Method for producing peptide-containing compound

Abstract

Disclosed is a method for producing a peptide-containing compound, the method including a step in which a compound represented by formula (I) (in the formula, X represents a monovalent organic group, provided that X is bonded to an oxygen atom directly bonded to X in the formula on an aromatic ring; R1 represents a C1-6 alkyl group or a C1-6 alkoxy group; and LG represents a monovalent leaving group) is brought into contact with a peptide, in which the C-terminus side is supported by a carrier, a primary or secondary amino group is at the N-terminus, and a side chain is optionally protected, so as to substitute one hydrogen atom on the amino group of the peptide with a group represented by formula (II) (in the formula, X and R1 are the same as those in formula (I), and the wavy line represents a bond to a nitrogen atom in the amino group).

Description

Method for producing peptide-containing compounds

[0001] This disclosure relates to a method for producing peptide-containing compounds. This disclosure also relates to fluorescent probes and libraries containing them, a method for screening fluorescent probes, and a method for evaluating enzyme activity.

[0002] Peptidases are broadly classified into exopeptidases and endopeptidases depending on the site where they recognize and break down peptide chains. Exopeptidases recognize the amino acid residue at the end of the peptide chain and hydrolyze the peptide bond at the base of the terminal residue. Endopeptidases recognize amino acid residues at locations other than the ends of the peptide chain and hydrolyze the peptide bond. Of these, exopeptidases are further classified into aminopeptidases and carboxypeptidases depending on whether the recognized terminal amino acid residue is located at the N-terminus or C-terminus. Aminopeptidases hydrolyze the peptide bond at the base of the amino acid residue at the N-terminus of the peptide chain. Carboxypeptidases hydrolyze the peptide bond at the base of the amino acid residue at the C-terminus of the peptide chain.

[0003] When the base of the N-terminal amino acid residue is hydrolyzed by aminopeptidase, the group located at that base is converted from an amide to an amino group. Here, the conversion from amide to an amino group causes a significant change in the electronic state of the base to which the nitrogen atom is bonded. Therefore, it has been relatively easy to develop functional molecules triggered by the conversion from amide to an amino group, such as fluorescent probes for detecting aminopeptidase activity whose fluorescence intensity increases upon hydrolysis, or prodrugs whose pharmacological activity is enhanced upon hydrolysis. For example, it has been reported that a fluorescent probe library for searching for fluorescent probes to detect aminopeptidase activity was constructed by first supporting a fluorescent dye on a solid phase and then modifying the aniline amino group with a peptide by solid-phase synthesis (Non-Patent Literature 1).

[0004] In contrast, when the base of the C-terminal amino acid residue is hydrolyzed by carboxypeptidase, the group located at that base is converted from an amide to a carboxyl group. However, the change in the electronic state that the carbonyl group attaches to during this amide-to-carboxyl conversion is smaller than the change caused by aminopeptidase. Therefore, developing functional molecules triggered by this amide-to-carboxyl conversion has been difficult until now.

[0005] Against this backdrop, in 2024, a fluorescent probe for detecting carboxypeptidase activity using a novel mechanism applying ProTide chemistry was reported (Non-Patent Literature 2). The structure of this fluorescent probe and its fluorescence mechanism are shown in the following reaction equation. In this fluorescent probe, the N-terminus of a dipeptide is bound to a phenolic hydroxyl group via a phosphoramidite structure. When carboxypeptidase (CP) recognizes and hydrolyzes the C-terminal amino acid residue, the carboxyl group of the remaining amino acid nucleophilically attacks the phosphorus atom, causing the remaining amino acid to also be eliminated, exposing the phenolic hydroxyl group. This mechanism makes it possible to link a structural change with a small change in electronic state, such as the conversion from amide to carboxy, to a structural change with a large change in electronic state, such as the conversion from a phosphoramidite structure to hydroxyl. Furthermore, a patent application for a fluorescent probe utilizing the same principle has also been filed (Patent Literature 1).

[0006] International Publication No. 2020 / 111279, U.S. Patent No. 9797837

[0007] Yugo Kuriki et al., "Development of a fluorescent probe library enabling efficient screening of tumour-imaging probes based on discovery of biomarker enzymatic activities.", Chem. Sci., 2022,13, 4474-4481.Yugo Kuriki et al., "Modular Design Platform for Activatable Fluorescence Probes Targeting Carboxypeptidases Based on ProTide Chemistry", J. Am. Chem. Soc., 146, 1, 521-531 (2024).Shingo Sakamoto et al., "Multiplexed single-molecule enzyme activity analysis for counting disease-related proteins in biological samples." Sci. Adv. 6, eaay0888 (2020).Yannick Rondelez et al., "Microfabricated arrays of femtoliter chambers allow single molecule enzymology", Nat. Biotech. 23(3), 361-365 (2005).

[0008] The organic synthesis of fluorescent probes described in Non-Patent Literature 2 was carried out by first substituting one chlorine atom of a substituted phosphonic acid dichloride with a fluorescent dye (Fluorophore), and then substituting the other chlorine atom with the N-terminal amino group of a peptide (Peptide), as shown in the following reaction equation.

[0009] When forming a phosphoramidite structure, due to reactivity constraints, it is necessary to first form a bond between the oxygen atom of the phenolic hydroxyl group and the phosphorus atom, and then form a bond between the nitrogen atom of the N-terminal amino group of the peptide and the phosphorus atom. Therefore, in the above method, in the liquid phase, the hydroxyl group of the fluorescent dye was first reacted with the phosphorus atom in a one-sided reaction, and then the N-terminal amino group of the peptide was further reacted with the phosphorus atom. Consequently, in the above method, after the reaction, it was necessary to isolate and purify only the target product from among the multiple products derived from the fluorescent dye in the reaction solution, which made the purification by chromatography complicated. Therefore, the above method tended to have a low yield based on the fluorescent dye (around 1-20%). In addition, because the above method required complicated purification for each probe, it was difficult to efficiently increase the variety of fluorescent probes.

[0010] To date, there have been no examples of organic synthesis by solid-phase synthesis of compounds in which a peptide has been modified via a phosphoramidite structure on a phenolic hydroxyl group.

[0011] This disclosure aims to provide a method for producing peptide-containing compounds.

[0012] The present inventors have discovered that by introducing a phosphoramidite structure to the N-terminal amino group of a peptide supported on a carrier on a solid phase, it is possible to produce compounds in which the peptide is modified via a phosphoramidite structure on a phenolic hydroxyl group without complicated purification.

[0013] This disclosure relates, for example, to the following: <1> A method for producing a peptide-containing compound, comprising: a peptide having a C-terminal side supported on a carrier, a primary or secondary amino group at the N-terminus, and which may have a protected side chain, to which the following formula (I): [In the formula, X represents a monovalent organic group, where X is bonded to an oxygen atom directly bonded to X in the formula and to an aromatic ring, R １ is C １－６ Alkyl or C １－６represents an alkoxy, and LG represents a monovalent leaving group.], a compound represented by the formula is brought into contact with one hydrogen atom on the amino group of the peptide to obtain the following formula (II): [wherein X and R １ are the same as those in formula (I), and the wavy line represents a bond with the nitrogen atom in the amino group.], a production method including a step of substituting with a group represented by the formula. <2> The X is a monovalent functional group (FG) or a precursor thereof, provided that a molecule represented by FG-OH is in a state where it does not exhibit a function when the hydrogen atom on the oxygen atom directly bonded to FG is substituted, and is in a state of a functional molecule that exhibits a function when the hydrogen atom is not substituted, The production method according to <1>. <3> The X is Z-Y-BN: [wherein, Y is an oxygen atom or N(-R ２ )(R ２ represents a hydrogen atom, C １－６ alkyl or C ２－６ alkenyl.], BN is, when Y is an oxygen atom, a group represented by the following formula which may have a substituent on the benzene ring: [wherein, * represents a bond with Y, and the wavy line represents a bond with the oxygen atom directly bonded to X in formula (I).], when Y is N(-R ２ ), a group represented by the following formula which may have a substituent on the benzene ring: [wherein, * represents a bond with Y, and the wavy line represents a bond with the oxygen atom directly bonded to X in formula (I).], Z represents a monovalent functional group (FG) or a precursor thereof, or hydroxy or a protected form thereof.], provided that a molecule represented by FG-YH is in a state where it does not exhibit a function when the hydrogen atom on Y is substituted, and is in a state of a functional molecule that exhibits a function when the hydrogen atom is not substituted, The production method according to <1> or <2>. <4> When Z represents hydroxy or a protected form thereof, the production method includes a step of converting Z into the monovalent functional group (FG) or a precursor thereof after the step of substitution, The production method according to <3>. <5> The functional molecule is -CO ２ H, -PO ３ H ２ and -SO３ A fluorescent molecule having at least one water-soluble substituent selected from the group consisting of H, a method for producing a fluorescent molecule according to any one of <2> to <4>. <6> The FG is the following formula (III): [In the formula, R ａ１ ~R ａ５ Each of these independently comprises a hydrogen atom; and a C atom which may have substituents. １－６ Alkyl, C ２－６ Alkenyl and C １－６ Alkoxy; halogen atom; nitro; hydroxyl; hydroxyl C which may have substituents on the carbon atom. １－３ Alkyl; -CO ２ H, -PO ３ H ２ and -SO ３ A water-soluble substituent selected from the group consisting of H; and C which may be substituted with the water-soluble substituent and further have substituents. １－６ Alkyl, C ２－６ Alkenyl and C １－６ Selected from the group consisting of alkoxys, where R ａ１ ~R ａ５ At least one of these may have the water-soluble substituent, or a substituent substituted with the water-soluble substituent. １－６ Alkyl, C ２－６ Alkenil or C １－６ It is an alkoxy, and the wavy line indicates a bond with an oxygen atom or Y. [See formula (IV):] [In the formula, SRG is represented by the following structural formula: A group represented by any of the following, R ｂ１ ~R ｂ５ and R ｃ１ ~R ｃ６ Each of these independently comprises a hydrogen atom; and a C atom which may have substituents. １－６ Alkyl, C １－６ Alkoxy and C ２－６ Alkenyl; halogen atom; nitro; hydroxyl; hydroxyl C which may have substituents on the carbon atom. １－３ Alkyl; the water-soluble substituent; and C which may have further substituents substituted with the water-soluble substituent. １－６ Alkyl, C ２－６ Alkenyl and C １－６Selected from the group consisting of alkoxys, where R ｂ１ ~R ｂ５ and R ｃ１ ~R ｃ６ At least one of them is the water-soluble substituent; or C which is substituted with the water-soluble substituent and may have further substituents. １－６ Alkyl, C ２－６ Alkenil or C １－６ It is an alkoxy, R ｄ１ and R ｄ２ Each of these independently may have a hydrogen atom or a substituent. １－６ Alkyl or C ２－６ C may be substituted with an alkenyl or the aforementioned water-soluble substituent and may further have substituents. １－６ Alkyl or C ２－６ Alkenil is here, R ｄ１ and R ｄ２ They become one, R ｄ１ and R ｄ２ It may form a 4- to 7-membered heterocycline containing a nitrogen atom to which is bonded, which may have substituents, and R ｄ１ and / or R ｄ２ These are, respectively, R ｃ５ or R ｃ６ In conjunction with R ｄ１ or R ｄ２ It may form a 5-7 member heterocycline or heteroaryl, which may have substituents, including the nitrogen atom to which it is bonded, R ｅ is an oxygen atom, Si(-R ｆ１ ) (-R ｆ２ ) or C(-R ｆ１ ) (-R ｆ２ ) shows, where R ｆ１ and R ｆ２ Each of these may independently have substituents, C １－６ Alkyl, C ２－６ Alkenil, C １－６ [This indicates an alkoxy or aryl compound, and the wavy line indicates a bond with an oxygen atom or Y.] or formula (V) below: [In the formula, R ｇ１ ~R ｇ６is, independently of each other, a hydrogen atom; C which may have a substituent １－６ alkyl, C １－６ alkoxy and C ２－６ alkenyl; a halogen atom; nitro; hydroxy; hydroxy C which may have a substituent on the carbon atom １－３ alkyl; the water-soluble substituent; and C which is substituted with the water-soluble substituent and may further have a substituent １－６ alkyl, C ２－６ alkenyl and C １－６ alkoxy selected from the group consisting of. The wavy line represents a bond to an oxygen atom or Y. ], a production method according to any one of <2> to <5>. <7> The following formula (VI) or the following formula (VII): A fluorescent probe represented by, wherein Y is an oxygen atom or N(−R ２ )[R ２ is a hydrogen atom, C １－６ alkyl or C ２－６ alkenyl. ], FG is the following formula (III): [In the formula, R ａ１ to R ａ５ are, independently of each other, a hydrogen atom; C which may have a substituent １－６ alkyl, C ２－６ alkenyl and C １－６ alkoxy; a halogen atom; nitro; hydroxy; hydroxy C which may have a substituent on the carbon atom １－３ alkyl; the water-soluble substituent; and C which is substituted with the water-soluble substituent and may further have a substituent １－６ alkyl, C ２－６ alkenyl and C １－６ alkoxy selected from the group consisting of, provided that at least one of R ａ１ to R ａ５ is the water-soluble substituent, or C which is substituted with the water-soluble substituent and may further have a substituent １－６ alkyl, C ２－６ alkenyl or C １－６ alkoxy, and the wavy line represents a bond to an oxygen atom or Y. ], the following formula (IV): [In the formula, SRG is the following structural formula: A group represented by any of the following, R ｂ１ ~R ｂ５ and R ｃ１ ~R ｃ６ Each of these independently comprises a hydrogen atom; and a C atom which may have substituents. １－６ Alkyl, C １－６ Alkoxy and C ２－６ Alkenyl; halogen atom; nitro; hydroxyl; hydroxyl C which may have substituents on the carbon atom. １－３ Alkyl; the water-soluble substituent; and C which may have further substituents substituted with the water-soluble substituent. １－６ Alkyl, C ２－６ Alkenyl and C １－６ Selected from the group consisting of alkoxys, where R ｂ１ ~R ｂ５ and R ｃ１ ~R ｃ６ At least one of them is the water-soluble substituent; or C which is substituted with the water-soluble substituent and may have further substituents. １－６ Alkyl, C ２－６ Alkenil or C １－６ It is an alkoxy, R ｄ１ and R ｄ２ Each of these independently may have a hydrogen atom or a substituent. １－６ Alkyl or C ２－６ C may be substituted with an alkenyl or the aforementioned water-soluble substituent and may further have substituents. １－６ Alkyl or C ２－６ Alkenil is here, R ｄ１ and R ｄ２ They become one, R ｄ１ and R ｄ２ It may form a 4- to 7-membered heterocycline containing a nitrogen atom to which is bonded, which may have substituents, and R ｄ１ and / or R ｄ２ These are, respectively, R ｃ５ or R ｃ６ In conjunction with R ｄ１ or R ｄ２ It may form a 5-7 member heterocycline or heteroaryl, which may have substituents, including the nitrogen atom to which it is bonded, R ｅis an oxygen atom, Si(-R ｆ１ ) (-R ｆ２ ) or C(-R ｆ１ ) (-R ｆ２ ) shows, where R ｆ１ and R ｆ２ Each of these may independently have substituents, C １－６ Alkyl, C ２－６ Alkenil, C １－６ [This indicates an alkoxy or aryl compound, and the wavy line indicates a bond with an oxygen atom or Y.] or formula (V) below: [In the formula, R ｇ１ ~R ｇ６ Each of these independently comprises a hydrogen atom; and a C atom which may have substituents. １－６ Alkyl, C １－６ Alkoxy and C ２－６ Alkenyl; halogen atom; nitro; hydroxyl; hydroxyl C which may have substituents on the carbon atom. １－３ Alkyl; the water-soluble substituent; and C which may have further substituents substituted with the water-soluble substituent. １－６ Alkyl, C ２－６ Alkenyl and C １－６ Selected from the group consisting of alkoxys, the wavy line indicates a bond with an oxygen atom or Y. The group is represented by ], however, molecules represented by FG-OH or FG-YH are fluorescent molecules in which the fluorescence intensity is greater when the oxygen atom directly bonded to FG or the hydrogen atom on Y is not substituted than when the hydrogen atom is substituted, and BN may have substituents on the benzene ring when Y is an oxygen atom, as shown in the following formula: [In the formula, * indicates a bond with Y, and the wavy line indicates a bond with the oxygen atom that directly bonds with BN in formula (VII).] This represents a group represented by, where Y is N(-R ２ If the following formula is true, the benzene ring may have substituents: [In the formula, * indicates a bond with Y, and the wavy line indicates a bond with an oxygen atom that directly bonds with BN in formula (VII).] This represents a group represented by ], where Pep is a peptide in which one hydrogen atom on the primary or secondary amino group at the N-terminus is substituted with a phosphorus atom in formula (VI) or formula (VII), a fluorescent probe. <8> A fluorescent probe library comprising at least five types of fluorescent probes as described in <7>. <9> A screening method for fluorescent probes, comprising contacting the at least five types of fluorescent probes contained in the fluorescent probe library described in <8> with a peptidase or a sample containing the same, and selecting fluorescent probes whose fluorescence intensity is greater after contact with the peptidase or the sample containing the same than when not in contact, as fluorescent probes targeting the peptidase and / or a disease that expresses the same, wherein the peptidase is at least one selected from the group consisting of carboxypeptidases and endopeptidases. <10> A method for evaluating enzyme activity, comprising the following steps (A) to (C): Step (A): a step of preparing an aqueous solution containing one molecule of peptidase, a first fluorescent probe, and a second fluorescent probe; Step (B): a step of catalyzing an enzymatic reaction in the aqueous solution prepared in step (A) with the peptidase; and Step (C): a step of measuring the fluorescence of the aqueous solution obtained in step (B); wherein the peptidase is at least one selected from the group consisting of carboxypeptidase and endopeptidase, the first fluorescent probe and the second fluorescent probe are each independently the fluorescent probes described in <7>, and the difference between the maximum absorption wavelength and / or maximum fluorescence wavelength of the corresponding fluorescent molecules represented by FG-OH or FG-YH is 30 nm or more.

