 ° C. for 20 hours. The reaction mixture was allowed to cool and then neutralized with cation exchange resin IR-120B NA. The resin was filtered off, and the resulting solution was concentrated under reduced pressure to obtain carboxylate 20 (10.3 mg, quant.) As a yellow solid.
  • Triphenylphosphine oxide (45.7 mg, 0.164 mmol) and trifluoromethanesulfonic anhydride (0.4 ml, 2.38 mmol) were added to a dichloromethane solution (1 ml) of amide 21 (44.5 mg, 0.0811 mmol) under an argon atmosphere at room temperature. For 40 minutes. Water (50 ml) was added to the reaction mixture, and the mixture was extracted with chloroform (50 ml) and ethyl acetate (2 ⁇ 50 ml). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure.
  • Triphenylphosphine oxide 124 mg, 0.446 mmol
  • trifluoromethanesulfonic anhydride 360 ⁇ l, 2.14 mmol
  • Water 50 ml was added to the reaction mixture, and the mixture was extracted with chloroform (50 ml) and ethyl acetate (2 ⁇ 50 ml).
  • the organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure.
  • the obtained residue was purified by column chromatography ⁇ silica gel 42 g; hexane-ethyl acetate (1: 2) ⁇ to obtain thiazoline 26 (44.2 mg, 71%) as a yellow solid.
  • Ethyl ester 28 (235 mg, 1.08 mmol) was dissolved in isopropyl alcohol (7 ml), and 1M aqueous sodium hydroxide solution (4 ml, 4 mmol) was added. After stirring at room temperature for 3 days, the mixture was neutralized with 1M hydrochloric acid. Water (50 ml) was added thereto, and the mixture was extracted with ethyl acetate (3 ⁇ 50 ml). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain carboxyl compound 29 (137.1 mg, 58%) as a yellow solid.
  • Triphenylphosphine oxide (44.9 mg, 0.161 mmol) and trifluoromethanesulfonic anhydride (17 ⁇ l, 0.110 mmol) are added to a solution of amide 30 (43.3 mg, 0.0751 mmol) in dichloromethane (5 ml) under an argon atmosphere at room temperature. Stir for minutes. Water (50 ml) was added to the reaction mixture, and the mixture was extracted with chloroform (50 ml) and ethyl acetate (2 ⁇ 50 ml). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure.
  • the obtained residue was purified by silica gel column chromatography ⁇ silica gel 85 g; chloroform-methanol (10: 1) ⁇ to obtain phenol 13 (448.8 mg, 74%) as a yellow solid.
  • the raw material (213.4 g, 1.12 mmol) was recovered.
  • Phenol compound 13 (20.0 mg, 0.114 mmol) and D-cysteine hydrochloride monohydrate (19.7 mg, 0.125 mmol) are dissolved in methanol: distilled water (2: 1) (3.0 ml), and carbonated under an argon atmosphere. Potassium (17.0 mg, 0.123 mmol) was added and stirred at room temperature for 30 minutes. The reaction mixture was acidified with 0.2 ml of 1M hydrochloric acid and then concentrated under reduced pressure. The resulting solid was filtered and washed with distilled water to give firefly luciferin (1) (23.5 mg, 74%) as a yellow solid.
  • Example 9 Measurement of 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 CHIRALCEL OD-RH (inner diameter 0.46 cm, length 15 cm) manufactured by Daicel Chemical Industries, Ltd.
  • Luminescence photon amount measurement It measured using Luminescencer-PSN AB-2200 by ATTO Corporation.
  • Emission spectrum measurement It measured using ATTO Co., Ltd. weak emission fluorescence spectrum apparatus AB-1850. All measured spectra are spectra corrected for the characteristics of the detector.
  • luciferase derived from the North American firefly Photinus pyralis
  • a recombinant type manufactured by Sigma or Promega was used.
  • the phosphate buffer solution is prepared by dissolving dipotassium hydrogen phosphate hydrate (special grade) and potassium dihydrogen phosphate hydrate (special grade) manufactured by Wako Pure Chemical Industries, Ltd. in ultrapure water and adjusting the pH. Used.
  • Potassium t-butoxide was manufactured by Tokyo Chemical Industry Co., Ltd.
  • Trifluoroacetic acid used was a product (special grade) manufactured by Wako Pure Chemical Industries, Ltd.
  • Enzyme solution The luciferase was diluted with Tris-HCl buffer solution (50 mM, pH 8.0) to 1 ⁇ g / ⁇ l, 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.
  • Bioluminescence spectrum In a 200 ⁇ L polystyrene tube, potassium phosphate buffer (0.5 M, pH 8.0, 20 ⁇ l), substrate solution (2.5 mM, 20 ⁇ l), enzyme solution (20 ⁇ l), then ATP-Mg solution (10 mM, 40 ⁇ l) The mixture was mixed and the emission spectrum was measured. The concentration of the enzyme solution used was 17 ⁇ M. However, firefly luciferin (1) used 1.7 ⁇ M, and phenol-type luciferin used 170 ⁇ M. The exposure time for emission spectrum measurement was 60 seconds. However, firefly luciferin was performed in 5 seconds.
  • a compound group having an emission wavelength over the entire visible region can be produced, such as the luciferin analog shown in FIG.

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  • Thiazole And Isothizaole Compounds (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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CN104262287A (zh) * 2014-09-02 2015-01-07 苏州罗兰生物科技有限公司 一种亚硫酸根比率荧光探针的制备及应用
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WO2013027770A1 (ja) * 2011-08-24 2013-02-28 国立大学法人電気通信大学 ルシフェラーゼの発光基質
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CN102532053B (zh) * 2011-12-30 2014-08-06 广东省微生物研究所 一种高纯度d-荧光素的制备和纯化方法
JP2015203019A (ja) * 2014-04-15 2015-11-16 Dic株式会社 シラン化合物およびその重合体、ならびにその重合体からなる液晶配向層
CN104262287A (zh) * 2014-09-02 2015-01-07 苏州罗兰生物科技有限公司 一种亚硫酸根比率荧光探针的制备及应用
CN104262287B (zh) * 2014-09-02 2016-06-08 苏州罗兰生物科技有限公司 一种亚硫酸根比率荧光探针的制备及应用
WO2021193069A1 (ja) * 2020-03-23 2021-09-30 黒金化成株式会社 新規複素環式化合物及びその塩、並びに発光基質組成物
JPWO2021193069A1 (enExample) * 2020-03-23 2021-09-30
CN117720477A (zh) * 2023-12-13 2024-03-19 广州博鹭腾生物科技有限公司 一种Akalumine盐酸盐的制备方法及其应用

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