WO2015174460A1 - 酵素特異的な細胞内滞留性蛍光化合物 - Google Patents
酵素特異的な細胞内滞留性蛍光化合物 Download PDFInfo
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- WO2015174460A1 WO2015174460A1 PCT/JP2015/063789 JP2015063789W WO2015174460A1 WO 2015174460 A1 WO2015174460 A1 WO 2015174460A1 JP 2015063789 W JP2015063789 W JP 2015063789W WO 2015174460 A1 WO2015174460 A1 WO 2015174460A1
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- enzyme
- hmder
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- IBXBYBOBCYDWDX-UHFFFAOYSA-N CCN(CC)c1ccc(C(c2ccccc2CO)c(ccc(O)c2C3OCCCO3)c2O2)c2c1 Chemical compound CCN(CC)c1ccc(C(c2ccccc2CO)c(ccc(O)c2C3OCCCO3)c2O2)c2c1 IBXBYBOBCYDWDX-UHFFFAOYSA-N 0.000 description 1
- HMPPXWHIRQSHBE-UHFFFAOYSA-N CC[N+](CC)=C1C=C2Oc(c(C3OCCCO3)c(cc3)[O-])c3C(c3ccccc3CO)=C2C=C1 Chemical compound CC[N+](CC)=C1C=C2Oc(c(C3OCCCO3)c(cc3)[O-])c3C(c3ccccc3CO)=C2C=C1 HMPPXWHIRQSHBE-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
- C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/40—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving amylase
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)
- G01N2333/938—Hydrolases (3) acting on glycosyl compounds (3.2) acting on beta-galactose-glycoside bonds, e.g. beta-galactosidase
Definitions
- the present invention relates to a novel fluorescent compound that stays in a target cell and can specifically act on the cell, and a method for specifically imaging a target cell in which a specific enzyme is expressed using the compound,
- the present invention relates to a probe used for imaging, and a detection kit, detection agent, diagnostic agent or kit containing the probe. More specifically, the present invention relates to a fluorescent compound that selectively visualizes cells expressing a reporter enzyme such as ⁇ -galactosidase, and an imaging method, imaging probe, detection agent, diagnostic agent or kit using the same.
- Non-patent Document 1 ⁇ -galactosidase enzyme-specific imaging probes are molecular tools for elucidating the aging mechanism of cells. It is also important. Furthermore, since it has been shown that ⁇ -galactosidase activity is increased in certain types of cancer cells (Non-Patent Document 2 and Non-Patent Document 3), an imaging probe specific for ⁇ -galactosidase enzyme is selected for cancer cells. It can be used as a typical fluorescent imaging probe.
- Non-patent Document 4 a method for imaging enzyme activity using X-Gal as a substrate has been widely used.
- X-Gal cannot be applied to living cells, it can be applied to living cells.
- Development of an imaging probe is desired.
- imaging probes applicable to many living cells have been developed.
- HMDER- ⁇ Gal and the like have been developed as ⁇ -galactosidase fluorescent probes applicable to living cells and living tissues that can be excited by visible light by controlling the spirocyclization reaction in the molecule (Non-patent Documents). 5, Patent Document 1).
- the enzyme reaction product leaks from the cells, or because cytotoxic UV light is used as excitation light, live cells etc. are clearly imaged at the single cell level. It was difficult to do.
- conventional cancer probes have a problem that they cannot be used for pathological diagnosis because they leak out of the cell by immobilizing a section for pathological diagnosis.
- the problem to be solved by the present invention is to generate fluorescence specifically for an enzyme activity, and at the same time, stay in the cell having the enzyme without fixing or fixing the cell.
- a fluorescent imaging probe using the fluorescent compound a detection method using the fluorescent probe, a detection kit, or a detection agent.
- an enzyme substrate having a substituent that changes a fluorescent dye to a quinone methide after reaction with a reporter enzyme into a fluorescent dye having a xanthene ring as a fluorophore It was discovered that by optimizing the structure and reacting with a reporter enzyme such as ⁇ -galactosidase, a fluorescent imaging probe that exhibits fluorescence for the first time and exhibits excellent intracellular retention can be obtained. . Based on this finding, the present invention has been completed.
- the present invention provides an enzyme-specific retention fluorescent compound containing a compound represented by the following formula (I) or a salt thereof.
- A represents a monovalent group cleaved by an enzyme
- R 1 represents 1 to 4 identical or different substituents bonded to a hydrogen atom or a benzene ring
- R 3 , R 4 , R 5 and R 6 each independently represent —CFR 10 R 11 or —CF 2 R 12 , or a hydrogen atom, a hydroxyl group, an alkyl group, or a halogen atom
- R 2 and R 7 each independently represent a hydrogen atom
- R 8 and R 9 each independently represent a hydrogen atom or an alkyl group
- R 10 , R 11 and R 12 each independently represent a hydrogen atom, an alkyl group or an alkenyl group
- X is an oxygen atom, Se, CR 13 R 14, or represents SiR 15 R 16
- R 13, R 14, a hydrogen atom or a to R 15 and R 16 are each independently Represents Kill group
- Y represents a C 1 -C 3 alkylene group
- Z represents an oxygen
- the enzyme-specific retention fluorescent compound is represented by the following formula. (In the formula, A, R 1 to R 9 , X and Y are the same as in formula (I).)
- At least one of R 3 , R 4 , R 5 , and R 6 is —CFR 10 R 11 .
- At least one of R 3 , R 4 , R 5 , and R 6 is —CH 2 F.
- A is a group that is cleaved by a hydrolase containing a reporter enzyme or an enzyme that is specifically expressed or activated in cancer cells, and more preferably, A is a galactopyranosyl group.
- the reporter enzyme is ⁇ -galactosidase.
- the enzyme-specific retention fluorescent compound or a salt thereof is a compound represented by (Ia) to (Ic) or a salt thereof.
- the present invention relates to a fluorescent probe containing an enzyme-specific retentive fluorescent compound represented by the formula (I), (I ′) or (Ia) to (Ic).
- the present invention expresses a specific enzyme containing an enzyme-specific retentive fluorescent compound represented by formula (I), (I ′) or (Ia)-(Ic)
- the present invention relates to a composition or a kit for detecting or visualizing a target cell.
- the target cell is a ⁇ -galactosidase-expressing cell, and more preferably the target cell is a cancer cell.
- the present invention expresses a specific enzyme using an enzyme-specific retention fluorescent compound represented by the formula (I), (I ′) or (Ia) to (Ic)
- the present invention relates to a method for detecting a target cell.
- an enzyme-specific retention fluorescent compound and an enzyme that is specifically expressed in the target cell are contacted in vitro or in vivo to detect a target cell in which the specific enzyme is expressed.
- the method includes a step of bringing an enzyme-specific retention fluorescent compound and an enzyme specifically expressed in the target cell into contact in vitro or in vivo, and a step of generating fluorescence by performing excitation light irradiation.
- the present invention relates to a method for detecting a target cell in which the specific enzyme is expressed. More preferably, in the method, the target cell is a ⁇ -galactosidase-expressing cell, and more preferably the target cell is a cancer cell.
- the present invention relates to a compound represented by the following formula (II), which is used for producing the formula (I).
