WO2005111229A1 - Substrate for enzymatic activity detection and method of detecting enzymatic activity therewith - Google Patents

Abstract

A substrate for enzymatic activity detection capable of detecting any enzymatic activity only by measuring the fluorescence intensity, etc. of substrate with the use of any change of fluorescence intensity, etc. as an index and excelling in operability, which substrate for enzymatic activity detection enables detection of enzymatic activity even when the amount of analyte solution is minute and does not need formation of cells for analyte solution incorporation, etc. to thereby attain striking enhancement of the integrity of detection part. There is provided a substrate for enzymatic activity detection, comprising a substrate, a first fluorescent group directly bonded to the substrate or bonded to the substrate via a first compound having its one end fixed to the substrate, and a second compound linked with the first fluorescent group by means of a peptide bond cleaved by enzymes.

Description

 Specification

 Substrate for detecting enzyme activity and method for detecting enzyme activity using the same

 Technical field

 The present invention relates to a substrate for detecting an enzyme activity for detecting an enzyme activity and a method for detecting an enzyme activity using the same.

 Background art

 [0002] In recent years, with the development of medical fields such as pathological diagnosis and research fields such as proteome analysis, it has become necessary to detect the activity of a plurality of enzymes. Various techniques for measuring in a solution using fluorescence or the like have been studied.

 For example, (Patent Document 1) describes "a fluorescent probe in which both ends of a substrate peptide cleaved specifically by caspase, which is one of proteolytic enzymes, are modified with a fluorescent group that undergoes fluorescence resonance energy transfer." ing.

 Patent Document 1: JP-A-2000-316598

 Disclosure of the invention

 Problems to be solved by the invention

[0003] However, the above-mentioned conventional technology has the following problems.

(1) The technique disclosed in (Patent Document 1) is that, as a result of the substrate peptide of a fluorescent probe placed in a solution being cleaved by an enzyme, the fluorescent groups at both ends of the substrate peptide generate fluorescence resonance energy, and the fluorescence wavelength and fluorescence Since the intensity changes, the enzyme activity is detected by measuring the intensity. As a method for measuring enzyme activity, there is a method in which a sample solution is injected into a cell having an opening formed in a microplate for fluorescence measurement, and high-speed measurement is performed by simultaneously measuring fluorescence on a plate reader. In this method, the opening area of the opening of the cell of the fluorescence measurement microplate needs to be formed large enough to inject the solution. With the development of medical and research fields in recent years, the need to measure the activities of multiple enzymes as quickly as possible and with a small amount of sample to increase research efficiency has increased. There is an increasing need to reduce the cell size of the microplate for fluorescence measurement as much as possible and increase the number of cells. Hara There is a limit to the cell size of the sample solution physically, and at present, there is a problem that the measurement can only be performed on a microplate having cells of a millimeter size such as 96 holes.

 (2) Therefore, when performing the fluorescence measurement, it takes time to replace the microplate, and there is a problem that the measurement efficiency cannot be increased and the measurement requires time.

[0004] The present invention solves the above-mentioned conventional problems, and it is possible to detect enzyme activity only by measuring the fluorescence intensity or the like of a substrate using a change in the fluorescence intensity or the like as an index. To provide a substrate for enzyme activity detection that can detect enzyme activity even with a sample solution of (1) and can dramatically increase the degree of integration of the detection section without forming a cell or the like into which the sample solution is injected. With the goal.

 In addition, the present invention can detect enzyme activity even in a small amount of a sample solution, can increase the detection sensitivity, can shorten the measurement time, can improve workability, and increase the measurement efficiency. Provided is a method for detecting an enzyme activity, wherein the enzyme activity of a sample solution containing a plurality of enzymes can be measured in a short time by using an enzyme activity detection substrate in which a fluorescent group is bonded to each of different types of peptides and the like. With the goal.

 Means for solving the problem

[0005] In order to solve the above-mentioned conventional problems, a substrate for enzyme activity detection and a method for detecting enzyme activity using the same according to the present invention have the following configurations.

 The substrate for enzyme activity detection according to claim 1 of the present invention comprises a substrate and a substrate directly bound to the substrate, or bound to the substrate via a first compound having one end fixed to the substrate. It has a configuration comprising: one fluorescent group; and a second compound bonded to the first fluorescent group with a peptide bond that is cleaved by an enzyme.

 With this configuration, the following operation is obtained.

(1) Since the first fluorescent group bonded to the substrate and the second fluorescent compound bonded to the first fluorescent group by a peptide bond that is cleaved by an enzyme, the first fluorescent group is reacted with the enzyme to form a peptide bond. Upon cleavage, the second compound is released. Since the fluorescence wavelength of the first fluorescent group from which the second compound has been released or the fluorescence intensity at a predetermined wavelength is different from that of the first fluorescent group in which the second compound is peptide-bonded, the enzyme activity is determined based on the change in the fluorescence intensity or the like. Can be detected. (2) Since a fluorescent group is bonded to the substrate, the enzyme activity can be detected simply by contacting a very small amount of a sample solution containing the enzyme and measuring the fluorescence intensity of the substrate, and the enzyme activity can be detected. The degree of integration of the detection unit can be dramatically increased.

 (3) Enzyme activity can be detected simply by contacting a very small amount of sample solution containing the enzyme, so that a large amount of sample solution is not required for measurement, and enzyme activity can be detected even with a small amount of sample solution. It can be carried out.

 [0006] Here, the substrate may be a chlorinated honolem, a halogenated hydrocarbon such as dichloromethane, an ester such as ethyl acetate, N, N-dimethylformamide, or dimethyl used in a condensation reaction for forming a peptide bond. Polar organic solvents such as sulfoxide, ethers such as dioxane and tetrahydrofuran, alcohols such as methanol and ethanol, and synthetic resins (polystyrene, etc.) insoluble in solvents such as pyridine, glass, etc. A sheet formed in a plane or the like is used. A commercially available carrier for solid phase organic synthesis such as Lantern Series (registered trademark) manufactured by Mimotobus Inc. can also be used.

 [0007] As the first compound and the second compound, amino acids, peptides in which two or more amino acids are peptide-bonded, and the like are used.

 The first compound and the second compound can be synthesized by immobilizing a C-terminal amino acid on a substrate and extending the peptide from the C-terminus using a conventional peptide synthesis method such as a solid phase method. In addition, a sequential extension method in which the target amino acid sequence is sequentially extended from the C-terminal side to the N-terminal side, and a fragment condensation method in which a plurality of short peptide fragments are synthesized and extended by coupling between the peptide fragments, etc. Can be used. Alternatively, the peptide can be synthesized using a peptide synthesizer by introducing 91-fluorenylmethyloxycarbonyl (Fmoc) amino acid, t_butyloxycarbonyl (Boc) amino acid, or the like. Furthermore, peptide bonds can be produced using a protease, or can be synthesized using genetic engineering.

[0008] As a condensation method for forming a peptide bond in the first compound or the second compound, a known method can be used, for example, an azide method, an acid chloride method, an acid anhydride method, a mixed acid anhydride. Method, DCC method, DCC_additive method, active ester method, carbonyldiimidazole method, redox method, method using Woodward reagent K, and the like. Before conducting the condensation reaction, do not participate in the reaction in amino acids or peptides by known means. It can protect carboxyl groups, amino groups, etc., and can also activate carboxynole groups and amino groups involved in the reaction.

 [0009] The first compound may be an amino acid or an amino acid sequence which is bound to the substrate by an amide bond, an ester bond, an ether bond, a thioether bond, a urethane bond or the like which is not cleaved by the enzyme, and which is not cleaved by the enzyme in the sample solution. A compound such as a peptide is used. By preventing the first compound from being cleaved by the enzyme, the first fluorescent group bound to the substrate via the first compound is released from the substrate by the action of the enzyme, and the first fluorescence having a change in fluorescence intensity or the like occurs. This is to prevent the group from drifting in the sample solution.

 [0010] As the first fluorescent group, those whose fluorescence wavelength or fluorescence intensity changes before and after the peptide bond between the first fluorescent group and the second compound is cleaved by the enzyme are used. In particular, when a peptide bond with the second compound is a non-fluorescent substance in a specific wavelength region, it emits fluorescence in the specific wavelength region when the peptide bond is cleaved to release the second compound. Fluorescent groups, for example, 4-methylcumaryl 7_amide (MCA), 7-amino_4_carboxymethylcoumarin (ACC), p-nitroalilide, α-naphthylamide, α-naphthyl ester and the like are preferably used.

 As the amino acid of the second conjugate which binds to the first fluorescent group, an amino acid in which the peptide bond at the C-terminal side is selectively cleaved by the enzyme is used. Thereby, the second compound bonded to the first fluorescent group can be released to change the fluorescence intensity of the first fluorescent group in a specific wavelength region.

 In addition, by forming the second compound with a peptide chain having a predetermined length (for example, about 15 mm) or more, a relatively long peptide chain is required for the cleavage action, rather than having high substrate specificity for each amino acid. Enzymes such as elastase can also be detected, and the types of enzymes that can be detected can be increased.

[0011] Examples of enzymes include serine proteases such as trypsin, chymotrypsin, thrombin, plasmin, kallikrein, perokinase, and elastase; aspartic proteases such as pepsin, cathepsin D, renin, and chymosin; Endopeptidase that cleaves internal peptide bonds of meta-oral proteases such as thermolysin and cystine proteases such as cathepsin B, H, L, and calpain; blood coagulation A system protease, a capture system protease, a hormone processing enzyme and the like are used.

 [0012] The substrate for enzyme activity detection according to claim 2 of the present invention comprises a substrate, and the substrate is introduced into the side chain of a third compound having one end fixed to the substrate, via the third compound. Or a fifth compound bound to the third compound or a peptide bond that is cleaved by the enzyme with the third compound or the second fluorescent group bound to the substrate and bound directly to the substrate. And a third fluorescent group that binds to the fifth compound and has the second fluorescent group and fluorescence resonance energy transfer.

