Fluorescent probe for detecting protein synthesis in mitochondria

Abstract

To provide a novel compound usable for protein detection in mitochondria, a fluorescent probe, and a method for detecting protein in mitochondria using the fluorescent probe.SOLUTION: A compound represented by the following formula (I) or a salt thereof.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a fluorescent probe. [Background technology]

[0002] Fluorescent probes for specific applications have been developed, each of which is made by combining a molecule that acts on a specific substance with a fluorescent dye.

[0003] Non-Patent Document 1 discloses a molecule called Fluorpuro, which is a conjugated fluorescent dye fluorescein and puromycin, and its synthesis. This molecule binds to the carboxy terminus of a protein in a cell-free protein synthesis system.

[0004] Non-Patent Document 2 discloses molecules in which the fluorescent dyes fluorescein (fluorescein, RhG, TAMRA, Cy3, Cy5) are linked to puromycin via various linkers, and their synthesis. This molecule is bound to the carboxy terminus of a protein in a cell-free protein synthesis system.

[0005] Non-Patent Document 3 discloses a molecule in which fluorescein and puromycin are linked via a linker with enhanced hydrophobicity, and its synthesis. This molecule penetrates the cell membrane and targets protein synthesis in the cytoplasm.

[0006] Non-Patent Document 4 reports the application of a conjugate of fluorescein and puromycin to the visualization of local protein synthesis in hippocampal neurons.

[0007] On the other hand, while most proteins in mammalian cells are synthesized by ribosomes in the cytoplasm, mitochondria have their own DNA and ribosomes and synthesize 13 types of proteins essential for energy production (electron transport chain and oxidative phosphorylation). [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] FEBS Letters 1999, 462, 43-46. DOI: 10.1016 / s0014-5793(99)01474-x [Non-patent document 2] Genome Research 2002, 12, 487-492. DOI: 10.1101 / gr.218802 [Non-patent document 3] Chemistry & Biology 2004, 11, 999-1008. DOI: 10.1016 / j.chembiol.2004.05.011 [Non-patent document 4] Neuron 2005, 45, 765-779. DOI: 10.1016 / j.neuron.2005.01.015 Summary of the Invention [Problem to be solved by the invention]

[0009] Abnormalities in mitochondrial protein synthesis lead to diseases such as mitochondrial diseases, so the development of methods to detect these abnormalities is important for the research and diagnosis of these diseases.

[0010] To detect protein synthesis in mitochondria, cycloheximide, which inhibits cytoplasmic protein synthesis, was added and radioisotopes [ 35 Although a method for quantifying protein labeling using [S]-labeled methionine is known, it requires complicated procedures and is toxic due to the inhibition of protein synthesis, making it difficult to apply to living cells or individuals.

[0011] Furthermore, as described in Non-Patent Documents 1-4, there have been cases in which molecular probes conjugated with fluorescent dyes and puromycin have been used to detect protein synthesis, but none of the fluorescent molecules described in Non-Patent Documents 1-4 target mitochondria.

[0012] An object of the present invention is to provide a novel compound and fluorescent probe that can be used to detect proteins in mitochondria, and a method for detecting proteins in mitochondria using the fluorescent probe. [Means for solving the problem]

[0013] In view of the above problems, and as a result of extensive research, the present inventors have developed a cell membrane-permeable compound having a structure in which a rhodamine compound, which is a mitochondria-selective fluorescent substance, and puromycin, which has the property of binding to the carboxy terminus of a peptide chain, are connected by a linker.

[0014] The present invention encompasses the embodiments described below. Section 1. A compound represented by the following formula (I) or a salt thereof: [ka] (In the formula, R 1 and R 2 are each independently hydrogen, optionally substituted C 1- C6 alkyl group, -OCH3, -OCF 3、 -COOH 、 -COOCH 3、 -COOC2H 5、 -CHOH or halogen, R 3 is a monovalent substituent present on a benzene ring, and n is 0 to 3; R 5 and R 6 are independently hydrogen, C 1- is a C6 alkyl group or -NH2, R 7 and R 8 are independently hydrogen, C 1- is a C6 alkyl group or -NH2, R 9 and R 12 are each independently a hydrogen atom, a halogen atom, or C 1- is a C6 alkyl group, R 10 and R 11 are each independently a hydrogen atom, a halogen atom, or C 1-6 is an alkyl group, R 5 and R 6 are both alkyl, R 5 The alkyl group represented by R 6 may be bonded to an alkyl group represented by R to form a 4- to 6-membered ring, and / or 7 and R 8 are all alkyl groups, R 7 The alkyl group represented by R 8 may be bonded to an alkyl group represented by the formula: R 5 and R 9 are all alkyl groups, R 5 The alkyl group represented by R 9 may be bonded to an alkyl group represented by R 6 and R 10 are all alkyl groups, R 6 The alkyl group represented by R 10 may be bonded to an alkyl group represented by the formula: R 7 and R 11 are all alkyl groups, R 7 The alkyl group represented by R 11 may be bonded to an alkyl group represented by R 8 and R 12 are all alkyl groups, R 8 The alkyl group represented by R 12 may be bonded to an alkyl group represented by the formula: X is oxygen, SiR 13 R 14 , GeR 13 R 14 , C.R. 13 R 14 , or POR 15 and R 13 and R 14 are each independently C 1- is a C6 alkyl group or an aryl group, and R15 is C 1- C6 alkyl group, C 2- C6 alkenyl group, C 2- a C6 alkynyl group or an aryl group, L is a linker Section 2. Item 2. The compound or salt thereof according to Item 1, wherein L is a linker moiety represented by the following formula (II): -(Z1) m -[X1] s -Y k -[X2] t -(Z2) n - (II) (In the formula, Z1 is -O-, -S-, -NR 18 -, -NR 18 -C(O)-, -C(O)NR 18 -, -OC(O)-, -C(O)-O-, a phosphate group, or -OP(=O)(-CH3)(-O-), X1 is C1-C 20 Alkylene, C2-C 20 Alkenylene, C2-C 20 Alkynylene, C3-C8 cycloalkylene, or (C2H5O) j (j is an integer from 1 to 10), Y is -O-, -S-, or -NR 18 -, -NR 18 -C(O)-, -C(O)NR 18 -, -OC(O)-, or -C(O)-O-, a triazolyl group; X2 is C1-C 20 Alkylene, C2-C 20 Alkenylene, C2-C 20 Alkynylene, C3-C8 cycloalkylene, or (C2H5O) j (j is an integer from 1 to 10), Z2 is -O-, -S-, -NR 18 -, -NR 18 -C(O)-, -C(O)NR 18 -, -OC(O)-, -C(O)-O-, a phosphate group, or -OP(=O)(-CH3)(-O-), R18 is hydrogen, C 1- C6 alkyl or aryl, k, m, n, s, and t are each independently 0 or 1, provided that k+m+n+s+t is 1 or greater. Section 3. The compound represented by formula (III) and the compound represented by formula (IV) are reacted with each other by the reaction of R 16 and R 17 Item 1. A method for producing the compound or a salt thereof according to Item 1, comprising reacting the compound or a salt thereof so that [ka] (In formula (III), R 1 and R 2 One of the groups is hydrogen, optionally substituted C 1- C6 alkyl group, -OCH3, -OCF 3、 -COOH 、 -COOCH 3、 -COOC2H 5、 -CHOH or halogen, R 3 is a monovalent substituent present on a benzene ring, and n is 0 to 3; R 5 and R 6 are independently hydrogen, C 1- is a C6 alkyl group or -NH2, R 7 and R 8 are independently hydrogen, C 1- is a C6 alkyl group or -NH2, R 9 and R 12 are each independently a hydrogen atom, a halogen atom, or C 1- is a C6 alkyl group, R 10 and R 11 are each independently a hydrogen atom, a halogen atom, or C 1- is a C6 alkyl group, R 5 and R 6 are both alkyl, R 5 The alkyl group represented by R 6may be bonded to an alkyl group represented by R to form a 4- to 6-membered ring, and / or 7 and R 8 are all alkyl groups, R 7 The alkyl group represented by R 8 may be bonded to an alkyl group represented by the formula: R 5 and R 9 are all alkyl groups, R 5 The alkyl group represented by R 9 may be bonded to an alkyl group represented by R 6 and R 10 are all alkyl groups, R 6 The alkyl group represented by R 10 may be bonded to an alkyl group represented by the formula: R 7 and R 11 are all alkyl groups, R 7 The alkyl group represented by R 11 may be bonded to an alkyl group represented by R 8 and R 12 are all alkyl groups, R 8 The alkyl group represented by R 12 may be bonded to an alkyl group represented by the formula: X is oxygen, SiR 13 R 14 , GeR 13 R 14 , C.R. 13 R 14 , or POR 15 and R 13 and R 14 are each independently C 1- is a C6 alkyl group or an aryl group, and R 15 is C 1- C6 alkyl group, C 2- C6 alkenyl group, C 2- is a C6 alkynyl group or an aryl group, R 16 is a reactive group.) [ka] (In formula (IV), R 17 is a reactive group.) Section 4. Item 3. A mitochondria-targeting fluorescent probe comprising the compound according to Item 1 or 2 or a salt thereof. Section 5. Item 5. A mitochondrial-targeted fluorescent probe according to Item 4, which is capable of detecting a protein. Section 6. Item 5. A mitochondria-targeted fluorescent probe according to Item 4, which is capable of detecting protein synthesis in mitochondria. Section 7. Item 5. The mitochondrial-targeted fluorescent probe according to Item 4, which is a fluorescent dye for bioimaging. Section 8. 1. A method for detecting proteins in mitochondria, comprising: Contacting cells having mitochondria with the mitochondria-targeted fluorescent probe according to item 4; detecting proteins bound by said mitochondria-targeted fluorescent probe in mitochondria within said cells. A method comprising: Section 9. Item 9. The method according to Item 8, wherein the step of detecting the protein comprises detecting protein synthesis based on the protein bands obtained by electrophoresis. [Effects of the Invention]