- R 3 , R 4 , R 5 , and R 6 each independently represent —C ( ⁇ O) H, a hydrogen atom, a hydroxyl group, an alkyl group, or a halogen atom
- R 2 and R 7 each represent Independently represents a hydrogen atom, a hydroxyl group, an alkyl group, or a halogen atom
- R 8 and R 9 each independently represent a hydrogen atom or an alkyl group
- X represents an oxygen atom or Se, CR 13 R 14 , or SiR 15 R
- 16 represents;
- R 13, R 14, R 15 and R 16 each independently represent a hydrogen atom or an alkyl group
- Y represents a C 1 -C 3 alkylene group, wherein, R 3, R 4, R 5 And at least one of R 6 represents —C ( ⁇ O) H.
- the compound of the formula (II) is a compound represented by the following formula (IIa) or (IIb).
- the enzyme-specific retentive fluorescent compound of the present invention changes the visible light absorption by an enzyme reaction, and at the same time uses the produced quinone methide to covalently bond to a protein that coexists in the cell, thereby providing excellent intracellular retentivity. Indicates. As a result of the combination of these effects, it becomes possible to visualize target cells expressing the enzyme in a detailed level such as a single cell level in a live cell state or in a fixed state.
- the enzyme-specific retention fluorescent compound of the present invention can be used as a molecular tool for elucidating the aging mechanism of cells, and can also be used as a selective fluorescent imaging probe in certain types of cancer cells.
- the imaging technique using the enzyme-specific retention fluorescent compound of the present invention can be performed with a microscope capable of normal cell imaging, and does not require any special equipment.
- fluorescence imaging at the single cell level is possible, it is possible to track changes in individual cells over time, and surgically without leaving cancer tissue using cancer cell selective fluorescence imaging It can also be excised.
- the industrial utility value and economic effect of the enzyme-specific retention fluorescent compound of the present invention are considered to be extremely large.
- FIG. 1 shows the intensity of fluorescence (a) generated by an enzyme reaction between 2-CHF 2 -HMDER- ⁇ Gal, which is an enzyme-specific retention fluorescent compound of the present invention, and ⁇ -galactosidase (a), change in absorption spectrum (b), It is the figure which showed the fluorescence spectrum (c).
- FIG. 2 shows the intensity (a) of fluorescence generated by the enzymatic reaction of 4-CHF 2 -HMDER- ⁇ Gal, which is the enzyme-specific retention fluorescent compound of the present invention, with ⁇ -galactosidase, change in absorption spectrum (b), It is the figure which showed the fluorescence spectrum (c).
- FIG. 1 shows the intensity of fluorescence (a) generated by an enzyme reaction between 2-CHF 2 -HMDER- ⁇ Gal, which is an enzyme-specific retention fluorescent compound of the present invention, and ⁇ -galactosidase (a), change in absorption spectrum (b), It is the figure which showed the fluorescence spectrum (c).
- FIG. 3 shows the fluorescence intensity (a), the change in absorption spectrum (b) generated by the enzymatic reaction between 4-CH 2 F-HMDER- ⁇ Gal, which is the enzyme-specific retention fluorescent compound of the present invention, and ⁇ -galactosidase, It is the figure which showed the fluorescence spectrum (c).
- FIG. 4 shows the enzyme-specific retention fluorescent compounds of the present invention, 2-CHF 2 -HMDER- ⁇ Gal, 4-CHF 2 -HMDER- ⁇ Gal, and 4-CH 2 F-HMDER- ⁇ Gal, and ⁇ -galactosidase. It is the figure which showed that the protein BSA which coexists in a solution can be fluorescence-labeled by making it react with an enzyme.
- (A) A fluorescence image obtained when an SDS-PAGE gel was excited with excitation light having a wavelength of 488 nm.
- Lane 1 2.5 ⁇ M 4-CH 2 F-HMDER- ⁇ Gal and 0.5 mg / mL BSA
- Lane 2 2.5 ⁇ M 4-CH 2 F-HMDER- ⁇ Gal, 0.5 mg / mL BSA and 5U ⁇ -galactosidase
- Lane 3 2.5 ⁇ M 4-CHF 2 -HMDER- ⁇ Gal, 0.5 mg / mL BSA and 5U ⁇ -galactosidase
- Lane 4 2.5 ⁇ M 2-CHF 2 -HMDER- ⁇ Gal, 0.5 mg / mL BSA And 5U ⁇ -galactosidase
- lane 5 2.5 ⁇ M HMDER- ⁇ Gal, 0.5 mg / mL BSA and 5U ⁇ -galactosidase.
- FIG. 5 shows that the enzyme-specific retention fluorescent compounds of the present invention, 2-CHF 2 -HMDER- ⁇ Gal, 4-CHF 2 -HMDER- ⁇ Gal, and 4-CH 2 F-HMDER- ⁇ Gal, inhibit intracellular proteins. It is a figure which shows carrying out fluorescence labeling specific to enzyme activity.
- A A fluorescence image obtained when an SDS-PAGE gel was excited with excitation light having a wavelength of 488 nm.
- Lane 1 2.5 ⁇ M 4-CH 2 F-HMDER- ⁇ Gal and 20 ⁇ L 1.5 mg / mL HEK cell lysate
- Lane 2 2.5 ⁇ M 4-CH 2 F-HMDER- ⁇ Gal and 20 ⁇ L 1.5 mg / mL HEK- lacZ cell lysate
- lane 3 2.5 ⁇ M 4-CHF 2 -HMDER- ⁇ Gal and 20 ⁇ L 1.5 mg / mL HEK-lacZ cell lysate
- lane 4 2.5 ⁇ M 2-CHF 2 -HMDER- ⁇ Gal and 20 ⁇ L 5 mg / mL HEK-lacZ cell lysate
- lane 5 2.5 ⁇ M HMDER- ⁇ Gal and 20 ⁇ L 1.5 mg / mL HEK-lacZ cell lysate.
- FIG. 6 is a diagram showing that 4-CH 2 F-HMDER- ⁇ Gal, which is an enzyme-specific retention fluorescent compound of the present invention, can be used for live cell fluorescence imaging at the single cell level.
- FIG. 7 is a diagram showing that 4-CH 2 F-HMDER- ⁇ Gal, which is an enzyme-specific retention fluorescent compound of the present invention, has excellent intracellular retention.
- FIG. 8 shows that by using 4-CH 2 F-HMDER- ⁇ Gal, which is an enzyme-specific retention fluorescent compound of the present invention, fluorescence imaging similar to that of living cells can be performed even in a sample subjected to fixation treatment.
- FIG. 9 shows that cells having different enzyme activities can be detected or distinguished using flow cytometry by utilizing 4-CH 2 F-HMDER- ⁇ Gal, which is an enzyme-specific retention fluorescent compound of the present invention.
- FIG. (A) Flow cytometry results of HEK cells, HEK-LacZ cells, and mixtures thereof reacted with 4-CH 2 F-HMDER- ⁇ Gal.
- (B) Flow cytometry results of HEK cells, HEK-LacZ cells, and mixtures thereof reacted with HMDER- ⁇ Gal.
- FIG. 10 is a diagram showing that 4-CH 2 F-HMDER- ⁇ Gal, which is an enzyme-specific retention fluorescent compound of the present invention, can be applied to fluorescence imaging of living tissues.
