WO2022260135A1 - 固相抽出を利用した蛍光プローブライブラリの調製方法、及びこれを用いた酵素活性計測方法 - Google Patents
固相抽出を利用した蛍光プローブライブラリの調製方法、及びこれを用いた酵素活性計測方法 Download PDFInfo
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- C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
- C07F9/65586—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system at least one of the hetero rings does not contain nitrogen as ring hetero atom
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- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/655—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms
- C07F9/6552—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms the oxygen atom being part of a six-membered ring
- C07F9/65522—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms the oxygen atom being part of a six-membered ring condensed with carbocyclic rings or carbocyclic ring systems
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- 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
- C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
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- 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
- C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
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- 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
- C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
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- C12Q2334/00—O-linked chromogens for determinations of hydrolase enzymes, e.g. glycosidases, phosphatases, esterases
- C12Q2334/20—Coumarin derivatives
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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)
- G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/70—Mechanisms involved in disease identification
- G01N2800/7023—(Hyper)proliferation
- G01N2800/7028—Cancer
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
Definitions
- the present invention provides a novel method for synthesizing a fluorescent probe, which enables easy synthesis of many types of fluorescent probes, a fluorescent mother compound that can be used in the method for synthesis, and a method for detecting enzyme activity obtained by the method for synthesis.
- the research group of the inventors has so far used microdevice-compatible enzyme activity detection fluorescent probes to detect enzymes in the blood at the single molecule level and perform activity-based profiling, and find new ways to treat diseases.
- body fluids such as blood and urine
- there are still many enzymatic activities in body fluids such as blood and urine that have not been reported to be related to diseases. Therefore, if it is possible to construct a fluorescent probe library that can comprehensively detect a wide variety of enzymatic activities with ultra-high sensitivity in microdevices, it will contribute to the search for enzymatic activities that will serve as new biomarkers.
- the fluorescent probes used to detect enzyme activity are usually synthesized and purified over several days to several weeks before being used for fluorescence assays. Conventionally, it has been difficult to prepare a group of fluorescent probes that comprehensively detect enzyme activity.
- a phos-tag that specifically captures a phosphate group is synthesized in a liquid phase using a fluorescent dye having a phosphonic acid, a phosphate ester, or a phosphate amide in the molecular skeleton as a core. Synthetic schemes are provided using the procedure of solid phase extraction with a carrier bound to. In addition, in this scheme, since the finally obtained fluorescent probe has phosphonic acid, it is possible to impart high water solubility required for assays using microdevices. considered to be optimal.
- A is an amino group (--NR 2 H) or a hydroxyl group (--OH);
- R 2 is a hydrogen atom and a branched, linear or cyclic substituted or unsubstituted alkyl group having 1 to 8 carbon atoms (one or more non-adjacent, non-terminal C atoms of the alkyl group are , O, S, CO or COO);
- R 1 if present, is the same or different monovalent substituents present on the benzene ring;
- S if present, is a linker;
- m is an integer from 0 to 3;
- a method of preparing a compound of formula (III) below, comprising: (In formula (III), B is an amide group (-NR 2 C( O)R, R is a hydrogen atom or an alkyl group having 1 to 8 carbon atoms), -NR 2 -CO-L (L represents a partial structure of an amino acid; ), a phosphate amide group (—NR 2 —PO(OR a )(OR b ), R a and R b are each independently a hydrogen atom, and a branched, linear or cyclic substituted group having 1 to 8 carbon atoms.
- a sulfonamide group (—NR 2 —SO 2 —R c , where R c is a hydrogen atom and a branched, linear or cyclic substituted or unsubstituted alkyl group having 1 to 8 carbon atoms (one or more of the alkyl groups is optionally replaced by O, S, CO or COO), an ester group, a phosphate group, a sulfate group, an ether group or -OL'(L' represents a sugar or a partial structure of a sugar); S, T, R 1 , m are as defined in Formula (I).
- A is an amino group (--NR 2 H) or a hydroxyl group (--OH);
- R 2 is a hydrogen atom and a branched, linear or cyclic substituted or unsubstituted alkyl group having 1 to 8 carbon atoms (one or more non-adjacent, non-terminal C atoms of the alkyl group are , O, S, CO or COO);
- R 1 if present, is the same or different monovalent substituents present on the benzene ring;
- S if present, is a linker;
- m is an integer from 0 to 3;
- [8] A method for detecting the activity of a plurality of enzymes in a biological sample, comprising contacting the biological sample with the compound of general formula (III) described in [6]. [9] The method according to [8], characterized in that a library of compounds of general formula (III) according to [6] is used. [10] The method according to [8] or [9], which uses a microdevice. [11] A method for testing an enzymatic assay of a composition comprising the compound of general formula (III) according to [6], wherein said composition and an organism containing or suspected of containing an enzyme that cleaves said compound The method comprising contacting with the sample.
- a method for screening a fluorescent probe capable of detecting a biomarker for a specific disease comprising: (1) adding a library of compounds of formula (III) according to [6] to a microdevice, such that at least one well of the microdevice comprises one compound of formula (III); add; (2) adding a solution containing a biological sample to the microdevice so as to create at least one well containing one molecule of enzyme in the microdevice, wherein the biological sample is A biological sample obtained from a patient with a specific disease or a biological sample obtained from a healthy subject; (3) contacting the compound of formula (III) with an enzyme to detect fluorescence in the wells of the microdevice, the step comprising: contacting the compound of formula (III) with a biological sample obtained from a patient with a specific disease, and measuring the fluorescence intensity (first fluorescence intensity) from the compound of formula (III); contacting the compound of formula (III) with a biological sample obtained from a healthy subject, and measuring the fluorescence intensity (second fluorescence intensity (second
- R 1 , S, T, and m are as described in detail in general formula (I).
- [15] (a) applying a fluorescent probe containing the compound of formula (IV) or a salt thereof to a clinical specimen of a subject, and (b) measuring a fluorescent image of the clinical specimen to which the fluorescent probe is applied.
- a method of diagnosing pancreatic cancer or predicting the likelihood that a subject from whom a biological sample is derived has pancreatic cancer.
- [16] (a) a step of contacting a biological sample obtained from a subject with a fluorescent probe containing the compound of formula (IV) or a salt thereof, and (b) fluorescence of the biological sample contacted with the fluorescent probe
- the diagnostic method or prediction method according to [15] or [16], wherein the compound of formula (VI) is the following compound: [18] A fluorescent probe for detecting pancreatic cancer for use in the method of [15] or [16], comprising the compound of formula (IV) or a salt thereof.
- a kit for detecting pancreatic cancer cells or tissues containing the compound of formula (IV) or a salt thereof.
- a method for diagnosing pancreatic cancer by detecting single-molecule enzymatic activity of CD13 in a biological sample obtained from a subject using a fluorescent probe containing a compound or a salt thereof, or a method from which the biological sample is derived A method for predicting the likelihood that a subject has pancreatic cancer.
- B is -NR 2 -CO-L, and the part of -CO-L is -Pro-Xaa (Pro is a proline residue, Xaa is glycine, serine, glutamic acid Representing amino acid residues such as), using a fluorescent probe containing the compound or a salt thereof according to [6], a single-molecule enzymatic activity of DPP4 in a biological sample obtained from a subject is detected.
- a method of diagnosing pancreatic cancer, or a method of predicting the likelihood that a subject from whom a biological sample is derived has pancreatic cancer, by [23] The method according to [6], wherein B is -NR 2 -CO-L and the portion of -CO-L is an alanine, lysine, arginine or methionine residue in general formula (III) using a microdevice.
- a fluorescent probe containing a compound or a salt thereof to detect single-molecule enzymatic activity of CD13 in a biological sample obtained from a subject; NR 2 -CO-L, wherein the -CO-L portion is a peptide of -Pro-Xaa (Pro represents a proline residue, Xaa represents an amino acid residue such as glycine, serine, glutamic acid, etc.) [6 ], a method for diagnosing pancreatic cancer by detecting single-molecule enzymatic activity of DPP4 in a biological sample obtained from a subject using a fluorescent probe containing the compound or a salt thereof, or A method of predicting the likelihood that a subject from whom a sample is derived has pancreatic cancer. [24] The method of any one of [21] to [23], wherein the biological sample is a pancreatic cancer patient, a patient suspected of having pancreatic cancer, or a healthy subject. It provides
- the present invention it is possible to provide a fluorescent probe synthesis scheme that takes advantage of liquid-phase synthesis and solid-phase synthesis.
- the present invention it is possible to provide for the synthesis of fluorescent probes that may allow solid phase synthesis and easy detachment from the solid phase.
- the synthesis scheme based on the present invention it is possible to synthesize and purify probes with a very simple scheme of only mixing and solution removal, and parallel processing is also possible, so that about 10 types of probes can be synthesized. Since probes can be prepared in a matter of days, the efficiency of probe development can be significantly improved.
- a library of compounds (fluorescent probes) of the present invention can be constructed by carrying out reactions in parallel in a plurality of reaction vessels according to the synthesis scheme based on the present invention.
- a compound of the present invention or a library thereof can be used to provide a method for detecting the activity of multiple enzymes in a biological sample.
- the compound library of the present invention includes several hundred types of compounds that can detect multiple enzymes, it can be used to detect biomarker candidate activities from biological samples in an ultrasensitive and exhaustive manner. Searching (screening) is possible, and biomarker activity detection fluorescent probes can also be screened.
- biomarker activity detection fluorescent probe obtained by the screening method of the present invention can be used to diagnose a disease by detecting a biomarker specific to a given disease.
- FIG. 1 is a perspective view schematically showing a microdevice according to one embodiment of the present invention;
- FIG. 1 shows a conceptual diagram of disease diagnosis using the present invention.
- FIG. 1 shows fluorescence microscopic images obtained by detecting the enzymatic activity of each probe in blood using a microdevice in Example 1.
- FIG. 1 shows fluorescence microscopic images obtained by detecting the enzymatic activity of each probe in blood using a microdevice in Example 1.
- FIG. 1 shows fluorescence microscopic images obtained by detecting the enzymatic activity of each probe in blood using a microdevice in Example 1.
- FIG. 2 shows fluorescence microscope images obtained using Arg-PMAC in Example 2.
- a representative fluorescence microscope image obtained in Example 3 is shown.
- the results of measuring the activity of PMUM-dCMP with a plate reader using the purified enzyme of ENPP3 are shown.
- 4 shows a fluorescence microscope image obtained in Example 6.
- FIG. 2 shows the results of comparing the number of wells with fluorescence intensity of 2500 AU or more detected in Example 6 between healthy subjects and pancreatic cancer patients.
- 4 shows an ROC curve created by performing determination based on the results of ENPP activity measurement in Example 6.
- FIG. 2 shows the results of comparing the number of wells with fluorescence intensity of 2
- halogen atom means a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom.
- alkyl may be straight chain, branched chain, cyclic, or an aliphatic hydrocarbon group consisting of a combination thereof.
- the number of carbon atoms in the alkyl group is not particularly limited . ) and 1 to 20 carbon atoms (C 1-20 ). When the number of carbon atoms is specified, it means “alkyl” having the number of carbon atoms within the specified range.
- C 1-8 alkyl includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neo-pentyl, n-hexyl, isohexyl, n-heptyl, n-octyl and the like are included.
- an alkyl group may have one or more optional substituents.
- substituents include, but are not limited to, alkoxy groups, halogen atoms, amino groups, mono- or di-substituted amino groups, substituted silyl groups, acyl, and the like.
- alkyl group When an alkyl group has more than one substituent, they may be the same or different.
- alkyl moieties of other substituents containing alkyl moieties eg, alkoxy groups, arylalkyl groups, etc.
- substituents include, but are not limited to, alkyl groups, alkoxy groups, hydroxyl groups, carboxyl groups, halogen atoms, sulfo groups, amino groups, alkoxycarbonyl groups, and oxo groups. These substituents may further have a substituent. Examples of such groups include, but are not limited to, halogenated alkyl groups, dialkylamino groups, and the like.
- alkoxy group refers to a structure in which the aforementioned alkyl group is bonded to an oxygen atom, and examples thereof include saturated alkoxy groups that are linear, branched, cyclic, or a combination thereof.
- methoxy, ethoxy, n-propoxy, isopropoxy, cyclopropoxy, n-butoxy, isobutoxy, s-butoxy, t-butoxy, cyclobutoxy, cyclopropylmethoxy, n- Pentyloxy group, cyclopentyloxy group, cyclopropylethyloxy group, cyclobutylmethyloxy group, n-hexyloxy group, cyclohexyloxy group, cyclopropylpropyloxy group, cyclobutylethyloxy group, cyclopentylmethyloxy group and the like are preferred. Examples include:
- Fluorophore nucleus One embodiment of the present invention is a compound represented by the following general formula (I) (hereinafter also referred to as "compound 1 of the present invention”).
- Compound 1 of the present invention plays a role as a scaffold molecule in the method for preparing the novel fluorescent probe of the present invention, which will be described later.
- the compound 1 of the present invention since the compound 1 of the present invention has a phosphonic acid, a phosphate ester group, or a phosphate amide group, the fluorescent probe finally obtained using the compound also has a phosphonic acid, a phosphate ester, or a phosphate amide group. Therefore, it is possible to impart high water solubility required for assays using microdevices.
- A is an amino group (--NR 2 H) or a hydroxyl group (--OH).
- R 2 is selected from the group consisting of a hydrogen atom and a branched, linear or cyclic substituted or unsubstituted alkyl group having 1 to 8 carbon atoms.
- Alkyl groups having 1 to 8 carbon atoms include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neo-pentyl, n-hexyl and isohexyl. , n-heptyl, n-octyl and the like.
- the alkyl group defined as R2 includes, for example, a case where a part of the alkyl group contains a polyethylene glycol chain, and a case where the entirety of R2 is a polyethylene glycol chain.
- substituents that the alkyl group of R 2 can have include an alkoxy group, a hydroxyl group, a carboxyl group, a halogen atom, a sulfo group, an amino group, an alkoxycarbonyl group, and an oxo group. It is not limited to these.
- R2 is a hydrogen atom.
- R 1 if present, are the same or different monovalent substituents present on the benzene ring.
- monovalent substituents for R 1 include halogen, substituted or unsubstituted alkyl groups having 1 to 8 carbon atoms, alkoxy groups, carboxyl groups, sulfonyl groups and aryl groups. wherein one or more non-adjacent, non-terminal C atoms of the alkyl group of R 1 may be replaced by O, S, CO or COO. Therefore, the alkyl group of R 1 includes, for example, a case where a part of the alkyl group contains a polyethylene glycol chain, and a case where the whole of R 1 is a polyethylene glycol chain.
- n is an integer of 0-3, preferably 0-1.
- a monovalent substituent for R 1 can be introduced at any position on the benzene ring of the coumarin skeleton.
- S is a linker that attaches the phosphonate, phosphate, or phosphate amide group of T to the coumarin.
- the linker include a substituted or unsubstituted hydrocarbon group, polyethylene glycol, a heterocyclic group, an amide group (including any group represented by -NHCO- or -CONH-), an aromatic ring having an amide group, and the like. Examples include, but are not limited to: Linkers that can be used for compound 1 of the present invention also include those in which two or more of the above groups are bonded.
- the hydrocarbon group includes an alkylene group having 1 to 10 carbon atoms, an alkynylene group having 1 to 10 carbon atoms, a cycloalkylene group, and an aromatic hydrocarbon.
- Polyethylene glycol is represented by —(C 2 H 4 —O) t — (t is an integer of 1 to 10), and either the ethylene group or the oxo group may be the side that binds to coumarin.
- a triazole group etc. are mentioned as a heterocyclic ring.
- An aromatic ring having an amide group can be represented by *--NHCO--Ar-- or *--Ar--CONH--- (Ar represents an aromatic ring and * represents a side that bonds to coumarin).
- polyethylene glycol, a heterocyclic group, an amide group, or an aromatic ring having an amide group has a substituted or unsubstituted hydrocarbon group, preferably an alkylene group having 1 to 10 carbon atoms, at one or both ends of the aromatic ring.
- the phosphonic acid group, phosphate ester group or phosphate amide group directly binds to coumarin.
- the phosphonic acid group, phosphate ester group or phosphate amide group is an alkylene group having 1 to 10 carbon atoms (e.g., methylene group, ethylene group), amide group, triazole It is attached to coumarin via a group.
- a phosphonic acid group, a phosphate ester group, or a phosphate amide group can be introduced at either the 3- or 4-position of the coumarin skeleton.
- One preferred embodiment of the present invention is a compound having the structure below wherein A is -NR 2 H in formula (I).
- a preferred embodiment of the invention are compounds of formula (I) wherein A is -NH2 .
- one preferred aspect of the compound represented by formula (Ia) is a compound having the following structure.
- R 1 , R 2 and m are the same as defined in formula (I), and n is an integer of 0-10.
- One preferred example of the compound represented by formula (Ia-1) is the following compound.
- Another preferred embodiment of the present invention is a compound having the following structure wherein A is -OH in formula (I).
- one preferred aspect of the compound represented by formula (Ib) is a compound having the following structure.
- R 1 and m are the same as defined in formula (I), and n is an integer of 0-10.
- One preferred example of the compound represented by formula (Ib-1) is the following compound.
- a method for producing a representative compound of compound 1 of the present invention is specifically shown in the examples of the present specification. Therefore, based on these explanations, those skilled in the art can appropriately select reaction materials, reaction conditions, reaction reagents, etc., and modify or modify these methods as necessary to achieve the present invention.
- Compound 1 can be made.
- Method for preparing a fluorescent probe (1) Method for preparing a compound represented by the general formula (III) Another embodiment of the present invention is a method for preparing a compound represented by the following formula (III). , which includes the following steps (1) to (5) (hereinafter also referred to as the “preparation method of the present invention”).
- A is an amino group (--NR 2 H) or a hydroxyl group (--OH);
- R 2 is a hydrogen atom and a branched, linear or cyclic substituted or unsubstituted alkyl group having 1 to 8 carbon atoms (one or more non-adjacent, non-terminal C atoms of the alkyl group are , O, S, CO or COO);
- R 1 if present, is the same or different monovalent substituents present on the benzene ring: S, if present, is a linker;
- m is an integer from 0 to 3;
- a fluorescent probe is synthesized in a liquid phase using a fluorescent dye having a phosphonic acid, a phosphate ester, or a phosphate amide in the molecular skeleton as a core, and then a phos that specifically captures the phosphate group.
- a procedure of performing solid-phase extraction using a carrier to which the -tag is attached can be employed.
- FIG. 1 shows a schematic diagram comparing the preparation method of the present invention with the conventional liquid phase synthesis method and solid phase synthesis method.
- the liquid phase synthesis method has high reaction efficiency, it is complicated because column chromatography or preparative HPLC is used for purification, and several hours/compound are required for purification. Further, the solid-phase synthesis method has a problem of low reaction efficiency, although the purification operation is simple.
- the preparation method of the present invention is based on the SAS (Synthesis-based on affinity separation) method, which combines the advantages of both methods, and can obtain the desired product (preferably with high purity) only by mixing and removing the solution. In the preparation method of the present invention, it is also possible to easily synthesize many kinds of fluorescent probes by parallel processing.
- Step (1) the following operation of protecting the T group of compound 1 of the present invention is carried out.
- A, S, T, R 1 and m are as defined above.
- a phosphonic acid group, a phosphate ester group, and a phosphate amide group, which are groups of T, can be protected by a method commonly used for protecting these protecting groups, for example, a silyl protecting group (e.g., tert- butyldiphenylsilyl group) or a tert-butyl group.
- a silyl protecting group e.g., tert- butyldiphenylsilyl group
- Reagents for protecting phosphonic acid groups, phosphate ester groups, and phosphate amide groups include TBDPS-Cl (tert-butyl(chloro)diphenylsilane), TBDMS-Cl (tert-butyldimethylsilyl chloride), isobutene, tert- Butanol and the like are preferably used.
- compound 1 of the present invention is dissolved in a solution containing imidazole (e.g., DMSO solution, etc.), and TBDPS-Cl is added to protect phosphonic acid, phosphoric acid ester, and phosphoric acid amide. can be done.
- imidazole e.g., DMSO solution, etc.
- Step (2) the A portion of the product obtained in step (1) is converted into a functional group that is cleaved upon contact with a substance to be measured such as an enzyme.
- the functional group that is cleaved upon contact with the substance to be measured is appropriately determined according to the type of biomolecule targeted in the measurement of enzymatic activity or the like using the final product, compound (III). is divided into the following (i) and (ii) depending on whether it is an amino group or a hydroxyl group.
- A is an amino group (--NR 2 H)
- amino acid can be any compound having both an amino group and a carboxyl group, including natural and non-natural compounds. It may be a neutral amino acid, a basic amino acid, or an acidic amino acid. In addition to amino acids that themselves function as transmitters such as neurotransmitters, physiologically active peptides (dipeptides, tripeptides, tetrapeptides, oligopeptides) and polypeptide compounds such as proteins can be used, for example, ⁇ -amino acids, ⁇ -amino acids, ⁇ -amino acids and the like. As the amino acid, it is preferable to use an optically active amino acid.
- amino acid N-terminus may be N-acylated (eg, N-acetylated) or N-carbamoylated.
- amino acid residue refers to a structure corresponding to a partial structure remaining after removing the hydroxyl group from the carboxyl group of an amino acid.
