Fluorescent dye for detecting γ-glutamyl transpeptidase 5 (GGT5)

Abstract

To provide a novel compound and a fluorescent dye that are usable for the detection of GGT5, and a method for detecting GGT5 using the compound or fluorescent dye.SOLUTION: A molecule is synthesized by conjugating a glutathione structure, which is the substrate recognition site of GGT5, with a moiety derived from a fluorescent dye, and is represented by the general formula (1) in the figure. (In the formula, X1 is S or O, X2 is O or NH, and A represents a moiety comprising a chromophore.)SELECTED DRAWING: Figure 8

Description

[Technical field]

[0001] The present invention relates to a fluorescent dye for detecting GGT5. [Background technology]

[0002] γ-Glutamyl transpeptidase (GGT) is an enzyme that cleaves the γ-glutamyl bond of γ-glutamyl compounds such as glutathione. GGT exists in vivo as a cell surface or membrane-bound enzyme, and is involved in the transfer of amino acids across cell membranes, exerting a physiological function of regulating the redox balance in the body. On the other hand, GGT is overexpressed in human tumors, promoting tumor progression, invasion, and drug resistance. One of the subtypes of the GGT family is GGT5.

[0003] GGT5 is an enzyme that metabolizes leukotriene C4 to leukotriene D4 in vivo, and is most abundantly expressed in the lungs in humans (Non-Patent Document 1). It has been reported that GGT5 is overexpressed in cancer-associated fibroblasts of lung adenocarcinoma, and is expressed in eosinophils, and is involved in inflammation (Non-Patent Document 2).

[0004] On the other hand, Non-Patent Document 3 reports on cancer imaging using a molecular probe (gGlu-HMRG) that emits fluorescence upon cleavage by γ-glutamyl transpeptidase 1 (GGT1).

[0005] Non-Patent Document 4 reports that in allergic inflammatory tissues, the concentration of leukotriene D4 (LTD4) increases due to lipid metabolism disorders caused by increased expression of GGT5 in eosinophils. The activity of GGT5 is indirectly detected by quantitatively analyzing its substrate (LTC4) and reaction product (LTD4) by LC-MS / MS. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Histochem. Cell. Biol. 2015, 143, 505-515 [Non-Patent Document 2] Aging 2020, 12, 13220-223 [Non-Patent Document 3] Science Translational Medicine 2011, 3, 110-119. DOI: 10.1126 / scitranslmed.3002823 [Non-Patent Document 4] Allergy 2019, 74, 1113-1124. DOI: 10.1111 / all.13726 Summary of the Invention [Problem to be solved by the invention]

[0007] The gGlu-HMRG described in Non-Patent Document 3 is not cleaved by GGT5 and therefore cannot be used as a probe for detecting GGT5 activity. In Non-Patent Document 4, GGT5 activity is not detected by fluorescence analysis, and quantification by LC-MS / MS is very costly and time-consuming.

[0008] GGT1 and GGT5 are enzymes that have the activity of cleaving the γ-glutamyl bond of γ-glutamyl compounds such as glutathione, but their substrate recognition is different. Although several molecular probes that emit fluorescence when cleaved by GGT1 are known, these molecular probes are not recognized as substrates of GGT5, and therefore no fluorescent probes capable of detecting GGT5 have been reported to date.

[0009] If there were an inexpensive and simple method for detecting GGT5, it would be useful for diagnosing patients with various allergies, including asthma, and for researching inflamed tissues.

[0010] An object of the present invention is to provide a novel compound and a fluorescent dye that can be used for detecting GGT5, and a method for detecting GGT5 using the compound or the fluorescent dye. [Means for solving the problem]

[0011] The present invention encompasses the embodiments described below. Section 1. General formula (1)

[0012] [ka]

[0013] (In the formula, R 1 is a direct bond or a divalent hydrocarbon group, X 1 is S or O, X 2 is O or NH, A is a moiety containing a chromophore. or a salt, hydrate or solvate thereof.

[0014] Section 2. Item 2. The compound according to item 1, wherein the chromophore has a xanthene skeleton, a silicon-substituted xanthene skeleton, a coumarin skeleton, a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene skeleton, a phenoxazine skeleton, a phenylbenzothiazole skeleton, a naphthalimide skeleton, a hemicyanine skeleton, or a merocyanine skeleton, or a salt, hydrate, or solvate thereof. Section 3. Item 2. The compound according to item 1, wherein the chromophore has a xanthene skeleton or a silicon-substituted xanthene skeleton, or a salt, hydrate, or solvate thereof.

[0015] Section 4. The compound according to item 1, wherein A is represented by formula (2), or a salt, hydrate or solvate thereof:

[0016] [ka]

[0017] (In the formula, R 5 , R 6 , R 7, R 8 , R 9 and R 10 are each independently hydrogen, halogen, cyano, -CF3, C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, C1-C6 alkylthio, C1-C6 alkoxy, sulfo, or carboxy; R 11 , R 12 , R 13 , and R 14 are each independently hydrogen, halogen, cyano, halogen, cyano, -CF3, C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, C1-C6 alkylthio, C1-C6 alkoxy, sulfo, or carboxy; 11 , R 12 , R 13 , and R 14 any alkyl moiety of may be further substituted with halogen, amino, hydroxy, cyano, C1-C6 alkyl, C1-C6 alkylamino, C1-C6 alkylthio, C1-C6 alkoxy, sulfo, or carboxy; Y is -O- or -MR 20 R 21 - (M is a silicon atom, a germanium atom, or a tin atom, R 20 and R 21 are each independently C1-C6 alkyl or aryl; Z is -C(O)-, -SO2- or CH2; B is -OR 2 (R 2 is hydrogen, optionally substituted alkyl, optionally substituted acyl), -NR 3 R 4 (R 3 and R 4 each independently represents hydrogen, alkyl, or cycloalkyl, or R 3 and R 4 together with the nitrogen atom to which they are attached form a monocyclic or bicyclic ring), or -OC(O)-R 1 -X 1 - (the group recognized by GGT5), * is X of the compound represented by formula (1).2 )

[0018] Section 5. Item 5. A fluorescent dye comprising the compound according to any one of items 1 to 4, or a salt, hydrate or solvate thereof. Section 6. Item 6. The fluorescent dye according to Item 5, which is a fluorescent dye for detecting GGT5-expressing cells. Section 7. Item 7. The fluorescent dye according to Item 6, wherein the GGT5-expressing cells include eosinophils.

