Fluorescent compound, method for producing the same, agent for detecting lipid peroxidation, and method for detecting lipid peroxidation

Abstract

To provide a fluorescent compound enabling selective detection of lipid peroxidation in cellular plasma membranes.SOLUTION: A fluorescent compound is represented by formula (1). R1 represents a linear aliphatic group having 4 to 22 carbon atoms, and R2 represents an aliphatic group having 1 to 3 carbon atoms, an aliphatic oxy group having 1 to 3 carbon atoms, an aliphatic thio group having 1 to 3 carbon atoms, or a heterocyclic group. L1 represents an alkylene group having 2 to 6 carbon atoms, and L2 represents an alkylene group having 1 to 6 carbon atoms. Ar represents an aromatic group, X represents a carboxyl group, a sulfonic acid group, or a phosphoric acid group. Y represents a trivalent linking group. i and j represent numbers of 0 to 2, and k represents a number of 2 to 4.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a fluorescent compound, a method for producing the same, an agent for detecting lipid peroxidation, and a method for detecting lipid peroxidation. [Background technology]

[0002] Ferroptosis, an intracellular iron-dependent cell death, is caused by the oxidation of lipids in biological membranes by reactive oxygen species, which accumulate as lipid peroxides. For example, Non-Patent Document 1 describes that lipid peroxidation in biological membranes can be detected using a specific fluorescent reagent. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Free Radic. Biol. Med., 2002, 33, 473-490. Summary of the Invention [Problem to be solved by the invention]

[0004] However, the fluorescent reagent described in Non-Patent Document 1 lacks biomembrane selectivity, making it difficult to clarify which biomembrane lipid peroxidation is involved in ferroptosis. One aspect of the present invention aims to provide a fluorescent compound that can selectively detect lipid peroxidation in cell plasma membranes. [Means for solving the problem]

[0005] Specific means for solving the above problems are as follows, and the present invention includes the following aspects. [1] A fluorescent compound represented by the following formula (1):

[0006] [ka]

[0007] In formula (1), R 1 represents a chain aliphatic group having 4 to 22 carbon atoms, and R 2 each independently represents an aliphatic group having 1 to 3 carbon atoms, an aliphatic oxy group having 1 to 3 carbon atoms, an aliphatic thio group having 1 to 3 carbon atoms, or a heterocyclic group; 2 may be linked to each other to form a ring, and L 1 represents an alkylene group having 2 to 6 carbon atoms, and L 2 represents an alkylene group having 1 to 6 carbon atoms, Ar represents an aromatic group, X represents a carboxy group, a sulfonic acid group, a phosphate group, or a salt thereof, and Y represents a trivalent linking group. i and j each independently represent a number from 0 to 2, and k represents a number from 2 to 4.

[0008] [2] The fluorescent compound according to [1], wherein Y in the formula (1) is a linking group represented by any one of the following:

[0009] [ka]

[0010] In the formula, R 3 represents an alkyl group having 1 to 3 carbon atoms, and * represents the bonding position.

[0011] [3] The fluorescent compound according to [1] or [2], which is represented by the following formula (1a):

[0012] [ka]

[0013] In formula (1a), R 1 represents a chain aliphatic group having 4 to 22 carbon atoms, and R 2a each independently represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; L 1 represents an alkylene group having 2 to 6 carbon atoms, and L 2 represents an alkylene group having 1 to 6 carbon atoms, Ar represents an aromatic group, and X represents a carboxy group, a sulfonic acid group, a phosphoric acid group, or a salt thereof.

[0014] [4] A lipid peroxidation detection agent comprising the fluorescent compound according to any one of [1] to [3].

[0015] [5] The detection agent according to [4], which detects lipid peroxidation in a cell plasma membrane.

[0016] [6] A method for detecting lipid peroxidation in a cell plasma membrane, comprising contacting a cell with the fluorescent compound according to any one of [1] to [3] and detecting a change in the wavelength of fluorescence emitted by the fluorescent compound.