- Amino acid residues include ⁇ -amino acid residues, ⁇ -amino acid residues, and ⁇ -amino acid residues.
- peptide refers to a structure in which two or more amino acids are linked by peptide bonds.
- the carboxyl group of the side chain of the amino acid is -NR H is combined with to form a carbonyl group, which is a part of an amino acid.
- amino acid partial structure of L is represented by the following formula (3).
- -NR 2 -CO-L is represented by the following formula. —NH—CO—CHR 3 —NHR 0 (1)
- R3 is hydrogen, a methyl group, or other natural amino acid (glycine, alanine, leucine, isoleucine, valine, lysine, cysteine, threonine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, serine, histidine , phenylalanine, methionine, tryptophan, tyrosine, proline).
- R3 can also form a ring with the nitrogen atom of NHR0 .
- R 3 also includes groups that constitute side chains of unnatural amino acids (citrulline, norvaline, etc.).
- R 3 also includes groups in which a part of the groups constituting the side chains of natural amino acids or non-natural amino acids are substituted or modified with other substituents.
- R 3 also includes groups other than groups constituting side chains of natural amino acids and unnatural amino acids, such as alkyl groups having various substituents.
- R 0 is an N-terminal protecting group (e.g., acetyl group (--COCH 3 ), succinamide (--CO--C 2 H 4 --COOH), Cbz (benzyloxycarbonyl group), etc.) , or a saturated or unsaturated alkyl group having 1 to 20 carbon atoms.
- N-terminal protecting group e.g., acetyl group (--COCH 3 ), succinamide (--CO--C 2 H 4 --COOH), Cbz (benzyloxycarbonyl group), etc.
- amino acid residues represented by —CO—CHR 3 —NHR 0 in formula (1) include glycine, alanine, leucine, isoleucine, valine, lysine, cysteine, threonine, arginine, asparagine, aspartic acid, glutamine, Glutamic acid, serine, histidine, phenylalanine, methionine, tryptophan, tyrosine, proline, pyroglutamic acid, Lys (Cbz) (a group in which the amino group of the side chain of lysine is substituted with a benzyloxycarbonyl group), citrulline, Met (O2) (methionine (S) in the side chain of sulfur dioxide (SO 2 ), sarcosine, 2-aminobutanoic acid, thioproline, azetidine carbonyl, Tyr (4-NO 2 ) (the hydroxyl group in the side chain of tyrosine is replaced by a
- -NR 2 -CO-L is represented by the following formula.
- R4 is hydrogen, a methyl group, or other natural amino acid (glycine, alanine, leucine, isoleucine, valine, lysine, cysteine, threonine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, serine, histidine , phenylalanine, methionine, tryptophan, tyrosine, proline).
- R4 can also form a ring with the NH nitrogen atom adjacent to the carbon to which it is attached.
- R 4 also includes groups that constitute side chains of unnatural amino acids (citrulline, norvaline, etc.).
- R 4 also includes a group in which a part of the group constituting the side chain of a natural amino acid or non-natural amino acid is substituted or modified with another substituent.
- R 4 also includes groups other than groups constituting side chains of natural amino acids and unnatural amino acids, such as alkyl groups having various substituents.
- R5 is hydrogen, a methyl group, or other natural amino acid (glycine, alanine, leucine, isoleucine, valine, lysine, cysteine, threonine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, serine, histidine , phenylalanine, methionine, tryptophan, tyrosine, proline).
- R5 can also form a ring with a nitrogen atom such as an amino group adjacent to the carbon to which it is attached.
- R5 also includes groups that form side chains of unnatural amino acids ( citrulline, norvaline, etc.).
- R5 also includes a group in which a part of the group constituting the side chain of a natural amino acid or non - natural amino acid is substituted or modified with another substituent.
- R5 also includes groups other than groups constituting side chains of natural amino acids and unnatural amino acids, such as alkyl groups having various substituents.
- formula (2) is an amino group, an N-acetylated amino group, a structure in which an amino group and an amino acid (which may be acetylated at the N-terminus) are bonded, or an amino group and multiple amino acids linked by peptide bonds It represents a structure bound to a peptide (the N-terminus of which may be acetylated, etc.).
- Examples of peptides represented by formula (2a) include Glu-Pro-, Suc-Ala-Ala-Pro-Abu-, Gly-Pro-, Lys-Ala, Phe-Met-, Cbz-Arg-Arg-, D-Ala-Leu-Lys-, Ac-Leu-Leu-Arg-, Lys-His-Leu-Tyr-, Phe-Thr-Thr-Tyr-, Suc-Leu-Leu-Val-Tyr-, Ac-Asp -Glu-Val-Asp-, Ac-Ile-Glu-Thr-Asp-, Ac-Ala-Ala-Pro-Val-, MeOSuc-Ala-Ala-Pro-Val-, Cbz-Gly-Val-Val-, Examples include, but are not limited to, Cbz-Gly-Pro-, Cbz-Ser-Lys-Leu-Gln-, Ac-Leu-Arg-Gly-Gly- and the like.
- the amino group (--NR 2 H) is converted to the group represented by formula (1)
- the appropriately protected amino acid is condensed with a dehydration condensation agent (COMU, HATU, etc.), acid anhydride or An amide bond is formed by preparing and reacting an acid chloride, and the protecting group of the amino acid side chain is deprotected.
- a dehydration condensation agent COMP, HATU, etc.
- An amide bond is formed by preparing and reacting an acid chloride, and the protecting group of the amino acid side chain is deprotected.
- the separately prepared side-chain protected peptide is condensed with a dehydration-condensation agent (COMU, HATU, etc.) to form the side chain. It is carried out by deprotecting the protecting group.
- a dehydration-condensation agent COMP, HATU, etc.
- Conversion of an amino group (--NR 2 H) to a phosphate amide group is carried out by a method using a reagent such as phosphoramidite.
- the ester group is represented by —COOR 3 , where R 3 is a substituted or unsubstituted alkyl group having 1 to 10 carbon atoms, —(C 2 H 4 ) s —CH 3 (s is 1 to 10 are integers).
- Phosphate ester groups include phosphate monoesters and phosphate diesters.
- R' is represented by, for example, WU-
- W is an organic base
- U is a sugar or It is a partial structure of its derivative, or a single bond.
- the U sugar or derivative thereof is ribose, deoxyribose, or derivatives thereof. Ribose, deoxyribose, or derivatives thereof are linked to an organic base at the 1'-position and phosphate-linked at the 5'-position.
- the organic base of W is preferably a nucleobase and derivatives thereof ; It is selected from the group consisting of an amino group represented by an amino group (R'' 2 N--) or an ammonium group.
- R'' is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms and may be the same or different.
- Nucleobases are selected from the group consisting of adenine, thymine, cytosine, guanine and uracil.
- Y is —O—(CH 2 ) n1 —, —O—(CH 2 ) n2 —Ar 1 —, —NH—(CH 2 ) n3 —, or —NH—(CH 2 ) n4 —Ar 2 — is.
- n1, n2, n3 and n4 are each independently an integer of 1-10.
- Ar 1 and Ar 2 are each independently a substituted or unsubstituted arylene group.
- the unsubstituted arylene group for Ar 1 and Ar 2 preferably has 6 to 14 carbon atoms, and specific examples include a phenylene group and a naphthylene group. Among them, a phenylene group is preferable as the unsubstituted arylene group for Ar 1 and Ar 2 .
- substituents possessed by the arylene group include halogen atoms and alkyl groups having 1 to 10 carbon atoms.
- a halogen atom is preferably a chlorine atom, a bromine atom or an iodine atom.
- the alkyl group having 1 to 10 carbon atoms a linear one is preferable, and a methyl group or an ethyl group is more preferable.
- the ether group is represented by —OR 4 , where R 4 is a substituted or unsubstituted alkyl group having 1 to 8 carbon atoms.
- the partial structure of the saccharide of L' in -OL' constitutes a saccharide or a part of the saccharide together with O to which L' is bound.
- Sugars include D-glucose, D-galactose, L-galactose, D-glucopyranose, D-xylose, D-mannose, D-fucose, L-fucose, D-arabinose, L-arabinose, DN-acetyl Glucosamine, DN-acetylgalactosamine, sialic acid, etc., preferably D-glucopyranose.
- a hydroxyl group When converting a hydroxyl group to an ester group, it is carried out by condensing an organic acid with a dehydration condensation agent (COMU, HATU, etc.), adjusting an acid anhydride or an acid chloride, and reacting.
- a dehydration condensation agent COMP, HATU, etc.
- step (1) When converting a hydroxyl group to a phosphate group, the product obtained in step (1) is reacted with a phosphate chloride such as phosphoryl chloride and an analog thereof.
- a phosphate chloride such as phosphoryl chloride and an analog thereof.
- Step (3) the protecting group of the T group of the product obtained in the step (2) is removed.
- the protective group is removed by, for example, exposing the reaction solution to acidic conditions such as trifluoroacetic acid, or using a reagent such as TBAF that gives fluorine ions for deprotection.
- a step of crude purification may be included after removal of the protective group for the T group.
- the step of crude purification includes, but is not limited to, ether precipitation, HPLC elution, and the like.
- Step (4) a compound represented by the following formula (II) is added to the product obtained in steps (4) and (3).
- the step (4) is characterized in that solid-phase extraction is performed using a carrier (compound represented by formula (II)) to which a phos-tag that specifically captures a phosphate group is bound. That is, as shown in FIG. 1, at the stage where the step (3) is completed, the liquid phase contains various reactants, reagents, and products, but the compound represented by the formula (II) can specifically capture a compound having a phosphonic acid group.
- a carrier compound represented by formula (II)
- M is Zn or Cu, preferably Zn.
- P is a carrier.
- any carrier used in solid-phase synthesis and solid-phase extraction can be used. Examples include agarose gel, resin (polystyrene beads, etc.), magnetic beads, and metal nanoparticles. It can be used preferably.
- step (5) the product obtained in step (4) is purified, and then the compound of formula (III) is eluted or eluted.
- wash buffer eg, a solution of 50% H 2 O and 50% MeCN containing Bis Tris-AcOH and NaCl
- a phosphonic acid containing compound typically phosphoric acid
- the pH can be adjusted using an appropriate acid or base.
- an aqueous NH3 solution, an aqueous ethylenediamine solution, or the like can be added to adjust the pH and elute the target compound.
- Another embodiment of the present invention is a compound represented by general formula (III) (hereinafter also referred to as “the compound of the present invention”).
- the compound represented by formula (III) is also referred to as "the fluorescent probe of the present invention”.
- the compound of the present invention can be used as one or more fluorescent probes for detecting enzyme activity, depending on the type of functional group introduced as B.
- the compound represented by formula (III) can be used as a fluorescent probe for detecting amidase activity.
- a carboxyl group etc. are mentioned as a substituent.
- Examples of the substituted or unsubstituted alkyl group having 1 to 8 carbon atoms for R include, but are not limited to, a hexyl group and an ethyl group having a carboxyl group.
- Amino acids, amino acid residues and peptides are as described in detail in step (2).
- the carboxyl group of the side chain of the amino acid is -NR
- a structure in which a carbonyl group is formed by bonding with 2 H to form a part of an amino acid is exemplified.
- amino acid partial structure of L is represented by the following formula (3).
- -NR 2 -CO-L is represented by the following formula. —NH—CO—CHR 3 —NHR 0 (1)
- R3 is hydrogen, a methyl group, or other natural amino acid (glycine, alanine, leucine, isoleucine, valine, lysine, cysteine, threonine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, serine, histidine , phenylalanine, methionine, tryptophan, tyrosine, proline).
- R 3 can also form a ring with the nitrogen atom of —NHR 0 .
- R 3 also includes groups that constitute side chains of unnatural amino acids (citrulline, norvaline, etc.). R 3 also includes groups in which a part of the groups constituting the side chains of natural amino acids or non-natural amino acids are substituted or modified with other substituents. R 3 also includes groups other than groups constituting side chains of natural amino acids and unnatural amino acids, such as alkyl groups having various substituents.
- R 0 is an N-terminal protecting group (e.g., acetyl group (--COCH 3 ), succinamide (--CO--C 2 H 4 --COOH), Cbz (benzyloxycarbonyl group), etc.) , or a saturated or unsaturated alkyl group having 1 to 20 carbon atoms.
- N-terminal protecting group e.g., acetyl group (--COCH 3 ), succinamide (--CO--C 2 H 4 --COOH), Cbz (benzyloxycarbonyl group), etc.
- amino acid residues represented by —CO—CHR 3 —NHR 0 in formula (1) include glycine, alanine, leucine, isoleucine, valine, lysine, cysteine, threonine, arginine, asparagine, aspartic acid, glutamine, Glutamic acid, serine, histidine, phenylalanine, methionine, tryptophan, tyrosine, proline, pyroglutamic acid, Lys (Cbz) (a group in which the amino group of the side chain of lysine is substituted with a benzyloxycarbonyl group), citrulline, Met (O2) (methionine sulfur dioxide (SO 2 ) in the side chain of sulfur (S)), sarcosine, 2-aminobutanoic acid, thioproline, azetidine carbonyl, Tyr (4-NO 2 ) (the hydroxyl group of the side chain of tyrosine is a
- the compound represented by formula (III) can be used as a fluorescent probe for detecting aminopeptidase or protease activity.
- -NR 2 -CO-L is represented by the following formula.
- R4 is hydrogen, methyl group, or other natural amino acid (glycine, alanine, leucine, isoleucine, valine, lysine, cysteine, threonine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, serine, histidine , phenylalanine, methionine, tryptophan, tyrosine, proline).
- R4 can also form a ring with the NH nitrogen atom adjacent to the carbon to which it is attached.
- R 4 also includes groups that constitute side chains of unnatural amino acids (citrulline, norvaline, etc.).
- R 4 also includes a group in which a part of the group constituting the side chain of a natural amino acid or non-natural amino acid is substituted or modified with another substituent.
- R 4 also includes groups other than groups constituting side chains of natural amino acids and unnatural amino acids, such as alkyl groups having various substituents.
- R5 is hydrogen, a methyl group, or other natural amino acid (glycine, alanine, leucine, isoleucine, valine, lysine, cysteine, threonine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, serine, histidine , phenylalanine, methionine, tryptophan, tyrosine, proline).
- R5 can also form a ring with a nitrogen atom such as an amino group adjacent to the carbon to which it is attached.
- R5 also includes groups that form side chains of unnatural amino acids ( citrulline, norvaline, etc.).
- R5 also includes a group in which a part of the group constituting the side chain of a natural amino acid or non - natural amino acid is substituted or modified with another substituent.
- R5 also includes groups other than groups constituting side chains of natural amino acids and unnatural amino acids, such as alkyl groups having various substituents.
- formula (2) is an amino group, an N-acetylated amino group, a structure in which an amino group and an amino acid (which may be acetylated at the N-terminus) are bonded, or an amino group and multiple amino acids linked by peptide bonds It represents a structure bound to a peptide (the N-terminus of which may be acetylated, etc.).
- Examples of peptides represented by formula (2a) include Glu-Pro-, Suc-Ala-Ala-Pro-Abu-, Gly-Pro-, Lys-Ala, Phe-Met-, Cbz-Arg-Arg-, D-Ala-Leu-Lys-, Ac-Leu-Leu-Arg-, Lys-His-Leu-Tyr-, Phe-Thr-Thr-Tyr-, Suc-Leu-Leu-Val-Tyr-, Ac-Asp -Glu-Val-Asp-, Ac-Ile-Glu-Thr-Asp-, Ac-Ala-Ala-Pro-Val-, MeOSuc-Ala-Ala-Pro-Val-, Cbz-Gly-Val-Val-, Examples include, but are not limited to, Cbz-Gly-Pro-, Cbz-Ser-Lys-Leu-Gln-, Ac-Leu-Arg-Gly-Gly- and the like.
- the compound represented by formula (III) can be used as a fluorescent probe for detecting peptidase or protease activity.
- B is a phosphate amide group (—NR 2 —PO(OR a )(OR b ), R a and R b are each independently a hydrogen atom and a branched, linear or cyclic substituted group having 1 to 8 carbon atoms or an unsubstituted alkyl group (one or more non-adjacent, non-terminal C atoms of said alkyl group may be replaced by O, S, CO or COO) selected from the group consisting of In some cases, the compound represented by formula (III) can be used as a fluorescent probe for detecting serine hydrolase activity.
- B is a sulfonamide group (—NR 2 —SO 2 —R c , where R c is a hydrogen atom and a branched, linear or cyclic substituted or unsubstituted alkyl group having 1 to 8 carbon atoms (one of the alkyl groups one or more non-adjacent, non-terminal C atoms may be replaced by O, S, CO or COO) is selected from the group consisting of) is represented by formula (III)
- the compound can be used as a fluorescent probe for detecting the activity of sulfur metabolism-related enzymes such as glutathione S-transferase.
- the ester group is represented by —COOR 3 , where R 3 is a substituted or unsubstituted alkyl group having 1 to 10 carbon atoms, —(C 2 H 4 ) s —CH 3 (s is 1 to 10 are integers).
- the compound represented by formula (III) can be used as a fluorescent probe for detecting esterase activity.
- the compound of formula (III) is a lipase It can be used as a fluorescent probe for activity detection.
- the compound represented by formula (III) can be used as a fluorescent probe for detecting phosphatase activity.
- R' is represented by, for example, WU-
- W is an organic base
- U is It is a partial structure of sugar or its derivative, or a single bond.
- the U sugar or derivative thereof is ribose, deoxyribose, or derivatives thereof. Ribose, deoxyribose, or derivatives thereof are linked to an organic base at the 1'-position and phosphate-linked at the 5'-position.
- the organic base of W is preferably a nucleobase and derivatives thereof ; It is selected from the group consisting of an amino group represented by an amino group (R'' 2 N--) or an ammonium group.
- R'' is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms and may be the same or different.
- Nucleobases are selected from the group consisting of adenine, thymine, cytosine, guanine and uracil.
- the phosphate monoester and phosphate diester may be bonded to the benzene ring via a linker (Y).
- Y is —O—(CH 2 ) n1 —, —O—(CH 2 ) n2 —Ar 1 —, —NH—(CH 2 ) n3 —, or —NH—(CH 2 ) n4 —Ar 2 — is.
- n1, n2, n3 and n4 are each independently an integer of 1-10.
- Ar 1 and Ar 2 are each independently a substituted or unsubstituted arylene group.
- the unsubstituted arylene group for Ar 1 and Ar 2 preferably has 6 to 14 carbon atoms, and specific examples include a phenylene group and a naphthylene group. Among them, a phenylene group is preferable as the unsubstituted arylene group for Ar 1 and Ar 2 .
- substituents possessed by the arylene group include halogen atoms and alkyl groups having 1 to 10 carbon atoms.
- the compound represented by formula (III) can be used as a fluorescent probe for detecting the activity of ENPPs.
- B is a sulfate ester
- the compound represented by formula (III) can be used as a fluorescent probe for detecting sulfatase activity.
- the ether group is represented by —OR 4 , where R 4 is a substituted or unsubstituted alkyl group having 1 to 10 carbon atoms.
- R 4 is a substituted or unsubstituted alkyl group having 1 to 10 carbon atoms.
- B is an ether group
- the compound represented by formula (III) can be used as a fluorescent probe for detecting oxidoreductase activity, such as Cytochrome P450.
- the partial structure of the saccharide of L' in -OL' constitutes a saccharide or a part of the saccharide together with O to which L' is bound.
- Sugars include D-glucose, D-galactose, L-galactose, D-glucopyranose, D-xylose, D-mannose, D-fucose, L-fucose, D-arabinose, L-arabinose, DN-acetyl Glucosamine, DN-acetylgalactosamine, sialic acid, etc., preferably D-glucopyranose.
- the compound represented by formula (III) can be used as a fluorescent probe for detecting ⁇ -glycosidase activity.
- Non-limiting examples of the fluorescent probes of the present invention are shown below.
- the compound represented by the general formula (III) may have one or more asymmetric carbon atoms depending on the type of substituent, and stereoisomers such as optical isomers or diastereoisomers are present. sometimes. All stereoisomers in pure form, any mixtures of stereoisomers, racemates, etc. are included within the scope of the present invention.
- the compound represented by general formula (III) or a salt thereof may exist as a hydrate or solvate, and any of these substances are included within the scope of the present invention.
- the type of solvent that forms the solvate is not particularly limited, but examples include solvents such as ethanol, acetone, and isopropanol.
- the phosphonic acid group is first protected with TBDPS-Cl, the amino group is amidated, then deprotected, and reacted with the phos-tag.
- purification, and pH adjustment to synthesize the fluorescent probe of the present invention is shown in FIG.
- the liquid phase contains various reactants, reagents and products as shown in the middle part of FIG.
- the compound of formula (II) phos-tag-bound carrier
- the compound specifically captures PMAC-AC having a phosphonic acid group, so that subsequent purification and pH adjustment are sufficient.
- the desired fluorescent probe of the present invention can be obtained (see FIG. 4).
- the preparation method of the present invention enables probe synthesis with simple experimental manipulations, and can be performed, for example, in a small plastic tube (eg, 1.5 mL plastic tube).
- a small plastic tube eg, 1.5 mL plastic tube.
- the fluorescent probe of the present invention which is the compound represented by the formula (III), includes one or more types of fluorescent probes for detecting enzyme activity, depending on the type of functional group introduced as B. can be used as Therefore, another embodiment of the present invention is a fluorescent probe for detecting enzyme activity, which contains a compound represented by general formula (III) or a salt thereof.