[0019] Section 8. A method for detecting GGT5 expressing cells, comprising: A method comprising: contacting a GGT5-expressing cell with the fluorescent dye according to any one of Items 5 to 7; and measuring fluorescence emitted by a compound produced by cleavage of a γ-glutamyl bond in the fluorescent dye. Section 9. Item 8. A cell detecting agent comprising the fluorescent dye according to any one of Items 5 to 7. Section 10. Item 8. A method for bioimaging of cells, comprising using the fluorescent dye according to any one of Items 5 to 7. Effect of the Invention

[0020] The compound represented by the general formula (1) of the present invention is useful as a fluorescent probe capable of measuring GGT5 because it does not substantially emit fluorescence in the absence of GGT5 but emits fluorescence under the catalysis of GGT5. Furthermore, the compound or a fluorescent dye containing the compound can be used to simply detect GGT5-expressing cells. [Brief description of the drawings]

[0021] [Figure 1] Each part of LTC4. [Diagram 2] Design of fluorescent probe for detecting GGT5. [Diagram 3] Proposed reaction mechanism of GGT5-Probe1 [Figure 4] UPLC graph of products in GGT5-Probe1 synthesis. [Diagram 5] UPLC graphs in the absence (top) and presence (bottom) of enzyme. [Figure 6] Photographs of solutions in the absence (left) and presence (right) of enzyme. [Figure 7] Reaction of recombinant human GGT5 with GGT5-Probe1. (A) Immediately after mixing, (B) after 30 min of incubation. [Figure 8] GGT5 concentration- and time-dependent changes in fluorescence intensity. [Figure 9] (A) Absorption spectrum (left) and fluorescence spectrum (right) of GGT5-Probe1 in the absence of GGT5. (B) Absorption spectrum (left) and fluorescence spectrum (right) of GGT5-Probe1 in the presence of GGT5. [Figure 10] Change in fluorescence intensity when GGT5-Probe1 was added to the culture medium of GGT5-expressing cells. [Figure 11] Confirmation of GGT5 expression by Western blotting. [Figure 12] Reaction of recombinant human GGT5 enzyme with each of GGT5-Probe1 to GGT5-Probe3. [Figure 13] Absorption spectrum (left) and fluorescence spectrum (right) of GGT5-Probe2 in the presence of GGT5. [Figure 14] Absorption spectrum (left) and fluorescence spectrum (right) of GGT5-Probe3 in the presence of GGT5. [Figure 15] Fluorescence image after adding GGT5-Probe3 to the culture medium of GGT5-expressing cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] In this specification, the terms "contain, include, and have" are concepts that also encompass "consist essentially of" and "consist only of."

[0023] In this specification, the prefix "C1-C6" of a functional group indicates that the functional group has 1 to 6 carbon atoms.

[0024] As used herein, the term "alkyl" refers to a monovalent hydrocarbon radical that is linear, branched, cyclic, or a combination of cyclic and linear or branched. For example, C1-C6 alkyl includes methyl, ethyl, n-propyl, 2-propyl, tert-butyl, pentyl, cyclopentyl, cyclopropylmethyl, hexyl, and the like.

[0025] As used herein, the term "alkenyl" refers to a straight or branched chain monovalent hydrocarbon group containing at least one double bond. For example, C2-C6 alkenyl includes ethenyl, propenyl, etc. When alkenyl has a substituent, the substituent may include unsubstituted C1-C6 alkyl, unsubstituted aryl, halogen (e.g., fluorine, chlorine, bromine, or iodine), hydroxy, carboxy, sulfo, amino, alkoxy, thioalkoxy, cyano, etc.

[0026] As used herein, the term "alkynyl" refers to a straight or branched chain monovalent hydrocarbon radical containing at least one triple bond. For example, C2-C6 alkynyl includes ethynyl, propynyl, and the like.

[0027] As used herein, the terms "alkoxy," "alkylamino," and "alkylthio" are used in their ordinary sense to refer to alkyl groups attached to the remainder of the molecule via an oxygen atom, an amino group, or a sulfur atom, respectively.

[0028] As used herein, the term "aryl" refers to any group that is unsubstituted or substituted with alkyl, cycloalkyl, cycloalkyl-alkyl, halo, cyano, hydroxy, alkoxy, amino, acylamino, mono-alkylamino, di-alkylamino, haloalkyl, haloalkoxy, -COR (where R is hydrogen, alkyl, cycloalkyl, cycloalkyl-alkyl, phenyl, phenylalkyl, aryl, or arylalkyl), -(CR 1 R 2 ) n -COOR (where n is an integer from 0 to 5, R 1 and R2 are independently hydrogen or alkyl, and R is hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, phenyl, phenylalkyl, aryl, or arylalkyl) or -(CR 1 R 2 ) n -CONR 3 R 4 (where n is an integer from 0 to 5, and R 1 and R 2 are independently hydrogen or alkyl, R is hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, phenyl, phenylalkyl, aryl, or arylalkyl, and R 3 and R 4 means a monovalent monocyclic or bicyclic aromatic hydrocarbon group of 6 to 10 ring atoms, independently substituted with 1 to 4 substituents selected from each independently being hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, phenyl, phenylalkyl, aryl, or arylalkyl. More specifically, aryl includes, but is not limited to, phenyl, biphenyl, 1-naphthyl, 2-naphthyl, and substituted forms thereof.

[0029] The present invention relates to a compound represented by general formula (1)

[0030] [ka]

[0031] (In the formula, R 1 is a direct bond or a divalent hydrocarbon group, X 1 is S or O, X 2 is O or NH, A is a moiety containing a chromophore. or a salt, hydrate or solvate thereof.

[0032] The compound represented by the above general formula (1) of the present invention is a molecule synthesized by binding a glutathione structure, which is the substrate recognition site of GGT5, to a moiety derived from a fluorescent dye.