[0017] [7] A method for producing a fluorescent compound represented by the following formula (4), comprising condensing an amine compound represented by the following formula (2) with a carboxylic acid derivative represented by the following formula (3):

[0018] [ka]

[0019] In the formula, R 1 represents a chain aliphatic group having 4 to 22 carbon atoms, and L 1 represents an alkylene group having 2 to 6 carbon atoms. 2 each independently represents an aliphatic group having 1 to 3 carbon atoms, an aliphatic oxy group having 1 to 3 carbon atoms, an aliphatic thio group having 1 to 3 carbon atoms, or a heterocyclic group, and two R 2 may be linked to each other to form a ring, and L 2 represents an alkylene group having 1 to 6 carbon atoms, Ar represents an aromatic group, X represents a carboxy group, a sulfonic acid group, a phosphate group or a derivative thereof, Z represents a hydroxy group, a halogen atom, an alkyloxy group having 1 to 3 carbon atoms or an N-succinimidoxy group, i and j each independently represent a number from 0 to 2, and k represents a number from 2 to 4. [Effects of the Invention]

[0020] According to one aspect of the present invention, a fluorescent compound capable of selectively detecting lipid peroxidation in cell plasma membranes can be provided. [Brief explanation of the drawings]

[0021] [Figure 1] FIG. 1 shows an example of detection of lipid peroxidation in the cell plasma membrane using ferroptosis-induced cells. DETAILED DESCRIPTION OF THE INVENTION

[0022] As used herein, the term "step" refers not only to an independent step, but also to a step that cannot be clearly distinguished from other steps, as long as the intended purpose of the step is achieved. Furthermore, the content of each component in a composition refers to the total amount of the multiple substances present in the composition, unless otherwise specified, when multiple substances corresponding to each component are present in the composition. Furthermore, the upper and lower limits of the numerical ranges described herein can be arbitrarily selected and combined from the numerical values ​​exemplified as numerical ranges. The following describes in detail embodiments of the present invention. However, the following embodiments are intended to embody the technical concept of the present invention, and are intended to exemplify fluorescent compounds and methods for producing the same, lipid peroxidation detection agents, and lipid peroxidation detection methods. The present invention is not limited to the fluorescent compounds and methods for producing the same, lipid peroxidation detection agents, and lipid peroxidation detection methods described below.

[0023] fluorescent compounds The fluorescent compound has a structure represented by the following formula (1). The fluorescent compound has excellent affinity with the cell plasma membrane (hereinafter simply referred to as "cell membrane"), which is the membrane surrounding the surface of the cell's protoplasm, among other biological membranes, and can specifically detect the presence of lipid peroxidation in the cell membrane. This is because, for example, the fluorescent compound 1 -YR 1This is thought to be because the fluorescent compound has a specific structure represented by the formula (hereinafter also referred to as the "membrane anchor site"), which gives it excellent affinity for biological membranes, particularly for cell plasma membranes. Here, lipid peroxidation refers to a phenomenon in which lipids constituting biological membranes are subjected to oxidative stress such as reactive oxygen species, resulting in the generation of lipid radicals, lipid peroxy radicals, etc. Relatively stable peroxides generated by lipid peroxidation are also called lipid peroxides. Note that lipid peroxy radicals generated by lipid peroxidation can become lipid peroxides through disproportionation. The fluorescent compound may be one that detects lipid peroxidation, or one that indirectly detects lipid peroxides.

[0024] [ka]

[0025] In formula (1), R 1 represents a chain aliphatic group having 4 to 22 carbon atoms. 2 each independently represents an aliphatic group having 1 to 3 carbon atoms, an aliphatic oxy group having 1 to 3 carbon atoms, an aliphatic thio group having 1 to 3 carbon atoms, or a heterocyclic group; 2 may be linked to each other to form a ring. 1 represents an alkylene group having 2 to 6 carbon atoms. 2 represents an alkylene group having 1 to 6 carbon atoms. Ar represents an aromatic group. X represents a carboxy group, a sulfonic acid group, a phosphate group, or a salt thereof. Y represents a trivalent linking group. i and j each independently represent a number from 0 to 2, and k represents a number from 2 to 4.

