Method for enzyme reaction using amyloid compound

Abstract

To provide a method that enables artificial control of regioselectivity including cleavage or modification in diverse enzyme reactions.SOLUTION: A method for controlling regioselectivity in an enzyme reaction, comprises a step of adding an enzyme to a substrate compound containing, within its molecule, an amyloid-binding motif having affinity for an amyloid compound, in the presence of the amyloid compound.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for controlling regioselectivity by performing an enzymatic reaction in the presence of an amyloid compound. [Background technology]

[0002] Amyloids are fibrous structures formed by stacking peptides and proteins, and have a cross-β-sheet structure in which β-sheets are aligned perpendicular to the fiber axis (e.g., Non-Patent Document 1). Accumulation of amyloids in vivo is known to cause various functional disorders (such diseases are collectively referred to as "amyloid diseases"). More than 35 proteins have been identified as causative agents of amyloid diseases. Examples of such amyloids include tau protein in Alzheimer's disease, α-synuclein in Parkinson's disease, amylin in diabetes, transthyretin in systemic amyloidosis, and huntingtin in Huntington's disease.

[0003] Enzymes are proteins that catalyze various chemical reactions in living organisms, and their precise, efficient, and sustainable substrate conversion has led to attempts at their application in many fields of material production. However, artificially controlling the regioselective conversion of substrates (promoting a chemical reaction only at a specific site when there are multiple sites with similar reactivity in the same substrate) is difficult and remains a challenge. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] F. Chiti and CM Dobson, Annu. Rev. Biochem., 2017, 86, 27-68. Summary of the Invention [Problem to be solved by the invention]

[0005] In view of the above problems of the prior art, an object of the present invention is to provide a method for artificially controlling the regioselectivity of cleavage, modification, etc. in various enzymatic reactions. [Means for solving the problem]

[0006] As a result of intensive research aimed at solving the above-mentioned problems, the present inventors have found that by introducing a site capable of binding to amyloid, which is formed by the aggregation of peptides having a specific sequence, into a substrate compound and then performing an enzymatic reaction in the presence of amyloid, the action of the enzyme is inhibited by the amyloid around the site where the amyloid binds, while the normal enzymatic reaction proceeds in the region of the same substrate where the amyloid does not bind, thereby enabling regioselective structural transformation of the substrate, and have completed the present invention.The present invention can be said to artificially control the regioselectivity of an enzymatic reaction by using such an amyloid-cooperative enzyme catalytic system.

[0007] That is, in one aspect, the present invention relates to a method for controlling regioselectivity in an enzymatic reaction using an amyloid, and more specifically, <1> A method for controlling regioselectivity in an enzymatic reaction, comprising the step of adding an enzyme to a substrate compound, in the presence of an amyloid compound, the substrate compound containing an amyloid-binding motif in the molecule that has affinity for the amyloid compound; <2> The amyloid-binding motif is one or more selected from the group consisting of an azobenzene structure, a benzothiazole structure, a xanthene structure, a phenothiazine structure, a boron dipyrromethene structure, a curcumin structure, and a porphyrin structure. <1> The method described in <3> The amyloid compound has a β-sheet structure and has the following sequence: Phe-X-Ala-Y wherein X is Phe or Ala; and Y is Ala-Leu-Leu, Ala-Ala-Leu, Ala-Leu-Ala, Ser-Leu-Leu, or Ala-Leu-NH. The above-mentioned peptide aggregates comprising <1> The method described in <4> The peptide consists of 7 to 40 amino acids containing the sequence. <3> The method described in <5> The position selectivity is the position of a functional group in the substrate compound that is cleaved or modified by the enzyme. <1> The method described in <6> The above, wherein cleavage or modification by the enzyme is inhibited in the region where the amyloid compound binds to the amyloid-binding motif. <1> The method described in <7> The product obtained by the enzyme reaction is a compound different from the product obtained by the enzyme reaction in the absence of the amyloid compound, or the composition ratio of the products is different. <1> and <8> The substrate compound is an oligopeptide, a polypeptide, an oligonucleotide, a polynucleotide, a sugar chain substance, a lipid molecule, or a low molecular weight compound. <1> The method This provides:

[0008] In another aspect, the present invention also relates to the above amyloid compounds and uses thereof, more particularly to <9> an amyloid compound which is an aggregate of peptides having a β-sheet structure and containing the following sequence: Phe-X-Ala-Y (wherein X is Phe or Ala; and Y is Ala-Leu-Leu, Ala-Ala-Leu, Ala-Leu-Ala, Ser-Leu-Leu, or Ala-Leu-NH2); <10> The above method for controlling the position selectivity in an enzymatic reaction <9> Use of the amyloid compounds described in This provides: [Effects of the Invention]