[0033] Since A contains a chromophore, in the presence of GGT5, the γ-glutamyl bond in the compound represented by general formula (1) is cleaved, triggering the formation of -CO-X 2 -(X 2 The cleavage of the ester or amide bond of the carboxyl group (O or NH) occurs, and the product having a chromophore emits fluorescence. By measuring this fluorescence, GGT can be detected qualitatively and quantitatively.

[0034] In certain embodiments, R 1 is a direct bond, and X 1 and the carbon of the carbonyl group are directly bonded.

[0035] In certain embodiments, R 1 R is a divalent hydrocarbon radical which is linear, branched, cyclic, or a combination of cyclic and linear or branched. 1 When R is a cyclic ring, it may be a saturated alicyclic ring or an aromatic ring. 1 The number of carbon atoms is not particularly limited, but is preferably 1 to 15, more preferably 1 to 10, and more preferably 1 to 6.

[0036] In certain embodiments, R 1 R is a divalent saturated hydrocarbon radical which is linear, branched, cyclic, or a combination of cyclic and linear or branched. 1 The number of carbon atoms is not particularly limited, but is preferably 1 to 15, more preferably 1 to 10, and more preferably 1 to 6.

[0037] In certain embodiments, R 1 R is a linear, divalent, saturated hydrocarbon group. 1 The number of carbon atoms is not particularly limited, but is preferably 1 to 15, more preferably 1 to 10, and more preferably 1 to 6.

[0038] In certain embodiments, the chromophore of A has a xanthene skeleton, a silicon-substituted xanthene skeleton, a coumarin skeleton, a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene skeleton (BODIPY skeleton), a phenoxazine skeleton, a phenylbenzothiazole skeleton, a naphthalimide skeleton, a hemicyanine skeleton, or a merocyanine skeleton. 2 is X in general formula (1). 2 X 2 is preferably O. The atoms bonded to the carbons forming each skeleton are not particularly limited.

[0039] [ka]

[0040] The xanthene skeleton includes a rhodamine skeleton and a fluorescein skeleton.

[0041] In certain embodiments, the chromophore of A has a xanthene backbone or a silicon-substituted xanthene backbone.

[0042] In certain embodiments, the chromophore backbone of A may have a group B attached thereto, where B is -OR 2 (R 2 is hydrogen, optionally substituted alkyl, optionally substituted acyl), -NR 3 R 4 (R 3 and R 4 each independently represents hydrogen, alkyl, or cycloalkyl, or R 3 and R 4 together with the nitrogen atom to which they are attached form a monocyclic or bicyclic ring), or -OC(O)-R 1 -X 1 - (a group recognized by GGT5).

[0043] The B value is X 2When the B group is attached to the 6-position of either the xanthene backbone, the silicon-substituted xanthene backbone, or the phenoxazine backbone, it is preferably attached to the 3-position of the backbone. Alternatively, the B group may be a functional group such as an alcohol or amino group attached to the backbone of the chromophore of a commercially available fluorescent dye.

[0044] B is -OR 2 Where R 2 When is an alkyl group which may have a substituent, the alkyl group may be substituted, for example, with halogen, amino, hydroxy, cyano, C1-C6 alkyl, C1-C6 alkylamino, C1-C6 alkylthio, C1-C6 alkoxy, sulfo, or carboxy.

[0045] B is -OR 2 Where R 2 When is an acyl which may have a substituent, the substituent of the acyl includes, for example, halogen, amino, hydroxy, cyano, C1-C6 alkyl, C1-C6 alkylamino, C1-C6 alkylthio, C1-C6 alkoxy, sulfo, or carboxy.

[0046] B is -OC(O)-R 1 -X 1 When it is -(a group recognized by GGT5), an example of the group having a moiety recognized by GGT5 includes a group represented by formula (3).

[0047] [ka]

[0048] (In the formula, * represents a bond to the carbon that forms the skeleton of the chromophore of A.)

[0049] In certain embodiments, A has the structure shown in formula (2).

[0050] [ka]

[0051] During the ceremony, R 5 , R 6 , R 7 , R 8 , R 9 and R 10 each independently is hydrogen, halogen, cyano, -CF3, C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, C1-C6 alkylthio, C1-C6 alkoxy, sulfo, or carboxy; R 11 , R 12 , R 13 , and R 14 are each independently hydrogen, halogen, cyano, halogen, cyano, -CF3, C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, C1-C6 alkylthio, C1-C6 alkoxy, sulfo, or carboxy; 11 , R 12 , R 13 , and R 14 any alkyl moiety of may be further substituted with halogen, amino, hydroxy, cyano, C1-C6 alkyl, C1-C6 alkylamino, C1-C6 alkylthio, C1-C6 alkoxy, sulfo, or carboxy; Y is -O- or -MR 20 R 21 - (M is a silicon atom, a germanium atom, or a tin atom, R 20 and R 21 are each independently C1-C6 alkyl or aryl; Z is -C(O)-, -SO2- or CH2; B is -OR 2 (R 2 is hydrogen, optionally substituted alkyl, optionally substituted acyl), -NR 3 R 4 (R 3 and R 4 each independently represents hydrogen, alkyl, or cycloalkyl, or R 3 and R 4together with the nitrogen atom to which they are attached form a monocyclic or bicyclic ring), or -OC(O)-R 1 -X 1 - (the group recognized by GGT5), * is X of the compound represented by formula (1). 2 It is a combination with.

[0052] B is -OR 2 Where R 2 is optionally substituted alkyl, B is -OR 2 Where R 2 is optionally substituted acyl, and B is -OC(O)-R 1 -X 1 Details of B when it is - (a group recognized by GGT5) are as explained above.

[0053] Although it is not intended that the present invention be bound by any particular hypothesis or theory, it is believed that since the compound represented by general formula (2) contains a chromophore bonded to a lactone ring, the compound of general formula (2) is a non-fluorescent or weakly fluorescent molecule, but when cleavage of the γ-glutamyl bond in the compound of general formula (2) by GGT5 is triggered, the lactone ring is opened by an intramolecular cyclization reaction due to a nucleophilic attack of the amino group generated by the cleavage, thereby releasing a fluorescent dye, and thus emitting stronger fluorescence than the compound of formula (2).