 With this configuration, the following operation is obtained in addition to the operation described in claim 1.

 (1) a second fluorescent group bound to the substrate by binding to the third or fourth compound, a fifth compound bound to the third or fourth compound by a peptide bond that is cleaved by an enzyme, Since it has a second fluorescent group that binds to the fifth compound and a third fluorescent group that exhibits fluorescence resonance energy transfer, when the peptide bond is cleaved by the enzyme, the second fluorescent group and the third fluorescent group are dissociated. As the distance increases, the fluorescence resonance energy transfer does not occur, and the enzyme changes the spectrum from the fluorescence spectrum from the second fluorescent group (or the third fluorescent group) to the fluorescent spectrum from the third fluorescent group (or the second fluorescent group). It can be used as an activity measurement index, whereby the enzyme activity can be detected using changes in fluorescence intensity or the like as an index.

 (2) By selecting the second fluorescent group and the third fluorescent group, it becomes possible to set the fluorescent wavelength of the second fluorescent group in the visible region, so that a visible light detection device such as a commercially available CCD camera can be used. It can be used for measurement and has excellent versatility.

 (3) By forming the fifth conjugate with a peptide chain having a predetermined length (for example, about 15 A) or more, a relatively long peptide chain having a high substrate specificity for each amino acid is cleaved. In addition, it is possible to detect enzymes such as elastase, which are required for the detection, and it is possible to increase the types of enzymes that can be detected and to increase the detection sensitivity.

[0013] Here, the fluorescence resonance energy transfer means that when two fluorescent compounds are present at a position close to each other in distance, the fluorescent spectrum of one of the two fluorescent compounds (called a donor) and the other (the donor) If the excitation spectrum overlaps with the excitation spectrum of the donor, the energy of the excitation wavelength of the donor is applied, and the fluorescence of the donor that should be observed is attenuated, and the fluorescence of the axceptor is observed instead. U. Here, as the second fluorescent group and the third fluorescent group, a combination of a donor and an acceptor in which fluorescence resonance energy transfer occurs can be used. For example, a third fluorescent group (or a second fluorescent group) which is an atomic group having an absorption band in a wavelength range overlapping with the fluorescent wavelength of the second fluorescent group (or the third fluorescent group) or the like is used. Specifically, a combination of dinitrophenyl (Dnp) with (7-methoxycoumarin-1-yl) acetyl (M〇Ac), anthraniloylbenzyl (ABz), N-methylanthranilic acid (Nma), etc. And EDANS (5- (2'_aminoethyl) aminonaphthalene_1-sulfonic acid), tryptophan (Trp) and 5-dimethylamino-1-naphthalenesulfonic acid (Dns), carboxydichlorofluorescein (CDCF) ) And carboxymethyl rhodamine (CTMR), a combination of carboxydichlorofluorescein (CDCF) and carboxy X-rhodamine (CXR), a combination of lucifer yellow (LY) and carboxymethyl rhodamine (CTMR), and the like.

 Any of these donor receptors may be the second fluorescent group or the third fluorescent group. This is because if the second fluorescent group undergoes an outer change, it can be used as an indicator for measuring the enzyme activity.

 [0015] When the second fluorescent group is directly bonded to the substrate, tributophan (Trp) or the like having a plurality of reaction points is used as the second fluorescent group, and the fourth fluorescent group is combined with the fluorescent resonance energy transfer. For example, 5-dimethylamino-1-naphthalenesulfonic acid (Dns) is used. This is because the second fluorescent group needs to bind to the substrate and the fourth compound.

 [0016] The third compound and the fourth compound are the same as the first compound described in claim 1, and thus the description is omitted. Further, the fifth compound is the same as the second compound described in claim 1, and the description is omitted.

 [0017] It is desirable that the length between the bonding portions of the second fluorescent group and the third fluorescent group bonded to the third compound or the fourth compound and the fifth compound, respectively, is 100A or less. As the distance between the bond between the second fluorescent group and the third fluorescent group increases, the fluorescence resonance energy transfer tends to decrease and the change in the fluorescence intensity and the like tends to decrease. Is extremely small, and the sensitivity is lowered.

[0018] The substrate for enzyme activity detection according to claim 3 of the present invention comprises a substrate, a sixth compound having one end immobilized on the substrate, and a fourth fluorescent group introduced into the sixth compound. The fourth firefly The seventh compound bound to the photogroup, the eighth compound bound to the seventh compound by a peptide bond cleaved by an enzyme, and the fourth fluorescent group bound to the eighth compound and fluorescence resonance energy transfer. And a fifth fluorescent group to be observed.

 With this configuration, the following effects can be obtained by replacing the functions described in claim 1 or 2 with the functions described above.

 (1) By forming the sixth compound with a peptide or the like of a predetermined length, the distance between the substrate and the enzyme action point (peptide bond between the seventh and eighth compounds) is optimized, Enzyme can be allowed to act without being affected, enzyme activity can be detected more accurately, and detection sensitivity can be increased.In addition, it has the ability to cleave relatively long peptide chains, which does not have high substrate specificity for individual amino acids It is also possible to detect enzymes such as elastase, which are required for the above, and it is possible to increase the types of enzymes that can be detected.

[0019] Here, as the fourth fluorescent group, tributophan (Trp) or the like having a plurality of reaction points and introduced into a side chain, a terminal, or the like of the sixth compound is used. For example, 5-dimethylamino_1_naphthalenesulfonic acid (Dns), which causes fluorescence resonance energy transfer with tributophan, is used. This is because the fourth fluorescent group requires a plurality of reaction points that bind to the sixth compound and the seventh compound.

 [0020] The sixth compound is the same as the first compound described in claim 1, and therefore the description is omitted. Further, the seventh and eighth compounds are the same as the second compound described in claim 1, and thus the description is omitted.

 [0021] The length between the bonding portions of the fourth fluorescent group and the fifth fluorescent group bonded to the seventh compound and the eighth compound, respectively, should be 100 A or less as described in claim 3. Is desirable.

 [0022] The invention according to claim 4 of the present invention is the substrate for detecting an enzyme activity according to claim 2 or 3, wherein the fifth compound or the eighth compound is the fourth compound or the second compound. The fourth compound is bonded to the second fluorescent group with a peptide bond cleaved by the enzyme, instead of being bonded to the conjugate by a peptide bond cleaved by the enzyme; or The seventh compound has a structure in which the seventh compound is bonded to the fourth fluorescent group by a peptide bond cleaved by the enzyme.

With this configuration, in addition to the function obtained in claim 2 or 3, the following function can be obtained. It is.

 (1) Since the fourth compound or the seventh compound is bonded to the second fluorescent group or the fourth fluorescent group by a peptide bond that is cleaved by an enzyme, the fourth compound and the fifth compound are not included in the sample solution. Sequences can be made with amino acids that are not cleaved by the enzyme, and the degree of design freedom can be increased.

 Here, the amino acid of the fourth compound that binds to the second fluorescent group and the amino acid of the seventh compound that binds to the fourth fluorescent group are selectively cleaved at the C-terminal peptide bond by the enzyme. Things can be done. As a result, the fourth and seventh compounds bound to the second and fourth fluorescent groups are cleaved by the enzyme and released, and the third and second fluorescent groups bound to the fifth compound, The fluorescence intensity of the second and fourth fluorescent groups is changed in such a manner that no fluorescence resonance energy transfer occurs between the fifth and fourth fluorescent groups bound to the eighth compound. Can be.

 The invention according to claim 5 of the present invention is the substrate for detecting enzyme activity according to any one of claims 1 to 4, wherein the second compound, the fifth compound, and the eighth The second fluorescent group introduced into the terminal group of the compound and / or the side chain of the third compound has an acetylated structure.

 With this configuration, the following operation is obtained in addition to the operation obtained in any one of claims 1 to 4.

 (1) Since the terminal groups of the second compound and the fifth compound and the second fluorescent group introduced into the side chain of the third compound are acetylated, aminopeptidase acting on the N-terminal peptide bond Can significantly reduce the activity of exo-peptidases such as endopeptidase, etc., even if these enzymes are contained in the sample solution. it can.

 [0025] Here, as an acetyl ligating agent for acetylating the N-terminal of the second fluorescent group such as the terminal group of the peptide of the second compound or the third compound or the tributophan introduced into the side chain of the third compound, etc. Acetic anhydride, N-hydroxysuccinimide acetate and the like can be used.

[0026] In the method for detecting an enzyme activity according to claim 6 of the present invention, the substrate for enzyme activity detection according to any one of claims 1 to 5 is contacted with a sample solution containing an enzyme to cause a reaction. The And a step of measuring the fluorescence of the substrate for enzyme activity detection.

 With this configuration, the following operation is obtained.

 (1) After contacting a very small amount of a sample solution containing an enzyme with a substrate, the enzyme activity can be detected simply by measuring the fluorescence intensity or the like of the substrate, so that the measurement time can be reduced. Workability can be increased and measurement efficiency can be increased.

 (2) Enzyme activity can be detected only by contacting a very small amount of a sample solution containing an enzyme.Therefore, a large amount of sample solution is not required for measurement, and enzyme activity can be detected even with a very small amount of sample solution. It can be carried out.

 (3) Since the enzyme activity is detected by fluorescence measurement, detection sensitivity and measurement accuracy can be improved.

 (4) By using a substrate for detecting enzyme activity in which a fluorescent group is bonded to each of different types of peptides, etc., and performing a wide range of image analysis with an image sensor, etc., the enzyme activity of a sample solution containing multiple enzymes can be determined. Measurement and analysis can be performed comprehensively in a short time, and measurement efficiency can be dramatically improved.

 Here, as the sample solution containing the enzyme, a solution adjusted to a pH at which the enzyme exhibits activity is used. As this pH adjuster, a buffer such as Tris_HCl, Hepes-K〇H or the like can be added as a reaction buffer. In addition, salts required for the expression of the enzyme activity and an activity protecting agent can be added.

 The invention's effect

 [0028] As described above, according to the enzyme activity detection substrate and the enzyme activity detection method using the same of the present invention, the following advantageous effects can be obtained.