- Another aspect of the present invention is a method for detecting the activity of a target enzyme in a cell, comprising the steps of (a) introducing the fluorescent probe of the present invention into the cell, and (b) the fluorescent probe a method comprising the step of measuring fluorescence emitted by reaction with the target enzyme in cells.
- the introduction of fluorescent probes into cells can be carried out by using cell lysate, cultured cells, or the like.
- the method of the present invention can further include observing the fluorescence response using fluorescence imaging means.
- a fluorometer having a wide measurement wavelength can be used, but the fluorescence response can also be visualized using fluorescence imaging means capable of displaying a two-dimensional image.
- fluorescence imaging By using the means of fluorescence imaging, the fluorescence response can be visualized in two dimensions, so that the target enzyme can be visualized instantaneously.
- a device known in the art can be used as the fluorescence imaging device.
- it is also possible to detect the reaction between the sample to be measured and the fluorescent probe by means of a change in the ultraviolet-visible absorption spectrum (for example, a change in absorbance at a specific absorption wavelength).
- the method of using the fluorescent probe of the present invention is not particularly limited, and it can be used in the same manner as conventionally known fluorescent probes.
- the compound of the present invention or a salt thereof is added to an aqueous medium such as physiological saline or a buffer solution, or a mixture of a water-miscible organic solvent such as ethanol, acetone, ethylene glycol, dimethylsulfoxide and dimethylformamide and an aqueous medium. is dissolved, the solution is added to an appropriate buffer containing cells or tissues, and the fluorescence spectrum is measured.
- the fluorescent probe of the present invention may be used in the form of a composition in combination with suitable additives. For example, it can be combined with additives such as buffers, solubilizers, and pH adjusters.
- the sample of cells to be measured in the step (a) can be cells expressing the target enzyme, but when such cells are cancer cells or cancer tissues expressing the target enzyme Furthermore, cancer cells and cancer tissues can be detected or visualized by the detection method of the present invention. That is, the fluorescent probe of the present invention, the composition containing the fluorescent probe, and the detection method of the present invention can also be used for prediction or diagnosis of cancer.
- cancer tissue means any tissue that contains cancer cells.
- tissue should be interpreted in the broadest sense to include an organ or a part or the whole of an organ, and should not be interpreted restrictively in any way.
- the cancer tissue is preferably a tissue expressing the target enzyme.
- diagnosis includes confirming the presence of a disease in any living body site, for example, confirming the presence of cancer tissue macroscopically or under a microscope in the case of cancer. should be interpreted in the broadest sense.
- the fluorescent probe of the present invention is usually prepared as a solution. It can also be applied by dissolving in distilled water for injection or an appropriate buffer solution.
- kit may contain other reagents and the like as necessary.
- additives such as dissolution aids, pH adjusters, buffers, tonicity agents, and the like can be used, and the amount of these additives can be appropriately selected by those skilled in the art.
- the target enzyme detection fluorescent probe of the present invention in the well of the microdevice described below, it can be used in a method for detecting the enzymatic activity of the target enzyme in a biological sample. That is, one preferred aspect of the present invention is a fluorescent probe for detecting a target enzyme containing the fluorescent probe of the present invention for use in microdevices.
- Microdevice A microdevice according to one embodiment of the present invention comprises the above-described fluorescent probe for enzyme detection of the present invention.
- the term "microdevice” includes microchamber devices, liposomes, droplets, and the like.
- the activity of the target enzyme in the biological sample can be detected with high quantification and sensitivity.
- the material of the microdevice is not particularly limited, and examples thereof include glass materials, silicon, plastics including dendritic polymers or copolymers, and the like.
- glass materials include soda-lime glass, Pyrex (registered trademark) glass, Vycol (registered trademark) glass, and quartz glass.
- resin polymers include poly(vinyl chloride), poly(vinyl alcohol), poly(methyl methacrylate), poly(vinyl acetate-co-maleic anhydride), poly(dimethylsiloxane) monomethacrylate, cyclic olefin polymers,
- Examples include fluorocarbon polymers, polystyrene, polypropylene, polyethyleneimine, and the like.
- Copolymers include, for example, poly(vinyl acetate-co-maleic anhydride), poly(styrene-co-maleic anhydride), poly(ethylene-co-acrylic acid), or derivatives thereof.
- the shape of the microdevice includes, for example, a multiwell plate in which an arbitrary number of wells (eg, microwells) are arranged, as shown in FIG. The number of wells per plate is, for example, 1 to 10,000,000, such as 10 to 500,000, such as about 100,000.
- the pore diameter of the wells of the microdevice may be, for example, 10 nm or more and 10 ⁇ m or less, for example, 100 nm or more and 10 ⁇ m or less, or for example, 1 ⁇ m or more and 10 ⁇ m or less.
- the well depth of the microdevice may be, for example, 10 nm or more and 100 ⁇ m or less, for example, 100 nm or more and 80 ⁇ m or less, or, for example, 200 nm or more and 70 ⁇ m or less.
- the microdevice may have one type of above-described enzyme-detecting fluorescent probe in one well.
- the fluorescence intensity of one kind of the fluorescent probe of the present invention can be detected for one target enzyme molecule in a biological sample, and the enzymatic activities of one target enzyme molecule can be compared.
- the amount of the fluorescent probe of the present invention contained in one well of the microdevice may be, for example, 100 nM or more and 1000 ⁇ M or less, for example, 1 ⁇ M or more and 1000 ⁇ M or less, for example, 10 ⁇ M or more and 1000 ⁇ M or less.
- a solution containing a biological sample is added to the microdevice. Sealing oil is then dripped to encapsulate the target enzyme in the biological sample within the microdevice well.
- the fluorescent probe of the present invention may be added to the solution containing the biological sample.
- Organic small-molecule fluorescent probes are excellent molecular tools that can detect the activity of enzymes by increasing fluorescence, and are particularly suitable for single-molecule enzymatic activity detection using the microdevices described above.
- the inventors' research has shown that it is possible to detect multiple enzyme activities in biological samples with ultra-high sensitivity by using , and to diagnose diseases by detecting abnormalities. (eg PCT/JP2020/22546, Science Advances 2020).
- fluorescent probes used for such activity detection are usually prepared by synthesizing and purifying a single compound over a period of several days to several months. It was difficult to prepare a group of fluorescent probes that comprehensively detect activity.
- the method for preparing the fluorescent probe of the present invention described above enables probe synthesis and purification in a very simple scheme of only mixing and solution removal, and parallel processing is also possible. Since it is possible to adjust the probes before and after the type in a few days, it is possible to greatly improve the efficiency of development such as probe improvement.
- Another embodiment of the present invention is by performing the following steps (1) to (5) in parallel in a plurality of reaction vessels, one type of compound represented by the following formula (III) is prepared for each vessel in the plurality of reaction vessels.
- m is an integer from 0 to 3;
- n is an integer from 0 to 1;
- step of converting (ii) when A is a hydroxyl group, the hydroxyl group is converted to an ester group, a phosphate group, a sulfate ester group, an ether group or -OL'(L' represents a saccharide or a partial structure of the saccharide); process; (3) a step of removing the protecting group for T of the product obtained in step (2), optionally including a step of crude purification after the removal of said protecting group; (4) adding a compound represented by the following formula (II) to the product obtained in step (3); (In formula (II), M is Zn or Cu; X is a linker group; P is a carrier. ) (5) A step of purifying the product obtained in step (4) and then eluting or eluting the compound of formula (III).
- the compound of formula (I) used in step (1) may be the same or different in each reaction vessel.
- the portion A is converted into a functional group that is cleaved upon contact with a substance to be measured such as an enzyme.
- a substance to be measured such as an enzyme.
- a plurality of small plastic tubes for example, 1 to 30 described in the fluorescent probe preparation method of the present invention are used in parallel. can be done.
- the compounds constituting the group of compounds to be obtained may all be different, or the same compounds may be partially included and most of them may be different. It is preferable that 80% or more of the compounds that constitute the compound group are different, more preferably 90% or more of the compounds that constitute the compound group are different, and 95% or more of the compounds that constitute the compound group are different. It is more preferable that they are different, and it is even more preferable that all the compounds constituting the compound group are different.
- preparation method 2 of the compound of the present invention By performing preparation method 2 of the compound of the present invention, it is possible to synthesize several tens of probes/day.
- various hydrolases (esterase, sulfatase, ENPPs, phosphatase, lipase, glycosidase, amidase, aminopeptidase, peptidase, protease, etc.) It is possible to prepare a group of fluorescent probes capable of detecting
- another aspect of the present invention is a method of constructing a library of compounds of formula (III) comprising multiple iterations of the preparation of compounds of formula (III) (hereafter referred to as “the library of the present invention”). (Also called “Rally construction method”).
- a method for detecting enzyme activity in a biological sample (hereinafter also referred to as the "detection method of the present invention").
- a microdevice can be suitably used in the detection method of the present invention.
- the microdevice used in the detection method of the present invention is equipped with the compound of formula (III) or its library.
- the activities of multiple enzymes in a biological sample can be detected with high quantification and sensitivity.
- microdevice that can be used in the detection method of the present invention are as detailed for the microdevice used in the fluorescent probe for enzyme detection of the present invention.
- the pore diameter of the wells of the microdevice may be, for example, 10 nm or more and 10 ⁇ m or less, for example, 100 nm or more and 10 ⁇ m or less, or for example, 1 ⁇ m or more and 10 ⁇ m or less.
- the well depth of the microdevice may be, for example, 10 nm or more and 100 ⁇ m or less, for example, 100 nm or more and 80 ⁇ m or less, or, for example, 200 nm or more and 70 ⁇ m or less.
- the microdevice may have one compound of formula (III) (fluorescent probe of the present invention) per well.
- the fluorescence intensity of one type of fluorescent probe of the present invention is detected for one molecule of a plurality of enzymes in a biological sample, and the enzymatic activities of each molecule of each of the plurality of enzymes and the compound of the present invention are compared. can do.
- the amount of the fluorescent probe of the present invention contained in one well of the microdevice may be, for example, 100 nM or more and 1000 ⁇ M or less, for example, 1 ⁇ M or more and 1000 ⁇ M or less, for example, 10 ⁇ M or more and 1000 ⁇ M or less.
- a solution containing a biological sample is added to the microdevice. Sealing oil is then dripped to encapsulate the enzymes in the biological sample within the wells of the microdevice.
- the fluorescent probe of the present invention may be added to the solution containing the biological sample.
- a method for detecting the activity of a plurality of enzymes in a biological sample comprises a biological sample (e.g., biological sample isolated from a subject, biopsy sample, body fluid sample, aqueous solution)
- a biological sample e.g., biological sample isolated from a subject, biopsy sample, body fluid sample, aqueous solution
- a method comprising contacting with a compound of (III).
- the biological sample can be a blood sample (eg, a serum sample, or a plasma sample).
- the method may further comprise measuring the fluorescence of the compound after contacting the compound with the aqueous solution. When a compound emits fluorescence, the presence or absence of fluorescence indicates the presence of enzyme activity in aqueous solution, and the intensity of fluorescence indicates the intensity of enzyme activity in aqueous solution.
- One aspect of the present invention is a method of detecting enzymatic activity in a biological sample, comprising contacting a compound of formula (III) with a plurality of enzymes.
- the contact is performed in the presence of serum.
- enzymatic activities of multiple enzymes in a biological sample can be detected. Details of the detection method of the present invention are shown below.
- a solution containing a biological sample is added to a microdevice comprising the compound of formula (III) described above or a library thereof.
- Biological samples include, for example, biological samples isolated from subjects, biopsy samples, body fluid samples, and aqueous solutions.
- the biological sample may be a blood sample (eg, serum sample or plasma sample) Any pH value may be selected for the solution containing the biological sample.
- the pH of the solution can be set to a value close to that in vivo, and in this case, for example, 6.0 or more and 8.0 or less.
- the pH of the solution can be acidified to the pH optimum in order to target specific groups of enzymes.
- a solution containing the biological sample and a solution containing the fluorescent probe of the present invention are separately prepared, the ratio of the biological sample and the fluorescent probe is appropriately adjusted, the two are mixed, and the mixed solution is applied to the microdevice. may be added.
- the protein concentration of the biological sample is usually, for example, 1 pM or more and 100 pM or less, such as 10 pM or more and 100 pM or less, as the concentration of the "target protein".
- the upper limit of the total protein concentration in the sample can be, for example, up to about 10 ⁇ M.
- a method for measuring the protein concentration of a biological sample for example, a method using an antibody-antigen reaction (eg, ELISA method, etc.) can be used as a method for measuring the concentration of the “target protein”.
- Methods for measuring the total protein concentration in a sample include colorimetric methods (e.g., bicinchoninic acid (BCA) method, Bradford method, Lowry method, biuret method, etc.) that utilize reactions between proteins and reagents. be done.
- the biological sample may be diluted with various aqueous solvents or the like so as to achieve the above concentrations.
- the aqueous solvent include water, physiological saline, phosphate buffered saline (PBS), Tris buffered saline (TBS), HEPES buffered saline, and the like. , but not limited to.
- Step 2 A drop of sealing oil is then applied to encapsulate the enzymes in the biological sample within the wells of the microdevice.
- the sealing oil any known oil that is usually used for encapsulating a sample in a microdevice may be used, and examples thereof include fluorine-based oil (FC-40, etc.).
- Step 3 Fluorescence within the wells of the microdevice is then detected using a fluorescence scanner, fluorescence microscope, or the like. Enzyme activity can be evaluated from the detected fluorescence intensity.
- what kind of enzyme is contained in the well of the microdevice where the enzyme activity is detected can be determined, for example, by comparing the separately prepared target protein with the enzyme activity pattern.
- one fluorescent probe of the present invention is brought into contact with a plurality of enzymes in a biological sample, whereby one molecule of a plurality of enzymes in the biological sample is It is possible to detect the fluorescence intensities of the different fluorescent probes of the present invention and compare the enzymatic activities of multiple single enzyme molecules.
- step 1 above it is possible to measure the enzymatic activity of one molecule of the enzyme by preparing wells (for example, multiple wells) containing one molecule of the enzyme and a plurality of fluorescent probes.
- each fluorescent probe contained in the fluorescent probe library of the present invention by contacting each fluorescent probe contained in the fluorescent probe library of the present invention with a plurality of enzymes in the biological sample, a plurality of enzymes in the biological sample are detected. It is possible to detect the fluorescence intensity of each fluorescent probe of the present invention for one molecule and compare the enzymatic activities of one molecule of a plurality of enzymes. It is also possible to screen for more useful fluorescent probes.
- the detection method of the present invention it is possible to detect various disease-related enzymes in the blood at the single-molecule level.
- Yet another aspect of the invention is a method of testing an enzymatic assay of a composition comprising a compound of formula (III), comprising or suspected of comprising said composition and an enzyme that cleaves said compound.
- the method comprising contacting with the biological sample.
- techniques similar to those described in the detection method of the present invention can be used.
- the library of the compound of formula (III) obtained by the library construction method of the present invention contains a plurality of, for example, several hundred types of compounds that can be detected by a plurality of enzymes. Biomarker candidate activities can be comprehensively searched (screened) from biological samples. More specifically, it is possible to screen fluorescent probes useful for diagnosing diseases and to identify enzymatic activities that serve as biomarkers from the results.
- another embodiment of the present invention is a method of using a library of compounds of formula (III) to screen fluorescent probes capable of detecting biomarkers of a particular disease. Microdevices are preferably used in the screening method of the present invention.
- One aspect of the present invention is A method of screening for fluorescent probes capable of detecting biomarkers of a specific disease, the method comprising: (1) adding a library of compounds of formula (III) to a microdevice such that at least one well of the microdevice is provided with one compound of formula (III); (2) adding a solution containing a biological sample to the microdevice so as to create at least one well containing one molecule of enzyme in the microdevice, wherein the biological sample is A biological sample obtained from a patient with a specific disease or a biological sample obtained from a healthy subject; (3) contacting the compound of formula (III) with an enzyme to detect fluorescence in the wells of the microdevice, the step comprising: Contacting the compound of formula (III) with a biological sample obtained from a patient with a specific disease, measuring the fluorescence intensity from formula (III) (first fluorescence intensity), and the compound of formula (III) and a biological sample obtained from a healthy subject, and measuring the fluorescence intensity (second fluorescence intensity) from the formula (
- biological samples examples include biological samples isolated from subjects, biopsy samples, body fluid samples, and aqueous solutions.
- a biological sample can also be a blood sample (eg, a serum sample, or a plasma sample).
- the biological sample is preferably a clinical sample.
- steps (1) to (2) of the screening method of the present invention and the procedure of measuring fluorescence in (3) are performed in the same manner as the procedure described for the detection method of the present invention. Further, in the step (2), as a specific condition for adding so that at least one well containing one molecule of the enzyme is generated, at least one well in one lane stochastically contains one molecule of the enzyme. Add at a concentration greater than or equal to that containing the enzyme. Further, in the screening method of the present invention, in steps (1) and (2), a solution containing the biological sample and a solution containing the compound of formula (III) are prepared separately, and the biological sample and the fluorescent probe are separated.
- the two are mixed, and the mixed solution (i.e., the solution containing the biological sample and the compound of formula (III)) is placed in each well in one lane of the microdevice. It may be added such that about one molecule of enzyme is distributed.
- body fluid samples including blood, urine, saliva, etc.
- microdevices are manufactured. Detect fluorescence in the wells.
- the compound of formula (III) added to the microdevice in the process is determined as a biomarker activity detection fluorescent probe.
- the enzyme activity is significantly different between body fluid samples derived from healthy subjects and diseased patients, the difference is found in diseased patients. It is likely that the biomarker candidate is present in the bodily fluid sample from which it is derived, as it is likely due to the activity of the biomarker candidate in the bodily fluid sample from which it was derived.
- the type of biomarker candidate can then be determined by identifying enzymes contained in the wells for which the assay results of both body fluid samples are significantly different.
- a body fluid sample derived from a diseased patient may contain a plurality of biomarker candidates. It is also possible to determine compounds as biomarker activity detection fluorescent probes.
- the fluorescent probe of the present invention which is the compound represented by formula (III), can be used as a fluorescent probe for detecting one or more enzymatic activities, depending on the type of functional group introduced as B.
- the fluorescent probes of the present invention can be used to detect biomarkers specific to a given disease, thereby diagnosing the disease. That is, another embodiment of the present invention is a method for diagnosing pathological conditions by detecting single-molecule enzymatic activity using the fluorescent probe of the present invention (hereinafter also referred to as "diagnostic method of the present invention").
- FIG. 6 shows a conceptual diagram of disease diagnosis using the present invention.
- the diagram on the left relates to the screening method of the present invention described above, and the diagram on the right shows an outline of a method for diagnosing pathological conditions by detecting single-molecule enzymatic activity.
- the present invention it is expected that it will be possible to diagnose diseases based on information such as the number of enzymes, activity, and activity fluctuations. Examples of fluorescent probes of the invention that can be used for such biomarker detection and non-limiting examples of diagnostic methods of the invention are provided below.
- another embodiment of the present invention is a method for detecting ENPP activity in a biological sample, comprising adding a compound of formula (IV) or a salt thereof and a biological sample in an aqueous solution. wherein an increase in fluorescence intensity in aqueous solution indicates the presence of ENPP activity.
- R 1 , S, T, and m are as described in detail in general formula (I).
- one preferred example of the biological sample is a biological sample from a patient with pancreatic cancer, a patient suspected of having pancreatic cancer, or a healthy subject.
- step (a) the step of applying a fluorescent probe containing the compound of formula (IV) or a salt thereof to a clinical specimen of a subject, and (b) the clinical specimen to which the fluorescent probe is applied
- a method for diagnosing pancreatic cancer, or a method for predicting the possibility that a subject from whom a biological sample is derived has pancreatic cancer hereinafter referred to as "diagnosis or prediction of the present invention (also called Method 1)
- diagnosis or prediction of the present invention also called Method 1
- the application of the fluorescent probe to the clinical sample in step (a) can be performed, for example, by locally spraying a solution of the fluorescent probe onto the clinical sample.
- the diagnostic or predictive method 1 of the present invention can be performed during surgical treatment of pancreatic cancer, or can be performed in vitro. Moreover, the diagnostic or predictive method 1 of the present invention also includes a mode in which medical practice is not included.
- a biological sample obtained from a subject is contacted with a fluorescent probe containing the compound of general formula (IV) or a salt thereof, and (b) the fluorescent
- a method of diagnosing pancreatic cancer comprising measuring the fluorescence intensity of a biological sample brought into contact with a probe, or a method of predicting the possibility that a subject from whom the biological sample is derived has pancreatic cancer (hereinafter referred to as Also referred to as “diagnostic or predictive method 2 of the present invention").
- the diagnostic or predictive methods 1 and 2 of the present invention are based on detecting ENPP enzymatic activity using the compound of formula (IV) or a salt thereof.
- ENPP it is preferable to detect the enzymatic activity of ENPP3, which is specifically found in pancreatic cancer.
- Diagnosis or prediction method 2 of the present invention can preferably be performed using a microdevice.
- Diagnosis or prediction method 2 of the present invention comprises preparing a solution containing a biological sample and a compound of general formula (IV) or a salt thereof, adding the solution to a microdevice, encapsulating the added solution into each well, It can be performed by incubating the microdevice enclosing the solution and then measuring the fluorescence intensity of each well with a fluorescence microscope.