- FIG. 11 is a diagram showing that 4-CH 2 F-HMDER- ⁇ Gal, which is an enzyme-specific retention fluorescent compound of the present invention, can be applied to single-cell fluorescence imaging.
- A Image of Drosophila (esg-lacZ) reacted with 4-CH 2 F-HMDER- ⁇ Gal. From left, fluorescence image and bright field image.
- FIG. 12 is a fluorescence spectrum image showing that 4-CH 2 F-HMDER- ⁇ Gal, which is an enzyme-specific retention fluorescent compound of the present invention, can be applied to cancer site-selective fluorescence imaging.
- White arrows indicate tumor location. Unmixed is an autofluorescence and fluorescence spectrum separated by a fluorescence spectrum.
- FIG. 13 is a diagram showing that 4-CH 2 F-HMDER- ⁇ Gal, which is an enzyme-specific retention fluorescent compound of the present invention, can be applied to fluorescence imaging of unfixed cells in a living body.
- FIG. 14 shows that the enzyme-specific retention fluorescent compound of the present invention, 4-CH 2 F-HMDER- ⁇ Gal, can be applied to single-cell fluorescence imaging of ⁇ -galactosidase activity that is randomly expressed in living tissue.
- FIG. 12 is a fluorescence spectrum image showing that 4-CH 2 F-HMDER- ⁇ Gal, which is an enzyme-specific retention fluorescent compound of the present invention, can be applied to cancer site-
- the alkyl group or alkenyl group may be any of an alkyl group or alkenyl group composed of linear, branched, cyclic, or a combination thereof.
- the number of carbon atoms of the alkyl group or alkenyl group is not particularly limited, but is, for example, about 1 to 6 carbon atoms, preferably about 1 to 4 carbon atoms, and more preferably about 1 or 2 carbon atoms.
- the alkyl group or alkenyl group may have one or more arbitrary substituents.
- substituents examples include an alkoxy group, a halogen atom (which may be a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom), an amino group, a mono- or di-substituted amino group, a substituted silyl group, and an acyl group.
- a group, an aryl group, and the like can be mentioned, but are not limited thereto.
- the alkyl group or alkenyl group has two or more substituents, they may be the same or different. The same applies to the alkyl part or alkenyl part of other substituents containing an alkyl part or alkenyl part (for example, an alkyloxy group or an aralkyl group).
- Enzyme-specific retention fluorescent compound is, in one embodiment, a compound having a structure represented by the following general formula (I).
- R 1 represents a hydrogen atom or 1 to 4 substituents bonded to a benzene ring.
- substituents include, but are not limited to, an alkyl group, an alkoxy group, a halogen atom, an amino group, a mono- or di-substituted amino group, a substituted silyl group, and an acyl group.
- R 1 is more preferably a hydrogen atom, a lower alkyl group or a lower alkoxy group.
- a hydrogen atom is particularly preferred.
- R 3 , R 4 , R 5 , and R 6 each independently represent —CFR 10 R 11 or —CF 2 R 12 , or represent a hydrogen atom, a hydroxyl group, an alkyl group, or a halogen atom, and R 10 , R 11 And R 12 each independently represents a hydrogen atom, an alkyl group or an alkenyl group, and further, at least one of R 3 , R 4 , R 5 , and R 6 is —CFR 10 R 11 or —CF 2 R 12 is represented. At least one of R 3 , R 4 , R 5 , and R 6 is preferably —CFR 10 R 11 . More preferably, at least one of R 3 , R 4 , R 5 , and R 6 is —CH 2 F.
- R 2 and R 7 each independently represent a hydrogen atom, a hydroxyl group, an alkyl group, or a halogen atom.
- R 2 and R 7 are preferably hydrogen atoms.
- R 8 and R 9 each independently represent a hydrogen atom or an alkyl group. When R 8 and R 9 both represent an alkyl group, they may be the same or different.
- R 8 and R 9 are each independently preferably a methyl group or an ethyl group, and more preferably R 8 and R 9 are both ethyl groups.
- X represents an oxygen atom, Se, CR 13 R 14 , or SiR 15 R 16 .
- R 13 , R 14 , R 15 and R 16 each independently represent a hydrogen atom or an alkyl group. Of these, an oxygen atom is preferred.
- the alkylene group may be a linear alkylene group or a branched alkylene group.
- a methylene group (—CH 2 —)
- an ethylene group (—CH 2 —CH 2 —)
- a propylene group (—CH 2 —CH 2 —CH 2 —)
- a branched alkylene group such as —CH ( CH 3 ) —, —CH 2 —CH (CH 3 ) —, —CH (CH 2 CH 3 ) — and the like can also be used.
- a methylene group or an ethylene group is preferable, and a methylene group is more preferable.
- Z represents an oxygen atom or NR 17
- R 17 represents a hydrogen atom or an alkyl group. Of these, an oxygen atom is preferred.
- the group A represents a monovalent group that is cleaved by an enzyme.
- a ⁇ -galactopyranosyl group an ⁇ -mannosyl group, a ⁇ -N-acetylglucosamyl group, a ⁇ -lactam ring
- examples thereof include, but are not limited to, phosphate esters, aminophenoxy groups, hydroxyphenoxy groups, and ⁇ -glutamic acid.
- Examples of the enzyme for cleaving A include a reductase, an oxidase, or a hydrolase.
- a reporter enzyme or an enzyme that is specifically expressed or activated in cancer cells more specifically, , ⁇ -galactosidase, ⁇ -lactamase, ⁇ -mannosidase, esterase, alkaline phosphatase, luciferase, peroxidase, cytochrome P450 oxidase, ⁇ -glucosidase, ⁇ -glucuronidase, ⁇ -hexosaminidase, lactase, ⁇ -glutamyltransferase, etc. It can be mentioned, but is not limited to these.
- ⁇ -galactosidase Preferred are ⁇ -galactosidase, ⁇ -lactamase, alkaline phosphatase, luciferase, ⁇ -hexosaminidase, peroxidase, or ⁇ -glutamyltransferase. Most preferred is ⁇ -galactosidase.
- the compound represented by the above formula (I) may exist as a salt.
- the salt include base addition salts, acid addition salts, amino acid salts and the like.
- the base addition salt include metal salts such as sodium salt, potassium salt, calcium salt, magnesium salt, ammonium salt, or organic amine salts such as triethylamine salt, piperidine salt, morpholine salt, and acid addition salt.
- Examples thereof include mineral acid salts such as hydrochloride, sulfate, and nitrate, and organic acid salts such as methanesulfonate, paratoluenesulfonate, citrate, and oxalate.
- Examples of amino acid salts include glycine salts.
- the salt of the compound of the present invention is not limited to these.
- the compound represented by the formula (I) may have one or more asymmetric carbons depending on the type of substituent, and there are stereoisomers such as optical isomers or diastereoisomers. There is a case. Pure forms of stereoisomers, any mixture of stereoisomers, racemates, and the like are all within the scope of the present invention.
- the compound represented by the formula (I) or a salt thereof may exist as a hydrate or a solvate, and any of these substances is included in the scope of the present invention.