[0009] According to the present invention, by using amyloids formed by the aggregation of peptides with specific sequences, it is possible to artificially control the regioselectivity of cleavage and modification in enzymatic reactions, which has been difficult to achieve until now, and to obtain products that could not be obtained by enzymatic reactions in the absence of amyloid. This amyloid-cooperative enzyme catalytic system is expected to be applied to new pharmaceuticals, biomaterials, and substance production methods. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 is a schematic diagram showing a typical enzyme reaction mechanism. [Figure 2] FIG. 2 is a schematic diagram showing the reaction mechanism of the present invention. [Figure 3] FIG. 3 is a graph showing the results of the reaction between p-nitrophenyl acetate (pNPA) and an enzyme (PLE) in the presence and absence of an amyloid compound (BL7). [Figure 4] FIG. 4 is a graph showing the results of the reaction between DPP-acetate (compound 1) and an enzyme (PLE) in the presence and absence of an amyloid compound (BL7). [Figure 5] FIG. 5 is a graph showing the results of the reaction between an oligopeptide substrate (compound 2) modified with the amyloid-binding motif DPP and an enzyme (trypsin) in the presence and absence of an amyloid compound (BL7). [Figure 6] FIG. 6 shows the results of the reaction between substrate compounds 2, 2a to 2g and an enzyme (trypsin) in the presence and absence of an amyloid compound (BL7). [Figure 7] FIG. 7 shows the relative activity (Arel) obtained in the reaction of substrate compound 2a with an enzyme (trypsin) using various amyloid compounds. [Figure 8] FIG. 8 is a graph showing the results of the reaction between substrate compound 3 and an enzyme (trypsin) in the presence and absence of an amyloid compound (BL7). [Figure 9]FIG. 9 is a graph showing the results of the reaction between substrate compound 3 and an enzyme (PAD) in the presence and absence of an amyloid compound (BL7). [Figure 10] FIG. 10 shows the reaction results (HPLC peaks) of substrate compound 4 with an enzyme (Glu-C) in the presence and absence of an amyloid compound (BL7). [Figure 11] FIG. 11 shows the results (HPLC peaks) of the enzymatic reaction (trypsin) of compound 8a, which formed an azobenzene amyloid-binding motif in situ, in the presence and absence of an amyloid compound (BL7). DETAILED DESCRIPTION OF THE INVENTION

[0011] The following describes embodiments of the present invention. The scope of the present invention is not limited to these descriptions, and other than the following examples, the present invention can be implemented with appropriate modifications within the scope that does not depart from the spirit of the present invention.

[0012] 1.Definition In this specification, the term "halogen atom" refers to a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom.

[0013] In this specification, the term "alkyl or alkyl group" may be any of a linear, branched, or cyclic aliphatic hydrocarbon groups, or a combination thereof. The number of carbon atoms in the alkyl group is not particularly limited, but for example, alkyl groups having 1 to 20 carbon atoms (C 1~20 ), 1 to 15 carbon atoms (C 1~15 ), 1 to 10 carbon atoms (C 1~10 In this specification, the alkyl group may have one or more optional substituents. For example, C 1~8Alkyl includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neo-pentyl, n-hexyl, isohexyl, n-heptyl, n-octyl, etc. Examples of the substituent include, but are not limited to, an alkoxy group, a halogen atom (which may be a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom), an amino group, a mono- or di-substituted amino group, a substituted silyl group, or an acyl group. When an alkyl group has two or more substituents, they may be the same or different. The same applies to the alkyl moiety of other substituents containing an alkyl moiety (e.g., an alkoxy group, an arylalkyl group, etc.).

[0014] As used herein, the term "aromatic ring" refers to a monocyclic or fused polycyclic conjugated unsaturated hydrocarbon ring structure, which may contain one or more heteroatoms (e.g., oxygen atom, nitrogen atom, sulfur atom, etc.) as ring-constituting atoms.