 According to the invention described in claim 1,

 (1) Since the fluorescent wavelength of the first fluorescent group from which the second compound has been released or the fluorescent intensity at a predetermined wavelength is different from that of the first fluorescent group in which the second compound is peptide-bonded, the enzyme is determined using the change in the fluorescent intensity as an index It is possible to provide a substrate for detecting enzyme activity which is excellent in versatility and capable of detecting an activity.

(2) Since a fluorescent group is bonded to the substrate, contact a trace amount of sample solution containing Enzyme activity can be detected simply by measuring the fluorescence intensity of the plate, etc., and the degree of integration of the detection section can be dramatically increased.The first fluorescence can be obtained without forming a cell or the like for injecting the sample solution. It is possible to realize a fluorescence measurement microplate having 10,000 or more detection units on a substrate having a size of 2 cm in length and 3 cm in width, for example, in a flat plate shape or the like having only groups bonded thereto.

 (3) Enzyme activity can be detected simply by contacting a very small amount of sample solution containing the enzyme, so that a large amount of sample solution is not required for measurement, and enzyme activity can be detected even with a small amount of sample solution. It is possible to provide a substrate for enzyme activity detection which can be performed and has excellent operability.

 [0029] According to the invention described in claim 2, in addition to the effect of claim 1,

 (1) When the peptide bond is cleaved by the enzyme, the distance between the second fluorescent group and the third fluorescent group is increased, so that the fluorescence resonance energy transfer does not occur, and the transfer from the second fluorescent group (or the third fluorescent group) occurs. The change in the spectrum from the fluorescence spectrum to the fluorescence spectrum from the third fluorescent group (or the second fluorescent group) can be used as an indicator of the enzyme activity. A substrate for enzyme activity detection which is excellent in versatility and can be detected.

 (2) By selecting the second fluorescent group and the third fluorescent group, it becomes possible to set the fluorescent wavelength of the second fluorescent group in the visible region, so that a visible light detection device such as a commercially available CCD camera can be used. This makes it possible to provide a substrate for detecting enzyme activity which is excellent in versatility.

 (3) By forming the fifth conjugate with a peptide chain having a predetermined length (for example, about 15 A) or more, a relatively long peptide chain having a high substrate specificity for each amino acid is cleaved. In addition, it is possible to provide an enzyme activity detection substrate that can detect enzymes such as elastase, which is required for the above, and can increase the types of enzymes that can be detected and can increase the detection sensitivity.

 [0030] According to the invention of claim 3, in addition to the effect of claim 1 or 2,

(1) By optimizing the distance between the substrate and the enzyme action point, the enzyme can act without being affected by the substrate, the enzyme activity can be detected more accurately, and the detection sensitivity can be increased. Rather than having high substrate specificity for each amino acid, it is also possible to detect enzymes such as elastase that require a relatively long peptide chain for cleavage, thereby increasing the types of enzymes that can be detected. A substrate for detecting enzyme activity can be provided.

 According to the invention described in claim 4, in addition to the effect of claim 2 or 3,

 (1) It is possible to provide a substrate for detecting enzyme activity, in which the fourth compound and the fifth compound can be arranged so as not to have enzyme specificity and the degree of freedom in design can be increased.

 [0032] According to the invention described in claim 5, in addition to the effect of any one of claims 1 to 4,

 (1) The activity of exopeptidases such as aminopeptidase that acts on the N-terminal peptide bond can be significantly reduced, and even if these enzymes are contained in the sample solution, an endo-substrate with high substrate specificity can be used. It is possible to provide a substrate for detecting an enzyme activity, which is capable of accurately detecting the activity of an enzyme such as a peptidase and has excellent detection accuracy.

 [0033] According to the invention described in claim 6,

 (1) After contacting a very small amount of a sample solution containing an enzyme with a substrate, the enzyme activity can be detected simply by measuring the fluorescence intensity or the like of the substrate, so that the measurement time can be reduced. It is possible to provide a method for detecting an enzyme activity that can enhance workability and enhance measurement efficiency.

 (2) Enzyme activity can be detected only by contacting a very small amount of a sample solution containing an enzyme.Therefore, a large amount of sample solution is not required for measurement, and enzyme activity can be detected even with a very small amount of sample solution. A method for detecting enzyme activity that can be performed can be provided.

 (3) Since the enzyme activity is detected by fluorescence measurement, a method for detecting the enzyme activity with high detection sensitivity can be provided.

 (4) By using a substrate for detecting enzyme activity in which a fluorescent group is bonded to each of different types of peptides, etc., and performing a wide range of image analysis with an image sensor, etc., the enzyme activity of a sample solution containing multiple enzymes can be determined. It is possible to provide a method for detecting an enzyme activity, which can comprehensively measure and analyze in a short time and can dramatically improve the measurement efficiency.

 Brief Description of Drawings

FIG. 1 is a schematic diagram showing the principle of detecting enzyme activity of a substrate for detecting enzyme activity in Embodiment 1. FIG. 2 is a schematic view showing the principle of detecting enzyme activity of a substrate for detecting enzyme activity in Embodiment 2.

 FIG. 3 is a schematic diagram of a substrate for detecting enzyme activity in Embodiment 3.

 FIG. 4 is a schematic diagram of an enzyme activity detection substrate according to a fourth embodiment.

 Explanation of symbols

[0035] 1, 10, 20, 30 Enzyme activity detection substrate

 2 Board

 3, 6 First fluorescent group

 4 Second compound

 5 enzymes

 11 Third compound

 12, 15 Second fluorescent group

 13 Fifth compound

 14 Third fluorescent group

 21 4th compound

 31 6th compound

 32 4th fluorescent group

 33 7th compound

 34 8th compound

 35 Fifth fluorescent group

 BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the best mode for carrying out the present invention will be described with reference to the drawings.

 (Embodiment 1)

 FIG. 1 is a schematic diagram showing the principle of detecting enzyme activity of the substrate for detecting enzyme activity according to Embodiment 1 of the present invention.

In the figure, 1 is a substrate for detecting the enzyme activity in Embodiment 1, 2 is a synthetic resin (polystyrene or the like) insoluble in solvents such as halogenated hydrocarbons and esters, glass, etc. Substrate 3 formed into a curved surface, etc., 3 is directly connected to substrate 2 by peptide bonds, etc. Before and after the peptide bond with the second compound 4 described below is cleaved by the enzyme 5 described below, one of the fluorescent groups that changes in the fluorescence wavelength and the fluorescence intensity, ie, 4-methylcoumaryl-7-amide ( (MCA) or the like, 4 is a second compound such as an amino acid or a peptide linked to the first fluorescent group 3 by a peptide bond cleaved by an enzyme 5 described below, and 5 is the first fluorescent group 3 and the second fluorescent group. An enzyme having a substrate specificity such as a serine protease that selectively cleaves a peptide bond with the conjugate 4, and 6 is a fluorescent wavelength due to the selective release of the second compound 4 by the enzyme 5. Is the changed first fluorescent group.

 [0037] The substrate 1 for enzyme activity detection configured as described above is prepared by fixing a C-terminal of an amino acid on the substrate 2 and elongating the peptide at the C-terminal, using a conventional peptide synthesis method such as a solid phase method. Using a sequential extension method in which the desired amino acid sequence is sequentially extended from the C-terminal side to the N-terminal side, a fragment condensation method in which multiple short peptide fragments are synthesized and extended by coupling between peptide fragments, and a peptide synthesizer. It can be synthesized using a method such as Fmoc method, Boc method, etc.

 The principle of detecting the enzyme activity of the substrate for enzyme activity detection of Embodiment 1 configured as described above will be described below.

 The first fluorescent group 3 such as 4-methylcoumaryl-17-amide (MCA) of the enzyme activity detection substrate 1 shown in FIG. 1 (a) is a non-fluorescent substance and does not show fluorescence in a specific wavelength region. When a sample solution containing the enzyme 5 is brought into contact with the enzyme activity detection substrate 1 and allowed to react, the enzyme 5 such as a serine protease having substrate specificity is formed between the first fluorescent group 3 and the second compound 4. It selectively cleaves peptide bonds (see Figure 1 (b)).

 The first fluorescent group 6 from which the second conjugated product 4 is released becomes a fluorescent substance such as 7-amino-methyl coumarin (AMC), and the fluorescent wavelength or the fluorescent intensity in the specific wavelength region is the same as that of the second compound 4. Since it is different from the peptide-bonded first fluorescent group 3, the enzyme activity can be detected using the change in fluorescence intensity or the like as an index (see FIG. 1 (c)).

As described above, since the enzyme activity detecting substrate according to Embodiment 1 is configured, the following effects can be obtained.

(1) Since a fluorescent group is bonded to the substrate, only a very small amount of a sample solution containing the enzyme is brought into contact with the substrate as a detection unit, and the fluorescence intensity of the substrate after a predetermined time is measured. A cell into which a sample solution is injected onto a substrate can detect enzyme activity depending on the amount of enzyme, the type of enzyme, etc., using the change in fluorescence intensity or the like corresponding to the number of molecules modified by the action of the enzyme as an index. Since the detection unit can be miniaturized without the necessity of forming such elements, the degree of integration of the detection unit on the substrate can be dramatically increased.

 (2) Enzyme activity can be detected only by contacting a very small amount of a sample solution containing an enzyme.Therefore, a large amount of sample solution is not required for measurement, and enzyme activity can be detected even with a very small amount of sample solution. It can be carried out.