- solvents such as ethanol, acetone, isopropanol, can be illustrated.
- cleavage of the group A and the opening of the spiro ring are caused by ⁇ -galactosidase as follows, and the fluorescent compound (III) covalently bound to the intracellular protein is produced.
- the fluorescent compound (III) covalently bound to the intracellular protein is produced. Details of the mechanism by which a compound similar to formula (III) emits fluorescence are known to those skilled in the art as described in WO 2005/024049.
- the compound represented by the general formula (I) or a salt thereof hardly emits fluorescence when irradiated with excitation light of, for example, about 440 to 550 nm in the neutral region, but the ring-opened compound produced by the enzyme activity is It has the property of emitting extremely strong fluorescence under the same conditions. Therefore, when the cell that has taken in the enzyme-specific retention fluorescent compound represented by formula (I) does not express an enzyme capable of cleaving the group represented by A, it is represented by formula (III). Such a ring-opening compound is not produced, and no fluorescent substance is produced in the cells.
- the compound represented by the formula (I) of the present invention enables detailed visualization at the single cell level without fixing the cells or after the fixing treatment.
- it has a wide range of uses such as a diagnostic agent and a diagnostic agent used for rapid pathological examination in a surgical field such as cancer.
- the intracellular retentive fluorescent compound of the present invention is selected from the cells of target cells expressing a specific enzyme. It can be used for a method of specific visualization. Specifically, the step of contacting the enzyme-specific retention fluorescent compound of formula (I) with an enzyme such as ⁇ -galactosidase that is specifically expressed in the target cell, and then fluorescence generated by irradiation with excitation light By performing the step of detecting, only target cells expressing ⁇ -galactosidase or the like can be specifically visualized.
- the intracellular retention fluorescent compound of the present invention As a means for bringing the intracellular retention fluorescent compound of the present invention into contact with an enzyme that is specifically expressed in a target cell, typically, a sample containing a solution containing the enzyme specific retention fluorescent compound is added, applied, or Although spraying is mentioned, it can select suitably according to the use.
- a sample containing a solution containing the enzyme specific retention fluorescent compound is added, applied, or Although spraying is mentioned, it can select suitably according to the use.
- the intracellular retention fluorescent compound of the present invention is applied to diagnosis or diagnosis assistance in an animal individual, or detection of a specific cell or tissue, the compound and an enzyme expressed in a target cell or tissue
- the means for contacting is not particularly limited, and for example, administration means common in the art such as intravenous administration can be used.
- the light irradiation performed on the target cell can be performed by irradiating the cell with light directly or via a waveguide (such as an optical fiber).
- a waveguide such as an optical fiber.
- any light source can be used as long as it can irradiate light including the absorption wavelength of the enzyme-specific stagnant fluorescent compound of the present invention after being subjected to enzyme cleavage. It may be appropriately selected depending on the environment to be implemented.
- the compound represented by the above general formula (I) or a salt thereof may be used as it is, but if necessary, an additive usually used for preparing a reagent may be used. You may mix
- additives such as a solubilizer, pH adjuster, buffer, and isotonic agent can be used as an additive for using the reagent in a physiological environment. Is possible.
- These compositions are generally provided as a composition in an appropriate form such as a mixture in powder form, a lyophilized product, a granule, a tablet, or a liquid, but distilled water for injection or an appropriate buffer at the time of use. It can be dissolved and applied in
- Purification by medium pressure column chromatography was performed using YFLC-AI580 (Yamazen Co., Ltd.). NMR measurements were performed using an AVANCE III 400 Nanobay (Bruker, Co. Ltd.).
- THF Tetrahydrofuran
- DMF N, N-dimethylformamide
- TFA Trifluoroacetic acid
- DAST N, N-diethylaminosulfur trifluoride
- PLC Thin layer plate for preparative
- MeOH (10 mL) was added under ice cooling to quench the reaction and the solvent was removed by evaporation.
- MeOH (28 mL) was added to the resulting residue to dissolve, and NaOMe (1.38 g, 25.5 mmol) was added and stirred at room temperature for 30 minutes.
- the solvent was removed by evaporation, CH 2 Cl 2 was added to the residue, celite filtration was performed, and the filtrate was concentrated by evaporation.
- leuco-4-CHO-HMDER 134 mg, 0.332 mmol
- anhydrous DMF 3 mL
- 2,3,4,6-tetra-O-acetyl- ⁇ -D-galactopyranoylyl bromide 705 mg, 1. 71 mmol
- Cs 2 CO 3 970 mg, 2.98 mmol
- Na 2 SO 4 390 mg, 2.75 mmol
- the solvent was removed by evaporation, CH 2 Cl 2 and saturated NH 4 Cl aqueous solution were added, liquid separation operation was performed 3 times, Na 2 SO 4 was added to the organic phase and dried, Celite filtration was performed, and the filtrate was evaporated.
- leuco-4-CHO-HMDER (249 mg, 0.616 mmol) was added to anhydrous DMF (6 mL), 2,3,4,6-tetra-O-acetyl- ⁇ -D-galactopyranosyl bromide (550 mg, 1. 34 mmol), Cs 2 CO 3 (1.50 g, 4.60 mmol) and Na 2 SO 4 (400 mg, 2.82 mmol) were added, and the mixture was stirred at room temperature for 2.5 hours.
- Ac 2 O 100 ⁇ L, 108 mg, 1.06 mmol was added to the reaction solution, and the mixture was further stirred at room temperature for 1 hour.
- the enzyme-specific retention fluorescent compound of the present invention has excellent properties as a fluorescent imaging probe.
- Fluorescence emission specific for enzyme reaction with ⁇ -galactosidase 2-CHF 2 -HMDER- ⁇ Gal, 4-CHF 2 -HMDER- ⁇ Gal and 4-CH 2 F-HMDER- ⁇ Gal are specifically treated with ⁇ -galactosidase. It was confirmed that fluorescence was generated.
- the enzyme-specific retention fluorescent compound of the present invention 2-CHF 2 -HMDER- ⁇ Gal, 4-CHF 2 -HMDER- ⁇ Gal or 4-CH 2 F-HMDER- ⁇ Gal, is reacted with ⁇ -galactosidase for 30 minutes. Changes in absorption spectrum and fluorescence spectrum (excitation wavelength: 550 nm) caused by the above were measured in the presence of 200 mM sodium phosphate buffer (pH 7.4). The measurement was performed using Shimadzu UV-2450 (Shimadzu Corp.) and Hitachi F-7000 (Hitachi Corp.).
- HEK cells that express ⁇ -galactosidase (HEK-lacZ cells) and normal HEK cells were used.
- HEK-lacZ cells HEK cells that express ⁇ -galactosidase
- normal HEK cells were used.
- 2.5 ⁇ M 4-CH 2 F-HMDER- ⁇ Gal and 20 ⁇ L 1.5 mg / mL HEK cell lysate (2) 2.5 ⁇ M 4-CH 2 F-HMDER- ⁇ Gal and 20 ⁇ L 1.5 mg / mL HEK-lacZ Cell Lysate (3)
- 2.5 ⁇ M 2-CHF 2 -HMDER- ⁇ Gal and 20 ⁇ L 1.5 mg / mL HEK-lacZ cell lysate (5) 2.5 ⁇ M HMDER- ⁇ Gal and 20 ⁇ L 1.5 mg / mL HEK
- reaction product was SDS- PAGE (running gel 10%, stacking gel 4%, and subjected to electrophoresis voltage 200V).