[0015] As used herein, the term "aryl or aryl group" refers to a monocyclic or fused polycyclic aromatic hydrocarbon group, or to an aromatic heterocycle containing one or more heteroatoms (e.g., oxygen, nitrogen, or sulfur) as ring constituent atoms. In this case, the group is referred to as a "heteroaryl group" or a "heteroaromatic group." Whether the aryl is a monocyclic or fused ring, it may be bonded at any available position. Non-limiting examples of monocyclic aryl groups include phenyl (Phe), thienyl (2- or 3-thienyl), pyridyl, furyl, thiazolyl, oxazolyl, pyrazolyl, 2-pyrazinyl, pyrimidinyl, pyrrolyl, imidazolyl, pyridazinyl, 3-isothiazolyl, 3-isoxazolyl, 1,2,4-oxadiazol-5-yl, or 1,2,4-oxadiazol-3-yl. Non-limiting examples of fused polycyclic aryls include 1-naphthyl, 2-naphthyl, 1-indenyl, 2-indenyl, 2,3-dihydroinden-1-yl, 2,3-dihydroinden-2-yl, 2-anthryl, indazolyl, quinolyl, isoquinolyl, 1,2-dihydroisoquinolyl, 1,2,3,4-tetrahydroisoquinolyl, indolyl, isoindolyl, phthalazinyl, quinoxalinyl, benzofuranyl, 2,3-dihydrobenzofuran- Examples of the aryl group include 1-yl, 2,3-dihydrobenzofuran-2-yl, naphthyridinyl, dihydronaphthyridinyl, tetrahydronaphthyridinyl, imidazopyridinyl, pteridinyl, purinyl, quinolidinyl, indolizinyl, tetrahydroquinolidinyl, and tetrahydroindolizinyl, 2,3-dihydrobenzothiophen-1-yl, 2,3-dihydrobenzothiophen-2-yl, benzothiazolyl, benzimidazolyl, fluorenyl, and thioxanthenyl groups. In the present specification, the aryl group may have one or more optional substituents on its ring. Examples of the substituents include, but are not limited to, alkoxy groups, halogen atoms, amino groups, mono- or di-substituted amino groups, substituted silyl groups, and acyl. When the aryl group has two or more substituents, they may be the same or different.The same applies to the aryl moiety of other substituents containing an aryl moiety (for example, an aryloxy group or an arylalkyl group). Examples include, but are not limited to, the following: When an aryl group has two or more substituents, they may be the same or different.

[0016] As used herein, "alkylamino" and "arylamino" refer to an amino group in which the hydrogen atom of the -NH group is substituted with one or two of the above alkyl or aryl groups. Examples include methylamino, dimethylamino, ethylamino, diethylamino, ethylmethylamino, and benzylamino.

[0017] In this specification, when a functional group is defined as "optionally substituted," the type, substitution position, and number of substituents are not particularly limited, and when two or more substituents are present, they may be the same or different. Examples of substituents include, but are not limited to, alkyl groups, alkoxy groups, hydroxyl groups, carboxyl groups, halogen atoms, sulfo groups, amino groups, alkoxycarbonyl groups, and oxo groups. These substituents may further contain substituents. Examples of such substituents include, but are not limited to, halogenated alkyl groups.

[0018] 2. Method of the Present Invention As described above, the method of the present invention for controlling regioselectivity in an enzymatic reaction comprises the step of adding an enzyme to a substrate compound containing an amyloid-binding motif in the molecule that has affinity for the amyloid compound in the presence of an amyloid compound, wherein the amyloid compound is formed by the aggregation of peptides having a specific sequence and has a β-sheet structure.

[0019] In general, naturally occurring enzymes catalyze chemical reactions with substrate selectivity by specifically forming an enzyme-substrate complex according to the lock-and-key model (Figure 1). Enzyme catalytic reactions proceed based on the proximity effect, which occurs when the substrate binds to the enzyme active site. Furthermore, recent advances in enzyme engineering have expanded the range of substrate structures and chemical reaction modes that can be covered by enzyme reactions. However, these existing technologies are limited to methods that focus on enzymes.

[0020] On the other hand, the present invention utilizes the fact that, as shown in the schematic diagram of Figure 2, when an amyloid compound binds to a specific region (amyloid-binding motif) of a substrate compound, enzyme action (cleavage of chemical bonds in the substrate and modification of functional groups) is inhibited around the amyloid-bound site. Here, "BG" in Figure 2 is the amyloid-binding motif. On the other hand, in the same substrate, in the region where the amyloid compound is not bound, enzyme action is not inhibited and normal enzymatic reactions proceed. Therefore, by introducing an amyloid-binding motif (BG) into a desired position in the substrate, enzyme action can be selectively inhibited, thereby enabling site-selective structural transformation of the substrate.

[0021] Without being bound by theory, it is thought that the inhibition of enzymatic reactions by amyloid compounds occurs because the amyloid-binding motif (BG) in the substrate binds to the cross-β sheet of the amyloid compound, physically blocking the relatively large amyloid compound from accessing the surrounding area (e.g., specific functional groups that are subject to cleavage by the enzyme).