[0054] In certain embodiments, the compound represented by formula (1) is R 1 is a direct bond or a linear, cyclic, or a combination of cyclic and linear divalent hydrocarbon group having 1 to 15 carbon atoms; X 1 is S or O, X 2 is O or NH, A has a structure represented by formula (2), B is -OR 2 (R 2 is hydrogen, optionally substituted alkyl, optionally substituted acyl), -NR 3 R4 (R 3 and R 4 each independently represents hydrogen, alkyl, or cycloalkyl, or R 3 and R 4 together with the nitrogen atom to which they are attached form a monocyclic or bicyclic ring), or -OC(O)-R 1 -X 1 - (a group recognized by GGT5).

[0055] In certain embodiments, the compound represented by formula (1) is R 1 is a direct bond or a linear, cyclic, or a combination of cyclic and linear divalent hydrocarbon group having 1 to 15 carbon atoms; X 1 is S, X 2 is O, A has a structure represented by formula (2), B is -OR 2 (R 2 is hydrogen, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 acyl), -NR 3 R 4 (R 3 and R 4 each independently represents hydrogen, C1-C6 alkyl, or C1-C6 cycloalkyl, or -OC(O)-R 1 -X 1 - (a group recognized by GGT5).

[0056] In certain embodiments, the salt of the compound represented by general formula (1) has an anion represented by general formula (4).

[0057] [ka]

[0058] (R 1 , X 1 , X 2 , A is as explained in relation to formula (1).

[0059] The anion represented by general formula (4) and its counter ion (cation) are not particularly limited, and examples thereof include base addition salts such as metal salts such as sodium salts, potassium salts, calcium salts, and magnesium salts; ammonium salts; and organic ammonium salts such as triethylammonium.

[0060] Furthermore, the compounds of the present invention may exist as hydrates or solvates, and all of these substances are included within the scope of the present invention.

[0061] The fluorescence emitted by the compound represented by formula (1) or a salt, hydrate or solvate thereof in the presence of GGT5 upon cleavage of the γ-glutamyl bond has an absorption wavelength and a fluorescence wavelength different from the fluorescence emitted by the compound represented by formula (1) or a salt, hydrate or solvate thereof in the absence of GGT5.

[0062] In a specific embodiment, the fluorescence emitted by a compound represented by formula (1) or a salt, hydrate or solvate thereof in the presence of GGT5, which is produced by cleavage of a γ-glutamyl bond, is stronger than the fluorescence emitted by a compound represented by formula (1) or a salt, hydrate or solvate thereof in the absence of GGT5.

[0063] In certain embodiments, the fluorescence intensity at the maximum fluorescence wavelength emitted by the compound represented by formula (1) or a salt, hydrate or solvate thereof in the presence of GGT5 is 5 or more, 10 or more, or 15 or more times the fluorescence intensity at the maximum fluorescence wavelength of the corresponding compound represented by formula (1) or a salt, hydrate or solvate thereof in the absence of GGT5.

[0064] Next, a method for producing the compound represented by formula (1) will be described.

[0065] Glutathione is dissolved in water or an aqueous solution, and a base is added to adjust the pH of the aqueous solution to about 7.0. Next, glutathione is reacted with a compound represented by formula (5) in a solvent (thiol-ene reaction), and purified to obtain a compound represented by formula (1). The compound represented by formula (5) may be dissolved in an organic solvent before reacting with glutathione. The organic solvent is preferably the same as the solvent used when reacting glutathione with the compound represented by formula (5). The reaction time and reaction temperature are not particularly limited, and for example, the reaction time is 30 minutes to 1 day, and the reaction temperature is within the range of 5 to 80°C. Purification can be performed by a known method such as chromatography. The compound represented by formula (5) may be a commercially available product, or may be synthesized by reacting acryloyl chloride or the like with a hydroxyl group or amino group of a functional group bonded to the skeleton of the chromophore of A, for example.

[0066] [ka]

[0067] (In the formula, X 2 and A are as described for the compound represented by formula (1), and R is a monovalent hydrocarbon group having a terminal carbon-carbon double bond or a monovalent hydrocarbon group having a terminal halogen.

[0068] In a specific embodiment, R is a monovalent hydrocarbon group having a terminal carbon-carbon double bond, which is linear, branched, cyclic, or a combination of cyclic and linear or branched. When R is cyclic, it may be a saturated alicyclic or aromatic ring. The number of carbon atoms in R is not particularly limited, but is preferably 1 to 15, more preferably 1 to 10, and more preferably 1 to 6.

[0069] In a specific embodiment, it is a monovalent hydrocarbon group having a halogen bonded to a terminal carbon, which is linear, branched, cyclic, or a combination of cyclic and linear or branched. The hydrocarbon group may be a saturated or unsaturated hydrocarbon group, but is preferably a saturated hydrocarbon group. The halogen may be fluorine, chlorine, bromine, or iodine. The number of halogens bonded to the terminal carbon may be 1 to 3. When R contains a ring, it may be a saturated alicyclic ring or an aromatic ring. The number of carbon atoms in R is not particularly limited, but is preferably 1 to 15, more preferably 1 to 10, and more preferably 1 to 6.

[0070] The present invention provides a fluorescent dye containing any of the above-described compounds represented by formula (1) or their salts, hydrates or solvates. The compound of the present invention or its salts, hydrates or solvates are designed to be recognized and cleaved by GGT5, and therefore can be used as a fluorescent dye for detecting GGT5.

[0071] In a specific embodiment, the fluorescent dye for detecting GGT5 is a fluorescent dye for detecting GGT5-expressing cells. Examples of GGT5-expressing cells include eosinophils, dendritic cells, macrophages, lung cancer cells, gastric cancer cells, and CAFs (cancer-associated fibroblasts). The fluorescent dye of the present invention can be used in disease diagnosis and research as a detection reagent for GGT5 activity of eosinophils contained in tissues or peripheral blood of patients with eosinophilic sinusitis and allergic diseases such as asthma.