- the diagnostic method of the present invention can include counting the number of wells in which the detected fluorescence intensity is equal to or higher than a predetermined intensity, that is, the number of wells in which ENPP is considered to be encapsulated.
- diagnosis or prediction method 2 of the present invention is performed, for example, as follows, but is not limited thereto.
- a compound of general formula (IV) is diluted in assay buffer to a predetermined concentration (eg, 200 ⁇ M).
- the composition of the assay buffer can be determined appropriately, and can be, for example, 100 mM Tris-HCl (pH 9.3), 1 mM MgCl 2 , 0.5% (w/v) CHAPS.
- a biological sample preferably a blood sample (e.g., serum sample or plasma sample) collected from a subject (including both pancreatic cancer patients and healthy subjects) is diluted with the above assay buffer (e.g., , about 250 times), and mixed with a diluted solution of the compound of general formula (IV) at a predetermined ratio (e.g., equal volume).
- a mixed solution of both was added to the microdevice, followed by the addition of sealing oil to seal the solution into each well.
- the solution-encapsulated microdevice is incubated under predetermined conditions (eg, 25° C. for 40 minutes), and then the fluorescence intensity of each well is measured with a fluorescence microscope.
- the number of wells in which the detected fluorescence intensity is equal to or higher than a predetermined intensity (for example, 2500 AU or higher), that is, the number of wells in which ENPP is considered to be encapsulated is counted.
- ROC curve for determination is prepared based on the results of ENPP activity measurement using the compound of formula (IV) or a salt thereof, and the possibility of pancreatic cancer is determined by the ROC curve.
- the compound of general formula (VI) is preferably the following compound.
- Another embodiment of the present invention is a fluorescent probe for detecting pancreatic cancer for use in diagnostic or prognostic methods 1 and 2 of the present invention, comprising a compound of general formula (IV) or a salt thereof.
- the method of using the fluorescent probe for pancreatic cancer detection is the same as described for the fluorescent probe of the present invention.
- Another embodiment of the present invention is a kit for detecting pancreatic cancer cells or tissues, containing the compound of general formula (IV) or a salt thereof.
- the compound of general formula (IV) or a salt thereof is usually prepared as a solution. It can also be applied by dissolving in distilled water for injection or an appropriate buffer at the time of use.
- kit may contain other reagents and the like as necessary.
- additives such as dissolution aids, pH adjusters, buffers, tonicity agents, and the like can be used, and the amount of these additives can be appropriately selected by those skilled in the art.
- the compound of general formula (VI) is preferably PMUM-dCMP described above.
- Another embodiment of the present invention is a method for detecting CD13 activity in a biological sample, wherein B is —NR 2 — in general formula (III)
- B is —NR 2 — in general formula (III)
- compound A of the present invention the compound of the present invention
- the portion of -CO-L is an alanine, lysine, arginine or methionine residue
- a biological sample are mixed in an aqueous solution.
- the above method comprising contacting, wherein an increase in fluorescence intensity in aqueous solution is indicative of the presence of CD13 activity.
- Another embodiment of the present invention uses a microdevice, and when B is -NR 2 -CO-L in general formula (III), the portion of -CO-L is alanine, lysine, arginine or methionine residue or This is a method for predicting the possibility that a subject from whom a biological sample is derived has pancreatic cancer (hereinafter also referred to as "diagnostic or predictive method A1 of the present invention").
- a biological sample obtained from a subject is contacted with a fluorescent probe containing the compound A of the present invention
- a method of diagnosing pancreatic cancer comprising measuring the fluorescence intensity of a biological sample brought into contact with a fluorescent probe, or a method of predicting the possibility that a subject from whom the biological sample is derived has pancreatic cancer (
- diagnosis or predictive method A2 of the present invention it is also referred to as “diagnostic or predictive method A2 of the present invention”.
- the diagnosis or prediction methods A1 and A2 of the present invention are based on detecting the enzymatic activity of one molecule of CD13 using the compound A of the present invention.
- Another embodiment of the present invention is a method for detecting the activity of DPP4 in a biological sample, wherein in general formula (III), when B is -NR 2 -CO-L, the -CO-L moiety is -Pro-Xaa (Pro represents a proline residue, Xaa represents an amino acid residue such as glycine, serine, glutamic acid, etc.) peptide of the present invention (hereinafter also referred to as "compound B of the present invention”) and a biological sample in an aqueous solution, wherein an increase in fluorescence intensity in the aqueous solution indicates the presence of DPP4 activity.
- Another embodiment of the present invention uses a microdevice, and when B is -NR 2 -CO-L in general formula (III), the portion of -CO-L is -Pro-Xaa (Pro is proline residue, Xaa represents an amino acid residue such as glycine, serine, glutamic acid, etc.), using a fluorescent probe containing the compound B of the present invention, one of DPP4 in a biological sample obtained from a subject.
- a method for diagnosing pancreatic cancer or a method for predicting the possibility that a subject from whom a biological sample is derived has pancreatic cancer by detecting molecular enzymatic activity hereinafter referred to as "diagnostic or predictive method of the present invention B1”).
- a biological sample obtained from a subject is contacted with a fluorescent probe containing the compound B of the present invention
- a method of diagnosing pancreatic cancer comprising measuring the fluorescence intensity of a biological sample brought into contact with a fluorescent probe, or a method of predicting the possibility that a subject from whom the biological sample is derived has pancreatic cancer ( Hereinafter, it is also referred to as “diagnosis or prediction method B2 of the present invention”).
- the diagnosis or prediction methods B1 and B2 of the present invention are based on detecting the enzymatic activity of one molecule of DPP4 using the compound B of the present invention.
- Another embodiment of the present invention uses a microdevice, and when B is -NR 2 -CO-L in general formula (III), the portion of -CO-L is an alanine, lysine, arginine or methionine residue , using a fluorescent probe containing the compound of the present invention to detect single-molecule enzymatic activity of CD13 in a biological sample obtained from a subject, and using a microdevice, in general formula (III)
- the portion of -CO-L is a peptide of -Pro-Xaa (Pro is a proline residue, Xaa is an amino acid residue such as glycine, serine, glutamic acid, etc.)
- a method for diagnosing pancreatic cancer by detecting the enzymatic activity of a single molecule of DPP4 in a biological sample obtained from a subject, using a fluorescent probe containing the compound of the present invention, or a method derived from a biological sample
- a solution containing the biological sample and the compound A (or B) of the present invention is prepared, the solution is added to the microdevice, and the added solution into each well, incubating the solution-encapsulated microdevice, and then measuring the fluorescence intensity of each well with a fluorescence microscope.
- the diagnostic or predictive method of the present invention can include counting the number of wells in which the detected fluorescence intensity is equal to or higher than a predetermined intensity, that is, the number of wells in which CD13 (or DPP4) is considered to be encapsulated. .
- Diagnostic or predictive methods A1, A2, B1 and B2 of the present invention, a combination of diagnostic or predictive methods A1 and B1 of the present invention, a combination of diagnostic or predictive methods A2 and B2 of the present invention, etc. also referred to as "diagnostic or predictive method"
- pancreatic cancer patients patients suspected of having pancreatic cancer (high risk of pancreatic cancer, diabetes patients, people with pancreatic cancer in the family, smokers, etc.) and healthy subjects.
- the diagnostic or predictive method of the present invention is not limited to pancreatic cancer patients or patients suspected of having pancreatic cancer, but also to healthy middle-aged and elderly humans (healthy subjects) to detect the possibility of pancreatic cancer. This is done for the purpose of confirmation, and for subjects with a high possibility of pancreatic cancer, imaging tests (precision tests) such as MRI can also contribute to finding early pancreatic cancer.
- the diagnostic or predictive method of the present invention can be performed during surgical treatment of pancreatic cancer, and can also be performed in vitro. Moreover, the diagnostic or predictive method of the present invention also includes a mode in which medical practice is not included.
- the diagnostic or predictive methods A1 and A2 of the present invention are performed, for example, as follows, but are not limited thereto.
- Compound A of the present invention is diluted in assay buffer to a given concentration (eg, 100 ⁇ M).
- the composition of the assay buffer can be determined appropriately, and can be, for example, 100 mM HEPES-NaOH (pH 7.4), 1 mM MgCl 2 , 1 mM CaCl 2 , 3 mM Triton X-100.
- a biological sample preferably a blood sample (e.g., serum sample or plasma sample) collected from a subject (including both pancreatic cancer patients and healthy subjects) is diluted with the above assay buffer (e.g., , about 10,000 times), and mixed with a diluted solution of the compound A of the present invention at a predetermined ratio (e.g., equal volume).
- a mixed solution of both was added to the microdevice, followed by the addition of sealing oil to seal the solution into each well.
- the solution-encapsulated microdevice is incubated under predetermined conditions (eg, 37° C. for 2 hours), and then the fluorescence intensity of each well is measured with a fluorescence microscope.
- the number of wells in which the detected fluorescence intensity is equal to or higher than a predetermined intensity (for example, 2500 AU or higher), that is, the number of wells in which CD13 is considered to be encapsulated is counted.
- ROC curve for determination is prepared based on the results of measuring CD13 activity using the compound A of the invention, and the possibility of pancreatic cancer is determined by the ROC curve.
- the diagnostic or predictive methods B1 and B2 of the present invention are performed, for example, as follows, but are not limited thereto.
- Compound B of the invention is diluted in assay buffer to a given concentration (eg, 100 ⁇ M).
- the composition of the assay buffer can be determined appropriately, and may be 100 mM HEPES-NaOH (pH 7.4), 1 mM MgCl 2 , 1 mM CaCl 2 , 3 mM Triton X-100.
- a biological sample preferably a blood sample (e.g., serum sample or plasma sample) collected from a subject (including both pancreatic cancer patients and healthy subjects) is diluted with the above assay buffer (e.g., , about 2,500 times), and mixed with a diluted solution of the compound B of the present invention at a predetermined ratio (e.g., equal volume).
- a mixed solution of both was added to the microdevice, followed by the addition of sealing oil to seal the solution into each well.
- the solution-encapsulated microdevice is incubated under predetermined conditions (eg, 25° C. for 2 hours), and then the fluorescence intensity of each well is measured with a fluorescence microscope.
- a histogram created based on the fluorescence intensity of each well is fitted to a curve consisting of two normal distributions. Wells with fluorescence intensity higher than the minimum value of the approximation curve are defined as the high activity group, and wells with low fluorescence intensity are defined as the low activity group.
- the compound B of the present invention is used to diagnose and predict the possibility that the subject from whom the biological sample is derived has pancreatic cancer from the proportion of the highly active group measured above.
- a ROC curve for determination is created based on the results of measuring DPP4 activity using the ROC curve, and the possibility of pancreatic cancer is determined based on the ROC curve.
- Non-limiting examples of compound A of the present invention are the following compounds.
- Non-limiting examples of compounds B of the present invention are the following compounds.
- the beads were washed five times with 1 mL of wash buffer (80% H 2 O and 20% MeCN, pH 6.8, containing 100 mM Bis Tris-AcOH). 7. The beads were washed three times with 1 mL washing solution (80% H2O and 20% MeCN). 8. Compounds were eluted three times with 300 ⁇ L of eluent (80% H 2 O and 20% MeCN, containing 1% ethylenediamine), the solution was neutralized by adding 500 ⁇ L of H 2 O (containing 2% AcOH), and then frozen. Dried.
- FIG. 2 shows the results of HPLC analysis of the compound composition in the solution during the process from .
- Load The composition of the solution added to the beads in the step of 6. 2 shows the solution composition of the supernatant removed after the target substance was retained on the beads at the stage of . 7. Wash. The solution composition of each supernatant washed three times at the stage of is shown. 8. Elute. The composition of the solution eluted at the stage of
- the residue was added to 1 mL of loading buffer (80% Bis Tris buffer (1 M, pH 6.8) and 20% MeCN) and loaded onto 500 ⁇ L of phos-tag beads. 7.
- the beads were washed five times with 1 mL of wash buffer (80% H 2 O and 20% MeCN, pH 6.8, containing 100 mM Bis Tris-AcOH).
- the beads were washed three times with 1 mL washing solution (80% H2O and 20% MeCN).
- Compounds were eluted three times with 300 ⁇ L of eluent (80% H 2 O and 20% MeCN, containing 1% ethylenediamine), the solution was neutralized by adding 500 ⁇ L of H 2 O (containing 2% AcOH), and then frozen. Dried.
- Assay Protocol1 Compounds 5 to 51, which are aminopeptidase, amidase, and protease activity detection fluorescent probes, were diluted with assay buffer so that the concentration of each compound was 100 ⁇ M.
- the assay buffer composition is 100 mM HEPES-NaOH (pH 7.4), 1 mM MgCl 2 , 1 mM DTT, 3 mM Triton X-100. 2.
- a human plasma sample (derived from a healthy subject or pancreatic cancer patient) was diluted 500-fold with the above-mentioned assay buffer and mixed with an equal volume of probe diluent.
- the final concentrations of both are 50 ⁇ M for the fluorescent probe and 1000-fold dilution for the human plasma sample. 3.
- a mixed solution of both was then added to the microdevice, followed by the addition of sealing oil to seal the solution into each well. 4.
- the fluorescence intensity of each well was measured with a fluorescence microscope. Here, an increase in fluorescence intensity was observed in the wells containing the enzyme that reacts with the fluorescent probe.
- FIGS. 7A and 7B Fluorescent microscope images obtained by performing the above assay for the fluorescent probes of compounds 5-51 are shown in FIGS. 7A and 7B.
- the image on the left side of each fluorescence microscope image in FIGS. 7A and 7B (labeled Healthy) is the result when using a human plasma sample derived from a healthy subject, and the image on the right side (labeled Tumor) is from a pancreatic cancer patient. of human plasma samples.
- the type of amino acid residue or peptide possessed by each fluorescent probe is indicated on the left side of each fluorescence microscope image.
- the synthetic scheme of the present invention can be useful for various fluorescent probes.
- Example 2 Measurement of Human Plasma Samples Using CD13 Probes in Microdevices
- Arg-PMAC and Met-PMAC which are CD13 activity detection probes, to biomarker detection was conducted by the following procedure. rice field.
- the CD13 probe, Arg-PMAC was diluted in assay buffer to 100 ⁇ M.
- the buffer composition is 100 mM HEPES-NaOH (pH 7.4), 1 mM MgCl 2 , 1 mM DTT, 3 mM Triton X-100.
- human plasma samples collected from 30 pancreatic cancer patients and 30 healthy subjects were diluted 500-fold with the above-mentioned assay buffer and mixed with a diluted solution of CD13 probe in an equal volume. At this time, the final concentrations of both are 50 ⁇ M for the CD13 probe and 1000-fold dilution for the human plasma sample. 3.
- a mixed solution of both was then added to the microdevice, followed by the addition of sealing oil to seal the solution into each well.
- FIG. 8 shows fluorescence microscope images obtained using Arg-PMAC. 5.
- a ROC curve was created for the number of wells in which the detected fluorescence intensity was above the separately measured background fluorescence Average + 3SD, that is, the number of wells in which CD13 was considered to be encapsulated. got the numbers. 6.
- Example 3 Measurement of Human Plasma Sample Using DPP4 Probe with Microdevice
- an experiment to verify the applicability of Glu-Pro-PMAC, a DPP4 activity detection probe, to biomarker detection was conducted by the following procedure.
- the DPP4 probe Glu-Pro-PMAC
- the buffer composition is 100 mM HEPES-NaOH (pH 7.4), 1 mM MgCl 2 , 1 mM DTT, 3 mM Triton X-100.
- human plasma samples collected from 30 pancreatic cancer patients and 30 healthy subjects were diluted 500-fold with the above-mentioned assay buffer and mixed with the diluent of the DPP4 probe in an equal volume. At this time, the final concentrations of both are 50 ⁇ M for the DPP4 probe and 1000-fold dilution for the human plasma sample. 3.
- the eluent is evaporated to remove the solvent, the residue is dissolved in 1 mL of loading buffer (80% H 2 O and 20% MeCN, pH 6.8, containing 100 mM bis tris-AcOH) and 500 ⁇ L of phos-tag carried on beads. 6. Wash the beads five times with 1 mL of wash buffer (80% H 2 O and 20% MeCN, pH 6.8, containing 100 mM bis tris-AcOH) and 1 mL of rinse solution (80% H 2 O and 20% MeCN). was washed twice with 7. Compounds were eluted with three 300 ⁇ L elution solutions (80% H 2 O and 20% MeCN, containing 1% ethylenediamine) and the solutions were lyophilized.
- loading buffer 80% H 2 O and 20% MeCN, pH 6.8, containing 100 mM bis tris-AcOH
- rinse solution 80% H 2 O and 20% MeCN
- Example 4 Evaluation of activity of PMUM-dCMP on ENPP The activity of PMUM-dCMP obtained in Synthesis Example 7 was measured using a plate reader using the purified ENPP3 enzyme. Assays were performed in Tris-HCl buffer (pH 9.3) containing 1 mM MgCl 2 , 0.5% w/w CHAPS, 10 ⁇ M PMUM-dCMP, and ENPP3 (recombinant, 9.9 ⁇ g/mL). The results are shown in FIG. As shown in FIG. 13, when ENPP3 was added, a significant increase in fluorescence intensity (FI) was observed compared to when ENPP3 was not added.
- FI fluorescence intensity
- Example 5 Investigation of fluorescent probes for detecting biomarker activity of pancreatic cancer
- plasma samples derived from 3 healthy subjects and 3 patients with pancreatic cancer were used for the purpose of searching for fluorescent probes for detecting biomarker activity of pancreatic cancer.
- PMUM-dCMP were used to perform enzymatic assays in microdevices.
- the protocol for the enzyme assay is as follows. 1. Compounds of formula (III) were diluted in assay buffer.
- the assay buffer composition is 100 mM HEPES-NaOH (pH 5-10), 1 mM MgCl 2 , 3 mM Triton X-100. 2.
- the human plasma sample was then diluted with the assay buffer described above and mixed in equal volumes with the probe diluent. At this time, the final concentrations of both are 10-100 ⁇ M for the probe and 500-1000 fold dilution for the human plasma sample. 3.
- a mixed solution of both was then added to the microdevice, followed by the addition of sealing oil to seal the solution into each well. 4.
- the fluorescence intensity of each well was measured with a fluorescence microscope. 5. Image analysis was performed, and there were compounds with different enzymatic activities in the presence or absence of disease.
- Example 6 Measurement of Human Plasma Samples Using ENPP Probes in Microdevices
- an experiment to verify the applicability of PMUM-dCMP to biomarker detection was performed by the following procedure.
- the assay buffer composition is 100 mM Tris-HCl (pH 9.3), 1 mM MgCl 2 , 0.5% (w/v) CHAPS.
- human plasma samples collected from 6 pancreatic cancer patients and 6 healthy subjects were diluted 250-fold with the above-mentioned assay buffer and mixed with the diluent of the ENPP probe in an equal volume. At this time, the final concentrations of both are 100 ⁇ M for the ENPP probe and 500-fold dilution for the human plasma sample. 3.
- a mixed solution of both was then added to the microdevice, followed by the addition of sealing oil to seal the solution into each well. 4.
- FIG. 14 shows a fluorescence microscope image. 5.
- pancreatic cancer As described above, measurement of the fluorescence intensity of human plasma samples using the ENPP probe PMUM-dCMP with a microdevice revealed a significant difference in pancreatic cancer patients compared to healthy subjects. From this result, it is possible to discover ENPP3 specifically found in pancreatic cancer by comparing the enzymatic activities of ENPP in biological samples derived from healthy and diseased subjects using PMUM-dCMP. It is possible. Specifically, in this example, a ROC curve for making determinations based on the enzymatic activity of ENPP determined based on the number of wells with AU2500 or higher was created, and the threshold was drawn at 23 wells with AU2500 or higher. , resulting in a sensitivity of 83% and a specificity of 100% (FIG. 16). Pancreatic cancer can be determined by such a ROC curve.