[0015] According to the present invention, the compound represented by formula (1) is useful as a fluorescent probe that targets mitochondria and can be used to detect proteins, particularly protein synthesis, within mitochondria. Furthermore, proteins within mitochondria can be easily detected using the compound or a fluorescent probe containing the compound. [Brief explanation of the drawings]

[0016] [Figure 1] The structure of puromycin. [Figure 2]Confocal laser scanning micrographs of cells. Each image is 60x magnification. (Top left) GFP staining (excitation wavelength 488 nm), (Top right) Probe 4 staining (excitation wavelength 555 nm), (Bottom left) bright field, (Bottom right) merged image. [Figure 3] An enlarged photograph of the area indicated by the dotted frame in the image at the bottom right of Figure 2. [Figure 4] (A) Electrophoresis gel before CBB staining (fluorescence detection: excitation wavelength 515-545 nm, emission wavelength 568-617 nm). (B) Electrophoresis gel after CBB staining. Lane 1: molecular weight marker, Lane 2: negative control without template DNA, Lane 3: reaction solution (with template DNA), Lane 4: reaction solution with probe 1 (10 μM), Lane 5: probe 1 (10 μM), Lane 6: reaction solution with probe 2 (10 μM), Lane 7: probe 2 (10 μM), Lane 8: reaction solution with probe 3 (10 μM), Lane 9: probe 3 (10 μM), Lane 10: reaction solution with probe 4 (10 μM), Lane 11: probe 4 (10 μM). DETAILED DESCRIPTION OF THE INVENTION

[0017] In this specification, the term "comprise" is a concept that encompasses "consist essentially of" and "consist only of."

[0018] In this specification, the prefix "C a- C b " means the number of carbon atoms is from a to b, including (a and b). For example, C 1- A C6 alkyl group refers to an alkyl group having 1 to 6 carbon atoms.

[0019] As used herein, "C 1-"C6 alkyl group" includes monovalent hydrocarbon groups that are linear, branched, cyclic, or a combination of cyclic and linear or branched groups. Examples of linear alkyl groups include methyl, ethyl, n-propyl, n-butyl, n-pentyl, and n-hexyl groups. Examples of branched alkyl groups include isopropyl, isobutyl, t-butyl, s-butyl, neopentyl, isohexyl, and 3-methylpentyl groups. "C 1- The "C6 alkyl group" preferably has 1 to 4 carbon atoms, and more preferably has 1 to 3 carbon atoms.

[0020] As used herein, "C 2- The term "C6 alkenyl group" includes straight-chain alkenyl groups and branched-chain alkenyl groups. Examples of straight-chain alkenyl groups include ethenyl, n-propenyl, n-butenyl, n-pentenyl, and n-hexenyl groups. Examples of branched-chain alkenyl groups include isopropenyl, isobutenyl, t-butenyl, s-butenyl, neopentenyl, isohexenyl, and 3-methylpentenyl groups. 2- The "C6 alkyl group" preferably has 2 to 4 carbon atoms.

[0021] As used herein, "C 2- The term "C6 alkynyl group" includes straight-chain alkynyl groups and branched-chain alkynyl groups. Examples of straight-chain alkyl groups include ethynyl groups, n-propynyl groups, n-butynyl groups, n-pentynyl groups, and n-hexynyl groups. Examples of branched-chain alkynyl groups include isopropynyl groups, isobutynyl groups, t-butynyl groups, s-butynyl groups, neopentynyl groups, isohexynyl groups, and 3-methylpentynyl groups. 2- The "C6 alkyl group" preferably has 2 to 4 carbon atoms.

[0022] As used herein, the term "aryl group" refers to a monocyclic or polycyclic aromatic group. The aryl group may have one or more heteroatoms (e.g., oxygen, nitrogen, or sulfur atoms) as ring-constituting atoms. When two or more heteroatoms are present, they may be the same or different. More specifically, aryl includes, but is not limited to, phenyl, biphenyl, 1-naphthyl, 2-naphthyl, and substituted forms thereof.

[0023] As used herein, the term "rhodamine compound" refers to a compound having a skeleton structure represented by the following formula (X):

[0024] [ka] (wherein X is oxygen, SiR 13 R 14 , GeR 13 R 14 , C.R. 13 R 14 , or POR 15 and R 13 and R 14 are each independently C 1- is a C6 alkyl group or an aryl group, and R 15 is C 1- C6 alkyl group, C 2- C6 alkenyl group, C 2- C6 alkynyl group or aryl group.

[0025] In one embodiment, the present invention provides a compound represented by the following formula (I) or a salt thereof:

[0026] [ka] (In the formula, R 1 and R 2 are each independently hydrogen, optionally substituted C 1- C6 alkyl group, -OCH3, -OCF 3、 -COOH, -COOCH 3、 -COOC2H 5、-CHOH or halogen, R 3 is a monovalent substituent present on a benzene ring, and n is 0 to 3; R 5 and R 6 are independently hydrogen, C 1- is a C6 alkyl group or -NH2, R 7 and R 8 are independently hydrogen, C 1- is a C6 alkyl group or -NH2, R 9 and R 12 are each independently a hydrogen atom, a halogen atom, or C 1- is a C6 alkyl group, R 10 and R 11 are each independently a hydrogen atom, a halogen atom, or C 1-6 is an alkyl group, R 5 and R 6 are both alkyl, R 5 The alkyl group represented by R 6 may be bonded to an alkyl group represented by R to form a 4- to 6-membered ring, and / or 7 and R 8 are all alkyl groups, R 7 The alkyl group represented by R 8 may be bonded to an alkyl group represented by the formula: R 5 and R 9 are all alkyl groups, R 5 The alkyl group represented by R 9 may be bonded to an alkyl group represented by R 6 and R 10 are all alkyl groups, R 6 The alkyl group represented by R 10 may be bonded to an alkyl group represented by the formula: R 7 and R 11 are all alkyl groups, R 7 The alkyl group represented by R 11may be bonded to an alkyl group represented by R 8 and R 12 are all alkyl groups, R 8 The alkyl group represented by R 12 may be bonded to an alkyl group represented by the formula: X is oxygen, SiR 13 R 14 , GeR 13 R 14 , C.R. 13 R 14 , or POR 15 and R 13 and R 14 are each independently C 1- is a C6 alkyl group or an aryl group, and R 15 is C 1- C6 alkyl group, C 2- C6 alkenyl group, C 2- a C6 alkynyl group or an aryl group, L is a linker

[0027] R 1 and R 2 may be the same or different.

[0028] R 1 and R 2 each of which is optionally substituted C 1- In the case of a C6 alkyl group, the substituents include halogen atoms (e.g., fluorine, chlorine, bromine, and iodine), hydroxyl groups, C 1- C6 alkoxy group, C 3- C7 cycloalkyl group, carbon number C 5- C 10 monocyclic unsaturated hydrocarbon groups, carbamoyl groups, (C 2- C7 acyl)oxy group, C as a substituent 1- C6 alkyl group or C 2- Examples include an amino group which may have a C7 acyl group. The number of substituents is not particularly limited, and is preferably 0 to 6, and more preferably 0 to 3.

[0029] R 1 and R2 each of which is optionally substituted C 1- If it is a C6 alkyl group, C 1- The C6 alkyl group may be either a straight-chain alkyl group or a branched-chain alkyl group.