- the gel obtained by SDS-PAGE was irradiated with excitation light at 488 nm, and fluorescence at 540-600 nm was observed at a PMT voltage of 1000 V (FIG. 5 (a)). After the observation, the gel was stained with Coomassie, and the position of BSA on the gel was confirmed (FIG. 5 (b)).
- HEK-LacZ cells incubated with 4-CH 2 F-HMDER- ⁇ Gal showed clear fluorescence (left side of FIG. 6). Further, in the mixture of HEK-LacZ cells and HEK cells, a clear difference was observed in the fluorescence level among individual cells, indicating that ⁇ -galactosidase activity for each cell can be detected and fluorescently imaged. On the other hand, when HMDER- ⁇ Gal was used, fluorescence imaging of ⁇ -galactosidase activity of individual cells could not be performed (right side of FIG. 6).
- HEK cells, HEK-lacZ cells, and mixtures of these cells were incubated with 1 ⁇ M 4-CH 2 F-HMDER- ⁇ Gal or HMDER- ⁇ Gal for 30 minutes (in the presence of 37 ° C., 5% CO 2 ). . These cells were analyzed with excitation light at 488 nm using a flow cytometer Accuri C6 (Accri Cytometers). (result) When a mixture of HEK-LacZ cells and HEK cells incubated with 4-CH 2 F-HMDER- ⁇ Gal was analyzed using flow cytometry, peaks corresponding to HEK-LacZ cells and HEK cells were clearly observed.
- Non-fixed fluorescence imaging of biological tissue having ⁇ -galactosidase activity It was confirmed that the enzyme-specific retention fluorescent compound of the present invention can be applied to fluorescent imaging of biological tissue.
- Drosophila esg-lacZ
- ⁇ -galactosidase in the midgut were prepared. The fly was dissected and fixed with 4% FPA, 4-CH 2 F-HMDER- ⁇ Gal was added, reacted for 10 minutes, washed, clarified with 80% glycerol, and fluorescence microscope ( TCS SP5: manufactured by Leica, controlled by LAS AF software.). As a control, Drosophila intestinal stem cells (esg-GFP) expressing GFP were observed. Observation conditions are as follows; excitation light: 514 nm, observation light: 535-595 nm (HyD2), 40 times.
- Ovarian cancer cells SHIN3 were seeded to prepare cancer model mice. In ovarian cancer cells, it is known that acid ⁇ -galactosidase activity is increased.
- fluorescence observation was performed using Maestro in-vivo imaging system (CRi). The observation conditions are as follows; excitation light: 490-530 nm, observation light: 550-800 nm (result)
- excitation light 490-530 nm
- observation light 550-800 nm (result)
- fluorescence derived from a fluorescent dye generated after the enzyme reaction was observed from a tissue site (indicated by a white arrow) considered to be a cancer site (FIG. 12).
- the observation conditions are as follows: (4-CH 2 F-HMDER- ⁇ Gal) excitation light: 514 nm, observation light: 525-585 nm, (mRFP1) excitation light: 594 nm, observation light: 610-700 nm, 63 Double. (result)
- 4-CH 2 F-HMDER- ⁇ Gal excitation light: 514 nm
- (mRFP1) excitation light: 594 nm
- observation light 610-700 nm
- a fat pad was dissected from a 3rd instar (final) fly and placed in a medium containing 10 ⁇ M 4-CH 2 F-HMDER- ⁇ Gal and 16 ⁇ M Hoechst 33342 (nuclear stain). Incubated for 20 minutes, washed with PBS and fixed in 80% glycerol. Further, in order to perform immunochemical staining, the dissected fat pad was immersed in 4% paraformaldehyde (PFA) -containing PBS for 20 minutes and fixed.
- PFA paraformaldehyde
- the fat pad was immersed in a mouse-derived monoclonal antibody against ⁇ -galactosidase (1: 250, Promega) for 30 minutes, and 10 ⁇ M 4-CH 2 F-HMDER- ⁇ Gal, 16 ⁇ M Hoechst 33342, Alexa 647 modification A secondary antibody was added, and observation was performed under a confocal fluorescence microscope (TCS SP5: manufactured by Leica, controlled by LAS AF software).
- the observation conditions are as follows: (Hoechst 33342) excitation light: 405 nm, observation light: 415-490 nm, (4-CH 2 F-HMDER- ⁇ Gal) excitation light: 514 nm, observation light: 525-600 nm, (Alexa 647 modified secondary antibody) Excitation light: 633 nm, observation light: 640-700 nm, 40 times. (result) Fluorescence imaging was performed using 4-CH 2 F-HMDER- ⁇ Gal, and it was confirmed that single-cell fluorescence imaging of ⁇ -galactosidase-active cells that were randomly expressed in living tissue was possible (FIG. 14). ). The above results demonstrate that by using the enzyme-specific retention fluorescent compound of the present invention, ⁇ -galactosidase active cells randomly expressed in living tissue can be visualized and discriminated by single cells. is there.
- fluorescence is emitted specifically with enzyme activity, and at the same time, it stays in a living cell having the enzyme, so that the cell can be selectively fixed at a single cell level without being fixed.
- Visualizable fluorescent compounds, fluorescent imaging probes using the fluorescent compounds, detection methods using the fluorescent probes, detection kits or detection agents are provided.
- the enzyme-specific retention fluorescent compound of the present invention and an imaging technique using the same can be used as a molecular tool for elucidating the aging mechanism of cells, and can be used as a cancer cell-selective fluorescent imaging probe for examination, diagnosis, etc. In a wide range of applications.
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Abstract
Description
(式中、R3、R4、R5、及びR6はそれぞれ独立に-C(=O)H、水素原子、ヒドロキシル基、アルキル基、又はハロゲン原子を表し;R2及びR7はそれぞれ独立に水素原子、ヒドロキシル基、アルキル基、又はハロゲン原子を表し;R8及びR9はそれぞれ独立に水素原子又はアルキル基を示し;Xは酸素原子又はSe、CR13R14、又はSiR15R16を表し;R13、R14、R15及びR16はそれぞれ独立に水素原子又はアルキル基を表し;YはC1-C3アルキレン基を表し、ここで、R3、R4、R5、及びR6の少なくとも一つは、-C(=O)Hを表す。)
本発明により提供される式(I)で表される酵素特異的滞留性蛍光化合物を細胞内に取り込ませた場合、Aで表される基を切断可能な酵素が発現している細胞では、該細胞内において、Aで表される基が切断されるとともに、R3、R4、R5、又はR6に位置する、-CFR10R11又は-CF2R12からフッ化水素が離脱し、キノンメチドが生成される。キノンメチドは急速に周囲の求核剤による攻撃を受けることから、細胞内でキノンメチドを生成した場合、周囲のタンパク質がもつ求核基と速やかに反応し、タンパク質に不可逆的に結合するものと考えられる。
上述の特性を示すため、本発明の細胞内滞留性蛍光化合物を、特定の酵素が発現している標的細胞の細胞を特異的に可視化する方法に用いることができる。具体的には、式(I)の酵素特異的滞留性蛍光化合物と標的細胞において特異的に発現するβ-ガラクトシダーゼ等の酵素とを接触させる工程、次いで、励起光照射を行うことで発生する蛍光を検出する工程を行うことによって、β-ガラクトシダーゼ等を発現している標的細胞のみを特異的に可視化することができる。
○使用した合成試薬、装置等
市販の原料は試薬メーカー(和光純薬株式会社、東京化成工業株式会社、Sigma-Aldrich Co. Ltd.)より購入した。
高速液体クロマトグラフィーによる精製に用いた装置およびカラム。
・ポンプ:PU-2080およびPU-2087(日本分光株式会社)
・検出器:MD-2010(日本分光株式会社)
・カラム:Inertsil ODS-3
(10 x 250 mm or 20 x 250 mm,
GL Science Inc.)