[0022] In the present invention, the "position" in "regioselectivity" refers to the position of the functional group in the substrate compound that is cleaved or modified by the enzyme. Therefore, "controlling regioselectivity" means that enzymatic cleavage or modification is inhibited in the region where the amyloid compound binds to the amyloid-binding motif in the substrate, resulting in a reaction different from a normal enzymatic reaction. As a result, the product obtained by the enzymatic reaction in the present invention can be a compound different from the product obtained by the original enzymatic reaction (enzymatic reaction in the absence of the amyloid compound). Alternatively, when multiple types of products exist, the present invention can be said to obtain the products in a composition ratio different from that obtained by the original enzymatic reaction.

[0023] In a preferred embodiment, the amyloid compound used in the method of the present invention is an aggregate of a peptide having a β-sheet structure and comprising the following amino acid sequence: Phe-X-Ala-Y

[0024] wherein X is Phe or Ala; and Y is Ala-Leu-Leu, Ala-Ala-Leu, Ala-Leu-Ala, Ser-Leu-Leu, or Ala-Leu-NH.

[0025] The above peptides can preferably consist of 7 to 40 amino acids containing the above sequence. For example, any 1 to 20 amino acids can be present on the left side (towards the N-terminus) of "Phe" in the above formula. Similarly, any 1 to 20 amino acids can be present on the right side (towards the C-terminus) of "Y" in the above formula. Specific examples of such peptides include the peptide groups listed in the Examples below.

[0026] The N-terminus of the peptide may be protected with a protecting group such as a benzoyl group (Bz), and the C-terminus of the peptide may be protected with a protecting group such as an amide group (-NH).

[0027] The β-sheet structure in the amyloid compound is preferably a cross-β-sheet structure in which the β-sheets are aligned perpendicular to the fiber axis.

[0028] The amyloid compound may have any aggregating property, but preferably has a relatively high aggregating property. The aggregating property can be evaluated, for example, by measuring the change in fluorescence intensity in an assay using thioflavin T dye, and the logarithm of the fluorescence intensity can be used as an index of amyloid aggregating property as the API value. The amyloid catalyst of the present invention typically has an aggregating property with an API value of 1 or more, more preferably 3 or more, and particularly preferably 3 or more.

[0029] The enzyme used in the method of the present invention is not particularly limited, and any enzyme known in the art can be used. As used herein, the term "enzyme" is broadly understood as a general term for polymeric compounds, primarily proteins, that catalyze any reaction and convert a substrate into a desired product, and mediate or promote chemical changes without changing or decomposing themselves. In some cases, purified enzymes can also be used.

[0030] Specific examples of such enzymes include hydrolases such as lipase, esterase, acylase, protease, amylase, saccharase, pepsin, trypsin, chymotrypsin, peptidase, carboxypeptidase, aminopeptidase, and phospholipase, and oxidoreductases such as dehydrogenase, reductase, oxidase, oxygenase, peroxidase, transhydrogenase, and catalase. Nucleases (e.g., DNase and RNase) and ATP-degrading enzymes such as adenosine phosphate deaminase, apyrase, alkaline phosphatase, acid phosphatase, hexokinase, adenosine triphosphatase, and phosphodiesterase can also be used. Typically, trypsin and endoproteinase Glu-C, as demonstrated in the Examples below, can be used.

[0031] Next, the substrate compound used in the present invention is not particularly limited, and any compound or substance that can serve as a substrate depending on the type of enzyme described above can be used. Such substrate compounds can include oligopeptides, polypeptides, oligonucleotides, polynucleotides, sugar chain substances, lipid molecules, or small molecular weight compounds. Examples of substrate compounds include, but are not limited to, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (including, for example, DNA such as cDNA and genomic DNA, and RNA such as mRNA), polysaccharides, oligosaccharides, lipids, small organic molecules (e.g., hormones, ligands, signaling substances, small organic molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals, etc.), and composite molecules thereof.

[0032] As described above, the substrate compound used in the present invention has an amyloid-binding motif having affinity for amyloid compounds introduced at any position, particularly at a position where it is desired to inhibit the action of an enzyme.

[0033] The amyloid-binding motif may be any motif known in the art, including, but not limited to, one or more selected from the group consisting of an azobenzene structure, a benzothiazole structure, a xanthene structure, a phenothiazine structure, a boron dipyrromethene structure, a curcumin structure, and a porphyrin structure. Preferably, the amyloid-binding motif is an azobenzene structure. Therefore, the substrate compound may be any derivative containing such an amyloid-binding motif within the molecule. The amyloid-binding motif, such as an azobenzene structure, in the substrate compound can also be introduced in situ in the reaction vessel to which the enzyme is added. For example, in situ azo coupling is known in the art.