[0026] R 1 The chain aliphatic group represented by may be a straight chain or a branched chain, preferably a straight chain. The chain aliphatic group may be a saturated aliphatic group or an unsaturated aliphatic group, preferably a saturated aliphatic group. The number of carbon atoms in the chain aliphatic group may be, for example, 4 or more and 22 or less, preferably 6 or more, 8 or more, or 10 or more, and may be 20 or less, 18 or less, 16 or less, or 14 or less. In one embodiment, R 1The chain aliphatic group represented by the formula (R) may be a linear saturated aliphatic group having 8 to 16 carbon atoms. 1 When is a chain aliphatic group having a specified number of carbon atoms, the hydrophobic interaction with the hydrophobic groups of the phospholipids that make up the cell membrane becomes stronger, and the affinity for the cell membrane tends to be further improved.

[0027] The trivalent linking group represented by Y is L 1 , L 2 and R 1 The linking group represented by Y may be, for example, a linking group represented by any one of (Ya), (Yb), and (Yc) below, and is preferably a linking group represented by (Ya).

[0028] [ka]

[0029] In the formula, R 3 represents an alkyl group having 1 to 3 carbon atoms, and * represents the bonding position. 3 The alkyl group represented by the formula (I) may be a straight chain or a branched chain, and may preferably have one carbon atom.

[0030] L 1 The alkylene group represented by the formula (I) may be a linear or branched alkanediyl group. The number of carbon atoms in the alkylene group may be, for example, 2 or more and 6 or less, and preferably 2 or more and 4 or less.

[0031] The functional group represented by X may be an anionic functional group. The functional group represented by X may be a carboxy group, a sulfonic acid group, a phosphate group, or a salt thereof, preferably a sulfonic acid group or a salt thereof. When X is a salt of a carboxy group, a sulfonic acid group, or a phosphate group, examples of the cation constituting the salt include alkali metal ions, alkaline earth metal ions, and ammonium ions. When X is an anionic functional group, the interaction with the cationic groups of the phospholipids constituting the cell membrane becomes stronger, and the affinity to the cell membrane is further improved.

[0032] L 2 The alkylene group represented by the formula (I) may be a linear or branched alkanediyl group. The number of carbon atoms in the alkylene group may be, for example, 1 or more and 6 or less, preferably 1 or more and 4 or less, or 1 or more and 3 or less.

[0033] R 2 R each independently represents an aliphatic group having 1 to 3 carbon atoms, an aliphatic oxy group having 1 to 3 carbon atoms, an aliphatic thio group having 1 to 3 carbon atoms, an aliphatic amino group having 1 to 3 carbon atoms, or a heterocyclic group. 2 The aliphatic group, aliphatic oxy group, aliphatic thio group, aliphatic amino group, and heterocyclic group represented by the formula (I) may each further have a substituent. The aliphatic group may be an alkyl group or an alkenyl group, and the same applies to the aliphatic groups of the aliphatic oxy group, aliphatic thio group, and aliphatic amino group. Specific examples of the aliphatic group include a methyl group, an ethyl group, a propyl group, an isopropyl group, and a vinyl group. R 2 Examples of the heterocyclic group represented by the formula (I) include a pyrrolyl group, a furyl group, a pyridyl group, and a thienyl group.

[0034] Two adjacent R 2 may be linked to each other to form a fused ring structure. The fused ring formed may be any of an aliphatic ring, an aromatic hydrocarbon ring, and an aromatic heterocyclic ring. 2 and a part of a pyrrole ring, there can be mentioned pyrrole, thiophene, thienothiophene, furan, benzene, and the like.