HPLCによる分離精製用いた溶媒。
A: 100 mM トリエチルアミン酢酸塩
B: 99% アセトニトリル、 1% milliQ
HPLC分離における送液は、それぞれ
25mL/min(ポンプ:PU-2087,カラム:20x250mm)5 mL/min(ポンプ:PU-2080,カラム:10x250mm)にて行った。
中圧カラムクロマトグラフィーによる精製はYFLC-AI580(山善株式会社)を用いて行った。
NMR測定はAVANCE III 400 Nanobay(Bruker,Co. Ltd.)を用いて行った。(400MHz for 1H NMR、101MHz for 13C NMR)
質量分析測定はMicrOTOF(ESI-TOF,Bruker,Co. Ltd.)を用いて行った。High-resolution MS(HRMS)測定については、外部標準物質としてギ酸ナトリウムを使用した。
THF: テトラヒドロフラン
DMF: N,N-ジメチルホルムアミド
TFA: トリフルオロ酢酸
DAST:三フッ化N,N-ジエチルアミノ硫黄
PLC: 分取用薄層プレート
2-CHF2-HMDER-βGal、4-CHF2-HMDER-βGalおよび4-CH2F-HMDER-βGalが、β-ガラクトシダーゼによる酵素処理特異的に蛍光を発生することを確認した。
本発明の酵素特異的滞留性蛍光化合物である2-CHF2-HMDER-βGal、4-CHF2-HMDER-βGalまたは4-CH2F-HMDER-βGalと、β-ガラクトシダーゼを30分間酵素反応させることによって生じる、吸収スペクトル変化及び蛍光スペクトル変化(励起波長550nm)を、リン酸ナトリウム緩衝液200mM存在下(pH7.4)で測定した。測定は、Shimadzu UV-2450(島津製作所)およびHitachi F-7000(日立製作所)を用いて行った。
(結果)
2-CHF2-HMDER-βGal、4-CHF2-HMDER-βGalおよび4-CH2F-HMDER-βGalは、β-ガラクトシダーゼと酵素反応することにより、蛍光を発生した(図1~3)。
2-CHF2-HMDER-βGal、4-CHF2-HMDER-βGalおよび4-CH2F-HMDER-βGalを用いて、β-ガラクトシダーゼによって切断されたこれらの化合物が、溶液中に共存するウシ血清アルブミンタンパク質(BSA)を蛍光ラベル化することを確認した。
(1)2.5μM 4-CH2F-HMDER-βGal および0.5mg,/mL BSA、(2)2.5μM 4-CH2F-HMDER-βGal,0.5mg/mL BSA および5U β-ガラクトシダーゼ (3)2.5 μM 4-CHF2-HMDER-βGal,0.5mg/mL BSA および5U β-ガラクトシダーゼ (4)2.5μM 2-CHF2-HMDER-βGal,0.5mg/mL BSA および5U β-ガラクトシダーゼ (5)2.5μM HMDER-βGal、0.5mg/mL BSA および5U β-ガラクトシダーゼを、それぞれ水溶液中(500mM リン酸ナトリウム緩衝液、pH7.4)にて反応させた後、反応産物をSDS-PAGE(ランニングゲル10%、スタッキングゲル4%、泳動電圧200V)に供した。SDS-PAGEによって得られたゲルに対して、488nm,の励起光を照射し、540-600nmの蛍光をPMT電圧 1000Vにて観察した(図4(a))。観察後、当該ゲルをクマシー染色し、ゲル上におけるBSAの位置を確認した(図4(b))。
(結果)
2-CHF2-HMDER-βGal、4-CHF2-HMDER-βGalおよび4-CH2F-HMDER-βGalと、β-ガラクトシダーゼを、BSA存在下で反応させることにより、SDS泳動後のBSAの位置に蛍光が確認された(図4のレーン2~4、75kDa付近のバンド)。β-ガラクトシダーゼを含まない試料(同レーン1)、またはHMDER-βGalを用いた試料(同レーン5)では、蛍光が確認されなかった。
2-CHF2-HMDER-βGal、4-CHF2-HMDER-βGalおよび4-CH2F-HMDER-βGalが、細胞内タンパク質を酵素活性特異的に蛍光ラベル化することを確認した。
β-ガラクトシダーゼを発現するHEK細胞(HEK-lacZ細胞)および通常のHEK細胞を用いた。
(1)2.5μM 4-CH2F-HMDER-βGal および20μL 1.5mg/mL HEK細胞ライセート(2)2.5μM 4-CH2F-HMDER-βGalおよび20μL 1.5mg/mL HEK-lacZ細胞ライセート(3)2.5 μM 4-CHF2-HMDER-βGalおよび20μL 1.5mg/mL HEK-lacZ細胞ライセート(4)2.5μM 2-CHF2-HMDER-βGalおよび20μL 1.5mg/mL HEK-lacZ細胞ライセート(5)2.5μM HMDER-βGalおよび20μL 1.5mg/mL HEK-lacZ細胞ライセートを、それぞれ、5%CO2存在下で37℃30分間インキュベートした後、反応産物をSDS-PAGE(ランニングゲル10%、スタッキングゲル4%、泳動電圧200V)に供した。SDS-PAGEによって得られたゲルに対して、488nm,の励起光を照射し、540-600nmの蛍光をPMT電圧 1000Vにて観察した(図5(a))。観察後、当該ゲルをクマシー染色し、ゲル上におけるBSAの位置を確認した(図5(b))。
(結果)
2-CHF2-HMDER-βGal、4-CHF2-HMDER-βGalおよび4-CH2F-HMDER-βGalを、HEK-lacZ細胞とインキュベートした試料において、細胞内タンパク質に蛍光が確認された(図5のレーン2~4)。β-ガラクトシダーゼを発現しないHEK細胞を用いた試料(同レーン1)、またはHMDER-βGalを用いた試料(同レーン5)では、蛍光が確認されなかった。
本願発明の酵素特異的滞留性蛍光化合物が、生細胞の蛍光イメージングに利用可能であることを確認した。
HEK細胞、HEK-lacZ細胞、およびこれらの細胞の混合物を、1μMの4-CH2F-HMDER-βGalまたはHMDER-βGalとともに、30分間インキュベート(37oC、5% CO2存在下)した後、そのまま、あるいは、培地により2回洗浄した後、共焦点顕微鏡を用いて、これらの細胞の蛍光画像および微分干渉画像(DIC)を取得した。また、4-CH2F-HMDER-βGalとインキュベーションした後、4%PFAを加えて室温で10分間インキュベーションすることで固定した細胞も、同様に観察した。共焦点顕微鏡には、白色レーザー光、および対物レンズHCX PL APO CS 40x/1.25(Leica社製)を備えた、TCS SP5X(Leica社製)を用い、LAS AF softwareにて制御した。観察条件は以下のとおり: ホワイトライトレーザー(WLL): 80%-25%,励起波長: 525nm,観測波長: 535-595nm,ゲイン: 800 V (PMT1)/350V(Scan-DIC),オフセット: 0%,ピンホール:67.88μM(1エアリーディスク).