[0034] The substrate compound and the amyloid-binding motif may be linked via any spacer. Such a spacer may be an alkyl chain, an ether chain, or a combination thereof, each of which may be substituted, or may have an amide group or ester group partially introduced therein. The spacer preferably has 1 to 20 carbon atoms, more preferably 1 to 10 carbon atoms, although the spacer is not limited thereto.

[0035] The substrate compound in the present invention may exist as a salt. Examples of such salts include base addition salts, acid addition salts, and amino acid salts. Examples of base addition salts include metal salts such as sodium salts, potassium salts, calcium salts, and magnesium salts, ammonium salts, and organic amine salts such as triethylamine salts, piperidine salts, and morpholine salts. Examples of acid addition salts include mineral acid salts such as hydrochlorides, sulfates, and nitrates, and organic acid salts such as carboxylates, methanesulfonates, paratoluenesulfonates, citrates, and oxalates. Examples of amino acid salts include glycine salts. However, the salts are not limited to these salts.

[0036] The substrate compound of the present invention may have one or more asymmetric carbon atoms depending on the type of substituent, and may exist as stereoisomers such as optical isomers or diastereoisomers. Pure stereoisomers, any mixture of stereoisomers, racemates, etc. are all included within the scope of the present invention. In addition, the substrate compound of the present invention or a salt thereof may exist as a hydrate or solvate. The type of solvent that forms the solvate is not particularly limited, but examples include solvents such as water, ethanol, acetone, and isopropanol.

[0037] The solvent in the method of the present invention is typically water, but a mixture of water and an organic solvent can be used depending on the circumstances. Examples of such organic solvents include protic solvents such as methanol and ethanol; and aprotic polar solvents such as DMSO. When water is used as the solvent, its pH can be adjusted using a buffer (buffer solution) known in the art, typically to a pH range of 4 to 7, preferably 5 to 6.

[0038] In another aspect, the present invention also relates to an amyloid compound used in the above method, which is an aggregate of peptides having a β-sheet structure and comprising the sequence of the above formula, as described above in detail.

[0039] From another perspective, the present invention can also be said to be the use of the above amyloid compound for controlling regioselectivity in an enzymatic reaction. [Example]

[0040] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0041] 1. Peptide synthesis Bz-Phe-Phe-Ala-Ala-Leu-Leu-NH2 (hereinafter referred to as "BL7") was synthesized as a peptide that forms an amyloid compound. The N-terminus of this peptide was protected with a benzoyl (Bz) group and the C-terminus with an NH2 group. This peptide was synthesized automatically from the C-terminus to the N-terminus using Rink amide resin using a CEM Liberty microwave peptide synthesizer using standard Fmoc solid-phase synthesis. Cycles including deprotection, washing, and coupling were performed automatically until synthesis was complete. The detailed procedure is as follows.

[0042] [Synthesis of BL7 (Bz-Phe-Phe-Ala-Ala-Leu-Leu-NH2)] Peptides were synthesized on Rink-amide resin (0.10 mmol scale) using the appropriate Fmoc amino acid and either benzoic acid or acetic acid at the N-terminus. The peptide resin was treated with a cocktail containing 95% TFA, 2.5% TIPS, and 2.5% HO for 1 hour. The filtrate collected by filtration was concentrated under reduced pressure and subsequently precipitated with diethyl ether. The resulting precipitate was collected by filtration and dissolved in DMSO. The mixture was purified by preparative HPLC to yield the purified peptide. Peptides were synthesized on Rink-amide resin (0.10 mmol scale) using the appropriate Fmoc amino acid and either benzoic acid or acetic acid as the N-terminal amino acid. The peptide resin was treated with a cocktail containing 95% TFA, 2.5% TIPS, and 2.5% HO for 1 hour. The filtrate collected by filtration was concentrated under reduced pressure and subsequently precipitated with diethyl ether. The resulting precipitate was collected by filtration and dissolved in DMSO. The mixture was purified by preparative HPLC to yield purified peptide BL7.

[0043] The resulting peptides were identified by liquid chromatography mass spectrometry (LCMS). LCMS (ESI) [M+H]+ m / z Calcd: 784.9 Found: 784.5

[0044] 2. Evaluation of inhibitory activity by amyloid First, to evaluate the inhibitory ability of amyloid compounds on enzyme reactions, the conversion reaction of ester groups by esterase enzymes was observed using model compounds having ester groups.