 In this embodiment, the case where the first fluorescent group 3 is directly bonded to the substrate 2 has been described. However, the first compound such as an amino acid or a peptide bonded to the substrate 2 has the first fluorescent group 3 attached thereto. In some cases, the first fluorescent group 3 is bonded to the substrate 2 via the first compound. Also in this case, the second compound 4 is bound to the first fluorescent group 3 by a peptide bond cleaved by the enzyme 5, as described in the first embodiment. This makes it possible to optimize the distance between the substrate 2 and the enzyme action point (the peptide bond between the first fluorescent group 3 and the second compound 4), thereby allowing the enzyme to act without being affected by the substrate. The effect is obtained that the activity can be detected more accurately. In this case, the first compound is synthesized with a peptide or the like having a sequence that is not cleaved by the enzyme in the sample solution. By preventing the first compound from being cleaved by the enzyme, the first fluorescent group 3 bonded to the substrate 2 via the first compound is released from the substrate by the action of the enzyme, and before and after contacting the enzyme. The first fluorescent group 3 in which the fluorescence intensity or the like has changed drifts in the sample solution, and the relationship between the binding location of the first fluorescent group 3 to the substrate 2 and the change in the fluorescence intensity or the like at that location is unclear. This is to prevent the As a result, if the sample solution is brought into contact with a substrate for enzyme activity detection in which peptides having different types of amino acid sequences are bound to a plurality of sites on a single substrate, the fluorescence intensity of the substrate can be simply measured. Patterns such as fluorescence intensity that differ depending on the type of enzyme are obtained, and enzyme activity characterized by the amino acid sequence can be detected.

 (Embodiment 2)

FIG. 2 is a schematic diagram illustrating the principle of detecting enzyme activity of the substrate for detecting enzyme activity according to Embodiment 2 of the present invention. Note that the same components as those in the first embodiment are denoted by the same reference numerals, and description thereof is omitted. In the figure, reference numeral 10 denotes a substrate for detecting an enzyme activity in Embodiment 2, 11 denotes a third compound such as an amino acid or peptide having one end immobilized on the substrate 2, and 12 denotes a side chain of the third conjugate 11. Introduced third fluorescent group 14 described below and fluorescence resonance energy transfer can be seen. Second fluorescent groups such as (7-methoxycoumarin_4_yl) acetyl (M〇Ac) and tryptophan (Trp), and 13 is the third fluorescent group Fifth compound such as amino acid and peptide linked by compound 11 and peptide bond cleaved by enzyme 5, and 14 is dinitrophenyl (Dnp), 5-dimethylamino-1-naphthalenesulfonic acid (Dns ) Is a third fluorescent group. The second fluorescent group 12 and the third fluorescent group 14 are bonded to each other at a distance (100 A or less) at which fluorescence resonance energy transfer is observed. Reference numeral 15 denotes a second fluorescent group whose fluorescence wavelength and the like have been changed by the selective release of the fifth conjugate 13 to which the third fluorescent group 14 is bonded by the enzyme 5.

The principle of detecting the enzyme activity of the substrate for enzyme activity detection of Embodiment 2 configured as described above will be described below.

 Since the second fluorescent group 12 and the third fluorescent group 14 of the enzyme activity detection substrate 10 shown in FIG. 2A are bonded to each other at a distance where fluorescence resonance energy transfer can be seen, the fluorescence of the second fluorescent group 12 The vector and the excitation spectrum of the third fluorescent group 14 overlap, and when the energy of the excitation wavelength of the second fluorescent group 12 is applied, the fluorescence of the second fluorescent group 12 that should be observed is attenuated, and The fluorescence of the third fluorescent group 14 is observed.

 When a sample solution containing the enzyme 5 is brought into contact with the substrate 10 for enzyme activity detection and allowed to react, the enzyme 5 having substrate specificity cleaves the peptide bond between the third compound 11 and the fifth compound 13 (FIG. 2). (b)).

 When the fifth fluorescent compound 13 to which the third fluorescent group 14 is bonded is released, no fluorescence resonance energy transfer is observed between the third fluorescent group 14 and the second fluorescent group 12, so that the second fluorescent group 12 When the energy of the excitation wavelength is applied, the fluorescence wavelength of the second fluorescent group 12, which should have been observed, is observed, and is different from the fluorescence wavelength before the reaction of the enzyme 5. Enzyme activity can be detected (see Fig. 2 (c)).

As described above, since the substrate for detecting an enzyme activity in the second embodiment is configured, the following operations can be obtained by adding the functions described in the first embodiment.

(1) By selecting the second fluorescent group and the third fluorescent group, the fluorescent wavelength of the second fluorescent group is Since it is possible to set the area, it is possible to use a visible light detection device such as a commercially available CCD camera to perform measurement, which is excellent in versatility.

 (2) By forming the fifth conjugate with a peptide chain having a predetermined length (for example, about 15 A) or more, a relatively long peptide chain having a high substrate specificity for each amino acid is cleaved. In addition, it is possible to detect enzymes such as elastase, which are required for the detection, and it is possible to increase the types of enzymes that can be detected and to increase the detection sensitivity.

 (Embodiment 3)

 FIG. 3 is a schematic view of a substrate for detecting enzyme activity according to Embodiment 3 of the present invention. Note that the same components as those of the second embodiment are denoted by the same reference numerals, and description thereof is omitted.

 In the figure, reference numeral 20 denotes a substrate for detecting an enzyme activity in Embodiment 3, and reference numeral 21 denotes a side chain or terminal of a second fluorescent group 12 such as tributophan (Τιρ) having a plurality of reaction points directly bonded to the substrate 2. This is the fourth compound such as amino acids and peptides introduced into the product. The fifth compound 13 is bonded to the fourth compound 21 by a peptide bond that is cleaved by the enzyme.

 The difference between the substrate for enzyme activity detection in the third embodiment and the second embodiment is that the second fluorescent group 12 directly bonded to the substrate 2 is bonded to the fourth compound 21.

 The principle of detecting the enzyme activity on the enzyme activity detecting substrate according to the third embodiment configured as described above is the same as that described in the second embodiment, and a description thereof will be omitted.

 [0045] As described above, the substrate for detecting enzyme activity in Embodiment 3 is configured. Therefore, after the operation described in Embodiment 2 is performed and the second fluorescent group is immobilized on the substrate, the normal operation is performed. It can be synthesized simply by elongating the fourth compound or the fifth compound using the peptide synthesis method, so that the operability of synthesizing the substrate for enzyme activity detection is excellent and the product yield can be increased. Is obtained.

In the present embodiment, the case where the fifth compound 13 is bonded to the fourth compound 21 by a peptide bond that is cleaved by an enzyme has been described. 4 In some cases, the compound 21 may have an amino acid sequence that is not cleaved by an enzyme, so that the second fluorescent group 12 and the fourth conjugate 21 are bound by a peptide bond that is cleaved by the enzyme. As a result, an effect is obtained that the degree of freedom of the amino acid sequence constituting the fourth compound and the fifth compound can be increased and the design can be facilitated. (Embodiment 4)

 FIG. 4 is a schematic view of a substrate for detecting an enzyme activity according to Embodiment 4 of the present invention. Note that the same components as those in the first embodiment are denoted by the same reference numerals, and description thereof is omitted.

 In the figure, 30 is a substrate for detecting enzyme activity in Embodiment 4, 31 is a sixth compound such as an amino acid or peptide having one end bonded to the substrate 2, and 32 is one reaction site bonded to the sixth conjugate 31. A fourth fluorescent group such as tributophan (Τι · ρ), 33 is a seventh compound such as an amino acid or a peptide to which another reactive site of the fourth fluorescent group 32 is bonded, and 34 is a seventh fluorescent compound 33 and an enzyme. The eighth compound 35, such as an amino acid or a peptide, bound by a peptide bond is bound by the eighth bond 34, and fluorescence resonance energy transfer is observed with the fourth fluorescent group 32. 5-Dimethylamino-1 It is a fifth fluorescent group such as naphthalenesulfonic acid (Dns).

 The principle of detecting the enzyme activity in the enzyme activity detecting substrate according to the fourth embodiment configured as described above is the same as that described in the second embodiment, and a description thereof will be omitted.

 [0048] As described above, the substrate for detecting an enzyme activity in Embodiment 4 is configured, and thus the following operation is obtained by adding the function described in Embodiment 2 to the substrate.

 (1) By forming the sixth compound 31 with a peptide or the like having a predetermined length, the substrate 2 and the enzyme action point (the peptide bond between the seventh and eighth conjugates 33 and 34) ), The enzyme can act without being affected by the substrate 2, the enzyme activity can be detected more accurately, the detection sensitivity can be increased, and the substrate specificity for each amino acid can be improved. It is also possible to detect enzymes such as elastase that require a relatively long peptide chain for the cleavage action, which can increase the number of detectable enzymes.

 [0049] In the present embodiment, the case where the eighth conjugated product 34 is bonded to the seventh conjugated product 33 by a peptide bond that is cleaved by an enzyme has been described. In some cases, the 34th compound and the seventh compound 33 have an amino acid sequence that is not cleaved by the enzyme, so that the fourth fluorescent group 32 and the seventh compound 33 are bound by a peptide bond cleaved by the enzyme. As a result, an effect is obtained that the degree of freedom of the amino acid sequence constituting the seventh compound and the eighth compound can be increased and the design can be facilitated.

 Example

Hereinafter, the present invention will be described specifically with reference to Examples. The present invention is not limited to these examples. It is not limited.

Amino acids, peptides, protecting groups, solvents, and the like described in this example use abbreviations commonly used in the art or abbreviations adopted by the IUPAC-IUB naming committee. For example, the following abbreviations are used. Ala: Alanine, Pro: Proline, Lys: Lysine, Phe: Phenylalanine, Aca: Aminocaproic acid, Ac: Acetyl, ACC: 7-Amino-4-carboxymethylcoumarin, Boc: t_Butyloxycarbonyl, Lys ( Boc): Side-chain t-butyloxycarbonyl-protected lysine, DCC: Dicyclohexylcarbodiimide, DCM: Dichloromethane, DIEA: N, N_diisopropylethylamine, DMF: N, N_Dimethinolehonolemamide, Et〇H : Ethanomono, Fmoc: 9-Fluorenylmethyloxycarbonyl, HATU: o_ (7-azabenzotriazonole-1-yl) _1,1,3,3-tetramethylperoniumhexaflu Olophosphate, HBTU: o_ (benzotriazono-1-yl) _1,1,3,3-tetramethylperonidium oxafluorophosphate, HOAt: 1-hydroxy-7— Hazobenzotriazole, H〇Bt: 1 —Hide Alkoxy benzotriazoles § tetrazole, TFA: Torifuruoro acetate, TYp: tryptophan, Dns: 5-di-methylamino-1 one naphthalenesulfonic acid.