[0014] In the manufacturing method described herein, a phosphoramidite structure is introduced on the solid phase to the N-terminal amino group of a peptide supported on a carrier. Therefore, by separating the carrier from the reaction solution after the reaction, by-products not supported on the solid phase are removed, and a compound in which a peptide is modified via a phosphoramidite structure on a phenolic hydroxyl group can be produced without complicated purification.

[0015] In the manufacturing method described herein, the purification of the target product is extremely simple. For example, the target product produced by the manufacturing method may have -CO at locations other than the peptide. ２ H, -PO ３ H ２ or -SO ３ Even compounds with highly reactive functional groups such as H can be produced without reducing yield or making purification excessively complicated. Such compounds are generally difficult to synthesize organically, as they are modified by peptides via a phosphoramidite structure on a phenolic hydroxyl group, and have -CO at a site other than the peptide. ２ H, -PO ３ H ２ or -SO ３ To date, there have been no reported cases of actually obtaining molecules having highly reactive functional groups such as H. Furthermore, the inventors have demonstrated that if such molecules are fluorescent probes, they can also be applied to single-molecule enzyme activity measurement systems.

[0016] The manufacturing method described herein eliminates the need for cumbersome purification for each target product and allows for a simple and highly efficient increase in the variety of target products, making it possible to construct, for example, a library of fluorescent probes. Using such a library of fluorescent probes, it becomes possible to screen for fluorescent probes whose fluorescence intensity increases upon enzymatic reaction with peptidases that perform hydrolysis in a way that leaves one residue at the N-terminus of a peptide, such as carboxypeptidases and endopeptidases.

[0017] In one embodiment of the present disclosure, a monovalent functional group (FG) is directly introduced to the N-terminal amino group of a peptide supported on a carrier by forming a phosphoramidite structure on a solid phase. The production method in this embodiment allows for the production of the target product in a small number of steps and without complicated purification. Therefore, this embodiment is suitable as a production method for scaling up useful target products found, for example, through screening, for experimental or industrial purposes.

[0018] In one embodiment of the present disclosure, a benzyl structure is introduced to the N-terminal amino group of a peptide supported on a carrier by forming a phosphoramidite structure on a solid phase, and then a monovalent functional group (FG) or its precursor is introduced to the benzyl structure in a liquid phase. In this embodiment, since the introduction of FG does not require a one-sided reaction, the target product can be produced in high yield based on FG, and the complexity of the reaction can be further reduced. Furthermore, in this embodiment, since FG or its precursor is introduced to a benzyl structure, which is a commonly used self-cleaving linker, the success or failure of the reaction and the yield are less dependent on the structure of FG. Therefore, this embodiment is suitable, for example, as a method for constructing a library by producing many variations of the target product using one type of FG, from the viewpoint of saving FG raw materials and from the viewpoint of obtaining the target product with high reliability.

[0019] Furthermore, in the production of compounds having structures other than peptides at the N-terminal amino group of a peptide, methods of synthesis in the liquid phase, such as those described in Non-Patent Document 2, require the use of amino acids or peptides in which the C-terminal carboxyl of the peptide is protected with an appropriate protecting group (e.g., tert-butyl) as building blocks. However, raw materials for amino acids with carboxyls protected with an appropriate protecting group are generally difficult to obtain, or if available, are very expensive. Therefore, conventional methods of synthesis in the liquid phase result in high production costs. In contrast, in a production method according to one aspect of this disclosure, the N-terminal amino group of a peptide with its C-terminus supported on a carrier is reacted, eliminating the need to use amino acids with protected C-terminal carboxyls as raw materials, as in conventional methods (in other words, the protecting group for the C-terminal carboxyl in conventional methods is replaced by the carrier). Therefore, by combining this method with general peptide synthesis methods such as the Fmoc solid-phase synthesis method, a variety of peptide-containing compounds can be synthesized without using hard-to-obtain raw materials.

[0020] This figure shows the structure, LC chart, and peak values ​​obtained by mass spectrometry of the probe obtained in Manufacturing Example 1. This figure shows the peptide sequence and LC chart of the probe obtained in Manufacturing Example 2. This figure shows the structure, LC chart, and peak values ​​obtained by mass spectrometry of the probe obtained in Manufacturing Example 3. This is a heatmap showing the fluorescence intensity of fluorescent probes having the peptide sequences shown in the second column when incubated with each recombinant enzyme shown in the second row in Test Example 1. This is a fluorescence image and histogram showing the results of detecting single-molecule enzyme activity in plasma samples from healthy individuals or pancreatic cancer patients when using one type of fluorescent probe, manufactured in Manufacturing Example 1, with the peptide being AR, in Test Example 2. This is a fluorescence image and fluorescence intensity plot showing the results of detecting single-molecule enzyme activity in plasma samples from pancreatic cancer patients when using two types of fluorescent probes, (I) manufactured in Manufacturing Example 1, with the peptide being AR, and (II) manufactured in Manufacturing Example 3, with the peptide being GK, in Test Example 2. This figure shows the LC chart of the probe obtained in Production Example 4 whose peptide is AY. This figure shows the LC chart of the probe obtained in Production Example 4 whose peptide is FH, HP, VL, FL, FM, AY, GQ, GY, GK, AH, AL, AM, AE, MK, RE, or KK. This figure shows the LC chart of the probe obtained in Production Example 4 whose peptide is FE, AG, AV, AS, AI, AP, T(OtBu)A, GR(Pbf), or ER(Pbf). This figure shows the LC chart of the probe obtained in Production Example 5 whose peptide is AY. This figure shows the LC chart of the probe obtained in Production Example 6 whose peptide is GF. This figure shows the structure and LC chart of the probe obtained in Production Example 7 whose peptide is AK. This figure shows the structure and LC chart of the probe obtained in Production Example 8 whose peptide is GL. This is a heatmap showing the fluorescence intensity of fluorescent probes having the peptide sequences shown in the "Sequence" row when incubated with each recombinant enzyme shown in the first column in Test Example 3.In Test Example 4, when plasma samples from healthy individuals or recombinant CPB1 were added, the following are scatter plots showing the fluorescence intensity per well for the probe (Green) where the peptide synthesized in Production Example 1 is AR and the probe (Red) where the peptide synthesized in Production Example 7 is GK, and an example of a fluorescence image when a plasma sample was added. In Test Example 4, when plasma samples from healthy individuals were added, the following are scatter plots showing the fluorescence intensity per well for the probe (Green) where the peptide synthesized in Production Example 5 is AY and the probe (Red) where the peptide synthesized in Production Example 8 is GL, and an example of a fluorescence image.

[0021] The following describes the forms for implementing this disclosure, but this disclosure is not limited to the following embodiments.

[0022] [Definitions and Terminology] In this disclosure, the "-" and "=" signs between two groups in a chemical formula represent a single bond and a double bond, respectively.

[0023] Alkyl is a group obtained by removing one hydrogen atom from an alkane. Alkyl may be a group obtained by removing one hydrogen atom from a linear or cyclic alkane, preferably a group obtained by removing one hydrogen atom from a linear alkane, and the linear alkane may be linear or branched. In one embodiment, the alkyl is C １－５ Alkyl is also acceptable. C １－５ Alkyl is an alkyl group having 1 to 5 carbon atoms. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, cyclopropyl, cyclobutyl, or cyclopentyl, and these are C １－５ It is alkyl.

[0024] An alkenyl is a group obtained by removing one hydrogen atom from an alkene. An alkenyl may be a group obtained by removing one hydrogen atom from a linear or cyclic alkene, preferably a group obtained by removing one hydrogen atom from a linear alkene, and the linear alkene may be linear or branched. An alkenyl according to one embodiment is C ２－４ Alkenil may also be used. C ２－４An alkenyl is an alkenyl having 2 to 4 carbon atoms. For example, an alkenyl may be vinyl, allyl, 2-butenyl, or 3-butenyl, and these are C ２－４ It is Alkenil.

[0025] An alkoxy is a monovalent group in which an alkyl group is bonded to a base oxygen atom. An alkoxy according to one embodiment may be an alkoxy containing a linear alkyl group. An alkoxy according to one embodiment is C １－５ Alkyl compounds may also be used. １－５ Alkoxy is an alkoxy having 1 to 5 carbon atoms. For example, alkoxy may be methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentoxy, isopentoxy, or neopentoxy, and these are C １－５ It is an alkoxy. In one embodiment, the alkoxy may be methoxy, ethoxy, propoxy, butoxy, or pentoxy, which are C2 containing a linear alkyl group. １－５ It is an alkoxy.

[0026] Alkoxymethyl is a group in which one hydrogen atom of a methyl group is substituted with an alkoxy group. In one embodiment, alkoxymethyl may be a group in which one hydrogen atom of a methyl group is substituted with an alkoxy group containing a linear alkyl group. In one embodiment, alkoxymethyl is C １－５ Alkylmethyl may also be used. １－５ Alkoxymethyl groups are groups in which one hydrogen atom of a methyl group is replaced by an alkoxy group having between 1 and 5 carbon atoms. Alkoxymethyl groups may also be, for example, methoxymethyl, ethoxymethyl, propoxymethyl, butoxymethyl, or pentoxymethyl, which are linear C groups. １－５ It is an alkyl-containing alkoxymethyl compound.

[0027] A halogen is an element belonging to Group 17 of the periodic table, and in one embodiment may be fluorine, chlorine, bromine, or iodine. A halogeno is a monovalent group corresponding to a halogen, and in one embodiment may be fluoro, chloro, bromo, or iodine.

[0028] The amino group is a group obtained by removing one hydrogen atom from the nitrogen atom of ammonia, a primary amine, or a secondary amine, preferably a group obtained by removing one hydrogen atom from the nitrogen atom of ammonia or a primary amine, and more preferably a group obtained by removing one hydrogen atom from the nitrogen atom of ammonia. In other words, the amino group may preferably be a primary or secondary amino group, and more preferably a primary amino group. When the amino group is a group obtained by removing one hydrogen atom from the nitrogen atom of a primary amine or a secondary amine, the substituent on the nitrogen atom may be, for example, an optionally substituted alkyl, an optionally substituted alkenyl, or an optionally substituted phenyl, and in one embodiment may be an alkyl.

[0029] A substituent is a monovalent group that substitutes for a hydrogen atom. In one embodiment, a substituent is a monovalent group that substitutes for a hydrogen atom on a carbon atom. A functional group that "may have substituents" may not have substituents, or may have one, two, three or more different substituents. In one embodiment, a functional group that "may have substituents" may not have substituents, or may have one substituent. In a preferred embodiment, a functional group that "may have substituents" may not have substituents. That is, in a preferred embodiment, a functional group that "may have substituents" may be the functional group itself.

[0030] The substituents may be, but are not limited to, alkyl, alkenyl, alkoxy, alkoxymethyl, halogen, amino, phenyl, or carboxyl groups, and these may have further substituents.

[0031] The phenolic hydroxyl group is a hydroxyl group in which the oxygen atom directly forms a single bond with the aromatic ring. In one embodiment, the aromatic ring may include a single six-membered ring structure, preferably a bicyclic structure in which two six-membered ring structures share one side, or a tricyclic structure in which, for example, one six-membered ring structure shares one side with another six-membered ring structure on each of two opposing sides. When the hydrogen atom is ionized from the phenolic hydroxyl group, the negative charge on the oxygen atom is delocalized on the aromatic ring.

[0032] The phosphoramidite structure is -P(=O)(-R １ In the structural formula )-, one of the two bonds of the group is bonded to the nitrogen atom and the other to the oxygen atom. １ is a monovalent substituent, and in one embodiment, C １－６ Alkyl or C １－６ It may represent an alkoxy, for example, ethoxy or tert-butyl.

[0033] In this disclosure, the protecting group for a functional group may be, for example, one of the protecting groups described in "GREEN'S PROTECTIVE GROUPS in Organic Synthesis" (5th edition, John Wiley & Sons, Inc., 2014).

[0034] In this disclosure, when the functional group to be protected is hydroxyl, the protecting group may be, for example, an ether protecting group, a silyl ether protecting group, an ester protecting group, or a carbonate protecting group. The ether protecting group protecting hydroxyl is a protecting group that forms an ether with the hydroxyl group to be protected, and may be, for example, methoxymethyl (MOM), 2-methoxyethoxymethyl (MEM), benzyloxymethyl, benzyl, p-methoxybenzyl (PMB), o-nitrobenzyl, or trityl, which may have substituents, and in a preferred embodiment, these groups may be unsubstituted. These protecting groups can be removed by deprotection methods commonly used by those skilled in the art. The silyl ether protecting group that protects the hydroxyl group is a protecting group that forms a silyl ether with the hydroxyl group to be protected. In one embodiment, the three methyl moieties of trimethylsilyl may each be independently substituted with an alkyl group, an alkenyl group, an alkoxy group, an alkoxymethyl group, or an alkoxymethyl group. In a preferred embodiment, it may be trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), tert-butyldimethylsilyl (TBDMS), or tert-butyldiphenylsilyl (TBDPS). These protecting groups can be easily deprotected by contact with fluoride ions. Fluoride ions can be supplied using reagents such as tetrabutylammonium fluoride (TBAF). The ester protecting group that protects the hydroxyl group is a protecting group that forms an ester with the hydroxyl group to be protected, and in one embodiment, the methyl portion of acetyl may be substituted with an alkyl group, an alkenyl group, an alkoxymethyl group, an alkoxymethyl group, an alkoxymethyl group, an alkoxymethyl group, or an alkoxymethyl group. In a preferred embodiment, it may be acetyl, pivaloyl, benzoyl, p-methoxybenzoyl, phenylacetyl, or trifluoroacetyl. These protecting groups can be removed by deprotection methods commonly used by those skilled in the art.A carbonate-based protecting group that protects a hydroxyl group is a protecting group that forms a carbonate with the hydroxyl group to be protected, and in one embodiment, the methyl portion of methoxycarbonyl may be substituted with an alkyl, alkenyl, alkoxymethyl, benzyl, or trityl group, and in a preferred embodiment, it may be methoxycarbonyl, tert-butoxycarbonyl, or benzyloxycarbonyl. These protecting groups can be removed by deprotection methods commonly used by those skilled in the art.

[0035] In this disclosure, if the functional group to be protected is a carboxyl group, the protecting group may be, for example, an ester protecting group. The ester protecting group protecting the carboxyl group is a protecting group that forms an ester with the carboxyl group to be protected, protecting the carboxyl group by substituting a hydrogen atom of the carboxyl group. The ester protecting group protecting the carboxyl group may be, for example, a hydrocarbon group which may have substituents, and in one embodiment may be an alkyl group which may have substituents, an alkenyl group which may have substituents, an alkoxymethyl group which may have substituents, an alkoxymethyl group which may have substituents, an alkoxymethyl group which may have substituents, or

[0036] In this disclosure, when the functional group to be protected is an amino group, the protecting group may be, for example, a carbamate protecting group, an amide protecting group, or an N-alkyl protecting group. A carbamate protecting group that protects an amino group is a protecting group that forms a carbamate with the amino group to be protected. Carbamate protecting groups that protect amino groups are, for example, groups in which the methyl portion of methoxycarbonyl is substituted with a hydrocarbon group which may be substituted. In one embodiment, the methyl portion of methoxycarbonyl may be substituted with an alkyl group which may be substituted, an alkenyl group which may be substituted, an alkoxymethyl group which may be substituted, a benzyl group which may be substituted, or a 9-fluorenylmethyl group which may be substituted. In a preferred embodiment, these may be tert-butoxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc), benzyloxycarbonyl (Cbz, Z), p-methoxybenzyloxycarbonyl (Cpz), 2-trimethylsilylethoxycarbonyl (Teoc), allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl (Troc), or methoxycarbonyl. These protecting groups can be removed by deprotection methods commonly used by those skilled in the art. Amide protecting groups that protect amino groups are protecting groups that form an amide with the amino group to be protected. The amide protecting group that protects the amino group may, in one embodiment, be a group in which the methyl portion of acetyl is substituted with an optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkoxymethyl, or optionally substituted benzyl, and in a preferred embodiment, it may be acetyl or trifluoroacetyl. These protecting groups can be removed by deprotection methods commonly used by those skilled in the art, such as base hydrolysis. The N-alkyl protecting group that protects the amino group is a protecting group that protects the amino group to be protected by N-alkylation, and is not limited to alkyl groups. The N-alkyl protecting group that protects the amino group may, for example, be optionally substituted benzyl, optionally substituted trityl, or allyl, and in a preferred embodiment, it may be benzyl, p-methoxybenzyl (PMB), trityl, or allyl.These protecting groups can each be removed by deprotection methods commonly used by those skilled in the art, such as hydrolysis or oxidation-reduction.

[0037] In this disclosure, when the functional group to be protected is phosphono or phospho, the protecting group may be, for example, a silyl phosphate protecting group or a phosphate ester protecting group. A silyl phosphate protecting group is a protecting group that forms a silyl phosphate ester with the phosphono or phospho to be protected. The silyl phosphate protecting group may be the same as the one described as a silyl ether protecting group that protects hydroxyl. A phosphate ester protecting group is a protecting group that forms a phosphate ester with the phosphono or phospho to be protected. The phosphate ester protecting group may be the same as the one described as an ester protecting group that protects carboxyl.