[0072] When any of the above-described compounds represented by formula (1) or salts, hydrates, or solvates thereof is dissolved in a solvent to prepare a solution, the organic solvent that can be used is not particularly limited, and both polar and nonpolar solvents can be used.

[0073] Examples of polar solvents include ether compounds (tetrahydrofuran, anisole, 1,4-dioxane, cyclopentyl methyl ether, etc.), alcohols (methanol, ethanol, allyl alcohol, etc.), ester compounds (ethyl acetate, etc.), ketones (acetone, etc.), halogenated hydrocarbons (dichloromethane, chloroform), dimethyl sulfoxide, amide solvents (N,N-dimethylformamide, dimethylacetamide, 1,3-dimethyl-2-imidazolidinone, N-methylpyrrolidone, etc.), and the like.

[0074] Examples of non-polar solvents include aliphatic organic solvents such as pentane, hexane, cyclohexane, and heptane; and aromatic solvents such as benzene, toluene, xylene, and mesitylene.

[0075] The present invention provides a method for detecting GGT5-expressing cells, comprising: Contacting a GGT5-expressing cell with any one of the fluorescent dyes described above; and and measuring the fluorescence emitted by a compound produced by cleavage of the γ-glutamyl bond of the compound or a salt, hydrate or solvate thereof in the fluorescent dye.

[0076] The present invention provides a cell detection agent containing any one of the fluorescent dyes described above. The cell detection agent may contain a buffer (Hepes buffer, Tris buffer, Tricine-sodium hydroxide buffer, phosphate buffer, phosphate buffered saline, etc.) to adjust the pH.

[0077] The present invention provides a method for bioimaging a cell, comprising any one of the fluorescent dyes described above.

[0078] The following examples are intended for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Unless otherwise specified, reagents are commercially available or are obtained or prepared according to conventional techniques or procedures known in the art. EXAMPLES

[0079] Example 1: Design of GGT5 detection fluorescent probe According to Wickham, S. et al., Anal. Biochem. 2011, 414, 209-214, GGT5 has higher substrate recognition strictness than GGT1. GGT1 can recognize a wide range of compounds containing γ-glutamyl bonds as substrates, and does not necessarily require a glutathione structure. In contrast, all structures reported so far as substrates for GGT5 contain a glutathione structure. However, various substituents can be bound to the sulfhydryl group of the cysteine ​​side chain contained in the glutathione structure. The types of substituents include fatty acids (the natural substrate LTC4), glutathione (oxidized glutathione: GSSG), 4-nitrobenzyl groups, and methyl groups. Based on the above findings, it was considered that the inclusion of glutathione is essential for structures that can be substrates for GGT5, but various substituents (including bulky ones) can be bound to the sulfhydryl group.

[0080] As shown in Figure 1, LTC4 is recognized as a substrate for GGT5 and is converted into a fluorescent molecule by an enzymatic reaction. Figure 2 shows the design of the GGT5 detection probe (GGT5-Probe1). The fluorescent dye structure was introduced into a portion that is less involved in GGT5 recognition and has a relatively high degree of freedom in design modification. FIG. 3 is a proposed reaction mechanism. Glutamine is released from GGT5-Probe1 by the catalytic action of GGT5, and then the free amine attacks the carbonyl group to form a seven-membered ring, producing fluorescein, which emits fluorescence.

[0081] Example 2 Synthesis of GGT5 probe 1. Synthesis of GGT5-Probe1 Glutathione (30 mg, 0.097 mmol) was dissolved in 5 mL of water and adjusted to pH 7.0 by adding 2M NaOH. Fluorescein o-acrylate (22 mg, 0.05 mmol) was dissolved in 0.5 mL of DMSO. The above solutions were mixed, and 4.5 mL of DMSO was added to make a total volume of 10 mL, and the mixture was stirred at room temperature for 5 hours. The target product was then purified by high performance liquid chromatography (HPLC) using a reverse phase column. Specifically, a mixed solution of solution A (water containing 0.1% TFA) and solution B (acetonitrile containing 0.1% TFA) was used as the mobile phase, and gradient elution was performed with the proportion of B from 10% (0 min) to 90% (45 min). The fraction containing the target product was lyophilized to obtain GGT5-Probe1 (48.9 mg, 0.070 mmol, y. 72.6%). In addition, the purity of GGT5-Probe1 was confirmed by ultra-performance liquid chromatography (UPLC) using a reverse-phase column (LaChromUltra C18 (2 μm)) and gradient elution with a mobile phase of a mixture of solution A (water containing 0.1% TFA) and solution B (acetonitrile containing 0.1% TFA) from 10% (0 min) to 90% (6.5 min) (Figure 4, detection wavelength 280 nm).

[0082] 11H NMR (500 MHz, DMSO-d-6): δ 10.2 (s, 1H), 8.45 (t, J = 5.6 Hz, 1H), 8.30 (d, J = 8.6 Hz, 1H), 8.24 (brs, 2H), 8.04 (d, J = 7.6 Hz, 1H), 7.83 (t, J = 7.5, 1H), 7.76 (t, J = 7.5 Hz, 1H), 7.34 (d, J = 7.7 Hz, 1H), 7.23 (d, J= 2.4 Hz, 1H), 6.91 (dd, J = 8.5, 2.4 Hz, 1H), 6.84 (d, J = 8.7 Hz, 1H), 6.73 (d, J = 1.8 Hz, 1H), 6.61 (brs, 2H), 4.55 (m, 1H), 3.97 (brs, 1H), 3.79 (dd, J = 5.8, 2.1 Hz, 2H), 2.89 (m, 5H), 2.66 (m, 1H), 2.37 (m, 2H), 2.01 (m, 2H) 13 13C NMR (125 MHz, DMSO-d6); δ 171.5, 171.4, 171.2, 171.1, 170.6, 169.0, 160.3, 158.7, 158.4, 152.8, 152.2, 152.0, 151.6, 136.3, 130.8, 129.6, 126.3, 125.4, 124.5, 118.6, 117.2, 113.5, 110.8, 109.5, 102.8, 82.5, 52.4, 52.1, 41.1, 34.7, 34.1, 31.1, 26.5, 26.4 HRMS (ESI + ) m / z: [M+H + calcd for C 33 H 31 N3O 12 , 694.1780; found, 694.1701

[0083]

Chem.