(Example 1)

In Example 1, the activity of the enzyme was measured by synthesizing a peptidyl fluorescent group-bonded spherical substrate as a substrate for detecting the enzyme activity. Hereinafter, the method will be described.

<Synthesis of spherical substrate bonded with peptidyl fluorescent group>

As the substrate, a spherical commercially available NH-PEGA-resin (manufactured by Watanabe Chemical Industry) was used. Solid phase synthesis

 2

Fix the vessel vertically, add NH-PEGA-resin (0.05 mmol / g, 0.5 g) and turn on the cock.

 2

DMF (10 ml) was flowed in the open state to replace the solvent. Next, close the vessel cock and dissolve Fmoc-Aca-OH (0.13 mmol, 44 mg), HBTU (0.13 mmol, 48 mg), and DIEA (0.13 mmol, 0.022 ml) in DMF (2 ml). Well, I made a reaction. After the reaction, the cock was opened and washed with DMF (10 ml) and methanol (10 ml) to obtain Fmoc-Aca_PEGA resin. Next, DMF (10 ml) was flown into the Fmoc-Aca-PEGA resin (0.05 mmol / g, 0.5 g) in the vessel with the cock open, and solvent replacement and washing were performed. The cock was closed, 20% piberidine / DMF was added, and the mixture was reacted for 30 minutes to remove Fmoc. Thereafter, the cock was opened to remove 20% pyridine ZDMF, and then washed with DMF (10 ml). Then I closed the cock In this state, Fmoc-Aca-OH (3 eq), HATU (3 eq), HOAt (3 eq), and DIEA (5 eq) were dissolved in DMF (2 ml), added, and reacted for 3 hours. Then, open the cock, wash with DMF (10 ml) and methanol (10 ml), and the first compound (Aca-Aca_) was bound to the substrate (PEGA resin).

Fmoc-Aca-Aca-PEGA resin was obtained.

 Next, the mixture was stirred with 20% piperidine ZDCM (1 mL) for 30 minutes to remove the Fmoc group. After washing three times with DCM (1 ml) and once with DMF (1 ml), it was dissolved in 1 ml of DMF

Fmoc-Lys (Boc) -ACC-OH (36 mg, 54 mmol), DCC (11 mg, 54 mmol), HOBt-H〇

(8.3 mg, 54 mmol) was added and reacted for 24 hours. After the reaction is complete, add 2x DMF (1 ml)

(1 ml) twice, twice with EtOH (1 ml), and twice with DCM (1 ml), and dried under reduced pressure to allow the first fluorescent group (ACC) to bind to the first compound (Aca-Aca- )

Fmoc-Lys (Boc) -ACC-Aca-Aca-PEGA resin was obtained.

 Thereafter, the resultant was washed once with DMF (1 ml), DIEA (32 ml, 183 mmol) and acetic anhydride (8.5 ml, 90 mmol) were diluted in DMF (1 ml), and the mixture was stirred for 1 hour. Thereafter, washing was performed three times with DMF (1 ml) and three times with DCM (1 ml), and the unreacted material was acetylated.

 Next, the mixture was stirred with 20% piperidine / DCM (1 ml) for 30 minutes to remove the Fmoc group. After washing with DCM (1 ml) three times with DMF (1 ml), Fmoc-Pro-OH (18 mg,

54 mmol), HATU (20 mg, 54 mmol), HOAt (7 mg, 54 mmol) and DIEA (16 ml, 90 mmol) were dissolved in DMF (1 ml) and reacted for 1 hour. Then, it was washed three times with DMF (1 ml) and three times with DCM (1 ml) to obtain Fmoc-Pro-Lys (Boc) -ACC-Aca-Aca-PEGA resin.

 Hereinafter, the same operation is repeated by using Fmoc-Ala-OH on Fmoc-Pro-Lys (Boc) -ACC-Aca-Aca-PEGA resin to extend the peptide, and the second compound (ACC) is added to the first fluorescent group (ACC).

Ala-Ala-Pro-Lys (Boc) -ACC_Aca-Aca-PEGA resin to which Ala-Ala-Pro-Lys was bound was obtained. After that, close the cock and dilute DIEA (2.5 mmol, 0.435 ml) and acetic anhydride (1.25 mmol, 0.117 ml) in DMF (2 ml), add them, and allow them to react for 1 hour. Acesila-Ala_Ala_Pro-Lys (Boc) -ACC-Aca_Aca_PEGA resin was obtained.

After adding Ac-Ala-Ala-Pro_Lys (Boc) -ACC-Aca-Aca-PEGA resin (0.05 mmol, 0.5 mg) to the vessel, DCM (10 ml) was poured with the cock open, and the solvent was replaced. Then, close the cock, add 25% TFA / DCM (2 ml), react for 30 minutes, remove Boc, then add DCM (10 ml), H After washing with O (10 ml), the desired peptidyl fluorescent group-bonded spherical substrate of Example 1

(Ac-Ala-Ala-Pro-Lys-ACC-Aca-Aca-PEGA resin) was obtained.

<Measurement of enzyme activity>

 Peptidinole fluorescent-group-bound spherical substrate as enzyme activity detection substrate of Example 1

 Ac-Ala-Ala-Pro-Lys-ACC-Aca-Aca-PEGA resin and the following solution were added, and the difference between the fluorescence value at the start of the experiment and the fluorescence value after 30 minutes was measured. Fluorescence values were measured using a WALLAC ARVOTM SX 1420 multilabel counter (manufactured by PerkinElmer) at an excitation wavelength of 370 nm and an emission wavelength of 460 nm.

 A: Ac-Ala-Ala-Pro-Lys-ACC-Aca-Aca-PEGA resin (wet) 5 mg, 20 mM Tris HC1 buffer (pH 7.2, 100 mM NaCl, 50 mM CaCl 2) 240 μl, trypsin (1 mg / 1 ml) to 10 μΐ

Β: Ac-Ala-Ala-Pro-Lys-ACC-Aca-Aca-PEGA resin (wet) 5 mg, 20 mM Tris HC1 buffer (pH 7.2, 100 mM NaCl, 50 mM CaCl) 240 μ1, chymotrypsin (1 mg / 1 ml)

10 μ \

 C: Ac-Ala-Ala-Pro-Lys-ACC-Aca-Aca-PEGA resin (wet) 5 mg, 20 mM Tris HC1 buffer (pH 7.2, 100 mM NaCl, 50 mM CaCl) 250 μ1

 D: 20 mM Tris HC1 buffer (pH 7.2, 100 mM NaCl, 50 mM CaCl) 250 μ1,

 The difference between Gael A and Gael B is the type of enzyme. The difference between Gael A (or Gael B) and Gael C is the presence or absence of the enzyme, and Gael A (or Gael B, C) and Gael. The difference from D is the presence or absence of a substrate for enzyme activity detection.

 The difference between the fluorescence value at the start of the experiment and the fluorescence value after 30 minutes for each well is shown in (Table 1).

[0053] [Table 1]

Comparing the fluorescence values of the wells A and B in Table 1 and the well C, the enzyme activity detection substrate of Example 1 was about 315-fold in the well A with trypsin and in the well B with chymotrypsin. It was confirmed that the difference was about 10 times. As a result, the substrate for enzyme activity detection of Example 1 It was shown that the enzyme activity can be detected based on the amount of the enzyme and the type of the enzyme. Further, comparing the fluorescence values of the wells A and B, the enzyme activity detection substrate of Example 1 shows that the fluorescence value of trypsin is about 30 times larger than that of chymotrypsin. Force S confirmed. This is the amino acid force S-lysine of the second conjugate which binds to the first fluorescent group of the substrate for enzyme activity detection of Example 1, and trypsin mainly selectively binds the peptide bond at the C-terminal side of lysine. It is presumed that the gene was expressed because of its specificity for cleavage. On the other hand, chymotrypsin mainly has the specificity of selectively cleaving the peptide bond at the C-terminal side of the aromatic amino acid residue, so it is presumed that the change in the fluorescence value before and after the reaction was small.

This indicates that the enzyme activity detection substrate of Example 1 has specificity depending on the enzyme, and that qualitative analysis of the enzyme having activity is possible. When a sample solution containing the same type of enzyme was brought into contact with the same type of enzyme activity detection substrate and the fluorescence value was measured after a predetermined time, the change in the fluorescence value was modified by the action of the enzyme. It was speculated that quantitative analysis of the enzyme was possible because it corresponds to the number of molecules.

(Example 2)

In Example 2, an enzyme activity was measured by synthesizing a peptidyl fluorescent group-bonded flat substrate as a substrate for enzyme activity detection. Hereinafter, the method will be described.

<Synthesis of planar substrate bonded with peptidyl fluorescent group>

As a substrate, a carrier made of a multi-plate synthetic resin for peptide synthesis (lanthanum series (registered trademark) manufactured by Mimotobus Co., Ltd.) was cut off by only one plate and made into a planar shape. One lantern as a substrate (D-series manufactured by Mimotops Co., introduction rate 18 mmol / unit) was placed in a screw tube, and the mixture was stirred with 20% piperidine ZDCM (1 mL) for 30 minutes to remove the Fmoc group. After washing three times with DCM (1 ml) and DMF (1 ml), Fmoc_Aca-〇H (19 mg 54 mmol), DCC (17 mg 81 mmol), HOBt-H〇 (8 mg 54 mmol) (1 ml) and add

 2

The reaction was performed for 24 hours. After the reaction is completed, wash twice with DMF (1 ml), twice with DCM (1 ml), twice with EtH (1 ml) and twice with DCM (1 ml), and dried under reduced pressure. I got Fmoc_Aca_lantern.