[0030] R 1 and R 2 When is a halogen, examples of the halogen atom include fluorine, chlorine, bromine, and iodine.

[0031] In certain embodiments, R 1 and R 2 are both hydrogen.

[0032] In certain embodiments, R 1 and R 2 is hydrogen and R 1 and R 2 The other is C 1- C6 alkyl group, -OCH3, -OCF 3、 -COOH, -COOCH 3、 -COOC2H 5、 -CH2OH or halogen.

[0033] In certain embodiments, R 1 and R 2 is hydrogen and R 1 and R 2 The other is C 1- C6 alkyl group, -OCH3, -OCF 3、 -COOCH 3、 -COOC2H 5、 -CH2OH or halogen.

[0034] In certain embodiments, R 1 and R 2 is hydrogen and R 1 and R 2 The other is C 1- It is a C6 alkyl group.

[0035] In certain embodiments, R 1 and R 2 are both C1- C6 alkyl group, -OCH3, -OCF 3、 -COOH, -COOCH 3、 -COOC2H 5、 -CH2OH or halogen.

[0036] In certain embodiments, R 1 and R 2 are both C 1- C6 alkyl group, -OCH3, -OCF 3、 -COOCH 3、 -COOC2H 5、 -CH2OH or halogen.

[0037] In certain embodiments, R 1 and R 2 are both C 1- C6 alkyl group, -OCH3, -OCF 3、 -COOCH 3、 -COOC2H 5、 -CHOH or halogen, and R 1 and R 2 are identical.

[0038] R 1 and R 2 can be appropriately selected by a person skilled in the art so that the compound represented by formula (I) or a salt thereof has a hydrophobicity suitable for permeating a cell membrane.

[0039] R 3 Examples of the monovalent substituent present on the benzene ring include C 1- C6 alkyl group or C 1- C6 alkylamino group, -OCH3, -OCF 3、 -COOCH 3、 -COOC2H 5、 Examples include —CH 2 OH or halogen.

[0040] In certain embodiments, R 3 is C 1- C6 alkyl group or C 1- C6 alkylamino group, -OCH3, -OCF 3、 -COOCH3、 -COOC2H 5、 -CH2OH or halogen, and n is 1 to 3.

[0041] In certain embodiments, R 3 In this case, n is 0.

[0042] R 5 and R 6 may be the same or different. In certain embodiments, R 5 and R 6 are the same and are both hydrogen or C 1- It is a C6 alkyl group.

[0043] R 7 and R 8 may be the same or different. In certain embodiments, R 7 and R 8 are the same and are both hydrogen or C 1- It is a C6 alkyl group.

[0044] R 9 and R 12 may be the same or different. In certain embodiments, R 9 and R 12 are the same and are both hydrogen or C 1- It is a C6 alkyl group.

[0045] R 10 and R 11 may be the same or different. In certain embodiments, R 9 and R 12 are the same and are both hydrogen or C 1- It is a C6 alkyl group.

[0046] In certain embodiments, R 5 and R 6 are all alkyl, and R 5 The alkyl group represented by R 6 and the alkyl group represented by R 7 and R8 are all alkyl groups, and R 7 The alkyl group represented by R 8 and an alkyl group represented by the formula (I) bond to form a 4- to 6-membered ring.

[0047] In certain embodiments, R 5 , R 6 , R 7 and R 8 are each independently hydrogen or C 1- C6 alkyl, and R 5 and R 6 , and R 7 and R 8 does not form a ring.

[0048] In certain embodiments, R 5 and R 9 are all alkyl, and R 5 The alkyl group represented by R 9 and the alkyl group represented by R 6 and R 10 are all alkyl groups, and R 6 The alkyl group represented by R 10 and an alkyl group represented by the formula (I) bond to form a 5- to 7-membered ring.

[0049] In certain embodiments, R 5 , R 9 R 6 and R 10 are each independently hydrogen or C 1- C6 alkyl, and R 5 and R 9 , and R 6 and R 10 does not form a ring.

[0050] In certain embodiments, R 7 and R 11 are all alkyl groups, and R 7 The alkyl group represented by R 11 and the alkyl group represented by R 8 and R 12 are all alkyl groups, and R 8The alkyl group represented by R 12 and an alkyl group represented by the formula (I) bond to form a 5- to 7-membered ring.

[0051] In certain embodiments, R 7 , R 11 , R 8 and R 12 are each independently hydrogen or C 1- C6 alkyl, and R 7 and R 11 , and R 8 and R 12 does not form a ring.

[0052] In certain embodiments, R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 are each independently hydrogen or C 1- It is a C6 alkyl and does not form a ring.

[0053] X can be oxygen or SiR 13 R 14 , GeR 13 R 14 , C.R. 13 R 14 , or POR 15 R 13 and R 14 C 1- When R is a C6 alkyl group or an aryl group, 13 and R 14 may be the same or different. An aryl group is, for example, phenyl.

[0054] The linker may be any linker capable of connecting the rhodamine compound and puromycin. In certain embodiments, L is a linker moiety represented by formula (II):

[0055] -(Z1) m -[X1] s -Y k-[X2] t -(Z2) n - (II) (In the formula, Z1 is -O-, -S-, -NR 18 -, -NR 18 -C(O)-, -C(O)NR 18 -, -OC(O)-, -C(O)-O-, a phosphate group, or -OP(=O)(-CH3)(-O-), X1 is C1-C 20 Alkylene, C2-C 20 Alkenylene, C2-C 20 Alkynylene, C3-C8 cycloalkylene, or (C2H5O) j (j is an integer from 1 to 10), Y is -O-, -S-, or -NR 18 -, -NR 18 -C(O)-, -C(O)NR 18 -, -OC(O)-, or -C(O)-O-, a triazolyl group; X2 is C1-C 20 Alkylene, C2-C 20 Alkenylene, C2-C 20 Alkynylene, C3-C8 cycloalkylene, or (C2H5O) j (j is an integer from 1 to 10), Z2 is -O-, -S-, -NR 18 -, -NR 18 -C(O)-, -C(O)NR 18 -, -OC(O)-, -C(O)-O-, a phosphate group, or -OP(=O)(-CH3)(-O-), R 18 is hydrogen, C 1- C6 alkyl or aryl, k, m, n, s, and t are each independently 0 or 1, provided that k+m+n+s+t is 1 or greater.

[0056] In certain embodiments, m, s, n are 1, and k and t are 0.

[0057] In certain embodiments, s, k, t are 1, and m and n are 0.

[0058] In certain embodiments, m, s, k are 1, and t and n are 0.

[0059] In certain embodiments, m, s, k, t, and n are 1.

[0060] The counter ion (cation) of the anion represented by formula (I) is 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.

[0061] The compound represented by formula (I) or a salt thereof may exist as a hydrate or solvate, and all of these substances are included in the scope of the present invention. The salt of the compound represented by formula (I) may be a pharmaceutically acceptable salt.

[0062] The method for producing the compound represented by formula (I) or a salt thereof is not particularly limited, and it can be produced by binding a rhodamine compound and puromycin using a known method.

[0063] In certain embodiments, the method for preparing a compound represented by formula (I) or a salt thereof includes reacting a compound represented by formula (III) and a compound represented by formula (IV) with a compound represented by formula (III) in the presence of R 16 and R 17 This includes reacting the two to bond together.

[0064] [ka] (In formula (III), R 1 and R 2 One of the groups is hydrogen, optionally substituted C 1- C6 alkyl group, -OCH3, -OCF 3、 -COOH, -COOCH 3、 -COOC2H5、 -CHOH or halogen, R 3 is a monovalent substituent present on a benzene ring, and n is 0 to 3; R 5 and R 6 are independently hydrogen, C 1- is a C6 alkyl group or -NH2, R 7 and R 8 are independently hydrogen, C 1- is a C6 alkyl group or -NH2, R 9 and R 12 are each independently a hydrogen atom, a halogen atom, or C 1- is a C6 alkyl group, R 10 and R 11 are each independently a hydrogen atom, a halogen atom, or C 1-6 is an alkyl group, R 5 and R 6 are both alkyl, R 5 The alkyl group represented by R 6 may be bonded to an alkyl group represented by R to form a 4- to 6-membered ring, and / or 7 and R 8 are all alkyl groups, R 7 The alkyl group represented by R 8 may be bonded to an alkyl group represented by the formula: R 5 and R 9 are all alkyl groups, R 5 The alkyl group represented by R 9 may be bonded to an alkyl group represented by R 6 and R 10 are all alkyl groups, R 6 The alkyl group represented by R 10 may be bonded to an alkyl group represented by the formula: R 7 and R 11 are all alkyl groups, R 7 The alkyl group represented by R11 may be bonded to an alkyl group represented by R 8 and R 12 are all alkyl groups, R 8 The alkyl group represented by R 12 may be bonded to an alkyl group represented by the formula: X is oxygen, SiR 13 R 14 , GeR 13 R 14 , C.R. 13 R 14 , or POR 15 and R 13 and R 14 are each independently C 1- is a C6 alkyl group or an aryl group, and R 15 is C 1- C6 alkyl group, C 2- C6 alkenyl group, C 2- is a C6 alkynyl group or an aryl group, R 16 is a reactive group.)