[0084] 2. Synthesis of GGT5-Probe2 Glutathione (30 mg, 0.097 mmol) was dissolved in 5 mL of water and adjusted to pH 7.0 by adding 2M NaOH. Fluorescein o,o-diacrylate (20 mg, 0.025 mmol) was dissolved in 0.5 mL of DMSO solution. The above solutions were mixed, and 4.5 mL of DMSO was added to make a total volume of 10 mL, and the mixture was stirred at room temperature for 5 hours. The target product was then purified by high performance liquid chromatography (HPLC) using a reversed-phase column. Specifically, a mixed solution of solution A (water containing 0.1% TFA) and solution B (acetonitrile containing 0.1% TFA) was used as the mobile phase, and gradient elution was performed with the proportion of B from 10% (0 min) to 90% (45 min). The fraction containing the target product was lyophilized to obtain GGT5-Probe2 (14.3 mg, 0.013 mmol, y. 52%).

[0085] 1 H NMR (500 MHz, DMSO-d6): δ8.41 (t, J = 5.9 Hz, 2H), 8.23 ​​(d, J = 8.6 Hz, 2H), 8.18 (br, 4H), 8.00 (d, J = 7.6 Hz, 1H), 7.77 (t, J = 7.5, 1H), 7.71 (t, J = 7.5 Hz, 1H), 7.34 (d, J = 7.7 Hz, 1H), 7.21 (d, J = 2.3 Hz, 2H), 6.89 (dd,J = 8.7, 2.2 Hz, 2H), 6.84 (d, J = 8.7 Hz, 2H), 4.47 (m, 2H), 3.86 (t, J = 6.4 Hz, 2H), 3.71 (m, 4H), 2.81 (m, 10H), 2.59 (m, 2H), 2.29 (m, 4H), 1.94 (m, 2H)LRMS (ESI + ) m / z: [M+H + ] calcd for C 46 H 50 NO 19 S, 1055.3; found, 1055.3

[0086] [ka]

[0087] 3. Synthesis of GGT5-Probe3 Hydroxymethyl-N,N'-diethylrhodol (HMDER) was synthesized with reference to the following literature. J. Am. Chem. Soc. 2011, 133, 12960-12963.

[0088] HMDER (61.4 mg, 0.164 mmol) was dissolved in dehydrated tetrahydrofuran (6 ml), and then acryloyl chloride (0.13 mL, 1.6 mmol) and triethylamine (0.27 mL, 1.9 mmol) were added and stirred at room temperature for 48 hours. The solvent was removed using a rotary evaporator, and the residue was purified by sequential column chromatography (silica gel, hexane:ethyl acetate=0:100-75:25) to obtain HMDER o-acrylate (5.0 mg, 0.012 mmol, 7%) as a colorless solid.

[0089] 1H-NMR (600 MHz, CDCl3) δ 7.37-7.33 (m, 2H), 7.26 (dd, J = 7.2, 1.8 Hz, 1H), 7.00 (d, J = 1.8 Hz, 1H), 6.94(d, J = 7.2 Hz, 1H), 6.94(d, J = 8.4 Hz, 1H), 6.77 (dd, J = 8.4, 1.8 Hz, 1H), 6.74 (d, J = 8.4 Hz, 1H), 6.59 (dd, J = 17.4, 1.2 Hz, 1H), 6.41 (d, J = 2.4 Hz, 1H), 6.38 (dd, J = 8.4, 2.4 Hz, 1H), 6.30 (dd, J = 17.4, 10.2 Hz, 1H), 6.00 (dd, J = 10.2, 1.2 Hz, 1H), 5.27 (d, J = 12 Hz, 1H), 5.24 (d, J = 12 Hz, 1H), 3.34 (q, J = 7.0 Hz, 4H), 1.15 (t, J = 7.0 Hz, 6H)

[0090] HMDER o-acrylate (5.0 mg, 0.010 mmol) was dissolved in 150 μL of DMSO and mixed with 150 μL of glutathione aqueous solution (77.8 mM). After stirring at room temperature for 1.5 hours, the target product was purified by high performance liquid chromatography (HPLC) using a reversed-phase column. Specifically, a mixed solution of solution A (water containing 0.1% TFA) and solution B (acetonitrile containing 0.1% TFA) was used as the mobile phase, and gradient elution was performed with the proportion of B from 10% (0 min) to 90% (45 min). The fraction containing the target product was lyophilized to obtain GGT5-Probe3 (4.2 mg, 0.005 mmol, y. 49%).

[0091] 1H NMR (500 MHz, DMSO-d6): δ 8.39 (s, 1H), 8.21 (d, J = 7.5 Hz, 1H), 8.12 (d, J = 4.3 Hz, 2H), 7.39 (d, J = 7.4 Hz, 1H), 7.30 (t, J = 6.7 Hz, 1H), 7.18 (t, J = 6.7, 1H), 6.94 (t, J = 2.4 Hz, 1H), 6.86 (d, J = 8.9 Hz, 1H), 6.74 (brs, 2H), 6.60 (d, J = 8.9Hz, 1H), 6.38 (d, J = 87.4 Hz, 1H), 6.31 (s, 1H), 5.16 (s, 2H), 4.46 (m,1H), 3.87 (brs, 1H), 3.70 (brs, 2H), 3.26 (dd, J = 6.8, 3.2 Hz ,5H), 2.81 (m, 5H), 2.57(m, 1H), 2.28 (m, 2H), 1.92 (m, 2H), 1.00 (t, J = 7.0, 6H) LRMS (ESI + ) m / z: [M+H + ] calcd for C 37 H 42 N4O 10 S, 735.3; found, 735.3

[0092]

change

[0093] Example 3 Reaction analysis of the combination of GGT5 and GGT5-Probe1 (1) GGT5-Probe1 synthesized in Example 2 was dissolved in HBSS(- / -) buffer to a final concentration of 100 μM, and recombinant human GGT5 (Bio-Techne, 7209-GT) was added to a final concentration of 67 pM. A control solution not containing GGT5 was also prepared in the same manner. These solutions were incubated overnight at 37 degrees, and then analyzed by UPLC using a reversed-phase column (LaChromUltra C18 (2 μm)) (detection wavelength: 280 nm). A mixed solution of solution A (water containing 0.1% TFA) and solution B (acetonitrile containing 0.1% TFA) was used as the mobile phase, and gradient elution was performed with the proportion of B from 10% (0 min) to 90% (6.5 min).