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Abstract
Description
また、このスキームにおいては、最終的に得られる蛍光プローブがホスホン酸を有するため、マイクロデバイスを用いたアッセイに求められる高い水溶性を付与することも可能であり、本スキームは本発明の目的に最適なものであると考えられる。
[1] 以下の一般式(I)で表される化合物。
(式(I)において、
Aは、アミノ基(-NR2H)又は水酸基(-OH)であり、
ここで、R2は、水素原子、及び炭素数1~8の分岐、直鎖又は環状の置換又は無置換のアルキル基(当該アルキル基の1つ以上の非隣接の、非末端のC原子は、O、S、CO又はCOOで置き換えられていてもよい)からなる群から選択され;
R1は、存在する場合は、ベンゼン環上に存在する同一又は異なる一価の置換基であり:Sは、存在する場合は、リンカーであり;
Tは、ホスホン酸基(-P(=O)(OH)2)、リン酸エステル基(-O-P(=O)(OH)2)又はリン酸アミド基(-NH-P(=O)(OH)2)から選択され;
mは、0~3の整数である。)
[2]Aが-NR2Hである、[1]に記載の化合物。
[3]Aが-OHである、[1]に記載の化合物。
[4](1)以下の式(I)で表される化合物のTの基を保護する工程;
(式(I)において、
Aは、アミノ基(-NR2H)又は水酸基(-OH)であり、
ここで、R2は、水素原子又は炭素数1~8の置換又は無置換のアルキル基であり;
R1は、存在する場合は、ベンゼン環上に存在する同一又は異なる一価の置換基であり:Sは、存在する場合は、リンカーであり;
Tは、ホスホン酸基(-P(=O)(OH)2)、リン酸エステル基(-O-P(=O)(OH)2)又はリン酸アミド基(-NH-P(=O)(OH)2)から選択され;
mは、0~3の整数である。)
(2)(1)の工程で得られる生成物について、
(i)Aがアミノ基(-NR2H)の場合は、当該アミノ基を、アミド基(-NR2C(=O)R、Rは水素原子又は炭素数1~8のアルキル基である)、-NR2-CO-L(Lは、アミノ酸の部分構造を表す)、リン酸アミド基(-NR2-PO(ORa)(ORb)、Ra及びRbは、各々独立に、水素原子、及び炭素数1~8の分岐、直鎖又は環状の置換又は無置換のアルキル基(当該アルキル基の1つ以上の非隣接の、非末端のC原子は、O、S、CO又はCOOで置き換えられていてもよい)からなる群から選択される)、又はスルホンアミド基(-NR2-SO2-Rc、Rcは、水素原子、及び炭素数1~8の分岐、直鎖又は環状の置換又は無置換のアルキル基(当該アルキル基の1つ以上の非隣接の、非末端のC原子は、O、S、CO又はCOOで置き換えられていてもよい)からなる群から選択される)に変換する工程、
(ii)Aが水酸基の場合は、当該水酸基をエステル基、リン酸エステル基、硫酸エステル基、エーテル基又は-O-L’(L’は、糖類又は糖類の部分構造を表す)に変換する工程;
(3)(2)の工程で得られる生成物のTの保護基を除去する工程であって、場合により、前記保護基の除去後に粗精製の工程を含んでもよい;
(4)(3)の工程で得られる生成物に、以下の式(II)で表される化合物を添加する工程;
(式(II)において、
Mは、Zn又はCuであり;
Xは、リンカー基であり;
Pは、担体である。)
(5)(4)の工程で得られる生成物を精製し、その後、式(III)の化合物を溶離又は溶出させる工程;
を含む、以下の式(III)で表される化合物を調製する方法。
(式(III)中、
Bは、アミド基(-NR2C(=O)R、Rは水素原子又は炭素数1~8のアルキル基である)、-NR2-CO-L(Lは、アミノ酸の部分構造を表す)、リン酸アミド基(-NR2-PO(ORa)(ORb)、Ra及びRbは、各々独立に、水素原子、及び炭素数1~8の分岐、直鎖又は環状の置換又は無置換のアルキル基(当該アルキル基の1つ以上の非隣接の、非末端のC原子は、O、S、CO又はCOOで置き換えられていてもよい)からなる群から選択される)、スルホンアミド基(-NR2-SO2-Rc、Rcは、水素原子、及び炭素数1~8の分岐、直鎖又は環状の置換又は無置換のアルキル基(当該アルキル基の1つ以上の非隣接の、非末端のC原子は、O、S、CO又はCOOで置き換えられていてもよい)からなる群から選択される)、エステル基、リン酸エステル基、硫酸エステル基、エーテル基又は-O-L’(L’は、糖類又は糖類の部分構造を表す)から選択され;
S、T、R1、mは、式(I)で定義した通りである。)
[5]複数の反応容器において並列的に以下の(1)~(5)の工程を行うことにより、前記複数の反応容器内で容器毎に1種類の以下の式(III)で表される化合物を調製する方法。
(式(III)中、
Bは、アミド基(-NR2C(=O)R、Rは水素原子又は炭素数1~8のアルキル基である)、-NR2-CO-L(Lは、アミノ酸の部分構造を表す)、リン酸アミド基(-NR2-PO(ORa)(ORb)、Ra及びRbは、各々独立に、水素原子、及び炭素数1~8の分岐、直鎖又は環状の置換又は無置換のアルキル基(当該アルキル基の1つ以上の非隣接の、非末端のC原子は、O、S、CO又はCOOで置き換えられていてもよい)からなる群から選択される)、又はスルホンアミド基(-NR2-SO2-Rc、Rcは、水素原子、及び炭素数1~8の分岐、直鎖又は環状の置換又は無置換のアルキル基(当該アルキル基の1つ以上の非隣接の、非末端のC原子は、O、S、CO又はCOOで置き換えられていてもよい)からなる群から選択される)、エステル基、リン酸エステル基、硫酸エステル基、エーテル基又は-O-L’(L’は、糖類又は糖類の部分構造を表す)から選択され;
S、T、R1、mは、式(I)で定義する通りである。)
(1)以下の式(I)で表される化合物のTの基を保護する工程;
(式(I)において、
Aは、アミノ基(-NR2H)又は水酸基(-OH)であり、
ここで、R2は、水素原子、及び炭素数1~8の分岐、直鎖又は環状の置換又は無置換のアルキル基(当該アルキル基の1つ以上の非隣接の、非末端のC原子は、O、S、CO又はCOOで置き換えられていてもよい)からなる群から選択され;
R1は、存在する場合は、ベンゼン環上に存在する同一又は異なる一価の置換基であり:Sは、存在する場合は、リンカーであり;
Tは、ホスホン酸基(-P(=O)(OH)2)、リン酸エステル基(-O-P(=O)(OH)2)又はリン酸アミド基(-NH-P(=O)(OH)2)から選択され;
mは、0~3の整数である。)
(2)(1)の工程で得られる生成物について、
(i)Aがアミノ基(-NR2H)の場合は、当該アミノ基を、アミド基(-NR2C(=O)R、Rは水素原子又は炭素数1~8のアルキル基である)、-NR2-CO-L(Lは、アミノ酸の部分構造を表す)、リン酸アミド基(-NR2-PO(ORa)(ORb)、Ra及びRbは、各々独立に、水素原子、及び炭素数1~8の分岐、直鎖又は環状の置換又は無置換のアルキル基(当該アルキル基の1つ以上の非隣接の、非末端のC原子は、O、S、CO又はCOOで置き換えられていてもよい)からなる群から選択される)、又はスルホンアミド基(-NR2-SO2-Rc、Rcは、水素原子、及び炭素数1~8の分岐、直鎖又は環状の置換又は無置換のアルキル基(当該アルキル基の1つ以上の非隣接の、非末端のC原子は、O、S、CO又はCOOで置き換えられていてもよい)からなる群から選択される)に変換する工程、
(ii)Aが水酸基の場合は、当該水酸基をエステル基、リン酸エステル基、硫酸エステル基、エーテル基又は-O-L’(L’は、糖類又は糖類の部分構造を表す)に変換する工程;
(3)(2)の工程で得られる生成物のTの保護基を除去する工程であって、場合により、前記保護基の除去後に粗精製の工程を含んでもよい;
(4)(3)の工程で得られる生成物に、以下の式(II)で表される化合物を添加する工程;
(式(II)において、
Mは、Zn又はCuであり;
Xは、リンカー基であり;
Pは、担体である。)
(5)(4)の工程で得られる生成物を精製し、その後、式(III)の化合物を溶離又は溶出させる工程。
[6]以下の一般式(III)で表される化合物又はその塩。
(式(III)中、
Bは、アミド基(-NR2C(=O)R、Rは水素原子又は炭素数1~8のアルキル基である)、-NR2-CO-L(Lは、アミノ酸の部分構造を表す)、リン酸アミド基(-NR2-PO(ORa)(ORb)、Ra及びRbは、各々独立に、水素原子、及び炭素数1~8の分岐、直鎖又は環状の置換又は無置換のアルキル基(当該アルキル基の1つ以上の非隣接の、非末端のC原子は、O、S、CO又はCOOで置き換えられていてもよい)からなる群から選択される)、スルホンアミド基(-NR2-SO2-Rc、Rcは、水素原子、及び炭素数1~8の分岐、直鎖又は環状の置換又は無置換のアルキル基(当該アルキル基の1つ以上の非隣接の、非末端のC原子は、O、S、CO又はCOOで置き換えられていてもよい)からなる群から選択される)、エステル基、リン酸エステル基、硫酸エステル基、エーテル基又は-O-L’(L’は、糖類又は糖類の部分構造を表す)から選択され;
R1は、存在する場合は、ベンゼン環上に存在する同一又は異なる一価の置換基であり:Sは、存在する場合は、リンカーであり;
Tは、ホスホン酸基(-P(=O)(OH)2)、リン酸エステル基(-O-P(=O)(OH)2)又はリン酸アミド基(-NH-P(=O)(OH)2)から選択され;
mは、0~3の整数である。)
[7][6]に記載の一般式(III)で表される化合物又はその塩を含む、酵素活性の検出用蛍光プローブ。
[8]生体試料中の複数の酵素の活性を検出する方法であって、生体試料と[6]に記載の一般式(III)の化合物とを接触させることを含む、該方法。
[9][6]に記載の一般式(III)の化合物のライブラリーを用いることを特徴とする、[8]に記載の方法。
[10]マイクロデバイスを用いることを特徴とする、[8]又は[9]に記載の方法。
[11][6]に記載の一般式(III)の化合物を含む組成物の酵素アッセイの試験方法であって、前記組成物と、前記化合物を切断する酵素を含む、または含むと疑われる生体試料と接触させることを含む、該方法。
[12]特定疾患のバイオマーカーを検出できる蛍光プローブをスクリーニングする方法であって、当該方法は、
(1)[6]に記載の式(III)の化合物のライブラリーをマイクロデバイスに添加する工程であって、当該マイクロデバイスの少なくとも1ウェルに1種類の式(III)の化合物を備えるように添加し;
(2)当該マイクロデバイスに生体試料を含む溶液を添加する工程であって、当該マイクロデバイスにおいて、1分子の酵素を含む少なくとも1つのウェルが生じるように添加し、ここで、前記生体試料は、特定疾患患者から得られた生体試料又は健常者から得られた生体試料であり;
(3)式(III)の化合物と酵素とを接触させて、マイクロデバイスのウェル内の蛍光を検出する工程であって、当該工程は、
式(III)の化合物と、特定疾患患者から得られた生体試料とを接触させて、式(III)の化合物からの蛍光強度(第1の蛍光強度)を測定すること、及び
式(III)の化合物と、健常者から得られた生体試料とを接触させて、式(III)の化合物からの蛍光強度(第2の蛍光強度)を測定すること、
を含み;
(4)前記第1の蛍光強度と前記第2の蛍光強度とを比較して差がある場合に、当該化合物が、前記蛍光プローブの候補であることが示されることにより、当該化合物をバイオマーカー活性検出蛍光プローブと判定する工程;
を含む、前記スクリーニング方法。
[13]生体試料においてENPPの活性を検出する方法であって、式(IV)の化合物又はその塩と生体試料とを水溶液中で接触させることを含み、水溶液における蛍光強度の増加は、ENPPの活性の存在を示す、前記方法。
式(IV)において、R1、S、T、mは、一般式(I)において詳述した通りである。
[14]前記生体試料が、すい臓がん患者、すい臓がんを有すると疑われる患者又は健常者の生体試料である、[13]に記載の方法。
[15](a)式(IV)の化合物又はその塩を含む蛍光プローブを被検体の臨床検体に適用する工程、及び(b)前記蛍光プローブを適用した臨床検体の蛍光像を測定することを含む、すい臓がんを診断する方法、または、生体試料が由来する被検者がすい臓がんである可能性を予測する方法。
[16](a)被検者から得られた生体試料を、式(IV)の化合物又はその塩を含む蛍光プローブと接触させる工程、及び(b)前記蛍光プローブと接触させた生体試料の蛍光強度を測定することを含む、すい臓がんを診断する方法、または、生体試料が由来する被検者がすい臓がんである可能性を予測する方法。
[17]式(VI)の化合物が以下の化合物である、[15]又は[16]に記載の診断方法又は、予測方法。
[18]式(IV)の化合物又はその塩を含む、[15]又は[16]に記載の方法において用いるためのすい臓がん検出用の蛍光プローブ。
[19]式(IV)の化合物又はその塩を含む、すい臓がん細胞又は組織の検出用キット。
[20]式(VI)の化合物が以下の化合物である、[18]に記載の蛍光プローブ。
[21]
マイクロデバイスを用いて、一般式(III)においてBが-NR2-CO-Lであって、-CO-Lの部分がアラニン、リジン、アルギニン又はメチオニン残基である、[6]に記載の化合物又はその塩を含む蛍光プローブを用いて、被検者から得られた生体試料中のCD13の1分子酵素活性を検出することにより、すい臓がんを診断する方法、または、生体試料が由来する被検者がすい臓がんである可能性を予測する方法。
[22]
マイクロデバイスを用いて、一般式(III)においてBが-NR2-CO-Lであって、-CO-Lの部分が-Pro-Xaa(Proはプロリン残基、Xaaはグリシン、セリン、グルタミン酸などのアミノ酸残基を表す)のペプチドである、[6]に記載の化合物又はその塩を含む蛍光プローブを用いて、被検者から得られた生体試料中のDPP4の1分子酵素活性を検出することにより、すい臓がんを診断する方法、または、生体試料が由来する被検者がすい臓がんである可能性を予測する方法。
[23]
マイクロデバイスを用いて、一般式(III)においてBが-NR2-CO-Lであって、-CO-Lの部分がアラニン、リジン、アルギニン又はメチオニン残基である、[6]に記載の化合物又はその塩を含む蛍光プローブを用いて、被検者から得られた生体試料中のCD13の1分子酵素活性を検出すること、及び
マイクロデバイスを用いて、一般式(III)においてBが-NR2-CO-Lであって、-CO-Lの部分が-Pro-Xaa(Proはプロリン残基、Xaaはグリシン、セリン、グルタミン酸などのアミノ酸残基を表す)のペプチドである、[6]に記載の化合物又はその塩を含む蛍光プローブを用いて、被検者から得られた生体試料中のDPP4の1分子酵素活性を検出することにより、すい臓がんを診断する方法、または、生体試料が由来する被検者がすい臓がんである可能性を予測する方法。
[24]前記生体試料が、すい臓がん患者、すい臓がんを有すると疑われる患者又は健常者の生体試料である、[21]~[23]のいずれか1項に記載の方法。
を提供するものである。
本発明の化合物又はそのライブラリーを用いて、生体試料中の複数の酵素の活性を検出する方法を提供することができる。
また、本発明の化合物1はホスホン酸、リン酸エステル基又はリン酸アミド基を有することにより、当該化合物を用いて最終的に得られる蛍光プローブもホスホン酸、リン酸エステル又はリン酸アミドを有するため、マイクロデバイスを用いたアッセイに求められる高い水溶性を付与することが可能となる。
ここで、当該アルキル基の1つ以上の非隣接の、非末端のC原子は、O、S、CO又はCOOで置き換えられていてもよい。従って、R2として規定するアルキル基には、例えば、アルキル基の一部にポリエチレングリコール鎖が含まれる場合や、R2の全体がポリエチレングリコール鎖である場合も含まれる。
また、R2のアルキル基が有することができる置換基としては、例えば、アルコキシ基、水酸基、カルボキシル基、ハロゲン原子、スルホ基、アミノ基、アルコキシカルボニル基、オキソ基などを挙げることができるが、これらに限定されることはない。
ここで、R1のアルキル基の1つ以上の非隣接の、非末端のC原子は、O、S、CO又はCOOで置き換えられていてもよい。従って、R1のアルキル基には、例えば、アルキル基の一部にポリエチレングリコール鎖が含まれる場合や、R1の全体がポリエチレングリコール鎖である場合も含まれる。
R1の一価の置換基は、クマリン骨格のベンゼン環上の任意の位置に導入することができる。
リンカーとしては、置換又は無置換の炭化水素基、ポリエチレングリコール、複素環基、アミド基(-NHCO-、-CONH-で表される何れの基も含む)、アミド基を有する芳香族環等が挙げられるが、これらに限定されない。また、本発明の化合物1に用いることができるリンカーには、上記で上げた2種類以上の基が結合したものも含まれる。
炭化水素基としては、炭素数1~10のアルキレン基、炭素数1~10のアルキニレン基、シクロアルキレン基、芳香族炭化水素が挙げられる。
ポリエチレングリコールは、-(C2H4-O)t-(tは1~10の整数である)で表され、エチレン基、オキソ基のいずれもがクマリンと結合する側であってもよい。
複素環としては、トリアゾール基等が挙げられる。
アミド基を有する芳香族環は、*-NHCO-Ar-又は*-Ar-CONH-(Arは、芳香族環を表し、*はクマリンと結合する側を表す)で表すことができる。
また、ポリエチレングリコール、複素環基、アミド基、アミド基を有する芳香族環の片方又は両方の端部に、置換又は無置換の炭化水素基、好ましくは炭素数1~10のアルキレン基を有していてもよい。
(1)一般式(III)で表される化合物を調製する方法
本発明のもう1つの実施態様は、以下の式(III)で表される化合物を調製する方法であって、以下の(1)~(5)の工程を含む、該方法である(以下「本発明の調製方法」とも言う)。
(1) 以下の式(I)で表される化合物のTの基を保護する工程;
(式(I)において、
Aは、アミノ基(-NR2H)又は水酸基(-OH)であり、
ここで、R2は、水素原子、及び炭素数1~8の分岐、直鎖又は環状の置換又は無置換のアルキル基(当該アルキル基の1つ以上の非隣接の、非末端のC原子は、O、S、CO又はCOOで置き換えられていてもよい)からなる群から選択され;
R1は、存在する場合は、ベンゼン環上に存在する同一又は異なる一価の置換基であり: Sは、存在する場合は、リンカーであり;
Tは、ホスホン酸基(-P(=O)(OH)2)、リン酸エステル基(-O-P(=O)(OH)2)又はリン酸アミド基(-NH-P(=O)(OH)2)から選択され;
mは、0~3の整数である。)
(2)(1)の工程で得られる生成物について、
(i)Aがアミノ基(-NR2H)の場合は、当該アミノ基を、アミド基(-NR2C(=O)R、Rは水素原子又は炭素数1~8のアルキル基である)、-NR2-CO-L(Lは、アミノ酸の部分構造を表す)、リン酸アミド基(-NR2-PO(ORa)(ORb)、Ra及びRbは、各々独立に、水素原子、及び炭素数1~8の分岐、直鎖又は環状の置換又は無置換のアルキル基(当該アルキル基の1つ以上の非隣接の、非末端のC原子は、O、S、CO又はCOOで置き換えられていてもよい)からなる群から選択される)、又はスルホンアミド基(-NR2-SO2-Rc、Rcは、水素原子、及び炭素数1~8の分岐、直鎖又は環状の置換又は無置換のアルキル基(当該アルキル基の1つ以上の非隣接の、非末端のC原子は、O、S、CO又はCOOで置き換えられていてもよい)からなる群から選択される)に変換する工程、
(ii)Aが水酸基の場合は、当該水酸基をエステル基、リン酸エステル基、硫酸エステル基、エーテル基又は-O-L’(L’は、糖類又は糖類の部分構造を表す)に変換する工程;
(3)(2)の工程で得られる生成物のTの保護基を除去する工程であって、場合により、前記保護基の除去後に粗精製の工程を含んでもよい;
(4)(3)の工程で得られる生成物に、以下の式(II)で表される化合物を添加する工程;
(式(II)において、
Mは、Zn又はCuであり;
Xは、リンカー基であり;
Pは、担体である。)
(5)(4)の工程で得られる生成物を精製し、その後、式(III)の化合物を溶離又は溶出させる工程。
液相合成法は、反応効率は高いが、精製操作にカラムクロマトグラフィーや分取HPLCを用いるため煩雑であり、精製に数時間/化合物が求められる。また、固相合成法は、精製操作は簡便であるが反応効率が低いという問題がある。本発明の調製方法は、両者の利点を組み合わせた、SAS(Synthesis-based on affinity separation)法に基づき、混和・溶液除去の操作のみで目的物を(好ましくは純度高く)得ることができる。本発明の調製方法では、並列処理で多種類の蛍光プローブを簡便に合成することも可能である。