[0035] The aromatic group represented by Ar may be an aromatic hydrocarbon group or an aromatic heterocyclic group, and examples of the aromatic group represented by Ar include a phenyl group, a naphthyl group, and a pyridyl group.

[0036] In one embodiment, the fluorescent compound may be a compound represented by the following formula (1a):

[0037] [ka]

[0038] In formula (1a), R 1 represents a chain aliphatic group having 4 to 22 carbon atoms. 2a Each of L independently represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms. 1 represents an alkylene group having 2 to 6 carbon atoms. 2 represents an alkylene group having 1 to 6 carbon atoms. Ar represents an aromatic group. X represents a carboxyl group, a sulfonic acid group, a phosphoric acid group, or a salt thereof.

[0039] R in Equation (1a) 1 , L 1 , L 2 The details of Ar and X are as follows: 1 , L 1 , L 2 , Ar and X are the same as above. 2a Examples of the alkyl group represented by the formula include a methyl group, an ethyl group, a propyl group, and an isopropyl group.

[0040] Specific examples of the fluorescent compound represented by formula (1) are shown below, but the present invention is not limited to these. In the following specific examples, Ph represents a phenyl group, and C 12 indicates a dodecyl group. Specific examples of the fluorescent compound represented by formula (1) include tautomers of the compounds shown below. .

[0041] [ka]

[0042] [ka]

[0043] The compound represented by formula (1) can be produced, for example, as follows: A fluorescent compound represented by formula (4) can be produced by condensing an amine compound represented by formula (2) with a carboxylic acid derivative represented by formula (3).

[0044] [ka]

[0045] In the formulas (2) to (4), R 1 represents a chain aliphatic group having 4 to 22 carbon atoms. 1 represents an alkylene group having 2 to 6 carbon atoms. 2 each independently represents an aliphatic group having 1 to 3 carbon atoms, an aliphatic oxy group having 1 to 3 carbon atoms, an aliphatic thio group having 1 to 3 carbon atoms, or a heterocyclic group; 2 may be linked to each other to form a ring. 2 represents an alkylene group having 1 to 6 carbon atoms. Ar represents an aromatic group. X represents a carboxy group, a sulfonic acid group, a phosphate group, or a derivative thereof. Z represents a hydroxy group, a halogen atom, an alkyloxy group having 1 to 3 carbon atoms, or an N-succinimidoxy group. i and j each independently represent a number from 0 to 2, and k represents a number from 2 to 4.

[0046] R in equations (2) to (4) 1 , R 2 , L 1 , L 2 The details of Ar and X are as follows: 1 , R 2 , L 1 , L 2 , Ar and X, respectively.

[0047] A commonly used condensation reaction can be applied to the condensation of the amine compound represented by formula (2) with the carboxylic acid derivative represented by formula (3). The condensation reaction can be appropriately selected depending on the type of Z. For example, when Z is a hydroxy group, a compound represented by formula (4) can be obtained using an appropriate condensation agent. Examples of condensation agents include dicyclohexylcarbodiimide (DCC), 1,3-diisopropylcarbodiimide (DIC), 1-(3-diethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU).

[0048] A base may be used in the condensation of the amine compound represented by formula (2) with the carboxylic acid derivative represented by formula (3). Examples of the base include organic bases such as triethylamine (TEA) and diisopropylethylamine (DIPEA).

[0049] Among the compounds represented by formula (1), a compound in which Y is represented by formula (Yb) can be produced by alkylating a compound in which Y is represented by formula (Ya) with an alkyl halide, etc. Furthermore, a compound in which Y is represented by formula (Yc) can be produced by utilizing a cycloaddition reaction between an azide and an alkyne (the so-called click reaction).