[0094] In the control solution, GGT5-Probe1 was almost stable, with a small amount of decomposition products due to hydrolysis (Figure 5, top). On the other hand, in the reaction solution containing the enzyme, GGT5-Probe1 was completely converted to the product (Figure 5, bottom). The retention time of the product was consistent with the retention time of fluorescein predicted from the proposed reaction mechanism (Figure 3). Furthermore, when each reaction solution was irradiated with ultraviolet light with a wavelength of 366 nm, it was confirmed that the control solution emitted only very weak fluorescence, whereas the solution containing the enzyme emitted strong fluorescence (Figure 6).

[0095] Example 4 Reaction analysis of GGT5-Probe1 with recombinant human GGT5 (2) GGT5-Probe1 synthesized in Example 2 was dissolved in HBSS(- / -) buffer to a final concentration of 100 μM, and recombinant human GGT5 (Bio-Techne, 7209-GT) was added to a final concentration of 67 pM. This solution was analyzed by UPLC using a reversed-phase column (LaChromUltra C18 (2 μm)) in the same manner as in Example 3 immediately after mixing or after incubation at 37°C for 30 minutes. By incubation for 30 minutes, GGT5-Probe1 was completely converted to the product (FIG. 7).

[0096] Example 5 Recombinant human GGT5 concentration-dependent reaction of GGT5-Probe1 GGT5-Probe1 was dissolved in HBSS(- / -) buffer to a final concentration of 1 μM, and recombinant human GGT5 (Bio-Techne, 7209-GT) was added to a final concentration of 0, 0.67, 6.7 or 67 pM. These solutions were reacted at 37°C for 60 minutes, and the fluorescence intensity was measured over time using a plate reader (excitation wavelength: 485 nm, emission wavelength: 535 nm). The fluorescence intensity increased in a GGT5 concentration- and time-dependent manner (FIG. 8).

[0097] Example 6 Changes in absorption and fluorescence spectra of GGT5-Probe1 by recombinant human GGT5 (A) GGT5-Probe1 was dissolved in HBSS(- / -) buffer to a final concentration of 2 μM and incubated in a cuvette with an optical path length of 0.5 cm at 37°C for 25 minutes. The changes in absorption and fluorescence spectra over time were measured using a UV-visible spectrophotometer and a fluorometer.

[0098] The results are shown in the left and right graphs of Figure 9(A), respectively. The excitation wavelength for the fluorescence spectrum was 490 nm. No changes were observed in either the absorption spectrum or the fluorescence spectrum during the measurement period. (B) GGT5-Probe1 was dissolved in HBSS(- / -) buffer to a final concentration of 2 μM, and recombinant human GGT5 (Bio-Techne, 7209-GT) was added to a final concentration of 6.7 pM. This solution was incubated in a cuvette with a path length of 0.5 cm at 37°C for 25 minutes, and the changes in absorption and fluorescence spectra over time were measured using a UV-Vis spectrophotometer and a fluorometer. The results are shown in the left and right graphs of Figure 9(B), respectively. The excitation wavelength of the fluorescence spectrum was 490 nm. Between 0 and 25 minutes, an increase in absorbance and fluorescence intensity over time was observed. The fluorescence intensity at the maximum fluorescence wavelength (530 nm) increased approximately 20-fold between 0 and 25 minutes.

[0099] Example 7 Experiment using GGT5 expressing cells 1 x 10 HEK293 cells 5 The cells were seeded at 1000 cells / well in a 24-well plate, and the next day, 1 μg / well of pCAGGS empty vector (mock), GGT5-WT-pCAGGS vector, which is a vector incorporating wild-type GGT5, or GGT5-T388A-pCAGGS vector, which is a vector incorporating a mutant protein in which tyrosine at position 388, the active center of GGT5, is mutated to alanine, was transfected using GeneJuice. After 48 hours, the medium was removed and the cells were washed with HBSS(- / -). GGT5-Probe1 (1 μM) dissolved in HBSS(- / -) was added to each well at 600 μg / well, and the cells were incubated at 37°C for 60 minutes. Immediately after the start of incubation and every 10 minutes from the start, 16 μL of the supernatant was transferred to a 384-well plate, and the fluorescence intensity was measured using a plate reader (excitation wavelength: 485 nm, emission wavelength: 535 nm).

[0100] As shown in FIG. 10, almost no increase in fluorescence intensity was observed in the mock and GGT5(T388A) mutant expressing cells, whereas an increase in fluorescence intensity over time was confirmed in the GGT5 expressing cells. The cell extract used in Figure 10 was collected and subjected to SDS-PAGE electrophoresis, and protein expression was confirmed by Western blotting using an anti-GGT5 antibody (Thermo; PA5-52514). GAPDH was also detected as a loading control using an anti-GAPDH antibody (Sigma; CB1001-500UGCN). As shown in Figure 11, in addition to the full-length protein at around 60 kDa, a light chain generated by self-cleavage at around 25 kDa was confirmed in GGT5-WT (the antibody used recognizes the light chain of GGT5). This self-cleavage is necessary for GGT5 enzyme activity, and it was confirmed that the T388A mutant does not have the self-cleavage activity (lacks GGT5 enzyme activity).

[0101] Example 8 Reaction analysis of GGT5-Probe1 to GGT5-Probe3 depending on the concentration of recombinant human GGT5 Each of GGT5-Probe1, GGT5-Probe2, and GGT5-Probe3 synthesized in Example 2 was dissolved in HBSS(- / -) buffer to a final concentration of 2 μM, and recombinant human GGT5 (Bio-Techne, 7209-GT) was added to the solution at 0.5 pM, 2.0 pM, and 8.0 pM. The fluorescence intensity during the reaction of these solutions at 37° C. for 600 minutes was measured over time using a plate reader (excitation wavelength: 485 nm, fluorescence wavelength: 535 nm).