(2)の工程では、(1)の工程で得られる生成物について、Aの部分を酵素などの測定対象物質との接触により切断される官能基に変換する。
測定対象物質との接触により切断される官能基は、最終生成物である化合物(III)を用いて行う酵素活性などの測定において標的とする生体分子の種類に応じて適宜に定められるが、Aがアミノ基である場合と水酸基である場合により、以下の(i)と(ii)に分けられる。
ここで、アミノ酸の部分構造とは、それが結合しているC=Oと一緒になって、アミノ酸、アミノ酸残基、ペプチド、アミノ酸の一部を構成していることを意味する。
また、アミノ酸のN末端は、Nアシル化(例えば、Nアセチル化等)、Nカルバモイル化されていてもよい。
アミノ酸残基には、αアミノ酸の残基、βアミノ酸の残基、γアミノ酸の残基が含まれる。
-NH-CO-CHR3-NHR0 (1)
式(1)において、R3は、水素、メチル基等の、天然アミノ酸(グリシン、アラニン、ロイシン、イソロイシン、バリン、リジン、システイン、トレオニン、アルギニン、アスパラギン、アスパラギン酸、グルタミン、グルタミン酸、セリン、ヒスチジン、フェニルアラニン、メチオニン、トリプトファン、チロシン、プロリン)の側鎖を構成する基を表す。R3は、NHR0の窒素原子と環を構成することもできる。
R3には、非天然アミノ酸(シトルリン、ノルバリンなど)の側鎖を構成する基も含まれる。また、R3は、天然アミノ酸や非天然アミノ酸の側鎖を構成する基の一部が他の置換基で置換又は修飾された基も含まれる。また、R3には、天然アミノ酸や非天然アミノ酸の側鎖を構成する基以外の基、例えば、種々の置換基を有するアルキル基なども含まれる。
式(1)の-CO-CHR3-NHR0で表されるアミノ酸残基は、L体、D体のいずれであってもよい。
は、アミノ基、Nアセチル化等されたアミノ基、アミノ基とアミノ酸(N末端がアセチル化等されていてもよい)が結合した構造、又は、アミノ基と複数のアミノ酸がペプチド結合により連なったペプチド(N末端がアセチル化等されていてもよい)と結合した構造を表す。
Uの糖又はその誘導体が、リボース、デオキシリボース、又はこれらの誘導体である。リボース、デオキシリボース、又はこれらの誘導体は、1’位で有機塩基と結合しており、5’位でリン酸結合している。
Wの有機塩基は、好ましくは、核酸塩基及びその誘導体;コリンの部分構造((CH3)3N+-C2H4-)、ジエチルアミノ基((CH3CH2)2N-)、及びアミノ基(R’’2N-)で表されるアミノ基又はアンモニウム基からなる群から選択される。ここで、R’’は、水素原子又は炭素数1~10のアルキル基であり、同一であっても異なっていてもよい。
核酸塩基は、アデニン、チミン、シトシン、グアニン及びウラシルからなる群から選択される。
Yは、-O-(CH2)n1-、-O-(CH2)n2-Ar1-、-NH-(CH2)n3-、又は、-NH-(CH2)n4-Ar2-である。
上記リンカーにおいて、左右いずれの方向の結合手がベンゼン環と結合してもよいが、好ましくは、O又はNHはベンゼン環に結合する。
n1、n2、n3及びn4はそれぞれ独立に1~10の整数である。
Ar1及びAr2はそれぞれ独立に置換又は無置換のアリーレン基である。
Ar1及びAr2における前記無置換のアリーレン基としては、炭素数6~14のものが好ましく、具体的にはフェニレン基、ナフチレン基等が挙げられる。中でも、Ar1及びAr2における前記無置換のアリーレン基としては、フェニレン基が好ましい。
アリーレン基が有する置換基としては、例えば、ハロゲン原子、炭素数1~10のアルキル基等が挙げられる。
ハロゲン原子としては、塩素原子、臭素原子又はヨウ素原子であることが好ましい。
炭素数1~10のアルキル基としては、直鎖状のものが好ましく、メチル
基又はエチル基がより好ましい。
糖類としては、D-グルコース、D-ガラクトース、L-ガラクトース、D-グルコピラノース、D-キシロース、D-マンノース、D-フコース、L-フコース、D-アラビノース、L-アラビノース、D-N-アセチルグルコサミン、D-N-アセチルガラクトサミン、シアル酸等が挙げられ、好ましくは、D-グルコピラノースである。
(3)の工程においては、(2)の工程で得られる生成物のTの基の保護基が除去される。
保護基の除去は、例えば、反応液をトリフルオロ酢酸などの酸性条件下にさらすことや、TBAFなどのフッ素イオンを与える試薬を用いて脱保護を行う。
ここで、粗精製の工程としては、エーテル沈殿、HPLCによる溶離などが挙げられるが、これらに限定されない。
リンカー基としては、例えば、C1-C6アルキレン基,アミノ基(-NH-),エーテル基(-O-),チオエーテル基(-S-)、カルボニル基(-C(=O)-)、チオニル基(-C(=S)-)、エステル基、アミド基、ウレア基(-NHC(=O)NH-)、チオウレア基(-NHC(=S)NH-)、ポリエチレングリコール;および、アミノ基、エーテル基、チオエーテル基、カルボニル基、チオニル基、エステル基、アミド基、ウレア基、チオウレア基、ポリエチレングリコールからなる群より選択される基を一端に有するC1-C6アルキレン基;アミノ基、エーテル基、チオエーテル基、カルボニル基、チオニル基、エステル基、アミド基、ウレア基、チオウレア基からなる群より選択される同一または異なった基を両端に有するC1-C6アルキレン基;およびこれら基からなる群より選択される2以上の基が直線状に結合された基を挙げることができる。
リンカー基として、好ましくは、アミド基を一端に有するC1-C6アルキレン基、ポリエチレングリコール、および、これらの構造をアミド結合やトリアゾール構造でつないだものである。
Pの担体としては、固相合成および固相抽出で用いられる担体であればいずれのものも使用することができるが、例えば、アガロースゲル、樹脂(ポリスチレンビーズ等)、磁気ビーズ、金属ナノ粒子を好適に用いることができる。
(5)の工程においては、(4)の工程で得られる生成物を精製し、その後、式(III)の化合物を溶離又は溶出させる。
pHの調整は、適宜の酸や塩基を用いて行うことができるが、例えば、NH3水溶液、エチレンジアミン水溶液等を添加してpHを調整し、目的化合物を溶離することができる。
置換基としては、カルボキシル基等が挙げられる。
Rの炭素数1~8の置換又は無置換のアルキル基としては、例えば、ヘキシル基、カルボキシル基を有するエチル基等が挙げられるが、これらに限定されない。
アミノ酸、アミノ酸残基及びペプチドについては、工程(2)において詳述した通りである。
-NH-CO-CHR3-NHR0 (1)
式(1)において、R3は、水素、メチル基等の、天然アミノ酸(グリシン、アラニン、ロイシン、イソロイシン、バリン、リジン、システイン、トレオニン、アルギニン、アスパラギン、アスパラギン酸、グルタミン、グルタミン酸、セリン、ヒスチジン、フェニルアラニン、メチオニン、トリプトファン、チロシン、プロリン)の側鎖を構成する基を表す。R3は、-NHR0の窒素原子と環を構成することもできる。
R3には、非天然アミノ酸(シトルリン、ノルバリンなど)の側鎖を構成する基も含まれる。また、R3は、天然アミノ酸や非天然アミノ酸の側鎖を構成する基の一部が他の置換基で置換又は修飾された基も含まれる。また、R3には、天然アミノ酸や非天然アミノ酸の側鎖を構成する基以外の基、例えば、種々の置換基を有するアルキル基なども含まれる。
式(1)の-CO-CH2R3-NHR0で表されるアミノ酸残基は、L体、D体のいずれであってもよい。
は、アミノ基、Nアセチル化等されたアミノ基、アミノ基とアミノ酸(N末端がアセチル化等されていてもよい)が結合した構造、又は、アミノ基と複数のアミノ酸がペプチド結合により連なったペプチド(N末端がアセチル化等されていてもよい)と結合した構造を表す。
Uの糖又はその誘導体が、リボース、デオキシリボース、又はこれらの誘導体である。リボース、デオキシリボース、又はこれらの誘導体は、1’位で有機塩基と結合しており、5’位でリン酸結合している。
Wの有機塩基は、好ましくは、核酸塩基及びその誘導体;コリンの部分構造((CH3)3N+-C2H4-)、ジエチルアミノ基((CH3CH2)2N-)、及びアミノ基(R’’2N-)で表されるアミノ基又はアンモニウム基からなる群から選択される。ここで、R’’は、水素原子又は炭素数1~10のアルキル基であり、同一であっても異なっていてもよい。
核酸塩基は、アデニン、チミン、シトシン、グアニン及びウラシルからなる群から選択される。
Yは、-O-(CH2)n1-、-O-(CH2)n2-Ar1-、-NH-(CH2)n3-、又は、-NH-(CH2)n4-Ar2-である。
上記リンカーにおいて、左右いずれの方向の結合手がベンゼン環と結合してもよいが、好ましくは、O又はNHはベンゼン環に結合する。
n1、n2、n3及びn4はそれぞれ独立に1~10の整数である。
Ar1及びAr2はそれぞれ独立に置換又は無置換のアリーレン基である。
Ar1及びAr2における前記無置換のアリーレン基としては、炭素数6~14のものが好ましく、具体的にはフェニレン基、ナフチレン基等が挙げられる。中でも、Ar1及びAr2における前記無置換のアリーレン基としては、フェニレン基が好ましい。
アリーレン基が有する置換基としては、例えば、ハロゲン原子、炭素数1~10のアルキル基等が挙げられる。
Bが硫酸エステルである場合は、式(III)で表される化合物はスルファターゼの活性検出用蛍光プローブとして用いることができる。
Bがエーテル基である場合は、式(III)で表される化合物はCytochrome P450などの酸化還元酵素の活性検出用蛍光プローブとして用いることができる。
糖類としては、D-グルコース、D-ガラクトース、L-ガラクトース、D-グルコピラノース、D-キシロース、D-マンノース、D-フコース、L-フコース、D-アラビノース、L-アラビノース、D-N-アセチルグルコサミン、D-N-アセチルガラクトサミン、シアル酸等が挙げられ、好ましくは、D-グルコピラノースである。
図2の上段に示すPMACのアミド化が終了した段階で、液相中には図2の中段に示すように様々な反応物、試薬、生成物が含まれている。ここで、式(II)の化合物(phos-tagの結合した担体)を添加すると、当該化合物はホスホン酸基を有するPMAC-ACを特異的に捕捉するため、その後、精製及びpH調整するだけで目的とする本発明の蛍光プローブを取得することができる(図4参照)。
上記の通り、式(III)で表される化合物である本発明の蛍光プローブは、Bとして導入した官能基の種類に応じて、1種以上の酵素活性検出用の蛍光プローブとして用いることができる。
従って、本発明のもう1つの実施態様は、一般式(III)で表される化合物又はその塩を含む酵素活性検出用の蛍光プローブである。
ここで、蛍光プローブを細胞内に導入するには、細胞破砕液や培養細胞等を使うことにより行うことができる。
「組織」の用語は臓器又は器官の一部又は全体を含めて最も広義に解釈しなければならず、いかなる意味においても限定的に解釈してはならない。がん組織としては標的酵素を発現している組織が好ましい。また、本明細書において「診断」の用語は任意の生体部位において疾患の存在を確認すること、例えば、がんの場合にはがん組織の存在を肉眼的又は顕微鏡下に確認することを含めて最も広義に解釈する必要がある。
即ち、本発明の1つの好ましい態様は、マイクロデバイス用である本発明の蛍光プローブを含む標的酵素検出用蛍光プローブである。
本発明の一実施形態に係るマイクロデバイスは、上述の本発明の酵素検出用蛍光プローブを備えるものである。
本明細書において、「マイクロデバイス」には、マイクロチャンバー型デバイス、リポソーム、ドロップレット等が含まれる。
また、マイクロデバイスの形状は、例えば、図5に示すように、任意の数のウェル(例えば、マイクロウェル)が配置されたマルチウェルプレート等が挙げられる。ウェルの数としては、プレート1枚当たり、例えば1個以上1000万個以下、例えば10個以上50万個以下、例えば10万個程度等が挙げられる。
また、マイクロデバイスのウェルの深さは、例えば10nm以上100μm以下であればよく、例えば100nm以上80μm以下であればよく、例えば200nm以上70μm以下であればよい。
孔径及び深さが上記範囲内であることにより、ウェル内に1分子の標的酵素を捕捉することができ、生体試料中の酵素1分子ずつの酵素活性を検出することができる。
これにより、生体試料中の標的酵素1分子に対して、1種類の本発明の蛍光プローブの蛍光強度を検出し、標的酵素1分子同士の酵素活性を比較することができる。
マイクロデバイスの使用方法としては、まず、生体試料を含む溶液をマイクロデバイスに添加する。次いで、マイクロデバイスのウェル内の生体試料中の標的酵素を封入するために、シーリングオイルを滴下する。
また、上記の生体試料を含む溶液には、本発明の蛍光プローブを添加してもよい。
有機小分子蛍光プローブは、酵素の活性を蛍光増大によって検出することが可能な優れた分子ツールであり、特に、上記したマイクロデバイスを用いた1分子酵素活性検出にこれを利用することで,生体サンプル中の複数の酵素活性を超高感度に検出し、その異常を検出することで疾患の診断をおこなうことが可能であることが発明者らの研究によって示されている(例えば、PCT/JP2020/22546、Science Advances 2020)。
一方、このような活性検出に用いる蛍光プローブは、通常、ひとつの化合物を数日~数ヶ月の期間をかけて合成、精製して調整されることから、血液中などに存在する多様な酵素の活性を網羅的に検出する蛍光プローブ群を調整することは困難であった。
これに対し,上記した本発明の蛍光プローブの調製方法は、混和、溶液除去だけの非常に簡便なスキームでプローブ合成・精製を行うことが可能であり、並列処理も可能であることにより、10種類前後のプローブを数日で調整することも可能であることから、プローブ改良等の開発の大幅な効率化が可能である。
複数の反応容器において並列的に以下の(1)~(5)の工程を行うことにより、前記複数の反応容器内で容器毎に1種類の以下の式(III)で表される化合物を調製する方法である。
(式(III)中、
Bは、アミド基(-NR2C(=O)R、Rは水素原子又は炭素数1~8のアルキル基である)、-NR2-CO-L(Lは、アミノ酸の部分構造を表す)、リン酸アミド基(-NR2-PO(ORa)(ORb)、Ra及びRbは、各々独立に、水素原子又は炭素数1~8のアルキル基である)、スルホンアミド基(-NR2-SO2-Rc、Rcは、水素原子又は炭素数1~8のアルキル基である)、エステル基、リン酸エステル基、硫酸エステル基、エーテル基又は-O-L’(L’は、糖類又は糖類の部分構造を表す)から選択され;
R1、m、nは、式(I)で定義する通りである。)
(1) 以下の式(I)で表される化合物のTの基を保護する工程;
(式(I)において、
Aは、アミノ基(-NR2H)又は水酸基(-OH)であり、
ここで、R2は、水素原子又は炭素数1~8の置換又は無置換のアルキル基であり;
R1は、存在する場合は、ベンゼン環上に存在する同一又は異なる一価の置換基であり:Tは、ホスホン酸基(-P(=O)(OH)2)、リン酸エステル基(-O-P(=O)(OH)2)又はリン酸アミド基(-NH-P(=O)(OH)2)から選択され;
mは、0~3の整数であり;
nは、0~1の整数である。)
(2)(1)の工程で得られる生成物について、
(i)Aがアミノ基(-NR2H)の場合は、当該アミノ基を、アミド基(-NR2C(=O)R、Rは水素原子又は炭素数1~8のアルキル基である)、-NR2-CO-L(Lは、アミノ酸の部分構造を表す)、リン酸アミド基(-NR2-PO(ORa)(ORb)、Ra及びRbは、各々独立に、水素原子又は炭素数1~8のアルキル基である)、又はスルホンアミド基(-NR2-SO2-Rc、Rcは、水素原子又は炭素数1~8のアルキル基である)に変換する工程、
(ii)Aが水酸基の場合は、当該水酸基をエステル基、リン酸エステル基、硫酸エステル基、エーテル基又は-O-L’(L’は、糖類又は糖類の部分構造を表す)に変換する工程;
(3)(2)の工程で得られる生成物のTの保護基を除去する工程であって、場合により、前記保護基の除去後に粗精製の工程を含んでもよい;
(4)(3)の工程で得られる生成物に、以下の式(II)で表される化合物を添加する工程;
(式(II)において、
Mは、Zn又はCuであり;
Xは、リンカー基であり;
Pは、担体である。)
(5)(4)の工程で得られる生成物を精製し、その後、式(III)の化合物を溶離又は溶出させる工程。
本発明の化合物の調製方法2で用いる工程(1)~(5)は、本発明の調製方法で詳述したのと同様である。
工程(1)で用いる式(I)の化合物が各反応容器で同じである場合は、工程(2)において、Aの部分を酵素などの測定対象物質との接触により切断される官能基に変換するには、各反応容器で異なる試薬を用いるのが好ましい。
化合物群を構成する化合物の80%以上が相違していることが好ましく、化合物群を構成する化合物の90%以上が相違していることがより好ましく、化合物群を構成する化合物の95%以上が相違していることが更に好ましく、化合物群を構成する全ての化合物が相違していることがより更に好ましい。
また、発明の化合物の調製方法2により、図3で模式的に示すように、様々な加水分解酵素(エステラーゼ、スルファターゼ、ENPP類、ホスファターゼ、リパーゼ、グリコシダーゼ、アミダーゼ、アミノペプチダーゼ、ペプチダーゼ、プロテアーゼ等)を検出することができる蛍光プローブ群を一度に調製することが可能である。
本発明のもう1つの実施態様は、式(III)の化合物(即ち、本発明の化合物)又はそのライブラリーを用いて、生体試料中の複数の酵素の活性を検出する方法である(以下「本発明の検出方法」とも言う)。
また、マイクロデバイスのウェルの深さは、例えば10nm以上100μm以下であればよく、例えば100nm以上80μm以下であればよく、例えば200nm以上70μm以下であればよい。
孔径及び深さが上記範囲内であることにより、ウェル内に1分子の酵素を捕捉することができ、生体試料中の酵素1分子ずつの酵素活性を検出することができる。
これにより、生体試料中の複数の酵素1分子に対して、1種類の本発明の蛍光プローブの蛍光強度を検出し、複数の酵素の各々と本発明の化合物について1分子同士の酵素活性を比較することができる。
マイクロデバイスの使用方法としては、まず、生体試料を含む溶液をマイクロデバイスに添加する。次いで、マイクロデバイスのウェル内の生体試料中の酵素を封入するために、シーリングオイルを滴下する。また、上記の生体試料を含む溶液には、本発明の蛍光プローブを添加してもよい。
本発明の検出方法について、以下に詳細を示す。
まず、上述の式(III)の化合物又はそのライブラリーを備えるマイクロデバイスに、生体試料を含む溶液を添加する。生体試料としては、例えば、被検者から単離された生体試料、生検試料、体液試料、水溶液が挙げられる。また、生体試料は、血液試料(例えば、血清試料、または血漿試料)であり得る
生体試料を含む溶液のpHは任意の値を選んでよい。例えば、溶液のpHは、生体内に近しい値にすることができ、この場合は、例えば6.0以上8.0以下であればよい。また、特定の酵素群に標的を絞るために、溶液のpHを至適pHに当たる酸性にすることもできる。
また、生体試料を含む溶液と、本発明の蛍光プローブを含む溶液を別々に調製して、生体試料と蛍光プローブの比率を適切に調整して両者を混合して、混合した溶液をマイクロデバイスに添加してもよい。
また、試料中の全体のタンパク質濃度としては、例えば上限を10μM程度までにすることもできる。
生体試料のタンパク質濃度の測定方法は、「目的とするタンパク質」の濃度の測定方法としては、例えば、抗体抗原反応を利用した方法(例えば、ELISA法等)を用いることができる。また、試料中の全体のタンパク質濃度の測定方法としては、タンパク質と試薬との反応を利用した比色法(例えば、ビシンコニン酸(BCA)法、ブラッドフォード法、ローリー法、ビウレット法等)が挙げられる。
生体試料は上記濃度となるように、各種水性溶媒等を用いて、希釈してもよい。前記水性溶媒としては、例えば、水、生理食塩水、リン酸緩衝生理食塩水(Phosphate buffered saline;PBS)、トリス緩衝生理食塩水(Tris Buffered Saline;TBS)、HEPES緩衝生理食塩水等が挙げられ、これらに限定されない。