[0065] [ka] (In formula (IV), R 17 is a reactive group.)

[0066] In formula (III), R 1 , R 2 , R 3 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , n, and X are as described for compounds of formula (I).

[0067] R 16 and R 17The combination of R may be any combination of reactive groups that react to form the linker L, and one skilled in the art can select an appropriate combination of reactive groups. 16 and R 17 is a combination of reactive groups that react to form the linker moiety represented by formula (II) above.

[0068] In certain embodiments, R 16 and R 17 is a combination of functional groups that generates an amide bond, a urethane bond, an ester bond, or an ether bond upon reaction. For example, R 16 Examples of R include a carboxy group, an amino group, and an isocyanate group. 16 When R is a carboxy group, an amino group, or an isocyanate group, 17 are respectively a functional group having an amino group at the terminal, a functional group having a carboxyl group at the terminal, and a functional group having a hydroxyl group at the terminal, then R 16 to form a linker.

[0069] In one aspect, the present invention provides a mitochondrial-targeted fluorescent probe comprising any of the compounds represented by formula (I) or salts thereof described above.

[0070] The compound represented by formula (I) or a salt thereof is a molecule having a structure in which a rhodamine compound, a mitochondria-selective fluorescent substance, and puromycin, which has the property of binding to the carboxy terminus of a peptide chain, are connected by a linker. As shown in Figure 1, puromycin has a structure similar to the 3' end of aminoacyl-tRNA, and has an adenosine-like structure, an amino acid-like structure, and an amide bond connecting these two structures. It is taken up into the ribosome and binds to the growing peptide chain, thereby terminating the elongation of the peptide chain.

[0071] Therefore, the compound represented by the above formula (I) or a salt thereof is localized in mitochondria after passing through the cell membrane, and its puromycin moiety binds to the carboxy terminus of a protein synthesized in a ribosome in the mitochondria, and can be used as a fluorescent probe that specifically detects proteins, particularly protein synthesis, in mitochondria.

[0072] The compound represented by formula (I) or a salt thereof and a fluorescent dye containing the compound have maximum absorption and fluorescence wavelengths in the range from visible light to near-infrared light. For example, the compound represented by formula (I) or a salt thereof and a fluorescent dye containing the compound have maximum absorption and fluorescence wavelengths in the range of 450 nm to 850 nm. Therefore, they are also suitable for bioimaging of fixed cells or tissues.

[0073] In one aspect, the present invention provides a method for detecting a protein in a mitochondria, the method comprising the steps of contacting a cell having mitochondria with the above-described mitochondria-targeted fluorescent probe, and detecting a protein bound by the mitochondria in the mitochondria within the cell.

[0074] According to the above method, a fluorescent dye containing the compound represented by formula (I) or a salt thereof can be used to selectively detect proteins in mitochondria.

[0075] In certain embodiments, the step of detecting the protein comprises detecting synthesis of the protein based on electrophoresed protein bands.

[0076] The fluorescent dye containing the compound represented by formula (I) or a salt thereof of the present invention, and the method for detecting proteins in mitochondria using the fluorescent dye, can be applied to research reagents and diagnostic agents for various diseases that cause abnormalities in intramitochondrial protein synthesis, such as mitochondrial diseases.

[0077] The disclosures of all patent applications and publications cited herein are hereby incorporated by reference in their entirety.

[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 techniques commonly used in the art or procedures described in published literature. In the following examples, the ratio of components when preparing liquid mixtures is expressed as a volume ratio (v / v). [Example]

[0079] Example 1 Synthesis of Fluorescent Probe 1

[0080] [ka]

[0081] Synthesis of Compound 1 A solution of N,N-diethyl-3-aminophenol (1.82 g, 11 mmol) in toluene (33 mL) was stirred at 60 °C for 5 min. Trimellitic anhydride (2.6 g, 14 mmol) was added to this solution and stirred under reflux for 24 h. After cooling to room temperature, the mixture was suction filtered and the residue was washed with toluene. Purification by column chromatography afforded crude compound 1 (2.4 g, crude).

[0082] Synthesis of compound 2 A solution of compound 1 (100 mg, 280 μmol) and N,N-diethyl-3-aminophenol (50.1 mg, 300 μmol) in methanesulfonic acid (1.2 mL) was stirred under reflux for 3 h. After cooling to room temperature, the mixture was neutralized with 2 M aqueous sodium hydroxide and purified by reverse-phase column chromatography (HO / CHCN, 0.1% TFA) to give compound 2 (43.4 mg, 72.3 μmol, 26%). 1H NMR (500 MHz, methanol-d4): δ 8.92 (d, J = 1.6 Hz, 1H), 8.44 (dd, J = 7.9 Hz, J = 1.6 Hz, 1H), 7.55 (d, J = 7.9 Hz, 1H), 7.13 (d, J = 9.5 Hz, 2H), 7.04 (dd, J = 9.5 Hz, J = 2.5 Hz, 2H), 6.99 (d, J = 2.5 Hz, 2H), 3.69 (q, J = 7.2 Hz, 8H), 1.31 (t, J = 7.2 Hz, 12H). 13 C NMR (126 MHz, methanol-d4): δ 167.9, 167.3, 159.9, 159.3, 157.2, 139.4, 134.5, 134.2, 133.5, 132.9, 132.2, 132.1, 115.5, 97.3, 95.9, 46.8, 12.8. HRMS (ESI) (m / z): calcd for C 29 H 29 N2O5[M-2H-CF3COO] - , 485.2077, found, 485.2049 (-2.8 mmu).

[0083] Synthesis of compound 3 Compound 2 (41.3 mg, 68.8 μmol) and 1-hydroxybenzotriazole monohydrate (21.0 mg, 138 μmol) were dissolved in N,N-dimethylformamide (3 mL). Diisopropylethylamine (60 μL, 344 μmol) and 11-azido-3,6,9-trioxaundecan-1-amine (14 μL, 68.8 μmol) were dissolved in N,N-dimethylformamide (4 mL) and added. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (26.4 mg, 138 μmol) was added and stirred at room temperature for 18 hours. The solvent was removed by rotary evaporation, and the residue was purified by reverse-phase column chromatography (HO / CHCN, 0.1% TFA) to give compound 3 (50.4 mg, 62.9 μmol, 92%). 1 H NMR (500 MHz, methanol-d4): δ8.79 (d, J = 1.6 Hz, 1H), 8.28 (dd, J = 7.9 Hz, J = 1.6 Hz, 1H), 7.54 (d, J = 7.9 Hz, 1H), 7.13 (d, J = 9.5 Hz, 2H), 7.04 (dd, J = 9.5 Hz, J = 2.5 Hz, 2H), 6.99 (d, J = 2.5 Hz, 2H), 3.62-3.76 (m, 24H), 1.31 (t, J = 7.2 Hz, 12H). LRMS (ESI): found, 687.

[0084] Synthesis of compound 4 A suspension of puromycin dihydrochloride (150 mg, 276 μmol) in dichloromethane (25 mL) was stirred at 0°C. Triethylamine (300 μL, 2.76 mmol) was added dropwise to the suspension and stirred until the solution became clear. Di-tert-butyl dicarbonate (301 mg, 1.38 mmol) was gently added to the solution and stirred at room temperature for 4 hours. The solvent was removed using a rotary evaporator, and the residue was purified by normal-phase column chromatography (CHCl / MeOH) to obtain compound 4 (155 mg, 271 μmol, quant.). 1H NMR (500 MHz, DMSO-d6): δ 8.45 (s, 1H), 8.23 ​​(s, 1H), 7.99 (d, J = 7.7 Hz, 1H), 7.20 (d, J = 8.5 Hz, 2H), 6.87 (d, J = 8.4 Hz, 1H), 6.83 (d, J = 8.5 Hz, 2H), 6.06 (d, J = 4.8 Hz, 1H), 6.00 (d, J = 2.9 Hz, 1H), 5.16 (t, J = 5.7 Hz, 1H), 4.44-4.54 (m, 2H), 4.17-4.24 (m, 1H), 3.91-4.00 (m, 1H), 3.72 (s, 3H), 3.65-3.71 (m, 1H), 3.20-3.65 (m, 7H), 2.91 (dd, J = 4.5 Hz, 13.9 Hz, 1H), 2.66-2.75 (m, 1H), 1.30 (s, 9H). 13 C NMR (126 MHz, DMSO-d6): δ 172.1, 157.8, 155.2, 154.3, 151.9, 149.7, 137.9, 130.3, 129.9, 119.6, 113.4, 89.3, 83.4, 78.1, 73.1, 60.9, 56.0, 55.0, 50.3, 36.9, 28.1. HRMS (ESI): calcd for C 27 H 37 N7Na1O7[M + Na] + , 594.2652 , found, 594.2667 (+ 1.5 mmu).