[0050] Lipid peroxidation detection agent The lipid peroxidation detection agent includes at least one fluorescent compound represented by formula (1). The fluorescent compound represented by formula (1) has a high affinity for the plasma membrane, one of the biological membranes that constitute cells, and is characterized by remaining in the plasma membrane upon contact with cells. Furthermore, the fluorescent compound represented by formula (1) is excited by light of 560 nm to 700 nm and emits red fluorescence with a peak emission wavelength in the wavelength range of 570 nm to 800 nm. Furthermore, when the fluorescent compound is oxidized and its molecular structure changes to an oxidized fluorescent compound, the peak emission wavelength shifts to a shorter wavelength, and the fluorescent compound emits green fluorescence with a peak emission wavelength in the wavelength range of 500 nm to 650 nm. Because the fluorescent compound is oxidized by radicals generated by lipid peroxidation, for example, and its structure changes to an oxidized fluorescent compound, the presence of lipid peroxidation in the cell plasma membrane can be detected by a change in the peak emission wavelength of the fluorescence.

[0051] The lipid peroxidation detection agent can be applied to cultured cells, cells derived from animal tissue, animal tissue, etc. to detect lipid peroxidation in cell plasma membranes. Animals subject to detection of lipid peroxidation include, for example, mammals and fish, and mammals include humans. Furthermore, subjects for detection of lipid peroxidation may also be non-human animals.

[0052] Lipid peroxidation detection kit In addition to the fluorescent compound represented by formula (1), the lipid peroxidation detection kit preferably further includes an instruction manual that describes how to contact cells with the fluorescent compound represented by formula (1) and detect lipid peroxidation by observing a change in the wavelength of the fluorescence emitted by the fluorescent compound. The instruction manual may be a document that is physically attached to the kit or a document that is provided online.

[0053] Methods for detecting lipid peroxidation The method for detecting lipid peroxidation in a cell plasma membrane includes a first step of contacting a cell with a fluorescent compound represented by formula (1), and a second step of detecting a change in the wavelength of fluorescence emitted by the fluorescent compound.

[0054] In the first step, cells are contacted with a fluorescent compound represented by formula (1). The fluorescent compound represented by formula (1) has excellent affinity for the cell plasma membrane, and therefore can be localized in the cell plasma membrane among the biological membranes that make up the cell. This allows for specific detection of lipid peroxidation in the cell plasma membrane.

[0055] The cells to be detected for lipid peroxidation are not particularly limited in terms of animal species, tissue of origin, etc., as long as they are animal cells having a cell membrane that can come into contact with a fluorescent compound. Specific examples of cells to be detected for lipid peroxidation include cultured cells such as established cell lines, cells derived from animal tissue immediately after collection from a living organism, and animal tissue itself. Animals to be detected for lipid peroxidation include, for example, mammals and fish, and mammals include humans. Furthermore, non-human animals may also be detected for lipid peroxidation.

[0056] An example of a method for contacting cells with a fluorescent compound is mixing live cells with a solution containing the fluorescent compound (hereinafter also referred to as a "detection reagent"). The detection reagent may be a solution in which the fluorescent compound is dissolved in a buffer solution, cell culture medium, or the like, appropriately selected depending on the cells to be detected. The concentration of the fluorescent compound in the detection reagent may be, for example, 0.1 μM or more and 5 μM or less, and preferably 1 μM or more and 2.5 μM or less.

[0057] In the second step, a change in the wavelength of the fluorescence emitted by the fluorescent compound is detected. When the fluorescent compound is oxidized by contact with radicals generated by lipid peroxidation, its structure changes to an oxidized fluorescent compound, and the peak wavelength of the fluorescence emission shifts to a shorter wavelength. By detecting the change in the wavelength of the fluorescence emitted by the fluorescent compound, the presence of lipid peroxidation in the cell plasma membrane can be detected. The change in the wavelength of the fluorescence emitted by the fluorescent compound may be detected by detecting a change in the peak wavelength of the fluorescence emission, or by visually observing the color tone of the fluorescence. Furthermore, the detection of the change in the wavelength of the fluorescence may be qualitative, quantitative, or semi-quantitative.