[0045] 2-1. Evaluation using p-nitrophenyl acetate (pNPA) 10 μL of a DMSO stock solution of p-nitrophenyl acetate (pNPA) and 25 μL of a DMSO-HCl aqueous stock solution of amyloid compound BL7 were added to 965 μL of 100 mM phosphate buffer, pH 8.0 (final amyloid concentration: 100 μM). The mixture was stirred and left at room temperature for 5 minutes. Next, 10 μL of porcine liver esterase (PLE) solution was added. After each incubation of the reaction mixture at room temperature, the solution was analyzed using UV-vis measurement. The concentration of the produced p-nitrophenol (pNP) was determined based on a calibration curve constructed from real samples of pNP.

[0046] The results are shown in Figure 3. As a result, pNPA was converted to pNP by the addition of PLE, even in the presence of amyloid.

[0047] 2-2. Evaluation using DPP-acetate (compound 1) (Figure 2) Next, the following ester compound (Compound 1) containing the amyloid-binding motif 4-(dimethylaminophenylazo)phenyl (DPP) was subjected to a similar measurement after adding PLE. [ka]

[0048] 1.0 μL of a DMSO stock solution of compound 1 and 2.5 μL of a 4 mM DMSO-HCl aqueous stock solution of amyloid compound BL7 were added to 96.5 μL of 100 mM phosphate buffer, pH 8.0 (final amyloid concentration: 100 μM). The mixture was vortexed and left at 37 °C for 5 minutes. Next, 1.0 μL of PLE solution was added. After incubating the reaction mixture at 37 °C, an aliquot (15 μL) of the solution was added to 15 μL of TFA to quench the reaction. A 20 μL aliquot of the mixture was analyzed using HPLC. The concentration of the resulting 4-hydroxy-4'-dimethylaminoazobenzene (1P) was determined based on the peak area as follows: where [1] is the initial concentration of compound 1. TIFF2025178805000002.tif15150

[0049] The results are shown in Figure 4. As a result, the production of 1P was reduced in the presence of amyloid compounds, suggesting that the reaction catalyzed by the enzyme PLE was inhibited by binding of amyloid compounds to the amyloid-binding motif DPP.

[0050] 3. Application of peptide substrates to enzymatic reactions Next, trypsin enzymatic reaction was carried out using an oligopeptide substrate (compound 2: BGs-RISVA (BGs-Arg-Ile-Ser-Val-Ala)) modified with the amyloid-binding motif DPP at its terminus.

[0051] 1 μL of a stock solution of compound 2 (TFA salt) dissolved in DMSO and 2.5 μL of a DMSO-HCl aqueous stock solution of amyloid compound BL7 (4 mM) were added to 96.5 μL of 100 mM phosphate buffer, pH 8.0 (final concentration of BL7: 0, 10, or 100 μM). The mixture was vortexed and left at room temperature for 5 minutes. Next, 0.5 μL of a 5-fold diluted trypsin (TG) stock solution was added to the solution. The reaction time was based on 1 minute, but if the reaction was slow or under diluted conditions, the reaction time was extended to 3–5 minutes to calculate the initial rate. After incubating the reaction mixture at 30°C for the appropriate time, 100 μL of TFA (100% v / v) was added, and a 10 μL aliquot was analyzed by HPLC. The concentration of the product, BR ([BR]), was determined based on the sum of the peak areas of compound 2 and BR. where [2] is the initial concentration of compound 2. TIFF2025178805000003.tif16145

[0052] Structure of compound BR: TIFF2025178805000004.tif26146

[0053] The results are shown in Figure 5. As a result, the addition of the amyloid compound suppressed the cleavage of arginine residues by trypsin, and inhibited the production of compound BR.

[0054] Similar measurements were also performed on substrate compounds 2a-2g, which have different amino acid sequences, as shown in Figure 6. Substrate compounds (2, 2a-2g) were purified by preparative HPLC to obtain TFA salts and dissolved in DMSO to prepare 1 mM stock solutions. 5.0 μL of the DMSO stock solution of the substrate and 2.5 μL of a DMSO-HCl aqueous solution of amyloid compound BL7 (0 mM, 0.8 mM, or 4 mM) were added to 193 μL of 100 mM phosphate buffer, pH 8.0 (final concentrations of substrate: 10 μM, BL7: 100 μM). The mixture was stirred and left at room temperature for 5 min. A diluted TG stock solution was added to the solution. After incubating the reaction mixture at 30 °C, an aliquot was added to TFA (20% v / v, or 100% v / v for compound 2), and a 10 μL aliquot was analyzed using HPLC. The recovery yield was determined based on the sum of the peak areas of the starting substrate (SM) and the product (P). TIFF2025178805000005.tif16157

[0055] In addition, the relative activity (A rel ) was calculated using the following formula: TIFF2025178805000006.tif13146

[0056] As shown in FIG. 6, in all substrates, cleavage of the arginine residue by trypsin was suppressed, and the production of compound P was inhibited.