[0085] Synthesis of compound 5 To a solution of compound 4 (142 mg, 248 μmol) in pyridine (3 mL), p-toluenesulfonic acid chloride (474 ​​mg, 2.48 mmol) was added and stirred at room temperature for 3 h. After removing the solvent using a rotary evaporator, the residue was purified by normal phase column chromatography (CHCl / MeOH) to give compound 5 (127 mg, 175 μmol, 70%). 1H NMR (500 MHz, CDCl3): δ8.19 (s, 1H), 7.73 (d, J = 1.9 Hz, 2H), 7.71 (s, 1H), 7.26 (d, J = 8.1 Hz, 2H), 7.10 (d, J = 8.5 Hz, 2H), 6.84 (d, J = 8.5 Hz, 2H), 6.64 (d, J = 5.1 Hz, 1H), 5.11 (d, J = 4.3 Hz, 1H), 4.15-4.49 (m, 6H), 3.17-4.00 (m, 9H), 2.86-3.07 (m, 2H), 2.41 (s, 3H), 2.04 (brs, 1H), 1.40 (s, 9H). 13 C NMR (126 MHz, CDCl3): δ172.3, 158.9, 155.6, 155.0, 151.9, 149.3, 145.2, 136.0, 132.7, 130.3, 130.0, 128.4, 128.1, 120.6, 114.4, 91.1, 82.1, 80.6, 74.2, 69.6, 56.3, 55.4, 53.4, 51.9, 38.0, 28.4, 21.8. HRMS (ESI): calcd for C 34 H 43 N7NaO9S [M + Na] + , 748.2741, found, 748.2778 (+ 3.7 mmu).

[0086] Synthesis of compound 6 A solution of compound 5 (28.8 mg, 39.7 μmol) in propargylamine (1.5 mL) was stirred at room temperature for 3 days. After removing the solvent using a rotary evaporator, the residue was purified by column chromatography. The resulting target product was dissolved in dichloromethane (1.5 mL) and stirred at 0°C. Trifluoroacetic acid (1.5 mL) was added dropwise to this solution, and the mixture was stirred at room temperature for 45 minutes. After removing the solvent using a rotary evaporator, the residue was purified by column chromatography (HO / CHCN, 0.1% TFA) to give compound 6 (9.3 mg, 12.6 μmol, 32%). 1H NMR (500 MHz, methanol-d4): δ 8.22 (s, 1H), 8.07 (s, 1H), 7.16 (dt, J = 8.7 Hz, 2.0 Hz, 2H), 6.87 (dt, J = 8.7 Hz, 2.0 Hz, 2H) 5.88 (d, J = 2.8 Hz, 1H), 4.73-4.76 (m, 1H), 4.63 (dd, J = 6.2 Hz, 2.9 Hz, 1H), 4.10-4.19 (m, 2H), 3.88-3.91 (m, 2H), 3.71 (s, 3H), 3.24-3.67 (m, 8H), 3.17 (t, J = 2.5 Hz, 1H), 2.96-3.10 (m, 2H). LRMS (ESI): found, 509.

[0087] Synthesis of probe 1 Compound 3 (4.6 mg, 5.7 μmol), compound 6 (3.0 mg, 4.1 μmol), tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (7.6 mg, 14 μmol), copper sulfate pentahydrate (11.9 mg, 47.5 μmol), and sodium ascorbate (9.4 mg, 47.5 μmol) were dissolved in purified water / acetonitrile / t-butyl alcohol (2 / 2 / 1) (2 mL) and stirred at room temperature for 19 h. The solution was diluted with purified water / acetonitrile (9 / 1) and purified by reverse-phase column chromatography (HO / CHCN, 100 mM triethylammonium acetate) and then further purified by reverse-phase column chromatography (HO / CHCN, 0.1% TFA) to give probe 1 (1.4 mg, 0.93 μmol, 23%). 1H NMR (500 MHz, methanol-d4): δ 8.72 (d, J = 1.7 Hz, 1H), 8.22 (dd, J = 1.9 Hz, 7.8 Hz, 1H), 8.13 (d, J = 3.2 Hz, 2H), 8.10 (s, 1H), 7.50 (d, J = 7.8 Hz, 1H), 7.25 (dt, J = 1.9 Hz, 8.5 Hz, 2H), 7.12 (d, J = 9.4 Hz, 1H), 7.02 (dd, J = 2.5 Hz, 9.4 Hz, 2H), 6.99 (d, J = 2.5 Hz, 2H), 6.96 (dt, J = 1.9 Hz, J = 8.5 Hz, 2H), 5.96 (d, J = 2.7 Hz, 1H), 4.89-4.92 (m, 1H), 4.68 (dd, J = 2.7 Hz, 6.3 Hz, 1H), 4.56-4.60 (m, 1H), 4.44 (d, J = 0.7 Hz, 2H), 4.23-4.29 (m, 2H), 3.87 (td, J = 1.2 Hz, 5.3Hz, 2H), 3.79 (s, 3H), 3.39-3.75 (m, 29 H), 3.05-3.19 (m, 2H), 1.31 (t, J = 6.8 Hz, 12 H). LRMS (ESI): found, 598.

[0088] Example 2 Synthesis of Yongguang プローブ2

[0089]

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[0090] Synthesis of compound 7 Compound 2 (30.2 mg, 50.3 μmol) and 1-hydroxybenzotriazole monohydrate (15.4 mg, 101 μmol) were dissolved in N,N-dimethylformamide (5 mL) and diisopropylethylamine (43 μL, 252 μmol) and propargylamine (3.2 μL, 50.3 μmol) were added. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (19.3 mg, 101 μmol) was added to this solution and stirred at room temperature for 18 hours. The solvent was removed by rotary evaporation, and the residue was purified by reverse-phase column chromatography (HO / CHCN, 0.1% TFA) to give compound 7 (11.8 mg, 18.5 μmol, 37%). 1 H NMR (500 MHz, methanol-d4): δ 8.79 (d, J = 1.6 Hz, 1H), 8.27 (dd, J = 7.9 Hz, J = 1.6 Hz, 1H), 7.54 (d, J = 7.9 Hz, 1H), 7.13 (d, J = 9.5 Hz, 2H), 7.04 (dd, J = 9.5 Hz, J = 2.5 Hz, 2H), 6.98 (d, J = 2.5 Hz, 2H), 4.25 (d, J = 2.5 Hz, 2H), 3.68 (q, J = 7.2 Hz, 8H), 2.67 (t, J = 2.5 Hz, 1 H), 1.31 (t, J = 7.2 Hz, 12H). LRMS (ESI): found, 524.

[0091] Synthesis of compound 8 Sodium azide (46 mg, 703 μmol) was gently added to a solution of compound 5 (102 mg, 141 μmol) in N,N-dimethylformamide (2 mL) and stirred at 90°C for 3 hours. After cooling to room temperature, the mixture was concentrated using a rotary evaporator and purified by column chromatography. A solution of the obtained target compound in dichloromethane (2 mL) was stirred at 0°C. Trifluoroacetic acid (2 mL) was added dropwise to this solution and stirred at room temperature for 30 minutes. After removing the solvent using a rotary evaporator, the residue was purified by reverse-phase column chromatography (HO / CHCN, 0.1% TFA) to give compound 8 (44.3 mg, 61.1 μmol, 43%). 1 H NMR (500 MHz, DMSO-d6): δ 8.48 (d, J = 7.5 Hz, 1H), 8.36 (s, 1H), 8.25 (s, 1H), 8.20 (m, 3H), 7.20 (dt, J = 2.0 Hz, 8.7 Hz, 2H), 6.91 (dt, J = 2.0 Hz, 8.7 Hz, 2H), 6.00 (d, J = 2.1 Hz, 1H), 4.62-4.70 (m, 2H), 4.09-4.17 (m, 1H), 3.82-3.88 (m, 1H), 3.74 (s, 3H), 3.11-3.71 (m, 7H), 2.92-3.04 (m, 2H). 13 C NMR (126 MHz, DMSO-d6): δ 168.9, 159.0, 158.6 (J C-F = 34 Hz), 154.6, 152.3, 150.2, 138.7, 131.1, 127.1, 120.1, 116.7 (J C-F = 294 Hz), 114.4, 90.1, 81.2, 73.0, 55.5, 53.9, 52.0, 52.0, 36.8. 22 H 29 N 10 O4[M-H-2CF3COO] + , 497.2373, found, 497.2349 (-2.4 mmu).