[0038] In this disclosure, when the functional group to be protected is sulfo, the protecting group may be, for example, a sulfoester protecting group. A sulfoester protecting group is a protecting group that forms a sulfoester with the sulfo to be protected. The sulfoester protecting group may be, for example, an optionally substituted hydrocarbon group, which in one embodiment may be an optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkoxymethyl, or optionally substituted phenyl, and in a preferred embodiment may be isopropyl, isobutyl, or neopentyl.

[0039] In this disclosure, unless otherwise specified, amino acids mean L-amino acids. For example, in this disclosure, "alanine" and "L-alanine" mean L-alanine, and "D-alanine" means D-alanine. The amino acids relating to this disclosure may be natural or unnatural amino acids, and in one embodiment, they may be natural amino acids. The amino acids relating to this disclosure may be, for example, L- or D-forms, and in one embodiment, they may be L-forms. The amino acids relating to this disclosure may be, for example, α-amino acids, β-amino acids, or γ-amino acids, and in one embodiment, they may be α-amino acids. In this disclosure, proline is treated as an α-amino acid. The amino acids relating to this disclosure may, for example, have an amino acid bonded to the α-carbon that is a primary amino acid or a secondary amino acid, and in one embodiment, a primary amino acid or N-C １－５ The amino acid may be an alkylamino acid, and in a preferred embodiment, it may be a primary amino acid. In a more preferred embodiment, the amino acid according to the disclosure may be selected from the group consisting of alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

[0040] The peptides relating to this disclosure contain two or more residues of the above-mentioned amino acids. The number of residues in the peptide relating to this disclosure may be 10 residues or less, 7 residues or less, 5 residues or less, 4 residues or less, 3 residues or less, or 2 residues or less. In one embodiment, the peptide may have 2 residues. In this disclosure, the amino acid residues of the peptide are listed from the N-terminus. For example, "AR" means a dipeptide in which the first residue from the N-terminus is alanine and the second residue is arginine.

[0041] [Method for Producing Peptide-Containing Compounds] The first embodiment of this disclosure relates to a method for producing peptide-containing compounds. Hereinafter, this embodiment will also be referred to as the "method for producing the first embodiment." In the method for producing the first embodiment, a phosphoramidite structure is introduced on a solid phase to the N-terminal amino group of a peptide supported on a carrier, thereby producing a compound in which a peptide is modified via a phosphoramidite structure on a phenolic hydroxyl group.

[0042] The manufacturing method of the first embodiment includes a substitution step. The substitution step involves adding formula (I): to a peptide (also referred to as the "contacted peptide") whose C-terminal end is supported on a carrier and which has a primary or secondary amino group at its N-terminus. By contacting the compound represented by (also referred to as "formula (I) compound"), one hydrogen atom on the amino group of the peptide is converted to the following formula (II): This is a substitution step in which a group represented by (also called the "(II) group") is substituted. In other words, the substitution step can be described as a step in which a phosphoramidite structure is introduced on a solid phase to the N-terminal amino group of a peptide supported on a carrier.

[0043] The carrier according to this disclosure is a carrier for solid-phase synthesis and has a group on its surface that can directly or indirectly capture the C-terminus of a peptide. In one embodiment, the carrier may have a group on its surface that can directly capture the carboxyl group at the C-terminus of a peptide. The carrier is not particularly limited as long as it can be used in solid-phase synthesis to capture the C-terminus of a peptide, and may be a carrier commonly used in the Fmoc solid-phase synthesis method, for example, one having chlorotrityl chloride on its surface, such as 2-chlorotrityl chloride resin. The carrier according to this disclosure may be, for example, bead-shaped, resin-shaped, or film-shaped, and in one embodiment may be a resin, but is not limited thereto.

[0044] The N-terminal amino group in the contacted peptide may be, for example, a primary amino group, or a primary amino group included in the peptide backbone. The number of amino acid residues in the contacted peptide may be, for example, 2 to 10 residues, 2 to 7 residues, 2 to 5 residues, 2 to 4 residues, 2 to 3 residues, or 2 residues, and in one embodiment, it may be 2 residues.

[0045] In one embodiment, the peptide to be contacted may be a peptide in which the peptide bond between the first amino acid from the N-terminus and the second amino acid from the N-terminus is hydrolyzed by an enzymatic reaction with a carboxypeptidase or endopeptidase. In this case, when the peptide-containing compound (hereinafter also referred to as the "final target product" or "target compound") is metabolized by a carboxypeptidase or endopeptidase, only the first amino acid from the N-terminus remains, and the remaining first amino acid is also eliminated by nucleophilic attack on its carboxylin atom. This makes it possible to replace the metabolism by the carboxypeptidase or endopeptidase with a structural change in which a phenolic hydroxyl group is exposed. When the target enzyme is a carboxypeptidase, the peptide to be contacted is preferably a dipeptide.

[0046] The side chain of the peptide to be contacted may be protected, and is preferable. Protection of the side chain of the peptide to be contacted means that the functional groups of the side chain are protected by protecting groups. For example, all or part of the hydroxy, amino, carboxy, and mercapto groups in the side chain of the peptide to be contacted may be protected by protecting groups; all or part of the hydroxy, amino, and carboxy groups in the side chain may be protected by protecting groups; all or part of the amino and carboxy groups in the side chain may be protected by protecting groups; or all of the amino and carboxy groups in the side chain may be protected by protecting groups. The protecting groups for the carboxyl groups in the side chain may be, for example, tert-butyl (t-Bu), benzyl (Bn), or paramethoxybenzyl (PMB). The protecting group of the amino group in the side chain may be, for example, tertial oxycarbonyl (Boc), benzyloxycarbonyl (Cbz), or 2,2,4,6,7-pentamethylhydrobenzofuran-5-sulfonyl (Pbf).

[0047] The peptide to be contacted can be prepared according to a conventional method based on a peptide solid-phase synthesis method. In such a method, after supporting the first C-terminal residue on a carrier, the N-terminus of the amino acid or peptide on the carrier is deprotected, and an amide bond (peptide bond) is formed between the N-terminal protected group of the second and subsequent amino acids. The preparation of the peptide to be contacted according to one embodiment may be carried out according to the Fmoc solid-phase synthesis method or the Boc solid-phase synthesis method, or it may be carried out according to the Fmoc solid-phase synthesis method. The Fmoc solid-phase synthesis method is a solid-phase synthesis method that uses 9-fluorenylmethyloxycarbonyl (Fmoc) as a protecting group for the N-terminal amino group of an amino acid. Deprotection of Fmoc can be carried out by contact with a secondary amine such as piperidine. The Boc solid-phase synthesis method is a solid-phase synthesis method that uses tertiary toxiccarbonyl (Boc) as a protecting group for the N-terminal amino group of an amino acid. Deprotection of Boc can be carried out by contact with an acid such as trifluoroacetic acid. The manufacturing method of this embodiment may include a step of preparing the peptide to be contacted by solid-phase synthesis (peptide synthesis step) before the substitution step.

[0048] In formula (I), X represents a monovalent organic group. Furthermore, X is bonded to the oxygen atom directly bonded to X in formula (I) on an aromatic ring. In other words, the monovalent group represented by the X-O- structural formula in the compound of formula (I) is the phenolic hydroxyl group of the compound represented by the X-OH structural formula, with the hydrogen atom removed. The structure of X will be described later.

[0049] In equation (I), R １ is C １－６ Alkyl or C １－６ It exhibits an alkoxy component, and in one embodiment, it exhibits tert-butyl or ethoxy. １ When R exhibits tert-butyl or ethoxy properties, the stability of the product tends to increase even after the formation of the phosphoramidite structure. １ If the compound exhibits tert-butyl or ethoxy properties, the synthesis is simplified.

[0050] In formula (I), LG represents a monovalent leaving group. LG may be a halogen or activated hydroxyl. Activated hydroxyl is an active group obtained by contacting hydroxyl with a coupling agent. The coupling agent may be one that is sometimes used to activate carboxyl groups in amide synthesis, for example, WSCD (EDC, 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide), HATU (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate), or COMU (1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate). LG is preferably a halogen, more preferably chloro, bromo, or iodine, and even more preferably chloro.

[0051] In the substitution step, LG is eliminated after a bond is formed between the phosphorus atom in formula (I) and the N-terminal amino group of the peptide being contacted. As a result, one hydrogen atom on the N-terminal amino group of the peptide being contacted is replaced by the group of formula (II). Thus, in formula (II), X and R １ This is similar to formula (I), and the wavy lines indicate the bonding with the nitrogen atom in the amino group. Thus, the compound of formula (I) acts as a building block having a phosphochloride structure in the formation of the phosphoramidite structure during the substitution process.

[0052] The substitution step is carried out by bringing the peptide to be contacted and the compound of formula (I) into contact in a solvent. The compound of formula (I) may be added to the solvent as the compound of formula (I). Alternatively, the compound of formula (I) may be formed by adding a compound having hydroxyl instead of LG in formula (I) and the above-mentioned coupling agent to the solvent, thereby activating the hydroxyl group in the solvent.

[0053] The solvent used in the substitution step can be any solvent that has high affinity for the peptide portion of the peptide to be contacted and that causes the formation of a phosphoramidite structure, for example, an organic solvent, and in one embodiment, a polar solvent, for example, N-methyl-2-pyrrolidone (NMP), N,N-dimethylformamide (DMP), or dimethyl sulfoxide (DMSO), with NMP being one example.

[0054] The solvent in the substitution step may further contain a dissolved base. The base can be any base that is soluble in the solvent and capable of forming a phosphoramidite structure, and may be an organic base, a tertiary amine, or, as an example, DIEA (diisopropylethylamine) or TEA (triethylamine).

[0055] The contact temperature in the substitution step may be any temperature that can cause the formation of the phosphoramidite structure. The contact temperature in the substitution step may be, for example, above 0°C. The inventors have found that even if the contact temperature exceeds 0°C, the phosphoramidite structure is formed without any problems. When the contact temperature exceeds 0°C, the manufacturing method of the first embodiment can be carried out easily and without constraints from the temperature surface of the carrier. The contact temperature in the substitution step may be, for example, 4°C to 35°C. The contact time in the substitution step may be any time that can cause the formation of the phosphoramidite structure, for example, 5 minutes to 168 hours or 15 minutes to 24 hours.

[0056] In the substitution process, by removing the reaction supernatant after contact, all substances other than the target solid-phase material are removed. Therefore, the purification of the target material after the substitution process can be carried out very simply by removing the reaction supernatant after contact and, if necessary, washing the support.

[0057] Next, we will explain X in formula (I), while also referring to the final product produced by the manufacturing method of the first embodiment. X is a monovalent organic group and does not need to hinder the formation of the phosphoramidite structure in the substitution step. The molecular weight (g / mol) of X may be, for example, 50 to 10,000 or 100 to 2,000. X may consist of at least one atom selected from the group consisting of, for example, hydrogen, boron, carbon, nitrogen, oxygen, fluorine, silicon, phosphorus, sulfur, and chlorine atoms.

[0058] In X, carboxy(-CO ２ H), amino group, hydroxy, phosphono(-PO ３ H ２ ), phospho and sulfo (-SO ３ Highly reactive functional groups such as H) may be protected, and are preferably protected.

[0059] In one embodiment, X may be a monovalent functional group (FG) or its precursor. FG will be described later. The precursor of FG may be a protected form of FG, a reduced or oxidized form of FG, or a protected form of a reduced or oxidized form of FG. The protected form of FG yields FG by deprotection. The reduced form of FG yields FG by oxidation (for example, oxidation using p-chloranil). The oxidized form of FG yields FG by reduction. The protected form of the reduced form of FG yields FG by deprotection and oxidation. The protected form of the oxidized form of FG yields FG by deprotection and reduction.

[0060] If X is FG or its precursor, a compound represented by the following formula (VI) can be obtained as the final target product by a simple method of recovering the product from the support after the substitution step and performing treatments such as deprotection and / or oxidation as necessary. １ This is similar to the explanation given for equation (I).

[0061] Pep is a peptide in which one hydrogen atom on the primary or secondary amino group at the N-terminus is substituted with the phosphorus atom in the formula. Pep may be a peptide in which the peptide bond between the first amino acid from the N-terminus and the second amino acid from the N-terminus is hydrolyzed by an enzymatic reaction with carboxypeptidase or endopeptidase. In this case, when the peptide-containing compound is metabolized with carboxypeptidase or endopeptidase, only the first amino acid from the N-terminus remains, and the remaining first amino acid is also eliminated by nucleophilic attack on its carboxyphosphorus atom. This makes it possible to replace the metabolism by carboxypeptidase or endopeptidase with a structural change in which a phenolic hydroxyl group is exposed. In other words, Pep can also be described as a contacted peptide in which the peptide portion is deprotected and a phosphoramidite structure is formed at the N-terminus amino group. The peptide sequence in Pep may be a sequence in which only one amino acid residue at the N-terminus remains after hydrolysis by carboxypeptidase and endopeptidase, or it may be a sequence in which this residue is unknown. The carboxypeptidase and endopeptidase may be, for example, those described in the evaluation method of the third embodiment below.

[0062] In another embodiment, X may be represented as Z-Y-BN, where Y is an oxygen atom or N(-R) ２ ) represents R ２ C is a hydrogen atom. １－６ Alkyl or C ２－６ It exhibits an alkenyl, preferably a hydrogen atom or C １－６ It may represent an alkyl group, more preferably a hydrogen atom, methyl, or ethyl, and even more preferably a hydrogen atom.

[0063] When Y is an oxygen atom, BN may have substituents on the benzene ring, as shown in the following formula: This is a group represented by [wherein * indicates a bond with Y, and the wavy line indicates a bond with the oxygen atom that is directly bonded to X in formula (I).]. In one embodiment, BN may represent the group represented by the same formula (i.e., the group in the above formula that does not have substituents on the benzene ring).

[0064] Y is N(-R) ２ If this is the case, BN may have substituents on the benzene ring, as shown below: This is a group represented by [wherein * indicates a bond with Y, and the wavy line indicates a bond with the oxygen atom that is directly bonded to X in formula (I).]. In one embodiment, BN may represent the group represented by the same formula (i.e., the group in the above formula that does not have substituents on the benzene ring).

[0065] Z may represent a monovalent functional group (FG) or its precursor, or a hydroxyl group or its protected derivative.

[0066] If Z is a monovalent functional group (FG) or its precursor, the final product can be obtained by a simple method of recovering the product from the support after the substitution step and performing treatments such as deprotection and / or oxidation as necessary, resulting in a compound represented by the following formula (VII). １ The explanation for Pep is the same as that for equation (I). The explanation for Pep is the same as that for equation (VI).

[0067] On the other hand, if Z is hydroxyl or a protected compound thereof, the manufacturing method of the first embodiment may include a step (conversion step) after the substitution step in which Z is converted to FG or its precursor. In this case, since Z is converted to FG or its precursor on a solid phase after the substitution step, the advantages of solid-phase synthesis, such as easy purification, can be enjoyed, and the compound represented by formula (VII) above can be obtained as the final target product. Furthermore, in this case, since FG or its precursor is introduced to a benzyl structure, which is a commonly used self-cleaving linker structure, the success and yield of the entire manufacturing method become less dependent on the structure of FG.

[0068] The conversion method in the conversion step is not particularly limited. The conversion step may include, for example, deprotecting the protecting group of Z as needed, converting hydroxyl to a leaving group, and converting the leaving group to FG or its precursor by a nucleophilic substitution reaction. The leaving group is the same as described for LG, and is preferably bromo or iodine, with bromo being more preferred. The method for converting hydroxyl to halogeno may involve reacting hydroxyl with phosphorus tribromide, phosphorus pentabromide, hydrogen bromide, hydrogen tetrabromide, elemental bromine, elemental iodine, lithium iodide, sodium iodide, or potassium iodide as a leaving group introduction reagent, for example, by reacting it with phosphorus tribromide.

[0069] Next, we will explain FG (monovalent functional group). FG is a group such that a molecule represented as FG-OH or FG-YH does not exhibit function when an oxygen atom or a hydrogen atom on Y is substituted, and exhibits function when the oxygen atom is not substituted.

[0070] The function of a functional molecule may be, for example, a pharmacological effect or a visualization effect. If the function of the functional molecule is a pharmacological effect, a prodrug that exerts its therapeutic effect only in the disease environment can be manufactured by employing a peptide that is eliminated in the presence of a disease-specific enzyme and / or in the disease environment as the Pep. A molecule having a visualization effect is a molecule that emits a detectable optical signal, such as a molecule that emits fluorescence (fluorescent molecule), a molecule that emits phosphorescence (phosphorescent molecule), a molecule that causes Raman scattering, or a molecule that produces chemiluminescence (luminescent molecule), with fluorescent molecules being preferred. If the function of the functional molecule is a visualization effect, a diagnostic probe that emits an optical signal only in the presence of a disease-specific enzyme and / or in the disease environment can be manufactured by employing a peptide that is eliminated in the presence of a disease-specific enzyme and / or in the disease environment as the Pep. For example, if the optical signal is a fluorescent signal, such a diagnostic probe is a fluorescent probe. Such a fluorescent probe can be used for both in vitro diagnostic methods using biological samples and in vivo diagnostic methods by direct administration to human subjects.

[0071] The molecular weight (g / mol) of FG may be, for example, 50 to 10,000 or 100 to 2,000. FG may consist of at least one atom selected from the group consisting of, for example, hydrogen, boron, carbon, nitrogen, oxygen, fluorine, silicon, phosphorus, sulfur, and chlorine atoms.

[0072] In one embodiment, FG is -CO ２ H, -PO ３ H ２ and -SO ３ It may have at least one water-soluble substituent selected from the group consisting of H. In other words, the above functional molecule is -CO ２ H, -PO ３ H ２ and -SO ３ It may have at least one water-soluble substituent selected from the group consisting of H. Hereafter, -CO ２ H, -PO ３ H ２ and -SO ３ A group selected from the group consisting of H is also simply referred to as a "water-soluble substituent." In one embodiment, the water-soluble substituent is -CO ２ H and -SO ３ The group may be selected from the group consisting of H. FG may have one, two, three, or four or more water-soluble substituents. FG according to one embodiment may have one water-soluble substituent. FG according to one embodiment may be a fluorescent molecule having at least one water-soluble substituent.