[0102] The results are shown in Figure 12. In the samples to which each probe was added, the fluorescence intensity increased in a GGT concentration-dependent manner and in a time-dependent manner. In the sample to which GGT5-Probe2 was added, the increase in fluorescence intensity in the absence of recombinant human GGT5 was small, and it was more stable in the buffer solution than GGT5-Probe1.

[0103] Example 9 Changes in absorption and fluorescence spectra of GGT5-Probe2 and GGT5-Probe3 by recombinant human GGT5 GGT5-Probe2 or GGT5-Probe3 was dissolved in HBSS(- / -) buffer to a final concentration of 2μM, and recombinant human GGT5 (Bio-Techne, 7209-GT) was added to a final concentration of 6.7pM. This solution was incubated in a cuvette with a path length of 0.5 cm at 37℃ for 25-30 minutes, and the changes in absorption and fluorescence spectra over time were measured using a UV-visible spectrophotometer and a fluorometer.

[0104] The results for GGT5-Probe2 are shown in the left and right graphs of Figure 13, and the results for GGT5-Probe3 are shown in the left and right graphs of Figure 14. The excitation wavelength of the fluorescence spectrum of GGT5-Probe2 was 490 nm, and the excitation wavelength of the fluorescence spectrum of GT5-Probe3 was 525 nm. Between 0 and 30 minutes, the absorbance and fluorescence intensity increased over time.

[0105] Example 10: Application experiment of GGT5-Probe3 to GGT5-expressing cells 1 x 10 HEK293 cells 5 The cells were seeded at 1000 cells / well on a 24-well polylysine-coated glass bottom dish, and the next day, the pCAGGS empty vector (mock) and the GGT5-WT-pCAGGS vector, which is a vector incorporating the wild type of GGT5, were transfected at 1 μg / well using GeneJuice. 48 hours after transfection, the medium was removed and washed with HBSS(- / -). GGT-Probe3 (probe concentration 2 μM) dissolved in HBSS(- / -) was added to the wells of GGT5 expressing cells and mock cells, and incubated at 37°C for 60 minutes. After incubation, the medium was removed, washed with HBSS(- / -), and MEM medium (phenol red free) was added, and the cells were observed under a confocal laser microscope (excitation wavelength: 561 nm, emission wavelength: 570-670 nm).

[0106] As shown in Figure 15, almost no fluorescence was observed in the sample without GGT5-Probe3 (Control) and the sample containing HEK293 cells transfected with a vector that does not express GGT5 (Probe3 Mock). GGT5-Probe3 is cell membrane impermeable, but the fluorescent substance (HMDER) generated when GGT5-Probe3 is cleaved by GGT5 was detected. Since HMDER is cell membrane permeable, in the sample in which GGT5-Probe3 was added using GGT5-expressing cells (Probe3 GGT5), fluorescence was observed in the cells at the same level as in the sample in which HMDER was added.

Claims

1. General formula (1) 【Chemistry 1】 (In the formula, R 1 is a direct bond or a divalent hydrocarbon group, X 1 is S or O, X 2 is O or NH, A is a moiety containing a chromophore. or a salt, hydrate or solvate thereof.

2. The compound according to claim 1, or a salt, hydrate or solvate thereof, wherein the chromophore has a xanthene skeleton, a silicon-substituted xanthene skeleton, a coumarin skeleton, a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene skeleton, a phenoxazine skeleton, a phenylbenzothiazole skeleton, a naphthalimide skeleton, a hemicyanine skeleton, or a merocyanine skeleton.

3. The compound according to claim 1 , or a salt, hydrate or solvate thereof, wherein the chromophore has a xanthene skeleton or a silicon-substituted xanthene skeleton.

4. The compound according to claim 1, wherein A is represented by formula (2), or a salt, hydrate or solvate thereof. 【Chemistry 2】 (In the formula, R 5 , R 6 , R 7 , R 8 , R 9 and R 10 are each independently hydrogen, halogen, cyano, or -CF 3 , C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, C1-C6 alkylthio, C1-C6 alkoxy, sulfo, or carboxy; R 11 , R 12 , R 13 , and R 14 are each independently hydrogen, halogen, cyano, halogen, cyano, -CF 3 , C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, C1-C6 alkylthio, C1-C6 alkoxy, sulfo, or carboxy, where R 11 , R 12 , R 13 , and R 14 any alkyl moiety of may be further substituted with halogen, amino, hydroxy, cyano, C1-C6 alkyl, C1-C6 alkylamino, C1-C6 alkylthio, C1-C6 alkoxy, sulfo, or carboxy; Y is -O- or -MR 20 R 21 - (M is a silicon atom, a germanium atom, or a tin atom, R 20 and R 21 are each independently C1-C6 alkyl or aryl; Z is -C(O)-, -SO 2 - or CH 2 and B is -OR 2 (R 2 is hydrogen, alkyl which may have a substituent, or acyl which may have a substituent), -NR 3 R 4 (R 3 and R 4 each independently represents hydrogen, alkyl or cycloalkyl, or R 3 and R 4 together with the nitrogen atom to which they are attached form a monocyclic or bicyclic ring), or —O—C(O)—R 1 -X 1 - (a group recognized by GGT5), * denotes X of the compound represented by formula (1). 2 It is a combination with

5. A fluorescent dye comprising the compound according to any one of claims 1 to 4, or a salt, hydrate or solvate thereof.

6. The fluorescent dye according to claim 5, which is a fluorescent dye for detecting GGT5 expressing cells.

7. The fluorescent dye according to claim 6 , wherein the GGT5 expressing cells include eosinophils.

8. A method for detecting GGT5 expressing cells, comprising: Contacting a GGT5 expressing cell with the fluorescent dye according to any one of claims 5 to 7; and measuring the fluorescence emitted by a compound produced by cleavage of the γ-glutamyl bond of said compound or a salt, hydrate or solvate thereof in said fluorescent dye.

9. A cell detection agent comprising the fluorescent dye according to any one of claims 5 to 7.

10. A method for bioimaging of cells, which comprises using the fluorescent dye according to any one of claims 5 to 7.

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