[0057] 4. Comparison using various amyloids Next, a similar experiment was performed using amyloid compounds formed by various peptides, as shown in Figure 7, instead of BL7. 2.5 μL of a DMSO stock solution of compound 2a (1 mM) and 2.5 μL of a DMSO-HCl aqueous stock solution (4 mM) of various amyloid compounds were added to 96.5 μL of 100 mM phosphate buffer, pH 8.0 (final concentrations of compound 2a: 10 μM, amyloid: 100 μM). The mixture was vortexed and left at room temperature for 5 minutes. Next, 1.0 μL of a 40-fold diluted TG stock solution was added. After incubating the reaction mixture at 30 °C for 30 minutes, an aliquot was added to TFA (20% v / v), and a 10 μL aliquot was analyzed using HPLC.

[0058] The relative activity (A rel ) is shown in Figure 7. As a result, it was found that the enzyme reaction was inhibited by all amyloid compounds. In particular, it was found that the amyloid compounds formed from peptides having the sequences 1, 4, and 9 to 12 in Figure 7 exhibited excellent inhibitory ability.

[0059] 5. Verification using substrate compounds with multiple reaction sites Next, an enzyme reaction was carried out using substrate compound 3, which has multiple reactive sites (arginine residues) within the molecule, and it was confirmed that the enzyme reaction was inhibited only at the reactive sites near the amyloid compound binding site.

[0060] [Trypsin] Substrate compound 3 was obtained as a TFA salt purified by preparative HPLC and dissolved in DMSO to obtain a 1 mM stock solution. 2.0 μL of the DMSO stock solution of the substrate and 2.5 μL of a DMSO-HCl aqueous stock solution of amyloid compound BL7 (0 mM or 4 mM) were added to 193 μL of 100 mM phosphate buffer, pH 8.0 (final concentrations of substrate: 10 μM, amyloid: 100 μM). The mixture was vortexed and left at room temperature for 5 minutes. Next, 2 μL of a 40-fold diluted TG stock solution was added. After incubating the reaction mixture at 30°C, an aliquot was added to TFA (20% v / v), and a 10 μL aliquot was analyzed using HPLC. Recovery or production yield was determined based on the sum of peak areas.

[0061] The results are shown in Figure 8. As a result, in the absence of amyloid, the reaction of cleaving "RN" in substrate compound 3 proceeded over reaction time (right panel), whereas in the presence of amyloid, cleavage of "RN" near the binding motif BG to which amyloid binds was inhibited, and cleavage of "RA" located at a position distant from BG proceeded (left panel). This result demonstrates that the enzymatic reaction is inhibited only near the binding site of amyloid, but proceeds normally at other reaction sites, i.e., it is possible to control the enzymatic reaction regioselectively.

[0062] [PAD] The same reaction was carried out using peptidylarginine deiminase (PAD), an enzyme that specifically converts arginine residues into citrulline, instead of trypsin.

[0063] 2.0 μL (1 mM) of a DMSO stock solution of substrate Compound 3 and 5 μL of a DMSO-HCl aqueous stock solution of amyloid compound BL7 (0 mM or 4 mM) were added to 193 μL of 50 mM HEPES buffer, pH 7.6, containing 10 mM CaCl2 and 5 mM DTT (final concentrations: substrate Compound 3: 10 μM; BL7: 100 μM). The mixture was vortexed and left at room temperature for 5 minutes. Next, 2 μL of PAD stock solution was added. After incubation of the reaction mixture at 30°C each time, an aliquot was added to TFA (20% v / v), and a 10 μL aliquot was analyzed using HPLC. Yields were determined based on the sum of peak areas.

[0064] The results are shown in Figure 9. As a result, in the absence of amyloid, the reaction proceeded with the passage of reaction time, in which the arginine (R) near the binding motif BG to which amyloid was bound in substrate compound 3 was converted to citrulline (right panel). On the other hand, in the presence of amyloid, citrulline conversion was inhibited at the arginine (R) near the binding motif BG to which amyloid was bound, while arginines distant from BG were preferentially converted to citrulline (left panel).

[0065] [Glu-C] Furthermore, the reaction of endoproteinase Glu-C was carried out using substrate compound 4, which has multiple glutamic acid residues (E). Glu-C is an enzyme that specifically cleaves the C-terminus of glutamic acid residues.