[0073] In one embodiment, FG may be a group represented by formula (III), formula (IV), or formula (V), and may be a group represented by formula (III) or formula (IV), or may be a group represented by formula (IV).

[0074] Formula (III) is as follows. The wavy line indicates a bond with an oxygen atom or Y. In this case, the compound represented by FG-OH is a derivative of the fluorescent dye umbelliferone. Also in this case, FG-N(-R ２The compound represented by )-H is a derivative of the fluorescent dye 7-aminocoumarin. When FG is a group represented by formula (III), molecules represented by FG-OH or FG-YH tend to have longer absorption and fluorescence wavelengths and higher fluorescence quantum yields when the oxygen atom or hydrogen atom on Y is not substituted, compared to when the oxygen atom or hydrogen atom on Y is substituted. Therefore, when selecting wavelengths suitable for the molecule in the state where the hydrogen atom is not substituted as the excitation wavelength and fluorescence detection wavelength for the target compound, the fluorescence signal is strengthened by the exposure of the phenolic hydroxyl group due to the decomposition of the phosphoramidite structure, or by the subsequent elimination of BN. Detailed design and organic synthesis of such FG can be carried out by referring to the knowledge disclosed in the publicly available literature.

[0075] R ａ１ ~R ａ５ Each of these independently comprises a hydrogen atom; and a C atom which may have substituents. １－６ Alkyl, C ２－６ Alkenyl and C １－６ Alkoxy; halogen atom; nitro; hydroxyl; hydroxyl C which may have substituents on the carbon atom. １－３ Alkyl; the water-soluble substituent; and C which may have further substituents substituted with the water-soluble substituent. １－６ Alkyl, C ２－６ Alkenyl and C １－６ Selected from the group consisting of alkoxys, where R ａ１ ~R ａ５ At least one of these may have the water-soluble substituent, or a substituent substituted with the water-soluble substituent. １－６ Alkyl, C ２－６ Alkenil or C １－６ It is an alkoxy.

[0076] In one embodiment, the group represented by formula (III) is R ａ４ is a carboxyl or its protected form, R ａ１ ~R ａ３ and R ａ５ However, each may independently have a hydrogen atom; or a substituent. １－６ Alkyl, C ２－６ Alkenyl and C１－６ Alkoxy; halogen atom; nitro; hydroxyl; hydroxyl C which may have substituents on the carbon atom. １－３ Alkyl; the water-soluble substituent; and C which may have further substituents substituted with the water-soluble substituent. １－６ Alkyl, C ２－６ Alkenyl and C １－６ The group may be selected from the group consisting of alkoxys. In another embodiment, the group represented by formula (III) is R ａ４ is a carboxyl or its protected form, R ａ１ ~R ａ３ and R ａ５ However, each may independently have a hydrogen atom; or a substituent. １－６ Alkyl, C ２－６ Alkenyl and C １－６ Alkoxy; halogen atoms; nitro; hydroxy and hydroxy C which may have substituents on the carbon atom. １－３ The group may be selected from the group consisting of alkyl groups. In a preferred embodiment, the group represented by formula (III) is R ａ４ is a carboxyl or its protected form, R ａ１ ~R ａ３ and R ａ５ However, each is independent of the hydrogen atom; C １－６ Alkyl; C ２－６ Alkenil; C １－６ Alkoxy; halogen atom; C １－６ Haloalkyl; nitro; hydroxy; and hydroxyC １－３ The group may be selected from the group consisting of alkyl groups. In one particular embodiment, the group represented by formula (III) is R ａ４ is a carboxyl or its protected form, R ａ１ ~R ａ３ and R ａ５However, the group may be a hydrogen atom. Such a group corresponds to the phenolic hydroxyl group of HCCA (Toru Komatsu et al., "Design and Synthesis of an Enzyme Activity-Based Labeling Molecule with Fluorescence Spectral Change", J. Am. Chem. Soc. 2006, 128, 50, 15946-15947) from which the hydrogen atom has been removed.

[0077] Formula (IV) is as follows. The wavy line indicates a bond with an oxygen atom or Y. When FG is the group represented by formula (IV), the molecule represented by FG-OH or FG-YH will have a higher fluorescence intensity when the oxygen atom is not substituted compared to when the oxygen atom or hydrogen atom on Y is substituted, for example, according to the following two principles: (Principle A) When the oxygen atom or hydrogen atom on Y is substituted, photo-induced electron transfer (PeT) occurs between SRG and the tricyclic moiety, resulting in a low fluorescence quantum yield. In contrast, when the oxygen atom is not substituted, photo-induced electron transfer (PeT) does not occur, resulting in a high fluorescence quantum yield. (Principle B) When an oxygen atom or a hydrogen atom on Y is substituted, the intramolecular nucleophile of SRG becomes relatively favored in an intramolecular cyclized state with the carbon atom to which SRG is bonded in formula (IV). As a result, part of the conjugated system of the tricyclic moiety is broken, resulting in shorter absorption and fluorescence wavelengths and a lower fluorescence quantum yield. In contrast, when the oxygen atom is not substituted, the intramolecular cyclized state becomes relatively unfavorable, resulting in longer absorption and fluorescence wavelengths and a higher fluorescence quantum yield. Therefore, the target compound is an activatable fluorescent dye because its fluorescence signal is strengthened by the exposure of the phenolic hydroxyl group due to the decomposition of the phosphoramidite structure, or by the subsequent elimination of BN. The detailed design and organic synthesis of such FG can be carried out by referring to the knowledge disclosed in the publicly available literature.

[0078] In formula (IV), SRG is a group represented by one of the following structural formulas.

[0079] R ｂ１ ~R ｂ５ and R ｃ１ ~R ｃ６ Each of these independently comprises a hydrogen atom; and a C atom which may have substituents. １－６ Alkyl, C １－６ Alkoxy and C ２－６ Alkenyl; halogen atom; nitro; hydroxyl; hydroxyl C which may have substituents on the carbon atom. １－３ Alkyl; the water-soluble substituent; and C which may have further substituents substituted with the water-soluble substituent. １－６ Alkyl, C ２－６ Alkenyl and C １－６ Selected from the group consisting of alkoxys, where R ｂ１ ~R ｂ５ and R ｃ１ ~R ｃ６ At least one of them is the water-soluble substituent; or C which is substituted with the water-soluble substituent and may have further substituents. １－６ Alkyl, C ２－６ Alkenil or C １－６ It is an alkoxy.

[0080] In one embodiment, R ｂ１ ~R ｂ５ Each of these independently comprises a hydrogen atom; and a C atom which may have substituents. １－６ Alkyl, C １－６ Alkoxy and C ２－６ Alkenyl; halogen atom; nitro; hydroxyl; hydroxyl C which may have substituents on the carbon atom. １－３ Alkyl; water-soluble substituent; and C which may have further substituents substituted with water-soluble substituents. １－６ Alkyl, C ２－６ Alkenyl and C １－６ Selected from the group consisting of alkoxys, where R ｂ１ ~R ｂ５ At least one of them is a water-soluble substituent; or C may be substituted with a water-soluble substituent and further have substituents. １－６ Alkyl, C ２－６ Alkenil or C １－６ It is an alkoxy, R ｃ１ ~R ｃ６Each of these independently comprises a hydrogen atom; and a C atom which may have substituents. １－６ Alkyl, C １－６ Alkoxy and C ２－６ Alkenyl; halogen atom; nitro; hydroxyl; hydroxyl C which may have substituents on the carbon atom. １－３ Alkyl; water-soluble substituent; and C which may have further substituents substituted with water-soluble substituents. １－６ Alkyl, C ２－６ Alkenyl and C １－６ Selected from the group consisting of alkoxys. In a preferred embodiment, R ｂ１ ~R ｂ５ Each of these is independently a hydrogen atom; C １－６ Alkyl; C １－６ Alkoxy; C ２－６ Alkenyl; halogen atom; nitro; hydroxy; hydroxyC １－３ Selected from the group consisting of alkyl and water-soluble substituents, however, R ｂ１ ~R ｂ５ At least one of them is a water-soluble substituent; or C is substituted with a water-soluble substituent. １－６ Alkyl, C ２－６ Alkenil or C １－６ It is an alkoxy, R ｃ１ ~R ｃ６ Each of these is independently a hydrogen atom; C １－６ Alkyl; C １－６ Alkoxy; C ２－６ Alkenyl; halogen atom; nitro; hydroxy; hydroxyC １－３ Selected from the group consisting of alkyl and water-soluble substituents. In a more preferred embodiment, R ｂ１ ~R ｂ５ At least one of them is -SO ３ It is H, and the remaining atoms are each independently hydrogen atoms, C １－６ Alkyl, C １－６ Alkoxy, hydroxy C １－３ Selected from the group consisting of alkyl and water-soluble substituents, R ｃ１ ~R ｃ６ These are, independently, hydrogen atoms and C １－６ Alkyl, C １－６ Selected from the group consisting of alkoxy and halogen atoms.

[0081] In one embodiment, R ｄ１ and R ｄ２ Each of these independently may have a hydrogen atom or a substituent. １－６ Alkyl or C ２－６ C may be substituted with an alkenyl or a water-soluble substituent and may have further substituents. １－６ Alkyl or C ２－６ Alkenil is here, R ｄ１ and R ｄ２ They become one, R ｄ１ and R ｄ２ It may form a 4- to 7-membered heterocycline containing a nitrogen atom to which is bonded, which may have substituents, and R ｄ１ and / or R ｄ２ These are, respectively, R ｃ５ or R ｃ６ In conjunction with R ｄ１ or R ｄ２ It may form a 5- to 7-membered heterocyclyl or heteroaryl, which may have substituents, including the nitrogen atom to which it is bonded. In a preferred embodiment, R ｄ１ and R ｄ２ These are, independently, hydrogen atoms and C １－６ Alkyl, C ２－６ C substituted with alkenyl or water-soluble substituents １－６ Alkyl or C ２－６ Alkenil is here, R ｄ１ and R ｄ２ They become one, R ｄ１ and R ｄ２ It may form a 4- to 7-membered heterocycline containing a nitrogen atom to which it is bonded, and R ｄ１ and / or R ｄ２ These are, respectively, R ｃ５ or R ｃ６ In conjunction with R ｄ１ or R ｄ２ It may form a 5-7 membered heterocyclyl or heteroaryl, which includes a nitrogen atom to which it is bonded.

[0082] In one embodiment, R ｅ is an oxygen atom, Si(-Rｆ１ ) (-R ｆ２ ) or C(-R ｆ１ ) (-R ｆ２ ) shows, where R ｆ１ and R ｆ２ Each of these may independently have substituents, C １－６ Alkyl, C ２－６ Alkenil, C １－６ It may represent an alkoxy or aryl. In a preferred embodiment, R ｅ is an oxygen atom, Si(-R ｆ１ ) (-R ｆ２ ) or C(-R ｆ１ ) (-R ｆ２ ) shows, where R ｆ１ and R ｆ２ Each of them is independent of C １－６ Alkyl, C ２－６ Alkenil, C １－６ This may represent an alkoxy or aryl group.

[0083] In a more preferred embodiment, the group represented by formula (IV) may be any of the groups shown in the following structural formulas.

[0084] In a particular embodiment, the group represented by formula (IV) may be any of the groups shown in the following structural formulas. These groups correspond to the hydroxyl groups of sTM and sTG described in Non-Patent Document 3, from which a hydrogen atom has been removed.

[0085] Formula (V) is as follows. The dashed line indicates a bond with an oxygen atom or Y. In this case, the compound represented by FG-OH is a derivative of resorphine, a fluorescent dye. When FG is a group represented by formula (III), molecules represented by FG-OH or FG-YH tend to have longer absorption and fluorescence wavelengths and higher fluorescence quantum yields when the oxygen atom or hydrogen atom on Y is not substituted, compared to when the oxygen atom or hydrogen atom on Y is substituted. Therefore, when selecting excitation and fluorescence detection wavelengths suitable for molecules with unsubstituted hydrogen atoms, the fluorescence signal becomes stronger due to the exposure of the phenolic hydroxyl group by the decomposition of the phosphoramidite structure, or subsequently the elimination of BN. Detailed design and organic synthesis of such FG can be carried out by referring to the knowledge disclosed in the public literature.

[0086] In one embodiment, R ｇ１ ~R ｇ６ Each of these independently comprises a hydrogen atom; and a C atom which may have substituents. １－６ Alkyl, C １－６ Alkoxy and C ２－６ Alkenyl; halogen atom; nitro; hydroxyl; hydroxyl C which may have substituents on the carbon atom. １－３ Alkyl; the water-soluble substituent; and C which may have further substituents substituted with the water-soluble substituent. １－６ Alkyl, C ２－６ Alkenyl and C １－６ Selected from the group consisting of alkoxys. In a preferred embodiment, R ｇ１ ~R ｇ６ Each of these is independently a hydrogen atom; C １－６ Alkyl; C １－６ Alkoxy; C ２－６ Alkenyl; halogen atom; nitro; hydroxy; hydroxyC １－３ Selected from the group consisting of alkyl and water-soluble substituents. In a particular embodiment, R ｇ１ ~R ｇ６ can represent a hydrogen atom. In this case, the group represented by formula (V) is the group obtained by removing a hydrogen atom from the hydroxyl group of resolphin.

[0087] The manufacturing method of the first embodiment may include a step (recovery step) in which the compound formed on the support is released from the support and recovered after the substitution step. The method for releasing the compound from the support in the recovery step may be any method that is commonly used by those skilled in the art in peptide solid-phase synthesis. For example, if the support is 2-chlorotrimethyl chloride resin, the release method may be contacting the support on which the formed compound is supported with an acid such as trifluoroacetic acid.

[0088] The manufacturing method of the first embodiment may include a step of further transforming the compound after the substitution step and / or after the recovery step. This allows the final product (i.e., the peptide-containing compound) to be obtained even if the compound of formula (I) is a compound containing a precursor of a group included in the final product (e.g., its protected form, its reduced form, its oxidized form, or a protected form of its reduced or oxidized form). More specifically, this step may include, for example, converting the precursor of FG to FG, and / or, if the peptide side chain protecting group is not removed in the recovery step, deprotecting the protecting group of the peptide side chain. By including this step, the final product can be obtained if the compound of formula (I) is a compound containing a precursor of FG (e.g., a protected form of FG, its reduced form, its oxidized form, or a protected form of its reduced or oxidized form). The transformation reaction in this process may be at least one selected from the group consisting of, for example, hydrolysis reactions, substitution reactions, addition reactions, elimination reactions, redox reactions, rearrangement reactions, cyclization reactions, and ring-opening reactions, and in one embodiment, it may be at least one selected from the group consisting of hydrolysis reactions and oxidation reactions.

[0089] In the manufacturing method of the first embodiment, purification may be performed as appropriate during and / or at the end of each step. The purification method can be selected as appropriate, for example, chromatography, liquid-liquid extraction, and recrystallization. In addition, in the manufacturing method of the first embodiment, solid-phase synthesis is performed, so in the solid-phase synthesis step, substances not captured by the support can be separated from the target product by simply removing the reaction supernatant. Therefore, compared to manufacturing methods that do not include solid-phase synthesis, the number of purification steps can be reduced, and the complexity of the purification process can be suppressed.

[0090] The form of the target compound produced by the manufacturing method of the first embodiment is not limited to the form of the molecular compound itself, but may be, for example, the molecular compound, a salt thereof, or a solvate thereof, or a metal complex containing thereof.

[0091] [Fluorescent Probe] The second embodiment of this disclosure is a fluorescent probe represented by formula (VI) or formula (VII), as described in the manufacturing method of the first embodiment. Hereinafter, this embodiment will also be referred to as the "fluorescent probe of the second embodiment." The form of the fluorescent probe of the second embodiment is not limited to the form of the molecule itself, but may be, for example, a salt of the molecule or a molecular compound thereof, or a solvate thereof. The salt may be, for example, a trifluoroacetate and / or a triethylamine salt. The solvate may be, for example, a hydrate. The fluorescent probe of the second embodiment is a diagnostic probe (fluorescent probe) that emits an optical signal in the presence of a disease-specific enzyme and / or only in a disease environment, and can be used for both in vitro diagnostic methods using biological samples and in vivo diagnostic methods administered directly to human subjects, and is particularly suitable for use in the evaluation method of the third embodiment described later.

[0092] [Method for Evaluating Enzyme Activity] The third embodiment of this disclosure relates to a method for evaluating enzyme activity. Hereinafter, this embodiment will also be referred to as the "evaluation method of the third embodiment." In the evaluation method of the third embodiment, the enzyme activity of peptidase is evaluated by metabolizing one molecule of peptidase with two types of fluorescent probes.

[0093] The peptidase in the evaluation method of the third embodiment is at least one selected from the group consisting of carboxypeptidases and endopeptidases. The EC classification shown hereafter is based on EC7, the EC classification established in 2019 by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB).