(結果)
試験化合物とインキュベートした後、細胞を培地で洗浄することなく観察した結果、4-CH2F-HMDER-βGalでインキュベートされたHEK-LacZ細胞は、鮮明な蛍光を示した(図6左側)。また、HEK-LacZ細胞とHEK細胞の混合物においては、個々の細胞における蛍光レベルに明確な差異が観察され、細胞毎のβ-ガラクトシダーゼ活性を検出・蛍光イメージング可能であることが示された。一方、HMDER-βGalを用いた場合には、個々の細胞のβ-ガラクトシダーゼ活性を蛍光イメージングすることはできなかった(図6右側)。
さらに、試験化合物とインキュベート後、細胞を培地により2回洗浄した後に観察した場合においても、4-CH2F-HMDER-βGalを用いた場合の蛍光強度には、ほとんど変化がなく、4-CH2F-HMDER-βGalとβ-ガラクトシダーゼとの酵素反応後に生成する蛍光色素が、ほとんど細胞から漏出しないことが示された(図7)。
また、4-CH2F-HMDER-βGalとインキュベーションした後に固定処理を行った試料においても、生細胞と同様の蛍光イメージングが可能であった(図8)。
本願発明の酵素特異的滞留性蛍光化合物を利用することで、生細胞の細胞毎の酵素活性を、フローサイトメトリーを用いて検出し得ることを確認した。
HEK細胞、HEK-lacZ細胞、およびこれらの細胞の混合物を、1 μMの4-CH2F-HMDER-βGalまたはHMDER-βGalとともに、30分間インキュベート(37oC 、5% CO2存在下)した。これらの細胞を、フローサイトメメーター Accuri C6(Accri Cytometers)を用いて、488nmの励起光で解析した。
(結果)
4-CH2F-HMDER-βGalとインキュベートされたHEK-LacZ細胞とHEK細胞の混合物を、フローサイトメトリーを用いて解析したところ、HEK-LacZ細胞、HEK細胞のそれぞれに対応するピークが明瞭に観察され、これらの細胞を、蛍光強度の差に基づいて明確に区別して検出可能であった(図9(a))。一方、HMDER-βGalとインキュベートされたHEK-LacZ細胞とHEK細胞の混合物では、これらの細胞をフローサイトメトリーの結果上区別することはできなかった(図9(b))。
本願発明の酵素特異的滞留性蛍光化合物を、生体組織の蛍光イメージングに適用し得ることを確認した。
β-ガラクトシダーゼを発現する(en-lacZ)ショウジョウバエ(Drosophila melanogaster)の羽原基(wing discs)を、20μMの4-CH2F-HMDER-βGal又はHMDER-βGalとともに、30分間室温にてインキュベーションした後、共焦点顕微鏡(TCS SP5:Leica社製、LAS AF software.にて制御)下で観察した。観察条件は以下のとおり;Ar:40%-25%,励起光:514nm,観測光:535-595nm(HyD2),20倍.
(結果)
4-CH2F-HMDER-βGalを用いて蛍光イメージングを行った場合、酵素反応生成物である蛍光色素は拡散しないため、β-ガラクトシダーゼ活性をもつ部位(後部)を選択的に蛍光イメージング可能であった(図10(a))。一方、HMDER-βGalを用いて蛍光イメージングを行った場合は、経時的に酵素反応生成物である蛍光色素が拡散し、β-ガラクトシダーゼ活性をもつ部位が判別できなかった(図10(b))。
本願発明の酵素特異的滞留性蛍光化合物を用いて、組織内の細胞を単一細胞レベルで蛍光イメージングすることが可能であることを確認した。
中腸にβ-ガラクトシダーゼを発現するショウジョウバエ(esg-lacZ)を作製した。当該ハエを解剖後4%FPAにより固定処理を行い、4-CH2F-HMDER-βGalを添加して10分間反応させた後洗浄し、80%グリセロールにて透明化処理を行い、蛍光顕微鏡(TCS SP5:Leica社製、LAS AF software.にて制御)にて観察を行った。対照として、GFPを発現するショウジョウバエ腸幹細胞(esg-GFP)を観察した。観察条件は以下のとおり;励起光:514nm,観測光:535-595nm(HyD2),40倍.
(結果)
ショウジョウバエ(esg-lacZ)を4-CH2F-HMDER-βGalと反応させることにより、組織内に蛍光を発する細胞を確認することができた(図11(a))。当該画像は、ショウジョウバエ腸幹細胞(esg-GFP)の蛍光画像(図11(b))と類似していた。
なお、固定処理を行っていない、β-ガラクトシダーゼ発現腸管細胞でも、同様の蛍光イメージングが可能であることを確認している。
本願発明の酵素特異的滞留性蛍光化合物を用いて、癌部位を選択的に蛍光イメージング可能であることを確認した。
卵巣癌細胞SHIN3を播種して、癌モデルマウスを作製した。卵巣癌細胞においては、酸性β-ガラクトシダーゼ活性が上昇していることが知られている。当該癌モデルマウスに、4-CH2F-HMDER-βGalを腹腔内注射して1時間後に、Maestro in-vivo imaging system(CRi)を用いて蛍光観察を行った。観察条件は以下のとおり;励起光:490-530nm,観測光:550-800nm
(結果)
蛍光観察の結果、癌部位と考えられる組織部位(白矢印で示す)から、酵素反応後に生成する蛍光色素由来の蛍光が観察された(図12)。蛍光スペクトルにより蛍光を分離する操作(unmix)を行ったところ、自家蛍光と蛍光スペクトルを分離することが可能であった。
本願発明の酵素特異的滞留性蛍光化合物を用いて、生体組織内にモザイク状に分布するβ-ガラクトシダーゼ活性細胞群を未固定で蛍光イメージングすることが可能であることを確認した。
(材料と方法)
雄のHis2Av-mRFP1, FRT80B/TM6Bをもつショウジョウバエ(Drosophila melanogaster)と雌のhs-flp;;arm-lacZ, FRT80Bをもつショウジョウバエを交配させ、その子が孵化して30時間後の一齢幼虫のときに37℃、1時間ヒートショックを与え、羽原基(wing discs)に以下の三種類の遺伝子型の細胞をモザイク状に発現させた;(1) β-ガラクトシダーゼのみが発現した細胞(arm-lacZ)、(2) 赤色蛍光タンパク質(mRFP1)のみが発現した細胞(His2Av-mRFP1)、(3) β-ガラクトシダーゼとmRFP1が共に発現した細胞(arm-lacZ/His2Av-mRFP1)。三齢(終齢)幼虫から解剖した羽原基を10 μMの4-CH2F-HMDER-βGalを含む培地中に30分間浸し、共焦点蛍光顕微鏡(TCS SP5:Leica社製、LAS AF softwareにて制御)下で観察した。観察条件は以下の通り;(4-CH2F-HMDER-βGal)励起光:514 nm、観測光:525-585 nm、(mRFP1)励起光:594 nm、観測光:610-700 nm、63倍。