[0092] Synthesis of probe 2 Compound 7 (1.7 mg, 2.7 μmol), compound 8 (1.5 mg, 2.1 μmol), L-histidine (3.6 mg, 23 μmol), copper sulfate pentahydrate (6.2 mg, 25 μmol), and sodium ascorbate (4.9 mg, 25 μmol) were dissolved in purified water / acetonitrile / t-butyl alcohol (2 / 2 / 1) (2 mL) and stirred at room temperature for 20 h. The solution was diluted with purified water / acetonitrile (9 / 1) and purified by reverse-phase column chromatography (HO / CHCN, 100 mM TEAA) followed by reverse-phase column chromatography (HO / CHCN, 0.1% TFA) to give probe 2 (2.1 mg, 1.5 μmol, 74%). 1 H NMR (500 MHz, methanol-d4): δ8.77 (d, J = 1.7 Hz, 1H), 8.22-8.26 (m, 2H), 7.95 (s, 1H), 7.86 (s, 1H), 7.51 (d, J = 8.0 Hz, 1H), 7.29 (dt, J = 2.0 Hz, 8.7 Hz, 2H), 7.10 (dd, J = 1.1 Hz, 9.4 Hz, 1H), 7.02 (dd, J = 2.3 Hz, 9.4 Hz, 2H), 6.99 (d, J = 2.3 Hz, 2H), 6.94 (dt, J = 2.0 Hz, J = 8.7 Hz, 2H), 5.94 (d, J = 2.4 Hz, 1H), 4.80-4.84 (m, 2H), 4.58-4.70 (m, 5H), 4.18-4.24 (m, 2H), 3.73 (s, 3H), 3.68 (q, J = 7.2 Hz, 8H), 3.40-3.61 (m, 6H), 3.15 (d, J = 7.9 Hz, 2H), 1.31 (t, J = 7.2 Hz, 12H). LRMS (ESI): found, 511.

[0093] Example 3 Synthesis of Fluorescent Probe 3

[0094] [ka]

[0095] Synthesis of compound 9 Compound 2 (48.7 mg, 81.1 μmol) and 1-hydroxybenzotriazole monohydrate (24.8 mg, 162 μmol) were dissolved in N,N-dimethylformamide (8 mL) and diisopropylethylamine (69 μL, 405 μmol) and 6-azidohexan-1-amine (11.5 μL, 81.1 μmol) were added. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (31.1 mg, 162 μmol) was added to this solution and stirred at room temperature for 17 hours. The solvent was removed by rotary evaporation, and the residue was purified by reverse-phase column chromatography (HO / CHCN, 0.1% TFA) to give compound 9 (17.7 mg, 24.4 μmol, 30%). 1 H NMR (500 MHz, methanol-d4): δ8.77 (d, J = 1.6 Hz, 1H), 8.25 (dd, J = 7.9 Hz, J = 1.6 Hz, 1H), 7.53 (d, J = 7.9 Hz, 1H), 7.13 (d, J = 9.5 Hz, 2H), 7.04 (dd, J = 9.5 Hz, J = 2.5 Hz, 2H), 6.98 (d, J = 2.5 Hz, 2H), 3.68 (q, J = 7.2 Hz, 8H), 3.45-3.51 (m, 2H), 1.60-1.76 (m, 4 H), 1.45-1.54 (m, 4H), 1.31 (t, J = 7.2 Hz, 12H). LRMS (ESI): found, 611.

[0096] Synthesis of probe 3 Compound 9 (5.6 mg, 7.7 μmol), compound 6 (4.0 mg, 5.4 μmol), L-histidine (10.0 mg, 64 μmol), copper sulfate pentahydrate (16.0 mg, 64 μmol), and sodium ascorbate (12.7 mg, 64 μmol) were dissolved in purified water / acetonitrile / t-butyl alcohol (2 / 2 / 1) (2 mL) and stirred at room temperature for 19 h. The solution was diluted with purified water / acetonitrile (9 / 1) and purified by reverse-phase column chromatography (HO / CHCN, 100 mM TEAA) followed by reverse-phase column chromatography (HO / CHCN, 0.1% TFA) to give probe 3 (2.8 mg, 2.0 μmol, 36%). 1H NMR (500 MHz, methanol-d4): δ8.75 (d, J = 1.9 Hz, 1H), 8.24 (dd, J = 1.9 Hz, 7.9 Hz, 1H), 8.15 (s, 1H), 8.11 (s, 1H), 8.07 (s, 1H), 7.52 (d, J = 7.9 Hz, 1H), 7.25 (dt, J = 2.0 Hz, 8.7 Hz, 2H), 7.12 (d, J = 9.4 Hz, 1H), 7.02 (dd, J = 2.5 Hz, 9.4 Hz, 2H), 6.99 (d, J = 2.5 Hz, 2H), 6.97 (dt, J = 2.0 Hz, J = 8.7 Hz, 2H), 5.96 (d, J = 2.6 Hz, 1H), 4.89-4.94 (m, 1H), 4.68 (dd, J = 2.5 Hz, 6.3 Hz, 5H), 4.40-4.48 (m, 4H), 4.26 (tt, J = 2.0 Hz, 7.5 Hz, 2H), 3.80 (s, 3H), 3.68 (q, J = 7.2 Hz, 8H), 3.38-3.63 (m, 10H), 3.17 (dd, J = 7.0 Hz, 14.7 Hz, 1H), 3.08 (dd, J = 7.0 Hz, 14.7 Hz, 1H), 1.94 (quint, J = 7.4 Hz, 2H), 1.66 (quint, J = 7.4 Hz, 2H), 1.35-1.50 (m, 4H), 1.31 (t, J = 7.2 Hz, 12H). LRMS (ESI): found, 560.

[0097] Example 4 Synthesis of Yongguang プローブ4

[0098]

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[0099] Synthesis of compound 10 A solution of 2,2',4,4'-tetrahydroxybenzophenone (1.00 g, 4.06 mmol) and sodium acetate trihydrate (132 mg, 0.97 mmol) in purified water (12 mL) was stirred at 150 °C for 17 hours. After cooling to room temperature, the residue after filtration was washed with purified water. The residue was collected and dried to obtain compound 10 (897 mg, 3.93 mmol, 97%). 1 H NMR (500 MHz, DMSO-d6): δ 10.81 (brs, 2H), 7.98 (d, J = 8.7 Hz, 2H), 6.86 (dd, J = 8.7, 2.2 Hz, 2H), 6.82 (d, J = 2.2 Hz, 2H). 13 C NMR (126 MHz, DMSO-d6): δ 174.4, 163.8, 157.9, 128.2, 114.5, 114.1, 102.6. HRMS (ESI): calcd for C 13 H7O4[M - H] - , 227.0344, found 227.0341 (-0.3 mmu).

[0100] Synthesis of compound 11 Compound 10 (500 mg, 2.19 mmol) was dissolved in anhydrous dichloromethane (11 mL) and pyridine (1.7 mL, 21.9 mmol) was added to the solution while cooling with ice and stirred for 5 minutes. Trifluoromethanesulfonic anhydride (1.1 mL, 6.57 mmol) was added dropwise to the solution, and the mixture was stirred at room temperature under an argon atmosphere for 19 hours. Purified water was added to quench the reaction, and the organic phase was recovered by liquid separation and extracted. This organic phase was washed with 1 M hydrochloric acid and saturated brine, respectively. The organic phase was dried over sodium sulfate, filtered, and the solvent was removed using a rotary evaporator. The residue was recrystallized from a dichloromethane / hexane solution to obtain compound 11 (1.02 g, 2.07 mmol, 94%). 1H NMR (500 MHz, CDCl3): δ8.44 (d, J = 8.8 Hz, 2H), 7.49 (d, J = 2.3 Hz, 2H), 7.35 (dd, J = 2.3 Hz, 8.8 Hz, 2H). 13 C NMR (126 MHz, CDCl3): δ174.7, 156.7, 153.5, 129.8, 121.5, 118.8 (q, J C-F = 320 Hz), 118.3, 111.6.