[0094] Carboxypeptidase (CP) is, for example, Carboxypeptidase A, Carboxypeptidase A2, Carboxypeptidase A3, Carboxypeptidase A4, Carboxypeptidase A5, Carboxypeptidase A6, Carboxypeptidase U, Carboxypeptidase B, Lysosomal carboxypeptidase A / Carboxypeptidase X, Carboxypeptidase E, Carboxypeptidase Z, Metallocarboxypeptidase D, Carboxypeptidase M, Tubulin-Tyr carboxypeptidase, Tubulin-glutamate Carboxypeptidase, Lysosomal Carboxypeptidase A / Serine carboxypeptidase A / Cathepsin A, Lysosomal Pro-Xaa Carboxypeptidase / Angiotensinase A, Carboxypeptidase N, Angiotensin It may be at least one selected from the group consisting of I-converting enzyme, Angiotensin-converting enzyme, and Glutamate carboxypeptidase II / Prostate specific membrane antigen. Carboxypeptidase A is EC classification 3.4.17.1 and is a CP encoded by the gene indicated by CPA1. Carboxypeptidase A2 is EC classification 3.4.17.15 and is a CP encoded by the gene indicated by CPA2. Carboxypeptidase A3 is EC classification 3.4.17.2 and is a CP encoded by the gene indicated by CPA3. Carboxypeptidase A4 is a CP with EC classification 3.4.17.4, encoded by the gene indicated as CPA4.Carboxypeptide A5 is a CP with EC classification 3.4.17.5, encoded by the gene indicated by CPA5. Carboxypeptide A6 is a CP with EC classification 3.4.17.6, encoded by the gene indicated by CPA6. Carboxypeptide U is a CP with EC classification 3.4.17.20, encoded by the gene indicated by CPU. Carboxypeptide B is a CP with EC classification 3.4.17.2, encoded by the gene indicated by CPB1. Lysosomal carboxypeptide A / Carboxypeptide X is a CP with EC classification 3.4.17.3, encoded by genes indicated by CPN1 and CPN2. Carboxypeptide E is a CP with EC classification 3.4.17.10, encoded by genes indicated by CPE. Carboxypeptide Z is a CP with EC classification 3.4.17.22, encoded by genes indicated by CPZ. Metallocarboxypeptide D is a CP with EC classification 3.4.17.22, encoded by genes indicated by CPD. Carboxypeptidase M is a CP encoded by the gene indicated by CPM, with an EC classification of 3.4.17.12. Tubulin-Tyr carboxypeptidase is a CP encoded by the genes indicated by VAS1 and VASH2, with an EC classification of 3.4.17.17. Tubulin-glutamate carboxypeptidase is a CP encoded by the gene indicated by AGBL1, with an EC classification of 3.4.17.18. Lysosomal hydrocarbon A / Serine hydrocarbon A / Cathepsin A is a CP encoded by the gene indicated by PRCP, with EC classification 3.4.16.5.Lysosomal Pro-Xaa carbohydrate peptide / angiotensinase A is a CP encoded by the gene indicated by PRCP, with an EC classification of 3.4.16.2. Carboxypeptidase N is a CP encoded by the gene indicated by CPN1, with an EC classification of 3.4.16.1. Angiotensin I-converting enzyme is a CP encoded by the gene indicated by ACE, with an EC classification of 3.4.15.1. Angiotensin-converting enzyme is a CP encoded by the gene indicated by ACE2, with an EC classification of 3.4.17.23. Glutamate hydrocarbon II / Prostate specific membrane antigen is classified as EC classification 3.4.17.21 and is a CP encoded by the gene indicated by FOLH1.

[0095] The endopeptidase may be, for example, Kallikreins, Cathepsins, Trypsin, Plasmin, Thrombin, Factor Xa, Furin, Urokinase, Enterokinase, Proteasome, Tryptase, Calpain, Chymotrypsin, Chimase, Renin, Caspase-1, Caspas, Granzyme B, Neurophyll elastase, Pancreatic elastase, FAPα, Amyloid A4 generating enzyme, MMPs, Neurolysin, or THOP1. The endopeptidases relating to this disclosure may be subtypes encoded by different genes within the class of enzymes described above, for example, KLK3 (Kallikrein related peptide 3, also known as prostate-specific antigen (PSA)) and KLK7 (Kallikrein related peptide 7), which are subtypes of Kallikreins.

[0096] The peptidase may be an enzyme contained in the sample or a purified enzyme, but in one embodiment, it may be an enzyme contained in the sample. The sample may be, for example, of human origin. The sample may be at least one selected from the group consisting of, for example, plasma, serum, urine, saliva, tears, cerebrospinal fluid, tissue lysates and cell lysates and their extracts, or at least one selected from the group consisting of plasma, serum, urine, saliva, tears and cerebrospinal fluid and their extracts, or at least one selected from the group consisting of plasma, serum and cerebrospinal fluid and their extracts. If the peptidase is an enzyme contained in the sample, the evaluation method of the third embodiment may also evaluate the sample itself, and for example, information for the diagnosis of the animal from which the sample originated (e.g., human) may be obtained.

[0097] In the evaluation method of the third embodiment, two types of fluorescent probes are used: a first fluorescent probe and a second fluorescent probe. The first fluorescent probe and the second fluorescent probe are each independently a fluorescent probe according to an aspect of the second embodiment of this disclosure. Because the fluorescent probe according to an aspect of the second embodiment of this disclosure has high water solubility, it can suitably detect the activity of a single molecule of peptidase even in single-molecule evaluation systems that require high water solubility for the fluorescent probe and its reaction products, such as the oil seal chamber system described later.

[0098] The first and second fluorescent probes have a difference of 30 nm or more between the maximum absorption wavelength and / or maximum fluorescence wavelength of their respective fluorescent molecules represented by FG-OH or FG-YH. This allows for the detection of fluorescence from two types of metabolites (i.e., fluorescent molecules represented by FG-OH or FG-YH) produced when these two fluorescent probes are metabolized by peptidase, even in a single solution, by appropriately setting the excitation wavelength and / or fluorescence detection wavelength. In this disclosure, the maximum absorption wavelength refers to the wavelength at which absorbance is maximum in the visible light region, and the maximum fluorescence wavelength refers to the wavelength at which fluorescence intensity is maximum in the visible light region. The visible light region refers to, for example, the wavelength range of 350 to 800 nm. The difference in the maximum absorption wavelengths of the first and second fluorescent probes, for their respective fluorescent molecules represented by FG-OH or FG-YH, may be, for example, 30 nm or more, 50 nm or more, 70 nm or more, or 90 nm or more. The difference in maximum fluorescence wavelength between the first fluorescent probe and the second fluorescent probe, where the respective fluorescent molecules represented by FG-OH or FG-YH are, for example, 30 nm or more, 50 nm or more, 70 nm or more, or 90 nm or more, may be such that.

[0099] The first and second fluorescent probes may be fluorescent probes in which Pep in formula (VI) or formula (VII) is a different peptide from each other. In this case, information on the substrate selectivity of a single peptidase molecule for multiple peptides can be obtained by the evaluation method of the third embodiment.

[0100] The evaluation method of the third embodiment includes the following steps (A) to (C): Step (A): A step of preparing an aqueous solution containing one molecule of peptidase, a first fluorescent probe, and a second fluorescent probe (preparation step); Step (B): A step of catalyzing an enzymatic reaction in the aqueous solution prepared in step (A) with the peptidase (reaction step); Step (C): A step of measuring the fluorescence of the aqueous solution obtained in step (B) (fluorescence measurement step).

[0101] The evaluation system in the evaluation method of the third embodiment is not particularly limited as long as it can form a closed reaction system with an aqueous solution containing one molecule of peptidase, a first fluorescent probe, and a second fluorescent probe, and can quantitatively measure the fluorescence derived from the metabolites of the first and second fluorescent probes (i.e., fluorescent molecules represented by FG-OH or FG-YH) generated within the closed reaction system. The closed reaction system may be spatially enclosed by, for example, a resin or oil, and the volume of the closed reaction system may be, for example, 5 fL to 100 fL. Furthermore, there may be multiple closed reaction systems, and step (B) may be carried out in each of them. A more specific evaluation system may be, for example, an evaluation system using a microdevice provided with wells with a volume of about 5 fL to 100 fL per well, in which case the portion of the surface of the aqueous solution (closed reaction system) in step (B) that is not covered by the wells may be covered with an organic solvent or a gas (e.g., air). If the portion of the aqueous solution surface not covered by the well in step (B) is covered by gas, the aqueous solution may remain in the well, for example, due to gravity and / or surface tension. Another specific evaluation system may be one in which droplets (e.g., liposomes) with a volume of about 5 fL to 100 fL each are present in the aqueous solution. In this case, the enzymatic activity of each peptidase can be evaluated by encapsulating an aqueous solution containing one molecule of peptidase, a first fluorescent probe, and a second fluorescent probe within each droplet.

[0102] In the evaluation method of the third embodiment, the evaluation system is, in a preferred embodiment, an evaluation system using a microdevice provided with wells having a volume of about 5 fL to 100 fL per well, and may be an evaluation system in which the portion of the surface of the aqueous solution (closed reaction system) in step (B) that is not covered by the wells is covered with an organic solvent, and this evaluation system will be referred to as the "oil seal chamber system" below. Such evaluation systems are described, for example, in Patent Document 1 and Non-Patent Document 1. In the oil seal chamber system, a closed reaction system can be realized by utilizing the property that hydrophobic solvents and aqueous solutions do not mix with each other. The hydrophobic solvent is not particularly limited as long as it does not mix with the aqueous solution, but for example, at least one selected from the group consisting of saturated hydrocarbons, unsaturated hydrocarbons, aromatic hydrocarbons, silicone oil, perfluorocarbons, halogenated solvents and hydrophobic ionic liquids, or a mixture containing the same, can be suitably used.

[0103] <Step (A): Preparation Step> Step (A) prepares an aqueous solution containing one molecule of peptidase, a first fluorescent probe, and a second fluorescent probe. In Step (A), typically, a solution (pre-encapsulation aqueous solution) containing one molecule of peptidase, a first fluorescent probe, and a second fluorescent probe is first prepared, and then the obtained pre-encapsulation aqueous solution is encapsulated in a microdevice or droplet, etc., such that there is one molecule of peptidase in each closed reaction system. That is, in one embodiment, Step (A) may also include encapsulating the pre-encapsulation aqueous solutions containing peptidase, a first fluorescent probe, and a second fluorescent probe in a space closed to the aqueous solution, such that there is one molecule of peptidase in each aqueous solution.

[0104] If step (A) includes sealing pre-encapsulation aqueous solutions containing peptidase, a first fluorescent probe, and a second fluorescent probe into a closed space relative to the aqueous solution such that each aqueous solution contains one molecule of peptidase, and if step (A) includes sealing aqueous solutions into a large number (e.g., 1000 or more) closed spaces, the average number of peptidase molecules contained in each of those aqueous solutions may be, for example, 0.0002 to 0.5, and may also be 0.0005 to 0.1, 0.001 to 0.07, or 0.003 to 0.05. If the average number of peptidase molecules contained in each aqueous solution is within the above range, the likelihood of each aqueous solution containing encapsulated peptidase being reduced to one molecule increases, making it easier to suppress the measurement of fluorescence originating from aqueous solutions containing two or more molecules of peptidase in step (C). This makes it easier to evaluate the enzymatic activity of the peptidase. The average number of peptidase molecules contained in each of the above aqueous solutions may be, for example, the value obtained by multiplying the concentration of peptidase in the aqueous solution before encapsulation [molecules / L] by the volume of the aqueous solution in step (B) (for example, the volume of the well) [L].

[0105] The preparation of the pre-encapsulation aqueous solution containing peptidase, a first fluorescent probe, and a second fluorescent probe is usually carried out by adding the peptidase, the first fluorescent probe, and the second fluorescent probe to a solvent. In this case, the first fluorescent probe and the second fluorescent probe may be added, for example, in the form of a salt, a solvate, or a solvate of a salt.

[0106] The concentration of peptidase in the aqueous solution before encapsulation is not particularly limited as long as fluorescence can be detected in the subsequent step (C) (fluorescence measurement step), but may be, for example, 5 fM to 20 pM, 10 fM to 10 pM, 30 fM to 3 pM, or 100 fM to 1 pM.

[0107] Furthermore, for example, in the evaluation method of the third embodiment, if the peptidase is a peptidase contained in plasma, the dilution ratio of the plasma in the aqueous solution before encapsulation is not particularly limited as long as fluorescence can be detected in the subsequent step (C) (fluorescence measurement step), but may be, for example, 10 to 1,000,000 times, 30 to 300,000 times, 100 to 100,000 times, 300 to 30,000 times, or 500 to 10,000 times.

[0108] The concentrations of the first and second fluorescent probes in the aqueous solution before encapsulation are not particularly limited as long as fluorescence can be detected in step (C) (fluorescence measurement step), but for example they may be 1 μM to 1000 μM each, 3 μM to 700 μM, 10 μM to 500 μM, or 30 μM to 300 μM. The concentrations of the first and second fluorescent probes in the aqueous solution before encapsulation are usually the same as the concentrations of the first and second fluorescent probes in the aqueous solution prepared in step (A), and the concentrations of the first and second fluorescent probes in the aqueous solution at the start of step (B).

[0109] The pH of the aqueous solution prepared in step (A) is not limited as long as it is the pH at which the enzymatic reaction by peptidase occurs. For example, it may be within the range of the optimal pH of peptidase ± 3.0, within the range of the optimal pH of peptidase ± 2.0, within the range of the optimal pH of peptidase ± 1.5, or within the range of the optimal pH of peptidase ± 1.0. Specifically, the pH of the aqueous solution prepared in step (A) may be, for example, 3.5 or higher, 4.0 or higher, 4.5 or higher, 5.0 or higher, 5.5 or higher, 6.0 or higher, 6.5 or higher, 7.0 or higher, 7.5 or higher, 8.0 or higher, 8.5 or higher, or 9.0 or higher, and may also be 9.5 or lower, 9.0 or lower, 8.5 or lower, 8.0 or lower, 8.0 or lower, 7.5 or lower, 7.0 or lower, 6.5 or lower, 6.0 or lower, 5.5 or lower, 5.0 or lower, 4.5 or lower, or 4.0 or lower.

[0110] Furthermore, the aqueous solution prepared in step (A) may contain other components in addition to peptidase, the first fluorescent probe, and the second fluorescent probe. Examples of other components include components of the sample from which the peptidase is derived, as well as buffers (e.g., phosphate buffers, HEPES buffers, and Tris buffers), pH adjusters (e.g., hydrochloric acid, citric acid, sodium hydroxide, sodium carbonate, and sodium bicarbonate), ion concentration adjusters (e.g., calcium chloride and magnesium chloride), osmotic pressure adjusters (e.g., sodium chloride), antioxidants (e.g., dithiothreitol (DTT)), and surfactants (e.g., Triton® X-100, CHAPS, and Tween® 20).

[0111] In the evaluation method of the third embodiment, if the evaluation system is an oil seal chamber system, step (A) is a step of filling a plurality of containments of a microchamber array having a hydrophobic surface with an aqueous solution containing peptidase, a first fluorescent probe, and a second fluorescent probe, and may include, in this order, pouring the aqueous solution containing peptidase, a first fluorescent probe, and a second fluorescent probe from the opening of the containment so that the number average number of peptidase molecules filled in each of the plurality of containment parts is 0.0002 to 0.5, and then blocking the opening with a hydrophobic solvent. As such a microchamber array, for example, those described in Patent Document 2 and Non-Patent Document 4 can be used, and more specifically, for example, Simoa® disk (Quanterx) can be used. The solution in each of the plurality of containment parts may be, for example, 1 fL to 500 fL, 5 fL to 100 fL, 10 fL to 75 fL, or 20 fL to 60 fL. Furthermore, the number average number of peptidase molecules filled in each of the above-mentioned multiple containment sections may be, for example, 0.0002 to 0.5, and may also be 0.0005 to 0.1, 0.001 to 0.07, or 0.003 to 0.05.

[0112] In the evaluation method of the third embodiment, when the evaluation system is an oil seal chamber system, fluorescent probes with high water solubility of the fluorescent probe and its metabolites (i.e., fluorescent molecules represented by FG-OH or FG-YH) can be suitably used as the first and second fluorescent probes from the viewpoint of suppressing leakage into hydrophobic solvents. As such fluorescent probes, for example, when a 1 μM of the compound is added to a two-layer system of octanol and pH 7.4 phosphate-buffered saline (PBS) in a volume ratio of 1:1, the proportion of the compound distributed in the PBS layer is 80% or more.

[0113] In one embodiment of the first aspect of this disclosure, when the evaluation system is an oil seal chamber system, a fluorescent probe having a product of the molar extinction coefficient [L / mol·cm] at the maximum absorption wavelength of the metabolite (i.e., a fluorescent molecule represented by FG-OH or FG-YH) and the fluorescence quantum yield of 10,000 or more can be suitably used, from the viewpoint of making fluorescence easier to observe. Similarly, a fluorescent probe having a maximum fluorescence wavelength of 450 nm or more for the metabolite can be suitably used.

[0114] <Step (B): Reaction Step> In Step (B), the peptidase is used to catalyze an enzymatic reaction in the aqueous solution prepared in Step (A).

[0115] The reaction temperature in step (B) is not particularly limited as long as it is a temperature at which an enzymatic reaction occurs with the peptidase, but may be, for example, 20°C to 50°C, or 23°C to 40°C. Specific examples of reaction temperatures in step (B) include, for example, 25°C and 37°C.

[0116] The reaction time in step (B) is not particularly limited as long as it is a time during which fluorescence can be quantitatively measured in the subsequent step (C) (fluorescence measurement step). For example, it may be between 1 minute and 72 hours, between 5 minutes and 48 hours, between 10 minutes and 30 hours, between 20 minutes and 24 hours, or between 30 minutes and 8 hours. Specific examples of reaction times in step (B) include, for example, 1 hour, 3 hours, 6 hours, and 12 hours.