Next, the mixture was stirred with 20% piperidine ZDCM (1 ml) for 30 minutes to remove the Fmoc group. . After washing with DCM (1 ml) three times with DMF (1 ml), Fmoc-Aca-OH (19 mg, 54 mmol), HATU (20 mg, 54 mmol), HOAt (7 mg, 54 mmol) ), DIEA (16 ml, 90 mmol) was dissolved in DMF (1 ml) and reacted for 1 hour. Then, the plate was washed three times with DMF (1 ml) and three times with DCM (1 ml), and Pmoc_Aca_Aca_lantern having one end of the first compound (Aca-Aca) immobilized on a substrate (lantern) was obtained.

Next, the mixture was stirred with 20% piperidine ZDCM (1 mL) for 30 minutes to remove the Fmoc group. The lantern was swollen by washing three times with DCM (1 ml) and once with DMF (1 ml), and Fmoc-Lys (Boc) -ACC-OH (36 mg, 54 mmol) dissolved in 1 ml of DMF was dissolved. ), DCC (11 mg, 54 mmol) and HOBt-HO (8.3 mg, 54 mmol) were added and allowed to react for 24 hours. After the reaction is completed, wash twice with DMF (1 ml), twice with DCM (1 ml), twice with EtOH (1 ml), and twice with DCM (1 ml), and then dried under reduced pressure for the first time. Compound (Aca-Aca) bound to the first fluorescent group (ACC)

Pmoc_Lys (Boc-A _Aca_Aca_lantern was obtained.

 Thereafter, the resultant was washed once with DMF (1 ml), DIEA (32 ml, 183 mmol) and acetic anhydride (8.5 ml, 90 mmol) were diluted in DMF (1 ml), and the mixture was stirred for 1 hour. Thereafter, washing was performed three times with DMF (1 ml) and three times with DCM (1 ml), and the unreacted material was acetylated.

 Next, the mixture was stirred with 20% piperidine / DCM (1 ml) for 30 minutes to remove the Fmoc group. After washing with DCM (1 ml) three times with DMF (1 ml), Fmoc-Pro-OH (18 mg, 54 mmol), HATU (20 mg, 54 mmol), HOAt (7 mg, 54 mmol) ), DIEA (16 ml, 90 mmol) was dissolved in DMF (1 ml) and reacted for 1 hour. Thereafter, the resultant was washed three times with DMF (1 ml) and three times with DCM (1 ml) to obtain Fmoc-Pro-Lys (Boc) -ACC-Aca-Aca-lantern.

 The same procedure was repeated twice with Fmoc-Ala-OH on Fmoc-Pro-Lys (Boc) -ACC-Aca-Aca-lantern to extend the peptide, and the second fluorescent group (ACC) Fmoc-Ala_Ala_Pro-Lys (Boc) _ACC-Aca_Aca-lantern to which the compound (Ala-Ala-Pro-Lys) was bound was obtained.

Thereafter, the mixture was stirred with 20% piperidine ZDCM (1 ml) for 30 minutes to remove the Fmoc group. Then, after washing with DMF (1 ml) three times with DCM (1 ml), DMF (1 mL), DIEA 32 ml (183 mmol) and acetic anhydride 8.5 ml (90 mmol) were added to DMF (1 mL). , And stirred for 1 hour. Then, wash the lantern 3 times with DMF (1 ml) and 3 times with DCM (1 ml), 25% The reaction was carried out using TFA / DCM for 30 minutes to remove the Boc group. Then, it is washed three times with DCM (1 ml), five times with H 0 (1 ml), three times with DCM (1 ml), dried under reduced pressure, and dried under reduced pressure.

Ala-Ala-Pro-Lys) terminal group was acetylated

 Ac-Ala-Ala-Pro-Lys (Boc) -ACC-Aca-Aca-lantern was obtained.

 The Boc group of the Lys side chain was removed using 25% TFA / DCM (lml) to obtain the desired substrate for detecting the enzyme activity (Ac-Ala-Ala-Pro-Lys-ACC_Aca-Aca_lantern) of Example 2. Was.

<Measurement of enzyme activity>

 On the substrate for enzyme activity detection of Example 2, 100 μl of 20 mM Tris HC1 buffer (pH 7.2, 100 mM NaCl, 50 mM CaCl), 100 μl of methanol, and 50 μl of trypsin (1 mg / 1 ml) were added. Addition

 2

 The fluorescence values before and after the reaction were measured. The fluorescence value was measured at an excitation wavelength of 370 nm and a fluorescence wavelength of 460 nm using a WALLAC ARVOTM SX 1420 multilabel counter (manufactured by PerkinElmer).

 As a result, the initial fluorescence value was 39,000, the fluorescence value after 18 hours of reaction was 145,000, and it was confirmed that the fluorescence value changed about 4-fold. As a result, it was shown that the enzyme having activity can be detected also in the enzyme activity detecting substrate of Example 2 using lantern as the substrate.

(Example 3)

 In Example 3, an enzyme activity was measured by synthesizing a peptidyl fluorescent group-bonded flat substrate as a substrate for detecting enzyme activity. Hereinafter, the method will be described.

 <Synthesis of planar substrate bonded with peptidyl fluorescent group>

 As a substrate, a carrier made of a multi-plate synthetic resin for peptide synthesis (lanthanum series (registered trademark) manufactured by Mimotobus Co., Ltd.) was cut off by only one plate and made into a planar shape. One lantern as a substrate (D_series, introduced by Mimotops Co., 18 mmol / unit) was placed in a screw tube, and the mixture was stirred with 20% piperidine / DCM (1 mL) for 30 minutes to cut out the Fmoc group. After washing with DCM (1 mix x 3), lantern was swollen with DMF (1 ml XI) and dissolved in 1 ml of DMF Fmoc-Aca-OH 20.0 mg (56.6 mmol), DCC 17.5 mg (84.5 mmol), HOBt -H〇

8.8 mg (57.5 mmol) was added and the mixture was stirred for 23 hours. DMF (1 ml X 2), DCM (1 ml X 2), DCM I EtOH = 1: 1 (1 mix 2), EtOH (1 ml X 2), DCM (1 ml XI), getyl ether (1 ml After washing the resin in X1) and drying, Fmoc-Aca-lantern was obtained.

 The mixture was stirred for 30 minutes using 20% piperidine / DCM (1 mL) to cut out the Fmoc group, washed with DCM (1 mix 3), and dissolved in 1 ml of DMF. Fmoc—Aca—OH 23.0 mg (65.1 mmol), 21.6 mg (56.8 mmol) of HATU, 8.1 mg (59.5 mmol) of HO At, and 15.7 ml (90.2 mmol) of DIEA were added and stirred for 1 hour. After washing with DMF (1 mix 3) and DCM (1 ml X 3), Fmoc-Aca-Aca-lantern having one end of the first compound (Aca-Aca) immobilized on a substrate (lantern) was obtained. Then, the mixture was stirred with 20% piperidine ZDCM (1 mL) for 30 minutes to remove the Fmoc group, and washed with DCM (1 mix 3) to obtain H-Aca-Aca-lantern.

[0059] Next, Fmoc-Phe-ACC-OH (33.4 mg 56.7 mmol), DCC 12.5 mg (60.6 mmol), HOBt-HO 8.9 mg (58.0 mmol) dissolved in 1 ml of DMF were added for 22 hours. Stirred. DMF

(1 mix x 2), DCM (1 ml X 2), DCM I EtOH = 1: 1 (1 ml X 2), EtOH (1 ml X 2), DCM (1 ml XI), getyl ether (1 mi After washing with x1), it was dried to obtain Fmoc-Phe-ACC-Aca-Aca-Lantern in which the first fluorescent group (ACC) was bonded to the first compound (Aca-Aca).

 Next, the mixture was stirred with 20% piperidine / DCM (1 mL) for 30 minutes to remove the Fmoc group, and washed with DCM (1 mix 3). Next, 24.7 mg (73.2 mmol) of Fmoc-Pro-OH dissolved in 1 ml of DMF, 21.4 mg (56.2 mmol) of HATU, 8.2 mg (60.2 mmol) of HOAt, and 15.7 ml (90.2 mmol) of DIEA were added and stirred for 1 hour. After washing, wash with DMF (1 ml X 3) and DCM (1 ml X 3).

 Fmoc_Pro_Phe_AC then _Aca_Aca_lantern got.

 Hereinafter, the same operation was performed to introduce Fmoc-Ala-OH twice, and the second compound (Ala-Ala-Pro-Phe) was bound to the first fluorescent group (ACC).

 Fmoc_Ala_Ala_Pro_Phe_A followed C_Aca_Aca_lantern.

 Then, the mixture was stirred with 20% piperidine ZDCM (1 mL) for 30 minutes to remove the Fmoc group, washed with DCM (1 mix 3), DMF (1 mL), DIEA 32 ml (183 mmol) and 8.5 ml (90 mmol) of acetic anhydride was added and stirred for 1 hour. After that, washing is performed with DMF (1 ml X 3) and DCM (1 ml X 3), the terminal group of the second compound (Ala_Ala_Pro-Phe) is acetylated, and the target substrate for enzyme activity detection of Example 3 is prepared. Peptidyl Fluorescent Group Bonded Flat Substrate

 Ac_Ala-Ala-Pro_Phe_ACC_Aca_Aca_lantem was obtained.

<Measurement of enzyme activity> The substrates for detecting the enzyme activity of Example 2 (introduction rate 2 μmol / substrate) were placed in the wells A and C of the microplate for fluorescence measurement, and the substrates for detecting the enzyme activity in Example 3 (introduction rate 2 μmol / substrate), add 100 μl of 20 mM Tris HC1 buffer (H 7.2, 100 mM NaCl, 50 mM CaCl), 100 μl of methanol, and add 50 μg of the following enzyme to each. Sa

2

 I let you.

 A: Chymotrypsin

 B: Chymotrypsin

 C: trypsin

 D: trypsin

 The measurement was performed using a WALLAC ARVOTM SX 1420 multilabel counter (PerkinElmer) to measure the fluorescence value before adding the enzyme and the fluorescence value 30 minutes after adding the enzyme at an excitation wavelength of 370 nm and an emission wavelength of 460 nm. And asked for the difference.