(結果)
4-CH2F-HMDER-βGalを用いて蛍光イメージングを行ったところ、上記三種類の遺伝子型をもつ細胞がモザイク状に存在する様子が鮮明に可視化された(図13)。
以上の結果は、本発明の酵素特異的滞留性蛍光化合物を用いることで、生体組織に存在するβ-ガラクトシダーゼ発現細胞を非固定で明確に可視化・判別可能であることを実証するものである。
本願発明の酵素特異的滞留性蛍光化合物を用いて、生体組織内にランダムに発現したβ-ガラクトシダーゼ活性を、単一細胞レベルで蛍光イメージングすることが可能であることを確認した。
(材料と方法)
ショウジョウバエ(Drosophila melanogaster)脂肪体のクローン解析を行うため、フリップアウト技術を使ってUAS-lacZをhs-flp122;Actin>y>Gal4に掛け合わせることによってβ-ガラクトシダーゼを過剰発現させた。生細胞蛍光イメージングを行うため、三齢(終齢)のハエから脂肪体を解剖し、10 μMの4-CH2F-HMDER-βGalと16 μMのHoechst 33342(細胞核染色剤)を含む培地に20分間インキュベーションし、PBSで洗浄し、80%グリセロール中に固定した。また、免疫化学染色を行うため、解剖した脂肪体を4%パラホルムアルデヒド(PFA)含有PBSに20分間浸し、固定処理を行った。ブロッキングの後、脂肪体をβ-ガラクトシダーゼに対するマウス由来モノクローナル抗体(1:250、プロメガ社)に30分間浸し、10 μMの4-CH2F-HMDER-βGal、16 μMのHoechst 33342、Alexa 647修飾二次抗体を加え、共焦点蛍光顕微鏡(TCS SP5:Leica社製、LAS AF softwareにて制御)下で観察した。観察条件は以下の通り;(Hoechst 33342)励起光:405 nm、観測光:415-490 nm、(4-CH2F-HMDER-βGal)励起光:514 nm、観測光:525-600 nm、(Alexa 647修飾二次抗体)励起光:633 nm、観測光:640-700 nm、40倍。
(結果)
4-CH2F-HMDER-βGalを用いて蛍光イメージングを行い、生体組織内にランダムに発現したβ-ガラクトシダーゼ活性細胞を単一細胞ずつ蛍光イメージングすることが可能であることを確認した(図14)。
以上の結果は、本発明の酵素特異的滞留性蛍光化合物を用いることで、生体組織内にランダムに発現したβ-ガラクトシダーゼ活性細胞を単一細胞ずつ可視化・判別可能であることを実証するものである。
Claims (18)
- 以下の式(I’)で表される化合物又はその塩を含む、酵素特異的滞留性蛍光化合物:
(式中、Aは酵素によって切断される一価の基を表し;R1は水素原子又はベンゼン環に結合する1個ないし4個の同一又は異なる置換基を表し;R3、R4、R5、及びR6はそれぞれ独立に-CFR10R11又は-CF2R12、若しくは水素原子、ヒドロキシル基、アルキル基、又はハロゲン原子を表し;R2及びR7はそれぞれ独立に水素原子、ヒドロキシル基、アルキル基、又はハロゲン原子を表し;R8及びR9はそれぞれ独立に水素原子又はアルキル基を表し;R10、R11及びR12はそれぞれ独立に水素原子、アルキル基又はアルケニル基を表し;Xは酸素原子、Se、CR13R14、又はSiR15R16を表し;R13、R14、R15及びR16はそれぞれ独立に水素原子又はアルキル基を表し;YはC1-C3アルキレン基を表し、ここで、R3、R4、R5、及びR6の少なくとも一つは、-CFR10R11又は-CF2R12を表す。) - 前記酵素が、レポーター酵素を含む加水分解酵素である、請求項1に記載の酵素特異的滞留性蛍光化合物。
- 前記酵素が、癌細胞で特異的に発現又は活性化する酵素である、請求項1に記載の酵素特異的滞留性蛍光化合物。
- 前記レポーター酵素がβ-ガラクトシダーゼであって、Aがガラクトピラノシル基である、請求項1~3のいずれかに記載の酵素特異的滞留性蛍光化合物。
- R3、R4、R5、及びR6の少なくとも一つは、-CFR10R11である、請求項1~4のいずれかに記載の酵素特異的滞留性蛍光化合物。
- R3、R4、R5、及びR6の少なくとも一つは、-CH2Fである、請求項1~4のいずれかに記載の酵素特異的滞留性蛍光化合物。
- 請求項1~7のいずれかに記載の酵素特異的滞留性蛍光化合物を含有する蛍光イメージングプローブ。
- 請求項1~7のいずれかに記載の酵素特異的滞留性蛍光化合物を含有する、特定の酵素が発現している標的細胞を検出するための、又は可視化するための組成物又はキット。
- 前記標的細胞が、β-ガラクトシダーゼ発現細胞である、請求項9に記載の組成物又はキット。
- 前記標的細胞が、癌細胞である、請求項9に記載の組成物又はキット。
- 請求項1~7のいずれかに記載の酵素特異的滞留性蛍光化合物を用いて、特定の酵素が発現している標的細胞を検出する方法。
- 請求項1~7のいずれかに記載の酵素特異的滞留性蛍光化合物と、当該標的細胞において特異的に発現する酵素とを、生体外において接触させ、特定の酵素が発現している標的細胞を検出する方法。
- 請求項1~7のいずれかに記載の酵素特異的滞留性蛍光化合物と、当該標的細胞において特異的に発現する酵素とを、生体外において接触させる工程、及び、励起光照射を行って蛍光を生じさせる工程を含むことを特徴とする、請求項12又は13のいずれかに記載の方法。
- 前記標的細胞が、β-ガラクトシダーゼ発現細胞である、請求項12~14のいずれかに記載の方法。
- 前記標的細胞が、癌細胞である、請求項12~15のいずれかに記載の方法。
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WO2018003686A1 (ja) * | 2016-06-30 | 2018-01-04 | 国立大学法人 東京大学 | 酵素特異的な細胞内滞留性赤色蛍光プローブ。 |
WO2020175688A1 (ja) * | 2019-02-28 | 2020-09-03 | 国立大学法人 東京大学 | 癌検出蛍光プローブ |
WO2020250998A1 (ja) * | 2019-06-14 | 2020-12-17 | 株式会社同仁化学研究所 | 細胞滞留性蛍光化合物並びにそれを用いた細胞の染色方法及び高感度検出方法 |
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EP3275889A4 (en) * | 2015-02-27 | 2018-11-21 | The University of Tokyo | Fluorescent probe for detecting calpain activity |
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