[0117] <Step (C): Fluorescence Measurement Step> In Step (C), the fluorescence of the aqueous solution obtained in Step (B) is measured and fluorescence data is acquired. Step (C) may be performed simultaneously with Step (B), and in this case, Step (C) may be performed multiple times during the process of Step (B) (so-called time-lapse measurement may be performed). Furthermore, the method for measuring fluorescence is not particularly limited as long as it is a method capable of measuring the fluorescence of the aqueous solution obtained in Step (B), and may be, for example, a method using a fluorescence microscope, and the fluorescence microscope may be, for example, an epi-illuminating microscope, a confocal microscope, or a total internal reflection microscope.

[0118] <Evaluation of Peptidase Enzyme Activity> Based on the fluorescence data obtained by the method described above, the enzyme activity of peptidase can be evaluated. The evaluation of peptidase enzyme activity may be based on the number of aqueous solutions in which fluorescence intensity above a certain threshold was observed among the aqueous solutions subjected to fluorescence measurement, the distribution (pattern) of fluorescence intensity of the aqueous solutions subjected to fluorescence measurement, or other indicators.

[0119] Specifically, if it is known that in aqueous solutions containing peptidases that meet certain conditions (e.g., conditions in which gene mutations have occurred, or conditions in which post-translational modifications have been made), the fluorescence intensity is above a threshold, while in aqueous solutions containing peptidases that do not meet certain conditions (e.g., conditions in which gene mutations have not occurred, or conditions in which post-translational modifications have not been made), then the enzyme activity of the peptidase may be evaluated based on whether or not the fluorescence intensity is above the threshold, and peptidases whose fluorescence intensity is above the threshold can be evaluated as peptidases that meet the specific conditions.

[0120] Specifically, for example, in an aqueous solution containing peptidase derived from a sample that meets certain conditions, the average fluorescence intensity of the aqueous solution is above a threshold, whereas in an aqueous solution containing peptidase derived from a sample that does not meet the conditions, the average fluorescence intensity of the aqueous solution is below the threshold. In this case, the sample itself can be evaluated through the evaluation of the peptidase enzyme activity, using whether or not the average fluorescence intensity of the aqueous solution containing peptidase derived from the target sample is above a threshold. For example, a sample whose average fluorescence intensity is above a threshold can be evaluated as a sample that meets the conditions. In this case, for example, if the sample that meets the conditions is a sample from a cancer patient and the sample that does not meet the conditions is a sample from a healthy person, the average fluorescence intensity of the aqueous solution containing peptidase derived from the sample is above a threshold can be used as a criterion to assist in the diagnosis of whether or not a person has cancer, and data can be obtained to be used in determining whether or not a person has cancer.

[0121] For example, if a sample that meets certain conditions has a predetermined percentage of aqueous solutions whose fluorescence intensity exceeds a threshold, while a sample that does not meet those conditions has a predetermined percentage of aqueous solutions whose fluorescence intensity exceeds a threshold, the sample itself can be evaluated by evaluating the enzyme activity of peptidases based on whether or not the percentage of aqueous solutions with fluorescence intensity above a threshold in the target sample is above a predetermined value. For example, a sample in which the percentage of aqueous solutions with fluorescence intensity above a threshold is above a predetermined value can be evaluated as a sample that meets certain conditions. In this case, for example, if a sample that meets certain conditions is a specimen from a cancer patient and a sample that does not meet certain conditions is a specimen from a healthy person, the percentage of aqueous solutions with fluorescence intensity above a threshold in the sample can be used as a criterion to assist in the diagnosis of whether or not a person has cancer, and data can be obtained to be used in determining whether or not a person has cancer.

[0122] Specifically, for example, if the distribution (pattern) of fluorescence intensity in an aqueous solution containing peptidase derived from a sample that satisfies certain conditions differs from the distribution (pattern) of fluorescence intensity in an aqueous solution containing peptidase derived from a sample that does not satisfy certain conditions, the sample itself can be evaluated through the evaluation of the enzyme activity of the peptidase, based on whether the distribution (pattern) of fluorescence intensity in the aqueous solution containing peptidase derived from the target sample is more similar to either distribution. For example, if the p-value obtained by performing a Student's t-test between a sample that satisfies certain conditions and the target sample is compared to the p-value obtained by performing a Student's t-test between a sample that does not satisfy certain conditions and the target sample, and the p-value obtained by performing a Student's t-test between a sample that satisfies certain conditions and the target sample is smaller, then the sample can be evaluated as a sample that satisfies certain conditions. In this case, for example, if a sample that meets certain conditions is derived from a cancer patient, and a sample that does not meet those conditions is derived from a healthy individual, then a Student's t-test can be performed between the sample that meets the conditions and the target sample. The p-value obtained from this test can be used as a criterion to assist in diagnosing whether or not a person has cancer, and data can be obtained to determine whether or not a person has cancer.

[0123] Specifically, if the fluorescence data is, for example, a fluorescence image (e.g., a fluorescence image obtained through evaluation in an oil seal chamber system), then a machine learning model trained to consider fluorescence images obtained using samples that meet specific conditions as samples that meet those conditions, and fluorescence images obtained using samples that do not meet those conditions as samples that do not meet those conditions can be used to evaluate the sample itself by taking the fluorescence image of the target sample as input and determining whether or not the target sample meets the specific conditions. This allows for evaluation of the sample itself through the evaluation of peptidase enzyme activity.

[0124] [Fluorescent Probe Library] The fourth embodiment of this disclosure relates to a fluorescent probe library including the fluorescent probe of the second embodiment. Hereafter, this embodiment will also be referred to as the "fluorescent probe library of the fourth embodiment".

[0125] The fluorescent probe library of the fourth embodiment may contain at least five types of the fluorescent probes of the second embodiment, and may also contain at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 100, at least 200, or at least 300 types. Each of the fluorescent probes of the second embodiment can be produced according to the manufacturing method of the first embodiment. The inventors have succeeded in producing such fluorescent probes by utilizing a solid-phase synthesis method, which allows for easy expansion of peptide variations and simple purification. Therefore, the fluorescent probe library of the fourth embodiment, which contains a large number of such fluorescent probes, can for the first time be constructed by the manufacturing method of the first embodiment.

[0126] Each fluorescent probe included in the fluorescent probe library of the fourth embodiment may have different FG and / or Pep structures. In one embodiment, the FG structure may be different, and the Pep structure may also be different. If the FG structure is different in each fluorescent probe included in the fluorescent probe library of the fourth embodiment, for example, in the screening method of the fifth embodiment described later, it is possible to find two sets of fluorescent probes suitable for the evaluation method of the third embodiment described above. If the Pep structure is different in each fluorescent probe included in the fluorescent probe library of the fourth embodiment, for example, in the screening method of the fifth embodiment described later, information on the ease of cleavage by peptidase depending on the difference in the Pep sequence can be obtained, and thus the structures of fluorescent probes and Pep that are easily hydrolyzed by the peptidase can be selected.

[0127] Furthermore, as a fluorescent probe library corresponding to the fluorescent probe library of the fourth embodiment, a fluorescent probe library containing at least five types in which the water-soluble functional group of FG in the fluorescent probe of the second embodiment is substituted with a hydrogen atom may also be an embodiment of this disclosure. Such a fluorescent probe library is suitable for selecting a Pep structure that is easily hydrolyzed for detection of a certain peptidase, for example, if the Pep structure differs in each fluorescent probe.

[0128] [Screening Method for Fluorescent Probes] The fifth embodiment of this disclosure relates to a screening method for fluorescent probes. Hereinafter, this embodiment will also be referred to as the "screening method of the fifth embodiment." In the screening method of the fifth embodiment, a desired fluorescent probe is selected from the fluorescent probe library of the fourth embodiment.

[0129] The screening method of the fifth embodiment includes contacting a fluorescent probe contained in the fluorescent probe library of the fourth embodiment with a peptidase or a sample containing the same (contact step), and selecting fluorescent probes whose fluorescence intensity has increased after contact with the peptidase or the sample containing the same as those that were not in contact, as fluorescent probes that target peptidase and / or diseases that express it (selection step).

[0130] The peptidase used in the screening method of the fifth embodiment is at least one selected from the group consisting of carboxypeptidases and endopeptidases, and is the same as that described in the evaluation method of the third embodiment. Furthermore, the sample containing the peptidase used in the screening method of the fifth embodiment is also the same as that described in the evaluation method of the third embodiment.

[0131] In the contact step, the fluorescent probe is brought into contact with peptidase or a sample containing it. In the contact step, for example, each of the fluorescent probes included in the fluorescent probe library of the fourth embodiment may be brought into contact with peptidase or a sample containing it. Alternatively, in the contact step, for example, two, three, four or more types of fluorescent probes included in the fluorescent probe library of the fourth embodiment may be brought into contact with peptidase or a sample containing it in the same system. In the latter case, the fluorescence derived from each metabolite (i.e., fluorescent molecules represented by FG-OH or FG-YH) may be detected separately by using a combination of excitation wavelength and fluorescence detection wavelength suitable for each metabolite.

[0132] The method for measuring the contact in the contact process and the fluorescence intensity of the resulting metabolites is not particularly limited as long as it allows for screening of the fluorescent probe, and may be, for example, a bulk system or a single-molecule enzyme activity evaluation system. The method using a bulk system may be, for example, by adding the fluorescent probe and peptidase to a solvent and measuring the fluorescence intensity of the solution, in which case the solvent, reaction conditions and measuring device may follow conditions generally used for measuring enzyme reactions and fluorescence intensity. The fluorescence intensity measuring device may be, for example, a fluorophotometer or a microwell plate reader. The method using a single-molecule enzyme activity evaluation system may be, for example, the method described in the evaluation method of the third embodiment, however, one, two or more types of fluorescent probes may be used.

[0133] In the selection process, fluorescent probes whose fluorescence intensity increases after contact with peptidase or a sample containing it compared to the non-contact state can be selected as fluorescent probes targeting peptidase and / or diseases that express it. In one embodiment, fluorescent probes that show the largest increase in fluorescence intensity compared to the non-contact state after contact with peptidase or a sample containing it may be selected as fluorescent probes targeting peptidase and / or diseases that express it. In another embodiment, in the single-molecule enzyme activity evaluation system, fluorescent probes that show superior quantity and / or quality of clusters in the fluorescence intensity distribution (pattern) after contact with peptidase or a sample containing it may be selected as a set of fluorescent probes targeting peptidase and / or diseases that express it.

[0134] Furthermore, in the screening method of the fifth embodiment, if multiple fluorescent probes having the same FG structure but different Pep structures exhibit different levels of fluorescence intensity increase and different amounts and / or qualities of clusters in the fluorescence intensity distribution (pattern), the Pep or set thereof belonging to a fluorescent probe that exhibits particularly excellent levels of increase and / or quality of clusters can be selected as a peptide sequence or set thereof suitable for use in functional molecules targeting peptidases and / or diseases that express them.

[0135] The present disclosure will be described in more detail below using examples, but the present disclosure should not be construed as being limited to the following examples.

[0136] The abbreviations used in this example are as follows: TBDMS: tert-butyldimethylsilyl AcCN: acetonitrile TBDMS-Cl: tert-butyldimethylsilyl chloride AcOEt: ethyl acetate brine: saturated saline Na ２ SO ４: Sodium sulfate Ar: Argon THF: Tetrahydrofuran DIEA: Diisopropylethylamine Fmoc: 9-Fluorenylmethyloxycarbonyl DCM: Dichloromethane NMP: N-methyl-2-pyrrolidone DMF: N,N-dimethylformamide HFIP: Hexafluoroisopropanol LC-MS: Liquid chromatography-mass spectrometry TBAF: Tetrabutylammonium fluoride PBr ３ : Phosphorus tribromide MeOH: Methanol H ２ O: Water DMSO: Dimethyl sulfoxide MPLC: Medium-pressure liquid chromatography TFA: Trifluoroacetic acid Et ２ O: Diethyl ether 4MU: 4-Methylumbelliferone SOCl ２ : Thionyl chloride Ac ２ O: Acetic anhydride PMB: Para-methoxybenzyl PMB-OH: Para-methoxybenzyl alcohol TEA: Triethylamine CaCl ２ Calcium chloride (MgCl) ２ Magnesium chloride (DTT): Dithiothreitol

[0137] <Manufacturing Example 1: Synthesis of a Fluorescent Probe Having a Benzyl Structure 1> [Step A: Synthesis of a Benzyl Building Block] The protection of the hydroxyl group of 4-Hydroxybenzol alcohol with TBDMS groups was carried out according to the method described in Non-Patent Document 2. 2.5 g (20 mmol; Tokyo Chemical Industry Co., Ltd.) of 4-Hydroxybenzol alcohol was dissolved in 30 mL of AcCN, and 4 g (60 mmol; Wako Pure Chemical Industries, Ltd.) of imidazole and 3 g (20 mmol; Tokyo Chemical Industry Co., Ltd.) of TBDMS-Cl were added and the mixture was stirred overnight at room temperature. After removing the reaction mixture under reduced pressure to reduce the AcCN, AcOEt was added. After washing several times with a bleed, Na ２ SO ４The product was dried to obtain the crude product. It was purified by column chromatography (silica gel, n-Hexane-AcOEt) to obtain the hydroxyl group TBDMS protected product. Under Ar substitution, 150 μL (1.26 mmol; Tokyo Chemical Industry Co., Ltd.) of ethyl phosphorodichloronitride was added dropwise to dry THF (5 mL) on an ice bath using a syringe. Then, 200 mg (0.42 mmol) of the TBDMS protected product obtained above was added dropwise in a solution of dry THF (4 mL) and DIEA (400 μL, 4.91 mmol). The reaction mixture was stirred on an ice bath, and after 30 minutes, it was loaded directly onto a silica gel column and purified using a Hexane-AcOEt 80:20-0:100 gradient to obtain 130 mg (0.36 mmol) of the target substance. The results of the mass spectrometry of the target substance are as follows, confirming that the desired benzyl building block was obtained. LRMS (ESI) ＋ ): m / z=365 (M+H) ＋

[0138] [Step B: Solid-phase synthesis of peptides] A two-amino acid peptide was extended using 60 mg of 2-chlorotrityl chloride resin by Fmoc solid-phase synthesis (Fmoc SPPS) according to a standard procedure. The detailed protocol is described below. Peptide extension was performed using the fully automated peptide synthesizer SyroI according to the standard protocol for Fmoc solid-phase synthesis. Specifically, with the side-chain protected amino acids supported on chlorotrityl resin, the peptide was extended one amino acid at a time from the C-terminus by repeating the following steps until the desired peptide sequence was obtained: (1) condensation, (2) washing, (3) deprotection of the Fmoc group, and (4) washing. For (1) condensation, the reaction was carried out for 40 min in 2 mL of DMF solution containing 0.16 M amino acid building blocks (Fmoc-AA-OH), 0.16 M HATU, and 0.64 M DIEA. (2) For washing, 2 mL of DMF was added and reacted for 1 min, then the solvent was removed, and this procedure was repeated three times. (3) For deprotection of the Fmoc group, 2 mL of 40% piperidine solution (DMF) was added and reacted for 3 min, then the solution was removed and 2 mL of 20% piperidine solution (DMF) was added and reacted for 3 min. (4) For washing, the same procedure as in (2) was performed six times.

[0139] [Step C: Introduction of benzyl building blocks to solid-phase peptides] After washing the resin twice with DCM following step B, 500 μL of NMP and 250 μL of DIEA were added. 5 mg of the benzyl building block obtained in step A was dissolved in 500 μL of NMP and gradually added dropwise. The mixture was stirred at room temperature for 30 minutes to allow the reaction to proceed, after which the resin was washed three times with DMF and three times with DCM. A portion of the resin was stirred in DCM-HFIP (4:1) for 1 hour, and a small amount of the compound was excised from the solid phase. The progress of the desired reaction was confirmed using LC-MS.

[0140] [Step D: Removal of TBDMS groups on the solid phase] A solution was prepared by mixing 500 μL of TBAF (1 M in THF; Tokyo Chemical Industry Co., Ltd.) and 40 μL of AcOH in 1.5 mL of THF. This solution was added to the resin obtained in step C and reacted at room temperature for 3 hours. The resin was then washed three times with DMF and three times with DCM. A portion of the resin was stirred in DCM-HFIP (4:1) for 1 hour, and a small amount of the compound was cleaved from the solid phase. The progress of the target reaction was confirmed using LC-MS.

[0141] [Step E: Conversion of hydroxyl to bromo on a solid phase] Add 2 mL of DCM and 100 μL of dry pyridine to the resin obtained in step D, and stir while adding PBr ３ After adding 50 μL (Wako Pure Chemical Industries), the mixture was stirred and reacted at room temperature for 10 minutes. After removing the reaction solution, the resin was washed three times with DCM and then once with MeOH.

[0142] [Process F: Cutting of phosphoramidite building block from the solid phase] The resin obtained in step E was stirred in DCM-HFIP (4:1) for 30 minutes, and the phosphoramidite building block synthesized on the solid phase was cleaved off. The reaction supernatant was collected. The remaining resin was washed with DCM and acetonitrite, and these were also added to the recovered solution. After adding more acetonitrite, the solvent was removed by vacuum distillation without heating. H was added to the residue. ２ O and DMSO were added, and the mixture was purified by preparative MPLC (reverse phase, 0.1% TFA system). The fraction containing the target substance was recovered, and acetonitrile was removed by vacuum distillation without heating, followed by H ２ O was added. The solution was instantly frozen and freeze-dried. Et was added to the residue after freeze-drying. ２ O was added, and the resulting precipitate was collected by centrifugation. The collected solid was stored as a phosphoramidite building block and used in the next step.