[0101] Synthesis of compound 12 Compound 11 (500 mg, 1.02 mmol) was dissolved in dimethyl sulfoxide (3.0 mL) and diethylamine (1.9 mL, 20.3 mmol) was added, followed by stirring at 90°C for 15 hours. After cooling to room temperature, purified water was added, and the target product was extracted with dichloromethane and washed with saturated brine. After drying over sodium sulfate, the extract was filtered, and the solvent was removed using a rotary evaporator. The residue was purified by normal phase column chromatography (CHCl / MeOH) to give compound 12 (170 mg, 0.502 mmol, 49%). 1 H NMR (500 MHz, CDCl3): δ8.09 (d, J = 9.0 Hz, 2H), 6.64 (dd, J = 2.4 Hz, 9.0 Hz, 2H), 6.44 (d, J = 2.4 Hz, 2H), 3.44 (q, J = 7.1 Hz, 8H), 1.23 (t, J = 7.1 Hz, 12H). 13 C NMR (126 MHz, CDCl3): δ174.8, 158.5, 152.0, 127.8, 111.5, 108.5, 96.3, 44.7, 12.6. HRMS (ESI): calcd for C 21 H 17 N2O2[M + H] + , 339.2073, found, 339.2116 (+4.3 mmu).

[0102] Synthesis of compound 13 A solution of 4-bromo-3-methylbenzoic acid (96.2 mg, 447 μmol) in anhydrous tetrahydrofuran was cooled to −78°C and stirred under an argon atmosphere for 30 minutes. A 1.6 M n-butyllithium hexane solution (500 μL) was added dropwise to this solution, and the mixture was stirred for 1.5 hours. A solution of compound 12 (15.0 mg, 44.3 μmol) in anhydrous tetrahydrofuran was added to this solution, and the mixture was stirred at room temperature for 2 hours. The reaction was quenched by the addition of 2 M hydrochloric acid, and the solvent was removed using a rotary evaporator. The residue was purified by reverse-phase column chromatography (HO / CHCN, 100 mM TEAA) and then by reverse-phase column chromatography (HO / CHCN, 0.1% TFA) to give compound 13 (20.0 mg, 35.1 μmol, 79%). 1 H NMR (500 MHz, methanol-d4): δ 8.14 (s, 1H), 8.08 (d, J = 7.8 Hz, 1H), 7.38 (d, J = 7.8 Hz, 1H), 7.14 (d, J = 9.5 Hz, 2H), 7.08 (dd, J = 2.3 Hz, J = 9.5 Hz, 2H), 7.00 (d, J = 2.3 Hz, 2H), 3.69 (q, J = 7.0 Hz, 8H), 2.12 (s, 3H), 1.31 (t, J = 7.0 Hz, 12H). 13 C NMR (126 MHz, methanol-d4): δ 168.9, 159.4, 157.6, 157.3, 137.9, 137.7, 133.7, 132.8, 132.3, 130.5, 128.4, 115.8, 114.2, 97.4, 46.8, 19.5, 12.7. HRMS (ESI): calcd for C 29 H 33 N2O3[M] + , 457.2491, found, 457.2505 (+1.4 mmu).

[0103] Synthesis of compound 14 Compound 13 (19.4 mg, 34.0 μmol) and 1-hydroxybenzotriazole monohydrate (10.4 mg, 68.0 μmol) were dissolved in N,N-dimethylformamide (2 mL) and diisopropylethylamine (58 μL, 340 μmol) and propargylamine (3.3 μL, 51.0 μmol) were added. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (13.0 mg, 68.0 μmol) was added to the solution and stirred at room temperature for 17 hours. The solvent was removed by rotary evaporation, and the residue was purified by reverse-phase column chromatography (HO / CHCN, 0.1% TFA) to give compound 14 (18.2 mg, 30.0 μmol, 88%). 1 H NMR (500 MHz, methanol-d4): δ 7.98 (brs, 1H), 7.91 (dd, 1.2 Hz, J = 8.1 Hz, 1H), 7.39 (d, J = 8.1 Hz, 1H), 7.16 (d, J = 9.5 Hz, 2H), 7.08 (dd, J = 2.4 Hz, J = 9.5 Hz, 2H), 7.01 (d, J = 2.4 Hz, 2H), 4.22 (d, J = 2.5 Hz, 2H) 3.70 (q, J = 7.3 Hz, 8H), 2.65 (t, J = 2.5 Hz, 1H), 2.14 (s, 3H), 1.32 (t, J = 7.3 Hz, 12H). 13 C NMR (126 MHz, methanol-d4): δ168.8, 159.5, 157.7, 157.4, 138.1, 136.8, 136.7, 132.4, 130.7, 130.6, 126.2, 115.9, 114.4, 97.5, 80.6, 72.2, 46.9, 30.1, 19.7, 12.8. HRMS (ESI): calcd for C 32 H 36 N3O2[M-CF3COO] + , 494.2808, found, 494.2817 (+0.9 mmu).

[0104] Synthesis of probe 4 Compound 14 (4.0 mg, 6.6 μmol), compound 14 (4.4 mg, 7.2 μmol), L-histidine (10.2 mg, 66 μmol), copper sulfate pentahydrate (16.0 mg, 66 μmol), and sodium ascorbate (13.0 mg, 66 μmol) were dissolved in purified water / dimethyl sulfoxide (1 / 1) (2 mL) and stirred at room temperature for 19 h. The solution was diluted with purified water / acetonitrile (9 / 1) and purified by reverse-phase column chromatography (HO / CHCN, 0.1% TFA) to give probe 4 (8.0 mg, 6.0 μmol, 91%). 1 H NMR (500 MHz, methanol-d4): δ8.28 (s, 1H), 8.08 (s, 1H), 7.95 (brs, 1H), 7.89 (dd, J = 1.1 Hz, 8.1 Hz, 1H), 7.84 (s, 1H), 7.37 (d, J = 8.1 Hz, 1H), 7.29 (dt, J = 2.1 Hz, 8.7 Hz, 2H), 7.13 (d, J = 9.5 Hz, 2H), 7.06 (dd, J = 2.4 Hz, 9.5 Hz, 1H), 7.01 (d, J = 2.4 Hz, 2H), 6.94 (dt, J = 2.1 Hz, 8.7 Hz, 2H), 5.98 (d, J = 2.2 Hz, 1H), 4.81 (dd, J = 6.0 Hz, 8.7 Hz, 1H), 4.69 (dd, J = 2.2 Hz, 6.0 Hz, 1H), 4.57-4.67 (m, 4H), 4.19-4.25 (m, 2H), 3.40-3.80 (m, 17H), 3.15 (d, J = 7.8 Hz, 2H), 2.12 (s, 3H), 1.32 (t, J = 7.2 Hz, 12H). 13C NMR (126 MHz, methanol-d4):δ170.3, 169.2, 162.2, 160.9. 159.5, 157.6, 157.4, 153.6, 150.3, 150.1, 146.2, 140.4, 138.0, 137.0, 136.6, 132.3, 131.8, 130.7, 130.6, 127.4, 126.2, 125.7, 121.6, 115.8, 115.6, 114.3, 97.5, 92.5, 81.7, 74.3, 57.6, 57.5, 57.3, 55.7, 53.8, 52.5, 46.9, 37.9, 36.2, 19.7, 12.8. HRMS (ESI): calcd for C 54 H 64 N 13 O6 [M-2H-3CF3COO] + 990.5103 , found 990.5059 (- 4.4 mmu).

[0105] Example 5 Co-staining HeLa cells were seeded onto glass-bottom dishes (35 mm) at 50,000 cells / dish and incubated at 3°C ​​under 5% CO2 for 5 hours. After confirming cell adhesion, the cells were incubated with CellLight TM 25 μL of Mitochondria-GFP, BacMam 2.0 was added and cultured at 37°C, 5% CO2 for 24 hours. After culture, the dish was washed twice with PBS (1 mL), and medium (1 mL) and probe 4 (final concentration 3 μM) were added. After incubation at 37°C, 5% CO2 for 30 minutes, the dish was washed twice with PBS (1 mL), medium (1 mL) was added, and the cells were observed under a confocal fluorescence microscope (Green: ex. 488 nm, em. 475-625 nm; Red: ex. 555 nm, em. 550-750 nm).

[0106] The results are shown in Figures 2 and 3.

[0107] The intracellular locations of the fluorescence of probe 4 and the fluorescence of the mitochondrial staining reagent nearly coincided, indicating that probe 4 was localized in mitochondria.