[0066] Substrate compound 4 was purified by preparative HPLC as the TFA salt and dissolved in DMSO to obtain a 1 mM stock solution. 0.5 μL of the DMSO stock solution of substrate compound 4 and 1.25 μL of a DMSO-HCl aqueous stock solution of amyloid compound BL7 (0 mM or 4 mM) were added to 48.25 μL of 100 mM phosphate buffer, pH 8.0 (final concentrations of substrate: 10 μM, amyloid: 100 μM). The mixture was vortexed and left at room temperature for 5 minutes. Next, 4 μL of Glu-C stock solution was added to the solution. The reaction mixture was incubated at 30 °C for 24 hours, after which an aliquot was added to 1.25 μL of TFA and 24.75 μL of DMSO. A 10 μL aliquot was analyzed using HPLC. The yield was determined based on the sum of the peak areas of substrate compound 4 and the product.

[0067] The results are shown in Figure 10. In the absence of amyloid, both compound BP2 (BGt-E), which is produced by cleavage of the glutamic acid residue near the amyloid-bound binding motif BG, and compound 7 (BGt-ERRNE), which is cleaved at a glutamic acid residue distant from BG, were produced. In contrast, the presence of amyloid reduced the production of BP2 (BGt-E).

[0068] 6. In situ azo coupling The terminally acetyl-protected peptide Ac-YGRISVA (compound 8) was dissolved in DMSO to obtain a 1 mM stock solution. Next, 10 μL of the DMSO stock solution of compound 8 was diluted with 90 μL of PB (pH 8.0). 1.0 μL of a 10 mM DMSO solution of 4-acetylbenzenediazonium hexafluorophosphate was added, and the mixture was incubated at room temperature for 1 hour. The resulting solution of the azo-linked peptide (compound 8a) was diluted 10-fold with PB (pH 8.0). To 97.5 μL of this solution, 2.5 μL of a DMSO-HCl aqueous stock solution of amyloid compound BL7 (0 mM or 4 mM) and 2.0 μL of a 5-fold diluted TG stock solution were added. The reaction mixture was incubated at 30°C for 1 hour, and then 10 μL aliquots were analyzed using HPLC.

[0069] The results are shown in Figure 11. As a result, it was demonstrated that the enzymatic reaction by trypsin can be inhibited even when azobenzene, an amyloid-binding motif, is formed in situ.

Claims

1. A method for controlling regioselectivity in an enzymatic reaction, comprising: A method comprising the step of adding an enzyme to a substrate compound, the substrate compound containing an amyloid-binding motif in the molecule that has affinity for the amyloid compound, in the presence of the amyloid compound.

2. 2. The method of claim 1, wherein the amyloid-binding motif is one or more selected from the group consisting of an azobenzene structure, a benzothiazole structure, a xanthene structure, a phenothiazine structure, a boron dipyrromethene structure, a curcumin structure, and a porphyrin structure.

3. The amyloid compound has a β-sheet structure and has the following sequence: Phe-X-Ala-Y wherein X is Phe or Ala; Y is Ala-Leu-Leu, Ala-Ala-Leu, Ala-Leu-Ala, Ser-Leu-Leu, or Ala-Leu-NH ２ It is.) The method of claim 1, wherein the peptide is an aggregate of peptides comprising:

4. The method of claim 3, wherein the peptide consists of 7 to 40 amino acids comprising the sequence.

5. The method of claim 1 , wherein the regioselectivity is the position of a functional group in the substrate compound that is cleaved or modified by the enzyme.

6. 2. The method of claim 1, wherein cleavage or modification by the enzyme is inhibited in the region where the amyloid compound binds to the amyloid-binding motif.

7. The method according to claim 1, wherein the product obtained by the enzymatic reaction is a compound different from the product obtained by the enzymatic reaction in the absence of the amyloid compound, or the composition ratio of the products is different.

8. The method of claim 1 , wherein the substrate compound is an oligopeptide, a polypeptide, an oligonucleotide, a polynucleotide, a sugar chain substance, a lipid molecule, or a low molecular weight compound.

9. an amyloid compound which is an aggregate of peptides having a β-sheet structure and comprising the following sequence: Phe-X-Ala-Y wherein X is Phe or Ala; Y is Ala-Leu-Leu, Ala-Ala-Leu, Ala-Leu-Ala, Ser-Leu-Leu, or Ala-Leu-NH ２ ).

10. Use of the amyloid compound according to claim 9 for controlling regioselectivity in an enzymatic reaction.

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