次いで、マイクロデバイスのウェル内の生体試料中の酵素を封入するために、シーリングオイルを滴下する。シーリングオイルとしては、通常マイクロデバイスにおいて試料の封入用途で用いられる公知のものであればよく、例えば、フッ素系オイル(FC-40等)等が挙げられる。
次いで、蛍光スキャナー、蛍光顕微鏡等を用いて、マイクロデバイスのウェル内の蛍光を検出する。検出された蛍光強度から、酵素活性を評価することができる。
本試験方法では、上記した本発明の検出方法で記載した内容と同様の技術を用いることができる。
本発明のライブラリー構築方法で得られる式(III)の化合物のライブラリーは、複数の酵素の検出が可能な複数、例えば数百種類の化合物を包含することから、これを用いて、生体試料からバイオマーカー候補活性を網羅的に検索(スクリーニング)することができる。より具体的には、疾患の診断に有用な蛍光プローブをスクリーニングし、その結果からバイオマーカーとなる酵素活性を同定することが可能である。
即ち、本発明のもう1つの実施態様は、式(III)の化合物のライブラリーを用いて、特定疾患のバイオマーカーを検出できる蛍光プローブをスクリーニングする方法である。本発明のスクリーニング方法では、マイクロデバイスを用いることが好ましい。
特定疾患のバイオマーカーを検出できる蛍光プローブをスクリーニングする方法であって、当該方法は、
(1)式(III)の化合物のライブラリーをマイクロデバイスに添加する工程であって、当該マイクロデバイスの少なくとも1ウェルに1種類の式(III)の化合物を備えるように添加し;
(2)当該マイクロデバイスに生体試料を含む溶液を添加する工程であって、当該マイクロデバイスにおいて、1分子の酵素を含む少なくとも1つのウェルが生じるように添加し、ここで、前記生体試料は、特定疾患患者から得られた生体試料又は健常者から得られた生体試料であり;
(3)式(III)の化合物と酵素とを接触させて、マイクロデバイスのウェル内の蛍光を検出する工程であって、当該工程は、
式(III)の化合物と、特定疾患患者から得られた生体試料とを接触させて、式(III)からの蛍光強度(第1の蛍光強度)を測定すること、及び
式(III)の化合物と、健常者から得られた生体試料とを接触させて、式(III)からの蛍光強度(第2の蛍光強度)を測定すること、
を含み;
(4)前記第1の蛍光強度と前記第2の蛍光強度とを比較して差がある場合に、当該化合物が、前記蛍光プローブの候補であることが示されることにより、当該化合物をバイオマーカー活性検出蛍光プローブと判定する工程;
を含む、前記スクリーニング方法である(以下「本発明のスクリーニング方法」とも言う)。
本発明のスクリーニング方法により、式(III)の化合物のライブラリーから、バイオマーカー活性を検出できる蛍光プローブの候補化合物を超高感度かつ網羅的に検索(スクリーニング)することが可能である。
また、(2)の工程において、1分子の酵素を含む少なくとも1つのウェルが生じるように添加するための具体的な条件としては、確率論的に1レーンのうち少なくとも1つのウェルが1分子の酵素を含むような濃度以上の濃度で添加する。
また、本発明のスクリーニング方法においては、(1)と(2)の工程について、生体試料を含む溶液と、式(III)の化合物を含む溶液を別々に調製して、生体試料と蛍光プローブの比率を適切に調整して両者を混合して、混合した溶液(即ち、生体試料と式(III)の化合物を含む溶液)をマイクロデバイスの1レーン内の各1ウェル内に当該生体試料中の約1分子の酵素が分布するように添加してもよい。
上記の通り、式(III)で表される化合物である本発明の蛍光プローブは、Bとして導入した官能基の種類に応じて、1種以上の酵素活性検出用の蛍光プローブとして用いることができるが、更に、本発明の蛍光プローブを、所定の疾患に特異的なバイオマーカーの検出に用いることにより疾患の診断をおこなうことができる。
即ち、本発明のもう1つの実施態様は、本発明の蛍光プローブを用いて、1分子酵素活性を検出することにより病態を診断する方法である(以下「本発明の診断方法」とも言う)。
本発明を用いた疾患診断の概念図を図6に示す。左側の図は、上記した本発明のスクリーニング方法に関するものであり、右側の図が1分子酵素活性を検出することにより病態を診断する方法の概略を示している。本発明を利用することで、酵素の数、活性、活性の揺らぎなどの情報に基づく疾患診断をおこなうことが可能であることが期待される。
このようなバイオマーカー検出に用いることができる本発明の蛍光プローブ の例、及び本発明の診断方法の非限定的な例を以下に示す。
即ち、本発明のもう1つの実施態様は、生体試料においてENPPの活性を検出する方法であって、式(IV)の化合物又はその塩と生体試料とを水溶液中で接触させることを含み、水溶液における蛍光強度の増加は、ENPPの活性の存在を示す、前記方法である。
式(IV)において、R1、S、T、mは、一般式(I)において詳述した通りである。
(a)の工程で蛍光プローブを臨床検体に適用するには、例えば、蛍光プローブの溶液を局所的に臨床検体にスプレーすることによって行うことができる。
また、本発明の診断方法は、検出された蛍光強度が所定の強度以上のウェル、すなわちENPPが封入されていると考えられるウェルの数を計測することを含むことができる。
一般式(IV)の化合物を、所定の濃度(例えば、200μM)となるようにアッセイバッファーで希釈する。アッセイバッファーの組成は適宜に定めることができるが、例えば、100mM Tris-HCl(pH9.3)、1mM MgCl2、0.5%(w/v)CHAPSであることができる。
次に、被検者(すい臓がん患者、健常者の何れも含む)から採取した生体試料、好ましくは、血液試料(例えば、血清試料、または血漿試料)を上記のアッセイバッファーで希釈し(例えば、250倍程度)、一般式(IV)の化合物の希釈液と所定の割合(例えば、等量)で混合する。
次に、両者の混合溶液をマイクロデバイスへと添加し、引き続いてシーリングオイルを添加することで、溶液を各ウェルへと封入した。
溶液を封入したマイクロデバイスを所定の条件(例えば、25℃で40分間)インキュベーションし、その後、蛍光顕微鏡で各ウェルの蛍光強度を測定する。
検出された蛍光強度が所定の強度以上(例えば、2500AU以上)のウェル、すなわちENPPが封入されていると考えられるウェルの数を計測する。
当該すい臓がん検出用の蛍光プローブの使用方法については、本発明の蛍光プローブについて説明したのと同様である。
本発明のもう1つの実施態様は、生体試料においてCD13の活性を検出する方法であって、一般式(III)においてBが-NR2-CO-Lの場合に、-CO-Lの部分がアラニン、リジン、アルギニン又はメチオニン残基である、本発明の化合物(以下「本発明の化合物A」とも言う)と生体試料とを水溶液中で接触させることを含み、水溶液における蛍光強度の増加は、CD13の活性の存在を示す、前記方法である。
マイクロデバイスを用いて、一般式(III)においてBが-NR2-CO-Lの場合に、-CO-Lの部分が-Pro-Xaa(Proはプロリン残基、Xaaはグリシン、セリン、グルタミン酸などのアミノ酸残基を表す)のペプチドである、本発明の化合物を含む蛍光プローブを用いて、被検者から得られた生体試料中のDPP4の1分子酵素活性を検出することにより、すい臓がんを診断する方法、または、生体試料が由来する被検者がすい臓がんである可能性を予測する方法である。
また、本発明の診断又は予測方法は、検出された蛍光強度が所定の強度以上のウェル、すなわちCD13(又はDPP4)が封入されていると考えられるウェルの数を計測することを含むことができる。
即ち、本発明の診断又は予測方法は、膵臓癌患者又は膵臓癌を有すると疑われる患者に限らずに、健康な中高年のヒト(健常者)に対して、膵臓癌の可能性があることを確認する目的で行い、膵臓癌の可能性が高い被験者はMRI等の画像検査(精密検査)を行うことで早期膵癌を見つけることにも寄与することができる。
本発明の化合物Aを、所定の濃度(例えば、100μM)となるようにアッセイバッファーで希釈する。アッセイバッファーの組成は適宜に定めることができるが、例えば、100mM HEPES-NaOH(pH7.4)、1mM MgCl2、1mM CaCl2、3mM Triton X-100であることができる。
次に、被検者(すい臓がん患者、健常者の何れも含む)から採取した生体試料、好ましくは、血液試料(例えば、血清試料、または血漿試料)を上記のアッセイバッファーで希釈し(例えば、10,000倍程度)、本発明の化合物Aの希釈液と所定の割合(例えば、等量)で混合する。
次に、両者の混合溶液をマイクロデバイスへと添加し、引き続いてシーリングオイルを添加することで、溶液を各ウェルへと封入した。
溶液を封入したマイクロデバイスを所定の条件(例えば、37℃で2時間)インキュベーションし、その後、蛍光顕微鏡で各ウェルの蛍光強度を測定する。
検出された蛍光強度が所定の強度以上(例えば、2500AU以上)のウェル、すなわちCD13が封入されていると考えられるウェルの数を計測する。
本発明の化合物Bを、所定の濃度(例えば、100μM)となるようにアッセイバッファーで希釈する。アッセイバッファーの組成は適宜に定めることができるが、100mM HEPES-NaOH(pH7.4)、1mM MgCl2、1mM CaCl2、3mM Triton X-100であることができる。
次に、被検者(すい臓がん患者、健常者の何れも含む)から採取した生体試料、好ましくは、血液試料(例えば、血清試料、または血漿試料)を上記のアッセイバッファーで希釈し(例えば、2,500倍程度)、本発明の化合物Bの希釈液と所定の割合(例えば、等量)で混合する。
次に、両者の混合溶液をマイクロデバイスへと添加し、引き続いてシーリングオイルを添加することで、溶液を各ウェルへと封入した。
溶液を封入したマイクロデバイスを所定の条件(例えば、25℃で2時間)インキュベーションし、その後、蛍光顕微鏡で各ウェルの蛍光強度を測定する。
各ウェルの蛍光強度に基づいて作成したヒストグラムを2つの正規分布からなる曲線に近似する。近似曲線の極小値よりも蛍光強度の高いウェルを高活性群、蛍光強度の低いウェルを低活性群とし、これらの合計のうち高活性群の占める割合を計測する。
有機合成のための試薬及び溶媒は東京化学工業(TCI)、和光純化工業又はアルドリッチケミカル社から供給され、それ以上精製することなく使用した。
プロトン核磁気共鳴(1H NMR)スペクトルをJEOL JMN-LA400装置で記録した。
質量スペクトルは、JEOL JMS-T100LP AccuTOFTM LC-plus4Gで測定した。
[合成実施例1]
化合物2の合成
NH2型の蛍光母核のホスホン酸を保護した化合物(化合物2)を以下の手順で合成した。
以下のスキームにより化合物1を合成した。
得られた化合物の1H-NMR、13C-NMR及び高分解能質量分析(high-resolution Mass Spectrometry;HR-MS)計による分析結果を以下に示す。
13C-NMR (100 MHz、 CDCl3) δ 161.0、 154.0、 149.4、 140.0、 126.8、 117.2、 116.3、 115.2、 115.1、 114.4、 107.9、 30.9.
HRMS (ESI-): Calcd. for [M-H]-、 350.00413、 Found、 350.00261 (-1.52 mmu).
以下のスキームにより化合物2を合成した。
得られた化合物の1H-NMR、13C-NMR及び高分解能質量分析(HR-MS)計による分析結果を以下に示す。
13C-NMR (100 MHz、 CDCl3) δ 161.2、 155.5、 147.9、 135.3、 131.7、 130.5、 129.1、 128.3、 128.0、 127.0、 112.5、 112.1、 101.8、 32.2、 26.4、 19.4.
HRMS (ESI-): Calcd. for [M-H]-、 492.13961、 Found、 492.13778 (-1.83 mmu).
化合物4の合成
OH型の蛍光母核のホスホン酸を保護した化合物(化合物4)を以下の手順で合成した。
得られた化合物の1H-NMR、13C-NMR及び高分解能質量分析(HR-MS)計による分析結果を以下に示す。
13C-NMR (100 MHz、 CD3OD) δ 162.1、 161.8、 155.6、 150.5、 127.3、 112.9、 111.8、 111.6、 102.1、 31.1.
HRMS (ESI+): Calcd. for [M+H]+、 257.02150、 Found、 257.02155 (+0.05 mmu).
化合物3(200mg、0.78mmol)及びイミダゾール(212mg、3.12mmol)のDMSO(2mL)の溶液にTBDPS-Cl(298μL、1.17mmol)を滴下追加した。反応混合物を空気中室温で15分撹拌した。混合物に、0.1M TEAA含有100%H2O及び0.1M TEAA含有100%CH3CNを加え、MPLC(溶離液:A/B=70/30>0/100、A:0.1M TEAA含有100%H2O、B:0.1M TEAA含有100%CH3CN)で精製した。溶離液を蒸発させて有機溶媒を留去し、ろ過した。残渣を乾燥して、無色固体の化合物4(182mg、0.37mmol、収率47%)を得た。
得られた化合物の1H-NMR、13C-NMR及び高分解能質量分析(HR-MS)計による分析結果を以下に示す。
13C-NMR (100 MHz、 CDCl3) δ 162.7、 162.2、 155.5、 153.2、 153.1、 135.7、 134.1、 129.8、 127.7、 114.0、 111.6、 110.7、 102.8、 37.1、 26.8、 19.5.
HRMS (ESI-): Calcd. for [M-H]-、 493.12363、 Found、 493.12158 (-2.05 mmu).
[合成実施例3]
アミダーゼプローブの合成例
NH2型の蛍光母核のホスホン酸を保護した化合物(化合物2)を用いて、以下の反応スキームに則り、以下の手順でアミダーゼプローブを合成した。
2.反応混合物を60℃で攪拌した。
3.300μLのTFA、5μLのトリエチルシラン及び5μLのH2Oを加え、反応混合物を攪拌した。
4.1mLのEt2Oを加え、混合物を遠心分離し(15、000rpm×10分、4℃)、上澄みを取り除いた。
5.残渣を1mLのロード緩衝液(80%Bis Tris緩衝液(1M、pH6.8)
及び20%MeCN)に溶解し、500μLのphos-tagビーズに担持させた。
6.ビーズを1mLの洗浄緩衝液(80%H2O及び20%MeCN、pH6.8、100mM Bis Tris-AcOH含有)で5回洗浄した。
7.ビーズを1mLの洗浄液(80%H2O及び20%MeCN)で3回洗浄した。
8.化合物を300μLの溶離液(80%H2O及び20%MeCN、1%エチレンジアミン含有)で3回溶離し、溶液に500μLのH2O(2%AcOH含有)を加えて中和したのち、凍結乾燥した。
Loadは、6.の段階でビーズに加えた溶液の組成、Flowthroughtは、6.の段階で目的物質をビーズに保持させた後、除去した上清の溶液組成を示す。Washは、7.の段階で3回洗浄を行ったそれぞれの上清の溶液組成を示す。Eluteは、8.の段階で溶離した溶液の組成を示す。
アミノペプチダーゼ、アミダーゼプローブの合成例
NH2型の蛍光母核のホスホン酸を保護した化合物(化合物2)を用いて、以下の反応スキームに則り、以下の手順でアミノペプチダーゼプローブを合成した。
2.反応混合物を60℃で攪拌した。
3.20μLのピペリジンを加え、反応混合物を攪拌した。
4.300μLのTFA、5μLのトリエチルシラン及び5μLのH2Oを加え、反応混合物を攪拌した。
5.1mLのEt2Oを加え、混合物を遠心分離し(15、000rpm×10分、4℃)、上澄みを取り除いた。
6.残渣を1mLのロード緩衝液(80%Bis Tris緩衝液(1M、pH6.8)
及び20%MeCN)に溶解し、500μLのphos-tagビーズに担持させた。
7.ビーズを1mLの洗浄緩衝液(80%H2O及び20%MeCN、pH6.8、100mM Bis Tris-AcOH含有)で5回洗浄した。
8.ビーズを1mLの洗浄液(80%H2O及び20%MeCN)で3回洗浄した。
9.化合物を300μLの溶離液(80%H2O及び20%MeCN、1%エチレンジアミン含有)で3回溶離し、溶液に500μLのH2O(2%AcOH含有)を加えて中和したのち、凍結乾燥した。
1H-NMR (400 MHz, D2O) δ 7.58 (d, 1H, J = 8.0 Hz), 7.41 (s, 1H), 7.21 (d, 1H, J = 7.8 Hz), 6.18 (d, 1H, J = 2.0 Hz), 3.1-2.9 (m, 5H), 1.9 (m, 2H), 1.5 (m, 2H).
HRMS (ESI+): calcd. for [M+Na]+, 434.1243, Found, 434.1205 (-3.8 mmu).
Met-PMAC was prepared using Fmoc-Met-OH as the building block.
1H-NMR (400 MHz, D2O) δ 7.69 (d, 1H, J = 7.8 Hz), 7.48 (s, 1H), 7.18 (d, 1H, J = 8.0 Hz), 6.1 (d, 1H, J = 2.0 Hz), 3.1-2.9 (m, 3H), 2.51 (d, 2H, J = 7.8 Hz), 2.12 (m, 2H), 1.93 (s, 3H).
HRMS (ESI+): calcd. for [M+Na]+, 409.0599, Found, 409.0578 (-2.1 mmu).
ペプチダーゼ、プロテアーゼプローブの合成例
NH2型の蛍光母核のホスホン酸を保護した化合物(化合物2)を用いて、以下の反応スキームに則り、以下の手順でペプチダーゼ、プロテアーゼプローブを合成した。
2.反応混合物を60℃で攪拌した。
3.a)300μLのTFA、5μLのトリエチルシラン及び5μLのH2Oを添加し、反応混合物を攪拌した。
b)(酸に弱い保護基を脱保護させたくない場合)300μLのMeCN及びTHF中5μLの1M TBAFを添加し、反応混合物を攪拌した。
4.1mLのEt2Oを加え、混合物を遠心分離し(15、000rpm×10分、4℃)、上澄みを取り除いた。
5.残渣を1mLのロード緩衝液(80%Bis Tris緩衝液(1M、pH6.8)
及び20%MeCN)に溶解し、500μLのphos-tagビーズに担持させた。
6.ビーズを1mLの洗浄緩衝液(80%H2O及び20%MeCN、pH6.8、100mM Bis Tris-AcOH含有)で5回洗浄した。
7.ビーズを1mLの洗浄液(80%H2O及び20%MeCN)で3回洗浄した。
8.化合物を300μLの溶離液(80%H2O及び20%MeCN、1%エチレンジアミン含有)で3回溶離し、溶液に500μLのH2O(2%AcOH含有)を加えて中和したのち、凍結乾燥した。
1H-NMR (400 MHz, D2O) δ 7.60 (d, 1H, J = 7.2 Hz), 7.44 (s, 1H), 7.18 (d, 1H, J = 7.2 Hz), 6.15 (d, 1H, J = 2.1 Hz), 4.29 (m, 1H), 3.6-3.5 (m, 2H), 3.06 (d, 2H, J = 20.4 Hz), 2.96 (m, 1H), 2.43 (t, 2H, J = 8.0 Hz), 2.24 (m, 1H), 2.1-1.7 (m, 5H).
HRMS (ESI+): calcd. for [M+H]+, 482.1328, Found, 482.1336 (+0.8 mmu).
1H-NMR (400 MHz, D2O) δ 7.68 (d, 1H, J = 8.4 Hz), 7.49 (s, 1H), 7.24 (d, 1H, J = 8.4 Hz), 6.20 (d, 1H, J = 2.0 Hz), 4.39 (m, 1H), 4.27 (m, 1H), 4.11 (m, 2H), 3.6-3.4 (m, 2H), 3.02 (d, 2H, J = 20.8 Hz), 2.4-2.3 (m, 5H), 2.1 (m, 1H), 1.8 (m, 2H), 1.7 (m, 2H), 1.2 (m, 6H), 0.8 (t, 3H, J = 8.4 Hz).