 The difference between the calculated fluorescence values in each well is shown in (Table 2).

[Table 2]

[0062] According to (Table 2), with respect to chymotrypsin, the enzyme activity of Example 3 in which the amino acid of the second compound bonded to the first fluorescent group is one kind of aromatic amino acid phenylalanine A large change in the fluorescence value was confirmed on the detection substrate. Also, with respect to trypsin, a large change in the fluorescence value was confirmed on the enzyme activity detection substrate of Example 2 in which the amino acid strength S-lysine of the second compound binding to the first fluorescent group was used. The difference between these changes is that chymotrypsin has the specificity of selectively cleaving peptide bonds mainly at the C-terminal side of aromatic amino acid residues, and trypsin is mainly at the C-terminal side of lysine. It is presumed that it was expressed because it has the specificity of selectively cleaving peptide bonds.

 As a result, it became clear that the ability to detect the activity with respect to the type of enzyme can be changed by changing the type of amino acid of the second compound that binds to the first fluorescent group.

(Example 4) In Example 4, the activity of the enzyme was measured by synthesizing a peptidyl fluorescent group-bonded flat substrate as a substrate for detecting the enzyme activity. Hereinafter, the method will be described.

<Synthesis of planar substrate bonded with peptidyl fluorescent group>

As a substrate, a carrier made of a multi-plate synthetic resin for peptide synthesis (lanthanum series (registered trademark) manufactured by Mimotobus Co., Ltd.) was cut off by only one plate and made into a planar shape. One lantern (D_series, Mimotops Co., 18 μmol I) was used as the substrate in the screw tube, and the mixture was stirred with 20% piperidine ZDCM (1 ml) for 30 minutes to remove the Fmoc group. . After washing 3 times with DCM (1 ml) and 2 times with DMF (1 ml), Fmoc-Aca_OH (19 mg, 54 μmol), DCC (17 mg, 81 μmol), HOBt-H〇 (8 mg , 54 mmol) in DMF (1 ml).

 2

And reacted for 24 hours. After completion of the reaction, wash twice with DMF (1 ml), twice with DCM (1 ml), twice with EtOH (1 ml), and twice with DCM (1 ml), and dried under reduced pressure.

Defended Fmoc-Aca-lantern.

 Next, the mixture was stirred with 20% piperidine / DCM (1 ml) for 30 minutes to remove the Fmoc group. After washing 3 times with DCM (1 ml) and 2 times with DMF (1 ml), Fmoc-Aca-OH (19 mg, 54 μmol), HATU (20 mg, 54 μmol), HO At (7 mg, 54 μmol) and DIEA (16 μ \, 90 βmol) were dissolved in DMF (1 ml), and the mixture was reacted for 1 hour. Then, it was washed three times with DMF (1 ml) and three times with DCM (1 ml) to obtain Fmoc-Aca-Aca-lantern.

Then, the mixture was stirred with 20% piperidine / DCM (1 ml) for 30 minutes to remove the Fmoc group. The lantern was swollen by washing three times with DCM (lml) and once with DMF (lml), and Fmoc-Lys (Ac-TYp (Boc)-)-OH (38 mg, 54 mmol), DCC (11 mg, 54 mmol) and HOBf HO (8.3 mg, 54 mmol) were added and reacted for 24 hours. After the reaction, DMF (1 ml)

 2

, Twice with DCM (1 ml), twice with EtOH (1 ml) and twice with DCM (1 ml), and then dried under reduced pressure (-Lys-Aca-Aca-) (Ac-TYp (Boc)-) was introduced into the chain

Fmoc_Lys (Ac—rrp (Boc) —Noichi Aca—Aca_lantern

 Next, the mixture was stirred with 20% piperidine ZDCM (1 ml) for 30 minutes to remove the Fmoc group. Then, after washing 3 times with DCM (1 ml) and 2 times with DMF (1 ml),

Fmoc-Lys (Boc) -OH (25 mg, 54 μmol), HATU (20 mg, 54 μmol), HO At (7 mg, 54 μmol), DIEA (16 μ1, 90 μmol) in DMF (1 ml), and the mixture was reacted for 1 hour. That Then, wash three times with DMF (1 ml) and three times with DCM (1 ml).

 Fmoc-Lys (Boc) _Lys (Ac_Trp (Boc)-)-Aca_Aca_lantern was obtained.

 Hereinafter, Fmoc_Pro_OH and Fmoc-Ala-OH are introduced into Fmoc-Lys (Boc) -Lys (Ac-Trp (Boc)-)-Aca-Aca-lantern to extend the peptide by repeating the same operation as in Example 3. By further reacting Dns_Cl

 Dns_Ala_Ala_Pro_Lys (Boc) _Lys (Ac_Trp (Boc) _) _ Aca_Aca_lantern.

 Next, the lantern was washed three times with DMF (1 ml) and three times with DCM (1 ml), and reacted with 25% TFA / DCM for 30 minutes to remove the Boc group. Then, the mixture was washed three times with DCM (1 ml), five times with H〇 (1 ml), and three times with DCM (1 ml), and dried under reduced pressure to allow the third and fifth compounds to bind (Ala -Ala-Pro_Lys-Lys-Aca-Aca) provided with Trp as a second fluorescent group in the side chain and Dns as a third fluorescent group at the end Dns for enzyme activity detection of Example 4 of interest -Ala-Ala-Pro-Lys-Lys (Ac-l rp—no Aca— Aca—lantern obtained.

<Measurement of enzyme activity>

 20 μm of 20 mM Tris-HCl buffer (pH 7.2, 100 mM NaCl, 50 mM CaCl 2), 100 μl of methanol 100 β \, 50 μl of trypsin (1 mg / 1 ml) were added to the substrate for detecting the enzyme activity of Example 4. 1

 2

 The fluorescence values before and after the reaction were measured. The fluorescence value was measured using an FP-6600 fluorescence spectrophotometer (manufactured by Jasco) at an excitation wavelength of 280 nm and a fluorescence wavelength of 350 nm.

 As a result, the initial fluorescence value was 37, the fluorescence value after 18 hours of reaction was 567, and it was confirmed that the fluorescence value changed about 15 times. Thus, it was shown that the enzyme having activity can be detected also in the enzyme activity detection substrate of Example 4.

(Example 5)

 In the same manner as in Example 4, one lantern (D-series, introduction rate: 18 μmol I) as a substrate was placed in a screw tube, and the Fmoc group was removed using piperidine / DCM. After washing with DCM and DMF, the lantern was swollen and dissolved in DMF

 Fmoc-TYp (Boc) -OH, DCC and HOBt-HO were added and reacted. After the reaction is completed, DMF,

After washing with DCM and EtOH and drying under reduced pressure, Fmoc-Trp (Boc) -lantern having (Fmoc-TYp (Boc)-) bonded to the substrate (lantern) was obtained.

Next, the Fmoc group was removed using piperidine ZDCM, and washed with DCM and DMF. , Fmoc-Lys (Boc) _OH, HATL ;, HOAt and DIEA were dissolved in DMF and reacted. Then, it was washed with DMF DCM to obtain Fmoc-Lys (Boc) -TYp (Boc) -lantern.

 Hereinafter, Fmoc_Lys (Boc) _Trp (the same procedure as in Example 3 in which Fmoc_Pro_uH and Fmoc_Ala_OH are introduced into Boc lantern) is repeated to elongate the peptide, and Dns-Cl is further reacted to obtain Dns-Ala-Ala- Pro—Lys (Boc) _T 卬 (Boc) -lantern was obtained.

 Next, the lantern was washed with DMF and DCM, and reacted with TFAZDCM to remove the Boc group. After that, the resultant was washed with DCM, H0, and DCM, and dried under reduced pressure.

 2

The substrate for enzyme activity detection of Example 5 which is provided with Trp as a second fluorescent group at the terminal of (Ala_Ala_Pro-Lys) to which the compound is bound and Dns at the terminal as a third fluorescent group

Dns_Ala_Ala_Pro_Lys_i'rp_ntern

When the enzyme activity detection ability of the substrate for enzyme activity detection of Example 5 was measured in the same manner as in Example 4, the detection ability of the substrate for enzyme activity detection of Example 5 was almost the same as that of Example 4. Was done. This indicates that the enzyme having activity can be detected also in the enzyme activity detection substrate of Example 5, and the substrate is bound to the substrate by using a fluorescent group such as tributophan having a plurality of reaction points. It has been clarified that the degree of freedom in designing an amino acid sequence of a peptide or the like can be further increased.

 (Example 6)

 One lantern as substrate (Mimotops D_series, introduction rate 18 μmol I) was placed in the screw tube, and Fmoc-Aca-OH was introduced into the substrate by the same operation as in Example 4.

Fmoc_Aca_Aca_lantern Get / Get.

Then, remove the Fmoc group using piperidine / DCM, wash with DCM and DMF to swell the lantern, and add Fmoc-TYp (Boc) -OH, DCC and HOBt-HO dissolved in DMF.

 2

Then, Fmoc-Trp (Boc) _Aca_Aca_lantern in which the sixth compound (Aca_Aca) was bound to the substrate (lantern) was protected.

 Hereinafter, Fmoc-Lys (Boc) _OH, Fmoc-Pro_OH, and Fmoc_Ala-OH were introduced to extend the peptide by repeating the same operation as in Example 3, and further reacted with Dns-Cl to obtain Dns-Ala-Ala-OH. Pro-Lys Boc ^ -l rp Bocy-Aca-Aca-lanterr ^^ Tr ^

Then, after washing with DMF and DCM and removing the Boc group using TFAZDCM, Washed with DCM, H0, DCM, dried under reduced pressure, and the seventh and eighth conjugates were combined.

2

 (Ala-Ala-Pro-Lys-) bound to T 卬 as the fourth fluorescent group and Dns as the fifth fluorescent group bound to the terminal

Dns-Ala-Ala-Pro-Lys-Ί rp-Aca-Aca_lantern.