[0058] In other aspects, the present invention also includes the use of a fluorescent compound represented by formula (1) in the manufacture of a detection agent for lipid peroxidation, the use of a fluorescent compound represented by formula (1) in a method for detecting lipid peroxidation, and the fluorescent compound represented by formula (1) for use in a method for detecting lipid peroxidation. [Example]

[0059] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

[0060] Example 1: Method for producing fluorescent compounds

[0061] [ka]

[0062] Synthesis of compound 2 Compound 2 is a known substance and was synthesized as follows with reference to a paper (Tetrahedron Lett., 2016, 57, 979-982) (1). A flame-dried, nitrogen-purged recovery flask was charged with 2.2 mL (21 mmol, 2 eq) of dimethylpyrrole and 30 mL of dehydrated dichloromethane. 0.86 mL (7 mmol, 1 eq) of methyl 4-chloro-4-oxobutyrate was added dropwise and stirred at room temperature for 20 hours in the dark. The reaction was monitored by thin-layer chromatography (TLC). After the disappearance of the starting material was confirmed, 4.2 mL (30 mmol, 5 eq) of triethylamine was added dropwise. After stirring at room temperature for 15 minutes, 5.3 mL (6 eq) of boron trifluoride diethyl etherate was added dropwise and stirred at room temperature for 1 hour. Since the disappearance of the starting material could not be confirmed by TLC, the mixture was stirred for an additional hour, but no clear disappearance of the starting material was observed. However, since there was no significant change in the quantity of the product spot, the reaction was stopped and the solvent was concentrated. The mixture was then purified by silica gel column chromatography. 1 The formation of compound 2 was confirmed by H-NMR measurement. Yield: 760 mg (31%)

[0063] 1 H-NMR(400MHz,CDCl3)δ=6.07(s,2H,CH),3.74(s,3H,O-CH3),3.31(t,J=8.78Hz,2H,CH2),2.61(t,J=8.78Hz,2H,CH2),2.52(s,3H,CH3),2.44(s,3H,CH3).

[0064] Synthesis of compound 3 A flame-dried, nitrogen-purged recovery flask was charged with 500 mg (1.43 mmol, 1 eq) of compound 2, 87.5 μL (0.715 mmol, 0.5 eq) of trans-cinnamaldehyde, 1250 μL of piperidine, 1250 μL of acetic acid, 100 mL of acetonitrile, and molecular sieves, and the mixture was stirred at 65°C for 15 minutes. The reaction was monitored by TLC, and since no disappearance of the starting materials was observed, 0.5 equivalents of trans-cinnamaldehyde was added to further advance the reaction. After 15 minutes, TLC showed no disappearance of the starting materials, but the formation of a dimer was confirmed. Therefore, the reaction was stopped, filtered, and the solvent was concentrated. The mixture was then purified by silica gel column chromatography. 1 The formation of compound 3 was confirmed by H-NMR and ESI-MS measurements. Yield: 30 mg (6.0%)

[0065] 1 H-NMR(400MHz,CDCl3)δ=7.45(d,J=7.36Hz,2H,CH),7.34(t,J=7.47Hz,2H,CH),7. 28(m,1H,CH),7.19(d,J=14.5Hz,1H,CH),7.07(m,2H,CH),6.75(d,J=14.1Hz,1H,C H),6.64(s,1H,CH),6.10(s,1H,CH),3.75(s,3H,CH3),3.33(t,J=8.91Hz,2H,CH2) ,2.62(t,J=9.11Hz,2H,CH2),2.55(s,3H,CH3),2.49(s,3H,CH3),2.45(s,3H,CH3).

[0066] MS(ESI), m / z Calcd. for C 27 H 27BF2N2O2Na:471.20,Found for 471.16[M+Na] + .