[0143] [Process G: Synthesis of fluorescent probes] The phosphoramidite building block obtained in step F (amount obtained using 60 mg as resin in step B) was dissolved in acetone or DCM, transferred to a 1.5 mL tube, and dried. sTG prepared according to the method described in Non-Patent Document 3 was added to the phosphoramidite building block in an amount of 2-3 equivalents, dissolved in a mixed solvent of 20 μL NMP and 10 μL DIEA. The mixture was stirred and reacted at room temperature for 3 hours, after which it was purified by preparative MPLC (reverse phase, 0.1% TFA system) under acidic conditions. The fraction containing the target product was recovered and freeze-dried. The residue after freeze-drying was treated with a 90% TFA aqueous solution (TFA 90% by volume and H2). ２ 500 μL of a mixed solution containing 10% by volume of O was added, and the mixture was stirred at room temperature for 1 hour to deprotect it. The reaction solution was then mixed with H ２ The solution was dissolved in O and purified by preparative MPLC (reverse phase, 0.1% TFA system). The fraction containing the target substance was recovered and freeze-dried to obtain the target substance.

[0144] Figure 1 shows the structure of the probe obtained as described above, the LC chart, and the peak values ​​obtained by mass spectrometry. The chromatogram in the LC chart shown in Figure 1 is obtained by using a 0.1% TFA aqueous solution as solution A and an 80% AcCN aqueous solution containing 0.1% TFA (AcCN 80% by volume and H ２ The absorbance at 440 nm is shown when the mixture solution (O20 volume%) was analyzed with a gradient of A:B = 99:1 to 5:95 over 3.5 min. For all compounds, high-purity target products were obtained simply by performing reverse-phase chromatography after the final step (step G) following solid-phase synthesis. Furthermore, for all compounds, mass spectrometry yielded m / z peaks corresponding to the removal of hydrogen atoms from the target substance.

[0145] <Manufacturing Example 2: Synthesis of Fluorescent Probes by Method of Introducing Fluorescent Dye on a Solid Phase 1> [Preparation of 4MU-ethyl-phosphochlorodate] Under Ar-substituted conditions, 400 μL (3 mmol) of ethyl phosphorodichlorolide was added dropwise to 5 mL of THF on an ice bath using a syringe. A solution of 400 mg (2.3 mmol; Tokyo Chemical Industry Co., Ltd.) of 4-methylumberiferone (4MU) dissolved in a mixed solvent of 5 mL of THF and 1 mL of DIEA was then added dropwise using a syringe, and the mixture was stirred at room temperature for 10 minutes to allow the reaction to proceed. The reaction solution was then loaded directly onto a silica gel column and purified by a gradient of AcOEt-Hexane 80:20 to 0:100 for 10 minutes to obtain the target substance (200 mg, 0.66 mmol). The results of mass spectrometry and NMR are shown below.

[0146] LRMS (ESI ＋ ): m / z=303(M+H) ＋ １ H-NMR (CDCl ３ ): δ7.56 (d, 1H, J = 7.2Hz), 7.16 (s, 1H), 7.15 (d, 1H, J = 7.2Hz), 6.21 (s, 1H), 4.22 (m, 2H), 2.40 (s, 3H), 1.36 (t, 3H, J = 7.2Hz).

[0147] [Introduction and excision of fluorescent dyes on a solid phase] 60 mg of the peptide-supported resin obtained in step B of Production Example 1 was washed three times with DMF and three times with DCM. 200 μL of NMP and 200 μL of DIEA were added to the washed resin. 50 μmol of 4MU-ethyl-phosphochlorodate dissolved in 200 μL of NMP was then added, and the mixture was stirred at room temperature for 2 hours. The reaction supernatant was removed, and the resin was washed three times with DMF and three times with DCM. 1 mL of 90% TFA aqueous solution was added to the washed resin, and the mixture was stirred at room temperature for 1 hour. The reaction supernatant was collected. The remaining resin was washed with 500 μL of Acetone and added to the collected solution. H2 ２ O was added to the solution to a total volume of approximately 5 mL, which was then purified by preparative MPLC (reverse phase, 0.1% TFA system). The fraction containing the target substance was recovered and freeze-dried to obtain the target substance.

[0148] The peptide sequences and LC charts of the probes obtained as described above are shown in Figure 2. The chromatograms in the LC charts shown in Figure 2 show the absorbance at 310 nm when solution A is a 0.1% TFA aqueous solution, solution B is an 80% AcCN aqueous solution containing 0.1% TFA, and analysis was performed with a gradient of A:B = 99:1 to 5:95 and 3.5 min. For all compounds, high-purity target products were obtained simply by performing reverse-phase chromatography after the final step following solid-phase synthesis.

[0149] <Manufacturing Example 3: Synthesis of Fluorescent Probes by Method of Introducing Fluorescent Dye on a Solid Phase 2> [Preparation of HCCA-PMB] 1200 mg of HCCA, prepared according to the method described in the literature (Toru Komatsu et al., "Design and Synthesis of an Enzyme Activity-Based Labeling Molecule with Fluorescence Spectral Change", J. Am. Chem. Soc. 2006, 128, 50, 15946-15947), was dissolved in 1.5 mL of pyridine and 3 mL of acetic anhydride, and the mixture was stirred at room temperature for 2 hours. To the resulting solution, toluene was added, and the solvent was removed by azeotrope. The residue was dissolved in 5 mL of toluene, and 1.5 mL of thionyl chloride (Wako Pure Chemical Industries) was added, and the mixture was stirred at 80°C for 2 hours. To the resulting solution, 20 mL of toluene was added, and the solvent was removed by azeotrope to obtain HCCA-Ac acid chloride as the residue. This was dissolved in 20 mL of DCM, 200 μL of pyridine was added, followed by 840 μL (6.75 μmol; Tokyo Chemical Industry Co., Ltd.) of p-methoxybenzone alcohol, and the mixture was stirred at room temperature for 2 hours to allow it to react. After that, the solvent was removed under reduced pressure, and then 2 mL of triethylamine and H2H ２ 5 mL of O was added and the mixture was stirred and reacted at room temperature for 18 hours. By purification using medium-pressure preparative separation (reverse phase; TFA acidic), 140 mg of the target substance, HCCA-PMB, was obtained. The results of mass spectrometry and NMR are shown below.

[0150] LRMS (ESI＋ ): m / z=327 (M+H) ＋ １ H-NMR (CDCl ３ ): δ11.07 (br, 1H), 8.64 (s, 1H), 7.72 (d, 1H, J = 8.4Hz), 7.35 (d, 2H, J = 8.8Hz), 6.9 1 (d, 2H, J=8.8Hz), 6.79 (d, 1H, J=8.4Hz), 6.68 (s, 1H), 5.18 (s, 2H), 3.68 (s, 3H).

[0151] [Preparation of HCCA-PMB-ethyl-phosphochlorodate] 50 mg (150 μmol) of HCCA-PMB was dissolved in a mixed solvent of 1 mL of THF and 500 μL of DIEA. This solution was then added dropwise to 80 mg (460 μmol; Tokyo Chemical Industry Co., Ltd.) of tert-Butylphosphonic dicloride dissolved in 2 mL of THF, and the mixture was stirred at room temperature for 18 hours. The reaction solution was then loaded onto a silica gel column and purified by column chromatography using an 80:20 to 0:100 gradient of AcOEt-Hexane for 10 mins to obtain 40 mg of the target substance, HCCA-PMB-ethylphosphochloride.

[0152] [Introduction and excision of fluorescent dyes on a solid phase] The target substance was obtained by following the same method as in Production Example 2 [Introduction and cleavage of fluorescent dye on a solid phase], except that HCCA-PMB-ethyl-phosphochlorodate was used instead of 4MU-ethyl-phosphochlorodate.

[0153] Figure 3 shows the structure of the probe obtained as described above, the LC chart, and the peak values ​​obtained by mass spectrometry. The chromatogram in the LC chart shown in Figure 3 shows the absorbance at 320 nm when solution A is a 0.1% TFA aqueous solution, solution B is an 80% AcCN aqueous solution containing 0.1% TFA, and analysis was performed with a gradient of A:B = 99:1 to 5:95 and 3.5 min. For all compounds, high-purity target products were obtained simply by performing reverse-phase chromatography after the final step following solid-phase synthesis. Furthermore, for all compounds, mass spectrometry yielded m / z peaks corresponding to the removal of hydrogen atoms from the target substance.

[0154] <Test Example 1: Investigation of Enzyme Activity Detection using a Fluorescent Probe Library 1> Carboxypeptidase (1 μg / mL) of each recombinant and fluorescent probe (10 μM) containing each peptide sequence were added to DPBS (pH 7.4, Thermo) containing 0.1% CHAPS. The mixture was mixed in a 384-well plate, and the increase in fluorescence intensity at 30 min was observed using a fluorescence plate reader (EnVision, PerkinElmer). A filter set for DAPI measurement was used as the filter set for excitation and fluorescence detection. The enzyme names, origins, and sources of the recombinant dyes used are shown in the table below. As for the fluorescent probes, 4MU synthesized in Production Example 2 was used as the fluorescent nucleus, and the peptide sequences are shown in the second column of Figure 4. The peptide sequences in Figure 4 are listed from the N-terminus (i.e., the side closest to 4MU).

[0155] Figure 4 is a heatmap showing the fluorescence intensity of fluorescent probes having each peptide sequence shown in the second column when incubated with each recombinant enzyme shown in the second row. The heatmap reflects the strength of the fluorescence intensity, with a whiter color indicating a faster increase in fluorescence intensity (i.e., suggesting high reactivity). According to Figure 4, for example, fluorescent probes having AR or AK as peptides were selected as suitable for detecting the activity of CPB1 and CPM. Thus, it has been demonstrated that a fluorescent probe library containing fluorescent probes produced by the manufacturing method of this disclosure can be used to screen for fluorescent probes useful for detecting the activity of various peptidases.

[0156] <Test Example 2: Application of Manufactured Fluorescent Probe to a Single-Molecule Enzyme Activity Detection System 1> Plasma sample (from a healthy person or from a pancreatic cancer patient) is mixed with enzyme reaction buffer (0.1 M HEPES-Na buffer (pH 7.4) with 1 mM CaCl2 ２ , 1 mM MgCl ２ The solution was diluted to 1 / 500–1 / 5000 with (a solution containing 100 μM DTT and 250 μM Triton X-100), and mixed with 60 μM of fluorescent probe dissolved in enzyme reaction buffer in a 1:1 volume ratio. 30 μL of the mixture was loaded into a Simoa Disk (Quanterix), and 60 μL of FC-70 was added and the disc was sealed. After incubation at room temperature for 18 hours, fluorescence imaging images were acquired using an epifluorescence microscope (Nikon Ti2) or a confocal fluorescence microscope (Nikon AX-Ti2).

[0157] Figure 5 shows fluorescence images and histograms of single-molecule enzyme activity detection in plasma samples from healthy individuals and pancreatic cancer patients, using one type of fluorescent probe, manufactured in Manufacturing Example 1, in which the peptide is AR. The results in Figure 5 were obtained using an epifluorescence microscope at a plasma sample dilution ratio of 1 / 500. As shown in Figure 5, more bright spots were observed in the plasma sample from pancreatic cancer patients than in the plasma sample from healthy individuals. Therefore, it has been demonstrated that the fluorescent probe manufactured by the manufacturing method of this disclosure can indeed be applied to peptidase activity detection using a single-molecule enzyme activity detection system.

[0158] Figure 6 shows the fluorescence images and fluorescence intensity plots for detecting single-molecule enzyme activity in plasma samples derived from pancreatic cancer patients, using two types of fluorescent probes: (I) one prepared in Production Example 1, with the peptide being AR, and (II) one prepared in Production Example 3, with the peptide being GK. The results in Figure 6 were obtained using a confocal fluorescence microscope at a plasma sample dilution ratio of 1 / 5000. In the fluorescence intensity plot, the vertical axis shows the fluorescence intensity at the wavelength corresponding to the fluorescent probe (I), and the horizontal axis shows the fluorescence intensity at the wavelength corresponding to the fluorescent probe (II). According to Figure 6, multiple clusters were observed in the fluorescence intensity plot. For example, a cluster of a minor subtype that recognizes AR but not GK was observed in the area enclosed in the upper left frame. Such clusters of minor subtypes would be obscured in evaluations of the bulk system. Therefore, it has been demonstrated that single-molecule enzyme activity detection using multiple fluorescent probes according to this disclosure allows for the evaluation of reactivity to multiple substrates at the single-molecule level of the enzyme, thereby enabling the detection of minor populations in enzyme activity.

[0159] <Production Example 4: Synthesis of Fluorescent Probes by Introducing Fluorescent Dyes on a Solid Phase 3> Following the same method as in Production Example 2, probes containing various peptides as partial structures were synthesized using 4MU-ethyl-phosphochlorodate as a starting material. Specifically, probes were synthesized in which the peptide in the structural formula shown as the target product in Production Example 2 is the dipeptide shown below. Probes with some protecting groups remaining, such as T(OtBu)A, GR(Pbf), and ER(Pbf), were synthesized by shortening the deprotection time in solid-phase synthesis to about 10 minutes. FH, HP, VL, FL, FM, AY, GQ, GY, GK, AH, AL, AM, AE, MK, RE, KK, FE, AG, AV, AS, AI, AP, T(OtBu)A, GR(Pbf), ER(Pbf)

[0160] Figure 7 shows the LC chart of the probe obtained in Production Example 4, where the peptide is AY. Figure 8 shows the LC chart of the probe obtained in Production Example 4, where the peptide is FH, HP, VL, FL, FM, AY, GQ, GY, GK, AH, AL, AM, AE, MK, RE, or KK. Figure 9 shows the LC chart of the probe obtained in Production Example 4, where the peptide is FE, AG, AV, AS, AI, AP, T(OtBu)A, GR(Pbf), or ER(Pbf). In this way, probes with an even wider range of structures could be manufactured using the same method as in Production Example 2.

[0161] <Production Example 5: Synthesis of a Fluorescent Probe with a Benzyl Structure 2> Using the same method as in Production Example 1, probes containing different peptides as substructures were synthesized. Specifically, probes were synthesized in which the peptide was AY in the structural formula shown as the target product in Production Example 1. Figure 10 shows the LC chart of the probe obtained in Production Example 5 in which the peptide was AY. In this way, probes with an even wider range of structures could be produced using the same method as in Production Example 1.

[0162] <Production Example 6: Synthesis of Fluorescent Probes by Introducing Fluorescent Dyes on a Solid Phase 4> Using the same method as in Production Example 3, probes containing different peptides as partial structures were synthesized. Specifically, probes in which the peptide was GF in the structural formula shown as the target product in Production Example 3 were synthesized. Figure 11 shows the LC chart of the probe obtained in Production Example 6 in which the peptide was GF. In this way, probes with an even wider range of structures could be produced using the same method as in Production Example 3.

[0163] <Manufacturing Example 7: Synthesis of a fluorescent probe with a resolphin as its fluorescent nucleus 1> Under argon purging, 220 mg of tert-Butylphosphonic Dichloride (Tokyo Chemical Industries, B5729, 1.26 mmol) was mixed with 4 mL of dry THF (Solution A). 90 mg of resolphin (Tokyo Chemical Industries, R0012, 0.42 mmol) was dissolved in a mixture of 2 mL of dry NMP and 400 μL (4.91 mmol) of DIEA, and this mixture was added dropwise to Solution A. After reacting at room temperature for 16 hours, the reaction mixture was loaded directly onto a silica gel column and purified by column chromatography (Hexane-AcOEt 80:20-0:100). The resulting compound (Phosphonochlorodate) was used directly in the next reaction.

[0164] 60 mg of the peptide-supported resin obtained in step B of Production Example 1 was washed three times with DMF and three times with DCM. 500 μL of NMP and 250 μL of DIEA were added to the washed resin. 50 μmol of Phosphonochlorodate obtained in the previous step was dissolved in 500 μL of NMP and gradually added dropwise to the resin-containing solution. After reacting at room temperature for 18 hours, the solution was washed three times with DMF and three times with DCM.

[0165] After drying the resin obtained in the previous step, add TFA (90%) and H to the resin. ２ Add 1 mL of a 10% O mixture and shake for 1 hour. Collect the solution and H ２After dilution with O, the solution was purified by preparative MPLC. The mobile phase for preparative MPLC was A = 0.01 M triethylamine acetate, B = AcCN, and a gradient from A:B = 99:1 to 0:100 was applied for 15 minutes. The fraction containing the target substance was freeze-dried as is.

[0166] Figure 12 shows the structure, LC chart, and mass spectrometry results of a representative probe obtained by the above method, in which the peptide is AK. The chromatogram in the LC chart shown in Figure 12 shows the absorbance at 450 nm when solution A is a 0.1% TFA aqueous solution, solution B is an 80% AcCN aqueous solution containing 0.1% TFA, and analysis was performed with a gradient of A:B = 99:1 to 5:95 and 3.5 min. Thus, probes with various fluorescent nuclei could be synthesized using the method of this disclosure.

[0167] <Manufacturing Example 8: Synthesis of a fluorescent probe with a resolphin as its fluorescent nucleus 2> [Step G1: Synthesis of a fluorescent probe] The phosphoramidite building block obtained in step F of Production Example 1 was dissolved in dichloromethane. After adding n-hexane to the solution, it was dried at room temperature. 3 mg of resolphin (Tokyo Chemical Industries, R0012) was dissolved in a mixture of 15 μL NMP and 10 μL DIEA. The resulting solution was sonicated and then added to the phosphoramidite building block dried above. The resulting mixture was then shaken at room temperature. After the phosphoramidite building block of the raw material had disappeared, it was purified by preparative MPLC (reverse phase, 0.1% TFA system). The fraction containing the target substance was recovered, and the target substance was obtained by distilling off AcCN under reduced pressure and then freeze-drying. The freeze-dried target substance was H ２ After dissolving in oxygen, the substance was recovered into the DCM layer by liquid-liquid separation using DCM as the organic layer. The solvent in the DCM layer was removed by reduced pressure distillation, and the target substance contained in the residue was used in subsequent test examples.