グリコシダーゼプローブの合成
OH型の蛍光母核のホスホン酸を保護した化合物(化合物4)を用いて、以下の反応スキームに則り、以下の手順でグリコシダーゼプローブを合成した。
1.試験管中で、PMAC-TBDPS(5μmol)及び塩化アセチルグリコピラノシル(25μmol)を250μLのMeCN及び500μLの2N Na2CO3水溶液に溶解した。
2.反応混合物を室温で攪拌した。
3.MeCN相を集めた。
4.THF中150μLの1M TBAFを加えて、攪拌した。
5.100μLの2N LiOH水溶液を加えて、10分間攪拌した。
6.400μLのロード緩衝液(80%H2O及び20%MeCN、pH6.8、100mM Bis Tris-AcOH含有)及び11.5μLのAcOHを中和のために添加し、混合物を100μLのAcOEtで洗浄した。
7.残渣を1mLのロード緩衝液(80%Bis Tris緩衝液(1M、pH6.8)及び20%MeCN)に溶解し、500μLのphos-tagビーズに担持させた。
8.ビーズを1mLの洗浄緩衝液(80%H2O及び20%MeCN、pH6.8、100mM Bis Tris-AcOH含有)で5回洗浄した。
9.ビーズを1mLの洗浄液(80%H2O及び20%MeCN)で3回洗浄した。
10.化合物を300μLの溶離液(80%H2O及び20%MeCN、1%エチレンジアミン含有)で3回溶離し、溶液に500μLのH2O(2%AcOH含有)を加えて中和したのち、凍結乾燥した。
合成実施例3~6で示した手順を用いて合成した化合物の蛍光プローブについて、マイクロデバイスを用いて血液中の酵素活性のアッセイを行った結果を以下に示す、
化合物5~51を用いた血液中酵素活性検出の結果
1.アミノペプチダーゼ、アミダーゼ、プロテアーゼ活性検出蛍光プローブである化合物5~51について、それぞれの化合物の濃度が100μMとなるようにアッセイバッファーで希釈した。アッセイバッファーの組成は、100mM HEPES-NaOH(pH7.4)、1mM MgCl2、1mM DTT、3mM TritonX-100である。
2.次いで、ヒト血漿サンプル(健常者由来あるいはすい臓がん患者由来)を上記のアッセイバッファーで500倍希釈し、プローブの希釈液と等量で混合した。この時、両者の終濃度は蛍光プローブが50μM、ヒト血漿サンプルが1000倍希釈である。
3.次いで、両者の混合溶液をマイクロデバイスへと添加し、引き続いてシーリングオイルを添加することで、溶液を各ウェルへと封入した。
4.溶液を封入したマイクロデバイスを37℃で4時間インキュベーションしたのち、蛍光顕微鏡で各ウェルの蛍光強度を測定した。ここで、蛍光プローブと反応する酵素の封入されているウェルにおいて、蛍光強度の上昇がみられた。
図7Aと図7B中の各蛍光顕微鏡画像の左側の画像(Healthyと付記)は健常者由来のヒト血漿サンプルを用いた場合の結果で、右側の画像(Tumorと付記)はすい臓がん患者由来のヒト血漿サンプルを用いた場合の結果である。また、各蛍光顕微鏡画像の左側に、各々の蛍光プローブが有するアミノ酸残基又はペプチドの種類を記載した。
マイクロデバイスでCD13プローブを用いたヒト血漿サンプルの測定
本実施例では、CD13活性検出プローブであるArg-PMACおよびMet-PMACのバイオマーカー検出への利用可能性を検証する実験を以下の手順で行った。
2.次いで、すい臓がん患者30名及び健常者30名から採取したヒト血漿サンプルを上記のアッセイバッファーで500倍希釈し、CD13プローブの希釈液と等量で混合した。この時、両者の終濃度はCD13プローブが50μM、ヒト血漿サンプルが1000倍希釈である。
3.次いで、両者の混合溶液をマイクロデバイスへと添加し、引き続いてシーリングオイルを添加することで、溶液を各ウェルへと封入した。
4.溶液を封入したマイクロデバイスを37℃で4時間インキュベーションしたのち、蛍光顕微鏡で各ウェルの蛍光強度を測定した。図8にArg-PMACを用いておこなった蛍光顕微鏡画像を示す。
5.検出された蛍光強度が別途計測したバックグラウンド蛍光のAverage+3SD以上、すなわちCD13が封入されていると考えられるウェルの数について、ROC曲線を作成したところ、図9に示すようにAUC=0.554という数字が得られた。
6.1~5の過程をMet-PMACを用いておこないROC曲線を作成したところ、図10に示すようにAUC=0.619という数字が得られた。
マイクロデバイスでDPP4プローブを用いたヒト血漿サンプルの測定
本実施例では、DPP4活性検出プローブであるGlu-Pro-PMACのバイオマーカー検出への利用可能性を検証する実験を以下の手順で行った。
2.次いで、すい臓がん患者30名及び健常者30名から採取したヒト血漿サンプルを上記のアッセイバッファーで500倍希釈し、DPP4プローブの希釈液と等量で混合した。この時、両者の終濃度はDPP4プローブが50μM、ヒト血漿サンプルが1000倍希釈である。
3.次いで、両者の混合溶液をマイクロデバイスへと添加し、引き続いてシーリングオイルを添加することで、溶液を各ウェルへと封入した。
4.溶液を封入したマイクロデバイスを25℃で2時間インキュベーションしたのち、蛍光顕微鏡で各ウェルの蛍光強度を測定した。図11に代表的な蛍光顕微鏡画像を示す。
5.検出された蛍光強度が別途計測したバックグラウンド蛍光のAverage+3SD以上、すなわちDPP4が封入されていると考えられるウェルの数について、2つのピークが観察され、これらのピークに属するウェルの数の比を取得し、ROC曲線を作成したところ、図12に示すようにAUC=0.724という数字が得られた。
2.反応溶液を105℃で撹拌した。
3.3mLのTBAF(1M、THF溶液)を加え、室温で攪拌した。
4.溶液をMPLC(溶離液:A/B=100/0>0/100、A:0.1%TFA含有100%H2O、B:0.1%TFA 含有100%MeCN)で粗精製した。
5.溶離液を蒸発させて溶媒を留去し、残渣を1mLのロード緩衝液(80%H2O 及び20%MeCN、pH6.8、100mM bis tris-AcOH含有)に溶解し、500μLのphos-tagビーズに担持させた。
6.ビーズを1mLの洗浄緩衝液(80% H2O及び20%MeCN、pH6.8、100mM bis tris-AcOH 含有)で5回洗浄し、1mLのリンス溶液(80%H2O及び20%MeCN)で2回洗浄した。
7.化合物を300μLの溶出溶液(80%H2O及び20%MeCN、1%エチレンジアミン含有)で3回溶離し、溶液を凍結乾燥した。
PMUM-dCMPのENPPに対する活性の評価
合成実施例7で得たPMUM-dCMPについて、ENPP3の精製酵素を用いてプレートリーダーで活性測定を行った。
アッセイは、1mM MgCl2、0.5%w/w CHAPS、10μM PMUM-dCMP、およびENPP3(組換え、9.9μg/mL)を含むTris-HClバッファー(pH9.3)で行った。結果を図13に示す。
図13で示されるように、ENPP3を添加した場合は、ENPP3未添加に比べて蛍光強度(FI)の顕著な増大が認められた。
すい臓がんのバイオマーカー活性検出蛍光プローブの検討
次に、すい臓がんのバイオマーカー活性検出蛍光プローブを探索する目的で、健常者3名、すい臓がん患者3名に由来する血漿サンプルを使用し、PMUM-dCMPを含む式(III)に類する複数の化合物を使用してマイクロデバイスでの酵素アッセイを行った。
酵素アッセイのプロトコルは以下の通りである。
1.式(III)に類する化合物をアッセイバッファーで希釈した。アッセイバッファーの組成は、100mM HEPES-NaOH(pH5-10)、1mM MgCl2、3mM TritonX-100である。
2.次いで、ヒト血漿サンプルを上記のアッセイバッファーで希釈し、プローブの希釈液と等量で混合した。この時、両者の終濃度はプローブが10-100μM、ヒト血漿サンプルが500-1000倍希釈である。
3.次いで、両者の混合溶液をマイクロデバイスへと添加し、引き続いてシーリングオイルを添加することで、溶液を各ウェルへと封入した。
4.溶液を封入したマイクロデバイスを37℃で1-12時間インキュベーションしたのち、蛍光顕微鏡で各ウェルの蛍光強度を測定した。
5.画像解析をおこない、疾患の有無において酵素活性が異なる化合物が存在した。
マイクロデバイスでENPPプローブを用いたヒト血漿サンプルの測定
本実施例では、PMUM-dCMPがバイオマーカー検出への利用可能性を検証する実験を以下の手順で行った。
2.次いで、すい臓がん患者6名及び健常者6名から採取したヒト血漿サンプルを上記のアッセイバッファーで250倍希釈し、ENPPプローブの希釈液と等量で混合した。この時、両者の終濃度はENPPプローブが100μM、ヒト血漿サンプルが500倍希釈である。
3.次いで、両者の混合溶液をマイクロデバイスへと添加し、引き続いてシーリングオイルを添加することで、溶液を各ウェルへと封入した。
4.溶液を封入したマイクロデバイスを25℃で40分間インキュベーションしたのち、蛍光顕微鏡で各ウェルの蛍光強度を測定した。図14に蛍光顕微鏡画像を示す。
5.検出された蛍光強度が2500AU以上のウェル、すなわちENPP3が封入されていると考えられるウェルの数について、健常者と比較してすい臓がん患者で有意な増加がみられた。図15にその結果を示す。
具体的には、本実施例において、AU2500以上のウェルの個数に基づいて決定したENPPの酵素活性に基づいて判定を行うROC曲線を作成し、AU2500以上のウェルが23個というところでthresholdを引くと、感度83%、特異度100%となる(図16)。このようなROC曲線によってすい臓がんを判定することが可能である。
Claims (24)
- 以下の一般式(I)で表される化合物。
(式(I)において、
Aは、アミノ基(-NR2H)又は水酸基(-OH)であり、
ここで、R2は、水素原子、及び炭素数1~8の分岐、直鎖又は環状の置換又は無置換のアルキル基(当該アルキル基の1つ以上の非隣接の、非末端のC原子は、O、S、CO又はCOOで置き換えられていてもよい)からなる群から選択され;
R1は、存在する場合は、ベンゼン環上に存在する同一又は異なる一価の置換基であり:Sは、存在する場合は、リンカーであり;
Tは、ホスホン酸基(-P(=O)(OH)2)、リン酸エステル基(-O-P(=O)(OH)2)又はリン酸アミド基(-NH-P(=O)(OH)2)から選択され;
mは、0~3の整数である。) - Aが-NR2Hである、請求項1に記載の化合物。
- Aが-OHである、請求項1に記載の化合物。
- (1)以下の一般式(I)で表される化合物のTの基を保護する工程;
(式(I)において、
Aは、アミノ基(-NR2H)又は水酸基(-OH)であり、
ここで、R2は、水素原子又は炭素数1~8の置換又は無置換のアルキル基であり;
R1は、存在する場合は、ベンゼン環上に存在する同一又は異なる一価の置換基であり:Sは、存在する場合は、リンカーであり;
Tは、ホスホン酸基(-P(=O)(OH)2)、リン酸エステル基(-O-P(=O)(OH)2)又はリン酸アミド基(-NH-P(=O)(OH)2)から選択され;
mは、0~3の整数である。)
(2)(1)の工程で得られる生成物について、
(i)Aがアミノ基(-NR2H)の場合は、当該アミノ基を、アミド基(-NR2C(=O)R、Rは水素原子又は炭素数1~8のアルキル基である)、-NR2-CO-L(Lは、アミノ酸の部分構造を表す)、リン酸アミド基(-NR2-PO(ORa)(ORb)、Ra及びRbは、各々独立に、水素原子、及び炭素数1~8の分岐、直鎖又は環状の置換又は無置換のアルキル基(当該アルキル基の1つ以上の非隣接の、非末端のC原子は、O、S、CO又はCOOで置き換えられていてもよい)からなる群から選択される)、又はスルホンアミド基(-NR2-SO2-Rc、Rcは、水素原子、及び炭素数1~8の分岐、直鎖又は環状の置換又は無置換のアルキル基(当該アルキル基の1つ以上の非隣接の、非末端のC原子は、O、S、CO又はCOOで置き換えられていてもよい)からなる群から選択される)に変換する工程、
(ii)Aが水酸基の場合は、当該水酸基をエステル基、リン酸エステル基、硫酸エステル基、エーテル基又は-O-L’(L’は、糖類又は糖類の部分構造を表す)に変換する工程;
(3)(2)の工程で得られる生成物のTの保護基を除去する工程であって、場合により、前記保護基の除去後に粗精製の工程を含んでもよい;
(4)(3)の工程で得られる生成物に、以下の式(II)で表される化合物を添加する工程;
(式(II)において、
Mは、Zn又はCuであり;
Xは、リンカー基であり;
Pは、担体である。)
(5)(4)の工程で得られる生成物を精製し、その後、式(III)の化合物を溶離又は溶出させる工程;
を含む、以下の一般式(III)で表される化合物を調製する方法。
(式(III)中、
Bは、アミド基(-NR2C(=O)R、Rは水素原子又は炭素数1~8の置換又は無置換のアルキル基である)、-NR2-CO-L(Lは、アミノ酸の部分構造を表す)、リン酸アミド基(-NR2-PO(ORa)(ORb)、Ra及びRbは、各々独立に、水素原子、及び炭素数1~8の分岐、直鎖又は環状の置換又は無置換のアルキル基(当該アルキル基の1つ以上の非隣接の、非末端のC原子は、O、S、CO又はCOOで置き換えられていてもよい)からなる群から選択される)、スルホンアミド基(-NR2-SO2-Rc、Rcは、水素原子、及び炭素数1~8の分岐、直鎖又は環状の置換又は無置換のアルキル基(当該アルキル基の1つ以上の非隣接の、非末端のC原子は、O、S、CO又はCOOで置き換えられていてもよい)からなる群から選択される)、エステル基、リン酸エステル基、硫酸エステル基、エーテル基又は-O-L’(L’は、糖類又は糖類の部分構造を表す)から選択され;
S、T、R1、mは、式(I)で定義した通りである。) - 複数の反応容器において並列的に以下の(1)~(5)の工程を行うことにより、前記複数の反応容器内で容器毎に1種類の以下の一般式(III)で表される化合物を調製する方法。
(式(III)中、
Bは、アミド基(-NR2C(=O)R、Rは水素原子又は炭素数1~8の置換又は無置換のアルキル基である)、-NR2-CO-L(Lは、アミノ酸の部分構造を表す)、リン酸アミド基(-NR2-PO(ORa)(ORb)、Ra及びRbは、各々独立に、水素原子、及び炭素数1~8の分岐、直鎖又は環状の置換又は無置換のアルキル基(当該アルキル基の1つ以上の非隣接の、非末端のC原子は、O、S、CO又はCOOで置き換えられていてもよい)からなる群から選択される)、又はスルホンアミド基(-NR2-SO2-Rc、Rcは、水素原子、及び炭素数1~8の分岐、直鎖又は環状の置換又は無置換のアルキル基(当該アルキル基の1つ以上の非隣接の、非末端のC原子は、O、S、CO又はCOOで置き換えられていてもよい)からなる群から選択される)、エステル基、リン酸エステル基、硫酸エステル基、エーテル基又は-O-L’(L’は、糖類又は糖類の部分構造を表す)から選択され;
S、T、R1、mは、式(I)で定義する通りである。)
(1)以下の式(I)で表される化合物のTの基を保護する工程;
(式(I)において、
Aは、アミノ基(-NR2H)又は水酸基(-OH)であり、
ここで、R2は、水素原子、及び炭素数1~8の分岐、直鎖又は環状の置換又は無置換のアルキル基(当該アルキル基の1つ以上の非隣接の、非末端のC原子は、O、S、CO又はCOOで置き換えられていてもよい)からなる群から選択され;
R1は、存在する場合は、ベンゼン環上に存在する同一又は異なる一価の置換基であり:Sは、存在する場合は、リンカーであり;
Tは、ホスホン酸基(-P(=O)(OH)2)、リン酸エステル基(-O-P(=O)(OH)2)又はリン酸アミド基(-NH-P(=O)(OH)2)から選択され;
mは、0~3の整数である。)
(2)(1)の工程で得られる生成物について、
(i)Aがアミノ基(-NR2H)の場合は、当該アミノ基を、アミド基(-NR2C(=O)R、Rは水素原子又は炭素数1~8のアルキル基である)、-NR2-CO-L(Lは、アミノ酸の部分構造を表す)、リン酸アミド基(-NR2-PO(ORa)(ORb)、Ra及びRbは、各々独立に、水素原子、及び炭素数1~8の分岐、直鎖又は環状の置換又は無置換のアルキル基(当該アルキル基の1つ以上の非隣接の、非末端のC原子は、O、S、CO又はCOOで置き換えられていてもよい)からなる群から選択される)、又はスルホンアミド基(-NR2-SO2-Rc、Rcは、水素原子、及び炭素数1~8の分岐、直鎖又は環状の置換又は無置換のアルキル基(当該アルキル基の1つ以上の非隣接の、非末端のC原子は、O、S、CO又はCOOで置き換えられていてもよい)からなる群から選択される)に変換する工程、
(ii)Aが水酸基の場合は、当該水酸基をエステル基、リン酸エステル基、硫酸エステル基、エーテル基又は-O-L’(L’は、糖類又は糖類の部分構造を表す)に変換する工程;
(3)(2)の工程で得られる生成物のTの保護基を除去する工程であって、場合により、前記保護基の除去後に粗精製の工程を含んでもよい;
(4)(3)の工程で得られる生成物に、以下の式(II)で表される化合物を添加する工程;
(式(II)において、
Mは、Zn又はCuであり;
Xは、リンカー基であり;
Pは、担体である。)
(5)(4)の工程で得られる生成物を精製し、その後、式(III)の化合物を溶離又は溶出させる工程。 - 以下の一般式(III)で表される化合物又はその塩。
(式(III)中、
Bは、アミド基(-NR2C(=O)R、Rは水素原子又は炭素数1~8の置換又は無置換のアルキル基である)、-NR2-CO-L(Lは、アミノ酸の部分構造を表す)、リン酸アミド基(-NR2-PO(ORa)(ORb)、Ra及びRbは、各々独立に、水素原子、及び炭素数1~8の分岐、直鎖又は環状の置換又は無置換のアルキル基(当該アルキル基の1つ以上の非隣接の、非末端のC原子は、O、S、CO又はCOOで置き換えられていてもよい)からなる群から選択される)、スルホンアミド基(-NR2-SO2-Rc、Rcは、水素原子、及び炭素数1~8の分岐、直鎖又は環状の置換又は無置換のアルキル基(当該アルキル基の1つ以上の非隣接の、非末端のC原子は、O、S、CO又はCOOで置き換えられていてもよい)からなる群から選択される)、エステル基、リン酸エステル基、硫酸エステル基、エーテル基又は-O-L’(L’は、糖類又は糖類の部分構造を表す)から選択され;
R1は、存在する場合は、ベンゼン環上に存在する同一又は異なる一価の置換基であり:
Sは、存在する場合は、リンカーであり;
Tは、ホスホン酸基(-P(=O)(OH)2)、リン酸エステル基(-O-P(=O)(OH)2)又はリン酸アミド基(-NH-P(=O)(OH)2)から選択され;
mは、0~3の整数である。) - 請求項6に記載の一般式(III)で表される化合物又はその塩を含む、酵素活性の検出用蛍光プローブ。
- 生体試料中の複数の酵素の活性を検出する方法であって、生体試料と請求項6に記載の一般式(III)の化合物とを接触させることを含む、該方法。
- 一般式(III)の化合物のライブラリーを用いることを特徴とする、請求項8に記載の方法。
- マイクロデバイスを用いることを特徴とする、請求項8又は9に記載の方法。
- 請求項6に記載の一般式(III)の化合物を含む組成物の酵素アッセイの試験方法であって、前記組成物と、前記化合物を切断する酵素を含む、または含むと疑われる生体試料と接触させることを含む、該方法。
- 特定疾患のバイオマーカーを検出できる蛍光プローブをスクリーニングする方法であって、当該方法は、
(1)請求項6に記載の式(III)の化合物のライブラリーをマイクロデバイスに添加する工程であって、当該マイクロデバイスの少なくとも1ウェルに1種類の式(III)の化合物を備えるように添加し;
(2)当該マイクロデバイスに生体試料を含む溶液を添加する工程であって、当該マイクロデバイスにおいて、1分子の酵素を含む少なくとも1つのウェルが生じるように添加し、ここで、前記生体試料は、特定疾患患者から得られた生体試料又は健常者から得られた生体試料であり;
(3)式(III)の化合物と酵素とを接触させて、マイクロデバイスのウェル内の蛍光を検出する工程であって、当該工程は、
式(III)の化合物と、特定疾患患者から得られた生体試料とを接触させて、式(III)の化合物からの蛍光強度(第1の蛍光強度)を測定すること、及び
式(III)の化合物と、健常者から得られた生体試料とを接触させて、式(III)の化合物からの蛍光強度(第2の蛍光強度)を測定すること、
を含み;
(4)前記第1の蛍光強度と前記第2の蛍光強度とを比較して差がある場合に、当該化合物が、前記蛍光プローブの候補であることが示されることにより、当該化合物をバイオマーカー活性検出蛍光プローブと判定する工程;
を含む、前記スクリーニング方法。 - 前記生体試料が、すい臓がん患者、すい臓がんを有すると疑われる患者又は健常者の生体試料である、請求項13に記載の方法。
- (a)一般式(IV)の化合物又はその塩を含む蛍光プローブを被検体の臨床検体に適用する工程、及び(b)前記蛍光プローブを適用した臨床検体の蛍光像を測定することを含む、すい臓がんを診断する方法、または、生体試料が由来する被検者がすい臓がんである可能性を予測する方法。
- (a)被検者から得られた生体試料を、一般式(IV)の化合物又はその塩を含む蛍光プローブと接触させる工程、及び(b)前記蛍光プローブと接触させた生体試料の蛍光強度を測定することを含む、すい臓がんを診断する方法、または、生体試料が由来する被検者がすい臓がんである可能性を予測する方法。
- 一般式(IV)の化合物又はその塩を含む、請求項15又は16に記載の方法において用いるためのすい臓がん検出用の蛍光プローブ。
- 一般式(IV)の化合物又はその塩を含む、すい臓がん細胞又は組織の検出用キット。
- マイクロデバイスを用いて、一般式(III)においてBが-NR2-CO-Lであって、-CO-Lの部分がアラニン、リジン、アルギニン又はメチオニン残基である、請求項6に記載の化合物又はその塩を含む蛍光プローブを用いて、被検者から得られた生体試料中のCD13の1分子酵素活性を検出することにより、すい臓がんを診断する方法、または、生体試料が由来する被検者がすい臓がんである可能性を予測する方法。
- マイクロデバイスを用いて、一般式(III)においてBが-NR2-CO-Lであって、-CO-Lの部分が-Pro-Xaa(Proはプロリン残基、Xaaはグリシン、セリン、グルタミン酸などのアミノ酸残基を表す)のペプチドである、請求項6に記載の化合物又はその塩を含む蛍光プローブを用いて、被検者から得られた生体試料中のDPP4の1分子酵素活性を検出することにより、すい臓がんを診断する方法、または、生体試料が由来する被検者がすい臓がんである可能性を予測する方法。
- マイクロデバイスを用いて、一般式(III)においてBが-NR2-CO-Lであって、-CO-Lの部分がアラニン、リジン、アルギニン又はメチオニン残基である、請求項6に記載の化合物又はその塩を含む蛍光プローブを用いて、被検者から得られた生体試料中のCD13の1分子酵素活性を検出すること、及び
マイクロデバイスを用いて、一般式(III)においてBが-NR2-CO-Lであって、-CO-Lの部分が-Pro-Xaa(Proはプロリン残基、Xaaはグリシン、セリン、グルタミン酸などのアミノ酸残基を表す)のペプチドである、請求項6に記載の化合物又はその塩を含む蛍光プローブを用いて、被検者から得られた生体試料中のDPP4の1分子酵素活性を検出することにより、すい臓がんを診断する方法、または、生体試料が由来する被検者がすい臓がんである可能性を予測する方法。 - 前記生体試料が、すい臓がん患者、すい臓がんを有すると疑われる患者又は健常者の生体試料である、請求項21~23のいずれか1項に記載の方法。
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