[0067] Synthesis of compound 4 100 mg (0.22 mmol, 1 eq) of compound 3, sodium hydroxide (5.18 mmol, 24 eq), and 10 mL of tetrahydrofuran were added to a recovery flask and stirred at room temperature. While monitoring the reaction by TLC, a total of 47 equivalents of sodium hydroxide was added and stirred for 5 hours. After confirming the disappearance of the raw materials by TLC, the mixture was neutralized, separated, and the organic phase was concentrated. 1 The formation of compound 4 was confirmed by H-NMR and ESI-MS measurements. Yield: 98 mg (99%)

[0068] 1H-NMR(400MHz,CD3OD)δ=7.51(d,J=7.32Hz,2H,CH),7.35(t,J=7.32Hz,2H,CH),7 .24(t,J=7.32Hz,1H,CH),7.19(m,1H,CH),7.12(d,J=4.03Hz,1H,CH),7.07(d,J=1 0.5Hz,1H,CH),6.85(d,J=16.1Hz,1H,CH),6.78(s,1H,CH),6.16(s,1H,CH),2.55( s,3H,CH3),2.51(s,3H,CH3),2.48(s,3H,CH3),2.55(s,3H,CH3),2.49(s,3H,CH3).

[0069] MS(ESI), m / z Calcd. for C 25 H 24 BF2N2O2:433.19,Found for 433.20[M] - .

[0070] Synthesis of Compound 1 A flame-dried, nitrogen-purged recovery flask was charged with 50 mg (0.11 mmol, 1 eq) of compound 4, 61 mg (0.20 mmol, 1.8 eq) of compound 5, 5 mL of dehydrated dimethylformamide, 38 μL (0.22 mmol, 2 eq) of N,N-diisopropylethylamine, and 65 mg (0.17 mmol, 1.5 eq) of HATU, and the mixture was stirred at room temperature. The reaction was monitored by TLC, and after 1 hour, the disappearance of the raw materials was observed. The reaction was stopped, and the solvent was concentrated. The mixture was then purified by silica gel column chromatography. The formation of compound 1 was confirmed by 1H-NMR and ESI-HRMS. Yield: 11.5 mg (12.3%)

[0071] 1H-NMR(400MHz,CD3OD)δ=7.51(d,J=7.98Hz,2H,CH),7.33(t,J=7.66Hz,2H,CH),7.25(d,J=7.12Hz,1H,CH),7 .21(d,J=10.8Hz,1H,CH),7.13(d,J=7.12Hz,1H,CH),7.09(d,J=10.4Hz,1H,CH),6.89(d,J=15.1Hz,1H,CH),6 .80(s,1H,CH),6.18(s,1H,CH),3.48(t,J=7.34Hz,2H,CH2),3.38(t,J=7.77Hz,4H,CH2,CH2),2.77(m,4H,CH2 ,CH2),2.49(s,3H,CH3),2.48(s,3H,CH3),2.45(s,3H,CH3),2.01(m,2H,CH2),1.52(m,2H,CH2),1.28(s,18H).

[0072] HRMS(ESI), m / z Calcd. for C 40 H 55 BF2N3O4S: 722.39744, Found for 722.39687 [MH] - .

[0073] Example 2 Detection of lipid peroxidation in the plasma membrane using ferroptosis-induced cells To confirm whether ferroptosis is related to plasma membrane oxidation, we performed staining experiments using HepG2 cells treated with erastin, a known inducer of ferroptosis. Elastin inhibits the cystine transporter, suppressing the uptake of cystine, a precursor to the antioxidant glutathione, and reducing glutathione levels. As a result, reactive oxygen species induced by intracellular iron produce and accumulate lipid peroxides, which induces ferroptosis.