[0168] As a representative probe obtained by the above method, the structure and LC chart of a probe in which the peptide is GL are shown in Figure 13. The chromatogram in the LC chart shown in Figure 13 shows the absorbance at 500 nm when solution A is a 0.1% TFA aqueous solution, solution B is an 80% AcCN aqueous solution containing 0.1% TFA, and analysis was performed with a gradient of A:B = 99:1 to 5:95 and 3.5 min. Thus, probes with various fluorescent nuclei could be synthesized by the method of this disclosure.

[0169] <Manufacturing Example 9: Synthesis of a fluorescent probe with a resolphin as its fluorescent nucleus 3> [Step F1: Introduction and excision of fluorescent dye on a solid phase] 10 mL of resolphin was dissolved in a mixture of 700 μL NMP and 300 μL DIEA. The solution was sonicated, and insoluble components were removed by centrifugation. The resulting solution was added to the compound-holding beads obtained in step E of Production Example 1. The resulting mixture was reacted at room temperature for 3 hours with vigorous stirring. After the reaction, the beads were collected and then washed several times with DMF and DCM, respectively. The beads were stirred in DCM-HFIP (4:1) for 1 hour to cleave the target product from the solid phase. After washing the beads with DCM and acetone, the solvent was removed by vacuum distillation. The resulting residue was purified by preparative MPLC (reverse phase, 0.1% TFA system). The LC chart of the target product thus obtained had a main peak at the same elution time as the original, as shown in Figure 13.

[0170] <Test Example 3: Investigation of Enzyme Activity Detection using a Fluorescent Probe Library 2> Fluorescent probes (10 μM) containing the carboxypeptidase (7.5 μg / mL) of each recombinant and the respective peptide sequence were added to DPBS (pH 7.4, Thermo) containing 0.1% CHAPS. After mixing in a 384-well plate, the mixture was allowed to stand at 25°C, and the increase in fluorescence intensity at 120 min was observed using a fluorescence plate reader (EnVision, PerkinElmer). A filter set for DAPI measurement was used as the filter set for excitation and fluorescence detection. The enzyme names, origins, and sources of the recombinant dyes used are shown in Table 1 of Test Example 1. The fluorescent probes used were those synthesized in Production Example 4 or those synthesized using the same protocol as Production Example 4 with 4MU as the fluorescent nucleus, and the peptide sequences are shown in the “Sequence” row of Figure 14. The peptide sequences in Figure 14 are listed from the N-terminus (i.e., the side closest to 4MU).

[0171] Figure 14 is a heatmap showing the fluorescence intensity of fluorescent probes having each peptide sequence shown in the "Sequence" row when incubated with each recombinant enzyme shown in the first column. The heatmap reflects the strength of the fluorescence intensity, with a whiter color indicating a faster increase in fluorescence intensity (i.e., suggesting high reactivity). According to Figure 14, for example, fluorescent probes having AY or GF as peptides were selected as suitable probes for detecting CPA1 activity. Also, according to Figure 14, for example, fluorescent probes having GK or AR as peptides were selected as suitable probes for detecting CPB1 activity.

[0172] Thus, it has been further demonstrated that a fluorescent probe library containing fluorescent probes produced by the manufacturing method of this disclosure can be used to screen for fluorescent probes useful for the activity of various peptidases.

[0173] <Test Example 4: Application of Manufactured Fluorescent Probes to a Single-Molecule Enzyme Activity Detection System 2> As fluorescent probes, a combination of a probe synthesized in Manufacturing Example 1 with the peptide AR and a probe synthesized in Manufacturing Example 7 with the peptide GK, or a combination of a probe synthesized in Manufacturing Example 5 with the peptide AY and a probe synthesized in Manufacturing Example 8 with the peptide GL was used. 10 μM of each of these fluorescent probes was used in CaCl ２ (1mM), MgCl ２ (1mM), ZnCl ２Recombinant CPB1 added to 0.1 ng / mL, or plasma samples added to 20,000-fold or 3,000-fold dilutions, were incubated at 25°C for 18 hours in HEPES buffer (100 mM, pH 8.5) containing (10 μM) and DTT (100 μM). 30 μL of the incubated solution was introduced into a microchamber array, Simoa® disk (Quanterix). Subsequently, 60 μL of Fluorinert® Simoa sealing oil (Quanterix, 102767) was introduced into the microchamber array as a hydrophobic solvent to remove aqueous solutions not introduced into the wells, forming W / O droplets in the wells. This introduction method probabilistically prepared wells containing one molecule of enzyme. After incubating the microchamber array at room temperature for 20 hours, fluorescence images were acquired using an epi-emission microscope Ti2 (Nikon) equipped with a 20x objective lens (Plan Apo 20×), an sCMOS camera (ORCA-Fusion C14440, Hamamatsu Photonics), a white light unit (X-Cite Xylis, OptoScience), and a motorized stage. The assay was performed using a solution containing IR-dye 800 (10 μM) as an internal standard, and the focus was adjusted using its fluorescence. All operations were controlled using NIS-Element software (Nikon). The filter sets used were the FITC filter set (mirror: 510 nm, excitation: 460-500 nm, detection: 510-560 nm), the mCherry filter set (mirror: 600 nm, excitation: 550-590 nm, detection: 608-683 nm), and the Cy7 filter set (mirror: 743 nm, excitation = 743 nm, emission = 767 nm).

[0174] Figure 15 shows a scatter plot of fluorescence intensity per well for a probe (Green) where the peptide synthesized in Production Example 1 is AR and a probe (Red) where the peptide synthesized in Production Example 7 is GK, when a plasma sample (20,000-fold dilution) derived from a healthy person or recombinant CPB1 is added, and an example of a fluorescence image when a plasma sample is added. Figure 16 shows a scatter plot of fluorescence intensity per well for a probe (Green) where the peptide synthesized in Production Example 5 is AY and a probe (Red) where the peptide synthesized in Production Example 8 is GL, when a plasma sample (3,000-fold dilution) derived from a healthy person is added, and an example of a fluorescence image. Thus, according to the manufacturing method and screening method of this disclosure, it was possible to find pairs of fluorescent probes for single-molecule measurement of carboxypeptidase activity with various substrate specificities.

Claims

1. A method for producing a peptide-containing compound, comprising: a peptide having its C-terminal end supported on a carrier, a primary or secondary amino group at its N-terminus, and which may have a protected side chain, and the following formula (I): [In the formula, X represents a monovalent organic group, where X is bonded to an oxygen atom directly bonded to X in the formula and to an aromatic ring, R １ is C １－６ Alkyl or C １－６ The compound represented by [which exhibits an alkoxy and LG represents a monovalent leaving group] is brought into contact with the peptide, and one hydrogen atom on the amino group of the peptide is released using the following formula (II): [In the formula, X and R １ The formula is the same as formula (I), and the dashed line indicates the bond with the nitrogen atom in the amino group. A manufacturing method comprising the step of substituting with a group represented by ].

2. The manufacturing method according to claim 1, wherein X is a monovalent functional group (FG) or a precursor thereof, wherein the molecule represented by FG-OH is in a state where it does not exhibit function when the hydrogen atom on the oxygen atom directly bonded to FG is substituted, and is in a state where it exhibits function when the hydrogen atom is not substituted.

3. X is Z-Y-BN: [where Y is an oxygen atom or N(-R ２ ), R ２ is a hydrogen atom, C １－６ alkyl or C ２－６ alkenyl.], BN is, when Y is an oxygen atom, a group which may have a substituent on the benzene ring, represented by the following formula: [where * represents a bond to Y, and the wavy line represents a bond to the oxygen atom directly bonded to X in formula (I).], and when Y is N(-R ２ ), a group which may have a substituent on the benzene ring, represented by the following formula: [where * represents a bond to Y, and the wavy line represents a bond to the oxygen atom directly bonded to X in formula (I).], Z represents a monovalent functional group (FG) or its precursor, or hydroxy or its protecting group.], provided that the molecule represented by FG-YH is in a state where it does not exhibit a function when the hydrogen atom on Y is substituted, and is in a state of a functional molecule that exhibits a function when the hydrogen atom is not substituted, the production method according to claim 1.

4. The manufacturing method according to claim 3, wherein, if Z represents a hydroxyl group or a protected compound thereof, the manufacturing method further comprises a step of converting Z to the monovalent functional group (FG) or its precursor after the substitution step.

5. The functional molecule is -CO ２ H, -PO ３ H ２ and -SO ３ A method for producing a fluorescent molecule according to any one of claims 2 to 4, wherein the fluorescent molecule has at least one water-soluble substituent selected from the group consisting of H.

6. The above FG is given by the following formula (III): [In the formula, R ａ１ ~R ａ５ Each of these independently comprises a hydrogen atom; and a C atom which may have substituents. １－６ Alkyl, C ２－６ Alkenyl and C １－６ Alkoxy; halogen atom; nitro; hydroxyl; hydroxyl C which may have substituents on the carbon atom. １－３ Alkyl; -CO ２ H, -PO ３ H ２ and -SO ３ A water-soluble substituent selected from the group consisting of H; and C which may be substituted with the water-soluble substituent and further have substituents. １－６ Alkyl, C ２－６ Alkenyl and C １－６ Selected from the group consisting of alkoxys, where R ａ１ ~R ａ５ At least one of these may have the water-soluble substituent, or a substituent substituted with the water-soluble substituent. １－６ Alkyl, C ２－６ Alkenil or C １－６ It is an alkoxy, and the wavy line indicates a bond with an oxygen atom or Y. [See formula (IV):] [In the formula, SRG is represented by the following structural formula: A group represented by any of the following, R ｂ１ ~R ｂ５ and R ｃ１ ~R ｃ６ Each of these independently comprises a hydrogen atom; and a C atom which may have substituents. １－６ Alkyl, C １－６ Alkoxy and C ２－６ Alkenyl; halogen atom; nitro; hydroxyl; hydroxyl C which may have substituents on the carbon atom. １－３ Alkyl; the water-soluble substituent; and C which may have further substituents substituted with the water-soluble substituent. １－６ Alkyl, C ２－６ Alkenyl and C １－６ Selected from the group consisting of alkoxys, where R ｂ１ ~R ｂ５ and R ｃ１ ~R ｃ６ At least one of them is the water-soluble substituent; or C which is substituted with the water-soluble substituent and may have further substituents. １－６ Alkyl, C ２－６ Alkenil or C １－６ It is an alkoxy, R ｄ１ and R ｄ２ Each of these independently may have a hydrogen atom or a substituent. １－６ Alkyl or C ２－６ C may be substituted with an alkenyl or the aforementioned water-soluble substituent and may further have substituents. １－６ Alkyl or C ２－６ Alkenil is here, R ｄ１ and R ｄ２ They become one, R ｄ１ and R ｄ２ It may form a 4- to 7-membered heterocycline containing a nitrogen atom to which is bonded, which may have substituents, and R ｄ１ and / or R ｄ２ These are, respectively, R ｃ５ or R ｃ６ In conjunction with R ｄ１ or R ｄ２ It may form a 5-7 member heterocycline or heteroaryl, which may have substituents, including the nitrogen atom to which it is bonded, R ｅ is an oxygen atom, Si(-R ｆ１ ) (-R ｆ２ ) or C(-R ｆ１ ) (-R ｆ２ ) shows, where R ｆ１ and R ｆ２ Each of these may independently have substituents, C １－６ Alkyl, C ２－６ Alkenil, C １－６ [This indicates an alkoxy or aryl compound, and the wavy line indicates a bond with an oxygen atom or Y.] or formula (V) below: [In the formula, R ｇ１ ~R ｇ６ Each of these independently comprises a hydrogen atom; and a C atom which may have substituents. １－６ Alkyl, C １－６ Alkoxy and C ２－６ Alkenyl; halogen atom; nitro; hydroxyl; hydroxyl C which may have substituents on the carbon atom. １－３ Alkyl; the water-soluble substituent; and C which may have further substituents substituted with the water-soluble substituent. １－６ Alkyl, C ２－６ Alkenyl and C １－６ A method for manufacturing according to any one of claims 2 to 4, wherein the group is selected from the group consisting of alkoxys, and the wavy line indicates a bond with an oxygen atom or Y.

7. The following formula (VI) or the following formula (VII): A fluorescent probe represented by the formula, wherein Y is an oxygen atom or N(−R ２ ) [R ２ represents a hydrogen atom, C １－６ alkyl or C ２－６ alkenyl.], FG is the following formula (III): [In the formula, R ａ１ to R ａ５ are each independently a hydrogen atom; C １－６ alkyl, C ２－６ alkenyl and C １－６ alkoxy which may have a substituent; a halogen atom; nitro; hydroxy; hydroxy C １－３ alkyl which may have a substituent on the carbon atom; the water-soluble substituent; and C １－６ alkyl, C ２－６ [[ID=2s]] alkenyl and C １－６ alkoxy substituted with the water-soluble substituent and optionally further having a substituent, selected from the group consisting of, provided that at least one of R ａ１ to R ａ５ is the water-soluble substituent, or C １－６ alkyl, C ２－６ alkenyl or C １－６ alkoxy substituted with the water-soluble substituent and optionally further having a substituent, and the wavy line represents a bond to an oxygen atom or Y.], the following formula (IV): [In the formula, SRG is a group represented by any of the following structural formulas: and R ｂ１ to R ｂ５ and R ｃ１ to R ｃ６ are each independently a hydrogen atom; C １－６ alkyl, C １－６ alkoxy and C ２－６ alkenyl which may have a substituent; a halogen atom; nitro; hydroxy; hydroxy C １－３ alkyl which may have a substituent on the carbon atom; the water-soluble substituent; and C １－６ alkyl, C ２－６ alkenyl and C １－６ Selected from the group consisting of alkoxys, where R ｂ１ ~R ｂ５ and R ｃ１ ~R ｃ６ At least one of the substituents is the water-soluble substituent; or C which is substituted with the water-soluble substituent and may have further substituents. １－６ Alkyl, C ２－６ Alkenil or C １－６ It is an alkoxy, R ｄ１ and R ｄ２ Each of these independently may have a hydrogen atom or a substituent. １－６ Alkyl or C ２－６ C may be substituted with an alkenyl or the aforementioned water-soluble substituent and may further have substituents. １－６ Alkyl or C ２－６ Alkenil is here, R ｄ１ and R ｄ２ They become one, R ｄ１ and R ｄ２ It may form a 4- to 7-membered heterocycline containing a nitrogen atom to which is bonded, which may have substituents, and R ｄ１ and / or R ｄ２ These are, respectively, R ｃ５ or R ｃ６ In conjunction with R ｄ１ or R ｄ２ It may form a 5-7 member heterocycline or heteroaryl, which may have substituents, including the nitrogen atom to which it is bonded, R ｅ is an oxygen atom, Si(-R ｆ１ ) (-R ｆ２ ) or C(-R ｆ１ ) (-R ｆ２ ) shows, where R ｆ１ and R ｆ２ Each of these may independently have substituents, C １－６ Alkyl, C ２－６ Alkenil, C １－６ [This indicates an alkoxy or aryl compound, and the wavy line indicates a bond with an oxygen atom or Y.] or formula (V) below: [In the formula, R ｇ１ ~R ｇ６ Each of these independently comprises a hydrogen atom; and a C atom which may have substituents. １－６ Alkyl, C １－６ Alkoxy and C ２－６ Alkenyl; halogen atom; nitro; hydroxyl; hydroxyl C which may have substituents on the carbon atom. １－３ Alkyl; the water-soluble substituent; and C which may have further substituents substituted with the water-soluble substituent. １－６ Alkyl, C ２－６ Alkenyl and C １－６ Selected from the group consisting of alkoxys, the wavy line indicates a bond with an oxygen atom or Y. The group is represented by ], however, molecules represented by FG-OH or FG-YH are fluorescent molecules in which the fluorescence intensity is greater when the oxygen atom directly bonded to FG or the hydrogen atom on Y is not substituted than when the hydrogen atom is substituted, and BN may have substituents on the benzene ring when Y is an oxygen atom, as shown in the following formula: [In the formula, * indicates a bond with Y, and the wavy line indicates a bond with the oxygen atom that directly bonds with BN in formula (VII).] This represents a group represented by, where Y is N(-R ２ If the following formula is true, the benzene ring may have substituents: [In the formula, * indicates a bond with Y, and the dashed line indicates a bond with the oxygen atom that directly bonds with BN in formula (VII).] This represents a group represented by [formula]. Pep is a fluorescent probe in which one hydrogen atom on the primary or secondary amino group at the N-terminus is substituted with the phosphorus atom in formula (VI) or formula (VII).

8. A fluorescent probe library comprising at least five types of fluorescent probes according to claim 7.

9. A screening method for fluorescent probes, comprising: contacting at least five types of fluorescent probes contained in the fluorescent probe library described in claim 8 with a peptidase or a sample containing the same; and selecting fluorescent probes whose fluorescence intensity increases after contact with the peptidase or the sample containing the same compared to when they were not in contact as fluorescent probes targeting the peptidase and / or diseases that express the same, wherein the peptidase is at least one selected from the group consisting of carboxypeptidases and endopeptidases.

10. A method for evaluating enzyme activity, comprising the following steps (A) to (C): Step (A): preparing an aqueous solution containing one molecule of peptidase, a first fluorescent probe, and a second fluorescent probe; Step (B): catalyzing an enzymatic reaction in the aqueous solution prepared in Step (A) with the peptidase; and Step (C): measuring the fluorescence of the aqueous solution obtained in Step (B); wherein the peptidase is at least one selected from the group consisting of carboxypeptidase and endopeptidase, the first fluorescent probe and the second fluorescent probe are each independently the fluorescent probe described in claim 7, and the difference between the maximum absorption wavelength and / or maximum fluorescence wavelength of the corresponding fluorescent molecules represented by FG-OH or FG-YH is 30 nm or more.

Images