[0108] Example 6: Functional evaluation of probes using a reconstituted cell-free protein synthesis system A reaction solution containing the dihydrofolate reductase (DHFR) gene as template DNA was prepared according to the protocol of a commercially available reconstituted cell-free protein synthesis kit (Purefrex 2.0). Probes 1–4 were also added to a final concentration of 10 μM. These were then incubated on an aluminum block at 37°C for 4 hours. After the reaction was completed, an equal volume (10 μL) of ultrapure water was added to the reaction solution. A 6x concentrated SDS-PAGE buffer containing a reducing agent (5 μL) was diluted with ultrapure water (5 μL) and the entire volume was added. The mixture was then incubated on an aluminum block at 95°C for 5 minutes to prepare the sample solution. Separately, probes 1–4 were diluted with ultrapure water (10 μL, final concentration 10 μM) to prepare a control containing only the probes without the reaction solution. This control was then supplemented with ultrapure water and 6x concentrated SDS-PAGE buffer containing a reducing agent (5 μL) to prepare the sample solution. These were electrophoresed on a 15% SDS-PAGE gel at 300 V for 25 minutes. After electrophoresis, the gel was observed using a gel imager (iBright, Thermo Fisher Scientific) and proteins were detected by fluorescence. The gel was then stained with CBB, destained, and observed.

[0109] The results are shown in Figures 4(A) and (B).

[0110] DHFR was expressed normally in the reaction solution to which DHFR DNA was added (lanes 3, 4, 6, 8, and 10). In the reaction solution to which probes 1 to 4 were added (lanes 4, 6, 8, and 10), fluorescence was observed in proteins of 19 kDa or less (the sum of the molecular weights of the probe and DHFR) (the molecular weight of full-length DHFR is 18 kDa). This indicates that probes 1 to 4 are recognized by ribosomes and bind to the elongating peptide chain.

Claims

1. A compound represented by the following formula (I) or a salt thereof: 【Chemical 1】 (In the formula, R 1 and R 2 are each independently hydrogen, optionally substituted C 1- C 6 Alkyl group, -OCH 3 , -OCF 3、 -COOH 、 -COOCH 3、 -COOC 2 H 5、 -CH 2 OH or halogen, R 3 is a monovalent substituent present on a benzene ring, and n is 0 to 3; R 5 and R 6 are independently hydrogen, C 1- C 6 Alkyl group or -NH 2 and R 7 and R 8 are independently hydrogen, C 1- C 6 Alkyl group or -NH 2 and R 9 and R 12 are each independently a hydrogen atom, a halogen atom, or C 1- C 6 is an alkyl group, R 10 and R 11 are each independently a hydrogen atom, a halogen atom, or C 1-6 is an alkyl group, R 5 and R 6 are both alkyl, R 5 The alkyl group represented by R 6 may be bonded to an alkyl group represented by R to form a 4- to 6-membered ring, and / or 7 and R 8 are all alkyl groups, R 7 The alkyl group represented by R 8 may be bonded to an alkyl group represented by the formula: R 5 and R 9 are all alkyl groups, R 5 The alkyl group represented by R 9 may be bonded to an alkyl group represented by R to form a 5- to 7-membered ring, and / or 6 and R 10 are all alkyl groups, R 6 The alkyl group represented by R 10 may be bonded to an alkyl group represented by the formula: R 7 and R 11 are all alkyl groups, R 7 The alkyl group represented by R 11 may be bonded to an alkyl group represented by R to form a 5- to 7-membered ring, and / or 8 and R 12 are all alkyl groups, R 8 The alkyl group represented by R 12 may be bonded to an alkyl group represented by the formula: X is oxygen, SiR 13 R 14 , GeR 13 R 14 , C.R. 13 R 14 , or POR 15 and R 13 and R 14 are each independently C 1- C 6 is an alkyl group or an aryl group, and R 15 is C 1- C 6 Alkyl group, C 2- C 6 Alkenyl group, C 2- C 6 an alkynyl group or an aryl group, L is a linker

2. 2. The compound or salt thereof according to claim 1, wherein L is a linker moiety represented by the following formula (II): (Z 1 ) m [X 1 ] s -Y k [X 2 ] t (Z 2 ) n (II) (In the formula, Z 1 are -O-, -S-, and -NR 18 -, -NR 18 -C(O)-, -C(O)NR 18 -, -OC(O)-, -C(O)-O-, phosphate group, or -OP(=O) (-CH 3 )(-O-), X 1 is C 1 -C 20 Alkylene, C 2 -C 20 Alkenylene, C 2 -C 20 Alkynylene, C 3 -C 8 Cycloalkylene, or (C 2 H 5 O) j (j is an integer from 1 to 10), Y is -O-, -S-, -NR 18 -, -NR 18 -C(O)-, -C(O)NR 18 -, -OC(O)-, or -C(O)-O-, a triazolyl group; X 2 is C 1 -C 20 Alkylene, C 2 -C 20 Alkenylene, C 2 -C 20 Alkynylene, C 3 -C 8 Cycloalkylene, or (C 2 H 5 O) j (j is an integer from 1 to 10), Z 2 -O-, -S-, -NR 18 -, -NR 18 -C(O)-, -C(O)NR 18 -, -OC(O)-, -C(O)-O-, phosphate group, or -OP(=O) (-CH 3 )(-O-), R 18 is hydrogen, C 1- C 6 alkyl or aryl, k, m, n, s, and t are each independently 0 or 1, provided that k+m+n+s+t is 1 or greater.

3. The compound represented by formula (III) and the compound represented by formula (IV) are reacted with each other by the reaction of R 16 and R 17 A method for producing the compound or salt thereof according to claim 1, comprising reacting the compound or salt thereof so that 【Chemistry 2】 (In formula (III), R 1 and R 2 One of the groups is hydrogen, optionally substituted C 1- C 6 Alkyl group, -OCH 3 , -OCF 3、 -COOH 、 -COOCH 3、 -COOC 2 H 5、 -CH 2 OH or halogen, R 3 is a monovalent substituent present on a benzene ring, and n is 0 to 3; R 5 and R 6 are independently hydrogen, C 1- C 6 Alkyl group or -NH 2 and R 7 and R 8 are independently hydrogen, C 1- C 6 Alkyl group or -NH 2 and R 9 and R 12 are each independently a hydrogen atom, a halogen atom, or C 1- C 6 is an alkyl group, R 10 and R 11 are each independently a hydrogen atom, a halogen atom, or C 1-6 is an alkyl group, R 5 and R 6 are both alkyl, R 5 The alkyl group represented by R 6 may be bonded to an alkyl group represented by R to form a 4- to 6-membered ring, and / or 7 and R 8 are all alkyl groups, R 7 The alkyl group represented by R 8 may be bonded to an alkyl group represented by the formula: R 5 and R 9 are all alkyl groups, R 5 The alkyl group represented by R 9 may be bonded to an alkyl group represented by R to form a 5- to 7-membered ring, and / or 6 and R 10 are all alkyl groups, R 6 The alkyl group represented by R 10 may be bonded to an alkyl group represented by the formula: R 7 and R 11 are all alkyl groups, R 7 The alkyl group represented by R 11 may be bonded to an alkyl group represented by R to form a 5- to 7-membered ring, and / or 8 and R 12 are all alkyl groups, R 8 The alkyl group represented by R 12 may be bonded to an alkyl group represented by the formula: X is oxygen, SiR 13 R 14 , GeR 13 R 14 , C.R. 13 R 14 , or POR 15 and R 13 and R 14 are each independently C 1- C 6 is an alkyl group or an aryl group, and R 15 is C 1- C 6 Alkyl group, C 2- C 6 Alkenyl group, C 2- C 6 an alkynyl group or an aryl group, R 16 is a reactive group.) 【Chemistry 3】 (In formula (IV), R 17 is a reactive group.)

4. A mitochondrial-targeted fluorescent probe comprising the compound according to claim 1 or 2 or a salt thereof.

5. The mitochondrial-targeted fluorescent probe according to claim 4, which is capable of detecting a protein.

6. The mitochondria-targeted fluorescent probe according to claim 4, which is capable of detecting protein synthesis in mitochondria.

7. The mitochondrial-targeted fluorescent probe according to claim 4, which is a fluorescent dye for bioimaging.

8. 1. A method for detecting proteins in mitochondria, comprising: Contacting a cell having mitochondria with the mitochondria-targeted fluorescent probe of claim 4; and detecting proteins bound by said mitochondria-targeted fluorescent probe in mitochondria within said cells. A method comprising:

9. 9. The method of claim 8, wherein the step of detecting the protein comprises detecting protein synthesis based on protein bands obtained by electrophoresis.

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