[0074] Ferroptosis was induced in HepG2 cells by adding 10 μM or 20 μM erastin and incubating for 24 hours. Compound 1 (also called "fluorescent probe 1"), prepared in HBSS at 1 μM, was added to the ferroptosis-induced HepG2 cells, and the cells were incubated for 5 minutes. The results are shown in Figure 1.

[0075] In non-treated cells, most of the plasma membrane showed red fluorescence, and green fluorescence was barely observed. On the other hand, in cells treated with erastin (10 μM or 20 μM), green fluorescence was observed in the plasma membrane. This indicates that fluorescent probe 1 was oxidized in the membrane.

[0076] This is thought to be due to the depletion of intracellular glutathione by erastin, which resulted in the formation of lipid peroxides in the plasma membrane, which then reacted with fluorescent probe 1. Although many reports have been published on the formation of lipid peroxides due to ferroptosis, it has not been identified which intracellular membranes are involved. Our results experimentally demonstrated that the plasma membrane is one of the membranes where lipid peroxidation occurs during the ferroptosis process.

Claims

1. A fluorescent compound represented by the following formula (1): 【Chemical 1】 (In formula (1), R １ represents a chain aliphatic group having 4 to 22 carbon atoms, R ２ each independently represents an aliphatic group having 1 to 3 carbon atoms, an aliphatic oxy group having 1 to 3 carbon atoms, an aliphatic thio group having 1 to 3 carbon atoms, or a heterocyclic group; ２ may be linked to each other to form a ring, and L １ represents an alkylene group having 2 to 6 carbon atoms; L ２ represents an alkylene group having 1 to 6 carbon atoms, Ar represents an aromatic group, X represents a carboxy group, a sulfonic acid group, a phosphate group or a salt thereof, and Y represents a trivalent linking group. i and j each independently represent a number from 0 to 2, and k represents a number from 2 to 4.

2. 2. The fluorescent compound according to claim 1, wherein Y in formula (1) is a linking group represented by any one of the following formulas: 【Chemistry 2】 (In the formula, R ３ represents an alkyl group having 1 to 3 carbon atoms, and * represents the bonding position.)

3. 2. The fluorescent compound according to claim 1, represented by the following formula (1a): 【Chemistry 3】 (In formula (1a), R １ represents a chain aliphatic group having 4 to 22 carbon atoms, R ２ａ each independently represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; L １ represents an alkylene group having 2 to 6 carbon atoms; L ２ represents an alkylene group having 1 to 6 carbon atoms, Ar represents an aromatic group, and X represents a carboxy group, a sulfonic acid group, a phosphoric acid group, or a salt thereof.

4. A detection agent for lipid peroxidation, comprising the fluorescent compound according to any one of claims 1 to 3.

5. The detection agent according to claim 4, which detects lipid peroxidation in a cell plasma membrane.

6. contacting a cell with a fluorescent compound according to any one of claims 1 to 3; and detecting a change in wavelength of the fluorescence emitted by the fluorescent compound.

7. A method for producing a fluorescent compound represented by the following formula (4), comprising condensing an amine compound represented by the following formula (2) with a carboxylic acid derivative represented by the following formula (3): 【Chemistry 4】 (In the formula, R １ represents a chain aliphatic group having 4 to 22 carbon atoms; L １ represents an alkylene group having 2 to 6 carbon atoms. ２ each independently represents an aliphatic group having 1 to 3 carbon atoms, an aliphatic oxy group having 1 to 3 carbon atoms, an aliphatic thio group having 1 to 3 carbon atoms, or a heterocyclic group; ２ may be linked to each other to form a ring, and L ２ represents an alkylene group having 1 to 6 carbon atoms, Ar represents an aromatic group, X represents a carboxy group, a sulfonic acid group, a phosphate group or a derivative thereof, Z represents a hydroxy group, a halogen atom, an alkyloxy group having 1 to 3 carbon atoms or an N-succinimidoxy group, i and j each independently represent a number from 0 to 2, and k represents a number from 2 to 4.

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