When the enzyme activity detection ability of the enzyme activity detection substrate of Example 6 was measured in the same manner as in Example 4, almost the same detection ability as that of Example 4 was confirmed on the enzyme activity detection substrate of Example 6. Was done. This shows that the enzyme having activity can be detected also in the enzyme activity detection substrate of Example 6, and the substrate is bound to the substrate by using a fluorescent group such as tributophan having a plurality of reaction points. It has been clarified that the degree of freedom in designing an amino acid sequence of a peptide or the like can be further increased.

 (Example 7)

In Example 7, the activity of the enzyme was measured by synthesizing a peptidyl fluorescent group-bonded flat substrate as a substrate for detecting the enzyme activity. Hereinafter, the method will be described.

<Synthesis of planar substrate bonded with peptidyl fluorescent group>

As a substrate, a carrier made of a multi-plate synthetic resin for peptide synthesis (lanthanum series (registered trademark) manufactured by Mimotobus Co., Ltd.) was cut off by only one plate and made into a planar shape. One lantern as a substrate (D-series manufactured by Mimotops, 18 μmol I introduction rate) was placed in a screw tube, and the mixture was stirred with 20% piperidine / DCM (1 mL) for 30 minutes to remove the Fmoc group. . After washing three times with DCM (1 ml) and DMF (1 ml), Fmoc-Aca-OH (19 mg, 54 / i mol), DCC (17 mg, 81 μππο1), HOBt-HO (8 mg , 54 / i mol) in DMF (1 ml)

 2

And reacted for 24 hours. After completion of the reaction, wash twice with DMF (1 ml), twice with DCM (1 ml), twice with EtOH (1 ml), and twice with DCM (1 ml), and dried under reduced pressure.

Defended Fmoc-Aca-lantern.

Next, the mixture was stirred with 20% piperidine ZDCM (1 ml) for 30 minutes to remove the Fmoc group. After washing 3 times with DCM (1 ml) and 2 times with DMF (1 ml), Fmoc_Aca_OH (19 mg, 54 μmol), HATU (20 mg, 54 μmol), HO At (7 mg , 54 μmol) and DIEA (16 μ \, 90 μmol) were dissolved in DMF (1 ml) and reacted for 1 hour. Then, it was washed three times with DMF (1 ml) and three times with DCM (1 ml) to obtain Fmoc-Aca-Aca-lantern. Next, the mixture was stirred with 20% piperidine / DCM (1 mL) for 30 minutes to remove the Fmoc group. The lantern was swollen by washing three times with DCM (1 ml) and once with DMF (1 ml), and Fmoc-Lys (Ac--TYp (Boc)-)-OH (38 mg) dissolved in 1 ml of DMF , 54 μmol), DCC (11 mg, 54 μmol), and HOBt-HO (8.3 mg, 54 μmol) were reacted for 24 hours. After the reaction is completed, wash twice with DMF (1 ml), twice with DCM (1 ml), twice with EtOH (1 ml), and twice with DCM (1 ml), and then dried under reduced pressure to remove Fmoc- Lys (Ac-TYp (Boc)-)-Aca_Aca-lantern was obtained.

 Next, the mixture was stirred with 20% piperidine ZDCM (1 ml) for 30 minutes to remove the Fmoc group. Then, after washing three times with DCM (1 ml) and twice with DMF (1 ml), Fmoc_Phe-〇H (21 mg, 54 μmol), HATU (20 mg, 54 μmol), HOAt (7 mg, 54 μmol) and DIEA (16 μ \, 90 μmol) were dissolved in DMF (1 ml), and the mixture was reacted for 1 hour. Thereafter, the resultant was washed three times with DMF (1 ml) and three times with DCM (1 ml) to obtain Fmoc_Phe_Lys (Ac_Trp (Boc)-)-Aca-Aca-lantern.

 M Introduction of moc-Pro-OH and Fmoc-Ala-OH into Fmoc-Phe-Lys (Ac-Trp (Boc)-)-Aca-Aca-iantern And by further reacting with Dns-Cl

 Dns_Ala_Ala_Pro_Pne_Lys (Ac_Trp (Boc) _ ノ 一 Aca_Aca_lantern

 The lantern was washed three times with DMF (1 ml) and three times with DCM (1 ml), and reacted with 25% TFA / DCM for 30 minutes to remove the Boc group. Then 3 times with DCM (1 ml) and 5 times with H 0 (1 ml)

After washing three times with DCM (1 ml) and drying under reduced pressure, the third compound and the fifth compound were combined (Ala-Ala-Pro-Phe-Lys-Aca-Aca). The substrate for enzyme activity detection of Example 7 having TYp as a second fluorescent group on the side chain and Dns as a third fluorescent group at the end.

 Dns_Ala_Ala_Pro_Pne_Lys (Ac_Trp _) _ Aca_Aca_lantern was protected.

<Measurement of enzyme activity>

 Prepare four wells (A-D) in the fluorescence measurement microplate, use the enzyme activity detection substrate of Example 4 (introduction rate 2 x molZ substrate) in wells A and C, and use Example 7 in wells B and D. Of the enzyme activity detection substrate (introduction rate 2 μmol / substrate), add 100 μl of 20 mM Tris HC1 buffer (pH 7.2, 100 mM NaCl, 50 mM CaCl) and 100 μl of methanol to each. Further below

 2

 The enzyme was reacted with 50 µg of enzyme.

A: Chymotrypsin B: Chymotrypsin

 C: trypsin

 D: trypsin

 Fluorescence values were obtained using an FP-6600 fluorescence spectrophotometer (manufactured by Jasco) at an excitation wavelength of 280 nm and a fluorescence wavelength of 350 nm. The fluorescence value before the enzyme was removed and the fluorescence value 20 minutes after the enzyme was removed. And the difference was determined.

 The difference between the calculated fluorescence values in each well is shown in (Table 3).

[Table 3]

According to (Table 3), for chymotrypsin, the substrate for enzyme activity detection of Example 7 in which the third compound and the fifth compound bound to each other had one kind of aromatic amino acid phenylalanine A large change in the fluorescence value was confirmed. Also, with respect to trypsin, a large change in the fluorescence value was confirmed on the enzyme activity detection substrate of Example 4 having lysine in the bound third and fifth compounds. The difference between these changes is that chymotrypsin mainly has the characteristic of selectively cleaving the peptide bond at the C-terminal side of the aromatic amino acid residue, as inferred in Example 3. Is presumed to be expressed mainly because it has the specificity of selectively cleaving the peptide bond at the C-terminal side of lysine.

 As a result, it became clear that the ability to detect the activity for the type of enzyme can be changed by changing the type of the amino acid sequence of the peptide or the like to which the fluorescent group is bound. In this example, an example was shown in which the result changes depending on a single amino acid residue in the sequence, such as chymotrypsin or trypsin. Since the amino acid sequence can be freely designed over the entire length of the sequence, it is possible to obtain an enzyme activity detection substrate that can express infinite substrate specificity and detect a wide variety of enzymes by designing the amino acid sequence. It was revealed.

 Industrial applicability

The present invention relates to a substrate for detecting an enzyme activity for detecting an enzyme activity, and an enzyme activity using the same. With regard to the detection method, the enzyme activity can be detected simply by measuring the fluorescence intensity of the substrate using the change in the fluorescence intensity etc. as an index, and the operability is excellent, and the enzyme activity is detected even with a small amount of sample solution. It is possible to provide a substrate for detecting enzyme activity, which can greatly increase the degree of integration of the detection section without having to form a well for injecting a sample solution and the like. Detection, the detection sensitivity can be increased, the measurement time can be shortened, the workability can be improved and the measurement efficiency can be increased. By using the enzyme activity detection substrate to which the enzyme is bound, it is possible to provide an enzyme activity detection method that can measure the enzyme activity of a sample solution containing a plurality of enzymes in a short time.

Claims

The scope of the claims

 [1] A substrate, a first fluorescent group directly bonded to the substrate, or bonded to the substrate via a first conjugate having one end fixed to the substrate, and the first fluorescent group. A substrate for detecting enzyme activity, comprising: a second compound bound by a peptide bond that is cleaved by an enzyme.

 [2] The substrate, which is introduced into a side chain of a third compound having one end immobilized on the substrate and bonded to the substrate via the third compound, or bonded to a fourth compound and bonded to the substrate. A second fluorescent group directly bonded to the third compound, a fifth compound bonded to the third compound or the fourth compound by a peptide bond that is cleaved by an enzyme, and a second fluorescent group bonded to the fifth compound. A substrate for enzyme activity detection, comprising: a third fluorescent group that exhibits resonance energy transfer.

 [3] a substrate, a sixth compound having one end immobilized on the substrate, a fourth fluorescent group introduced into the sixth compound, a seventh compound bonded to the fourth fluorescent group, An eighth compound bonded to the compound by a peptide bond that is cleaved by an enzyme; and a fifth fluorescent group bonded to the eighth compound and having the fourth fluorescent group and fluorescence resonance energy transfer observed. A substrate for detecting enzyme activity.

 [4] Instead of the fifth compound or the eighth compound being linked to the fourth compound or the seventh compound by a peptide bond that is cleaved by the enzyme, the fourth compound is Wherein the second fluorescent group is bound by a peptide bond cleaved by the enzyme, or the seventh compound is bound to the fourth fluorescent group by a peptide bond cleaved by the enzyme. 4. The substrate for detecting an enzyme activity according to claim 2 or 3, wherein

 [5] The terminal group of the second compound, the fifth compound, the eighth compound, and / or the second fluorescent group introduced into a side chain of the third compound is acetylated. The substrate for detecting an enzyme activity according to any one of claims 1 to 4, characterized in that:

 [6] A step of contacting an enzyme-containing sample solution with the substrate for enzyme activity detection according to any one of claims 1 to 5 to cause a reaction, and performing a fluorescence measurement of the substrate for enzyme activity detection. And a method for detecting enzyme activity.