Method for producing cyclic compound, method for producing compound library, screening method, and cyclic compound

Abstract

The present disclosure provides a method for producing a cyclic compound represented by formula (I), the production method involving: causing a macrocyclic enzyme to act on a peptide represented by formula (II): LP-X-(Xd)m-Xa'-(Xe)n-Xb-Y-Xc-Z to form a nitrogen-containing six-membered ring A while eliminating LP when LP is present, thereby forming a cyclic compound represented by formula (I'); and causing a prenylated enzyme to act on the cyclic compound represented by formula (I') to subject at least one amino acid at least included in any one of Xa', Xb, Xc, and Z to prenylation to form the cyclic compound represented by formula (I). In the formula, Xa, Xb, Xc, m Xds, and n Xes are each independently an amino acid or an analog thereof, Z may or may not be present, Z is a peptide including 1-100 amino acids and / or an analog thereof when Z is present, m is an integer selected from 0 to 39, n is an integer selected from 0 to 39, the sum of m and n is an integer selected from 1 to 39, the ring A is a nitrogen-containing six-membered ring which may have a substituent, B1 is an oxygen atom, a sulfur atom, or an NH group, R1 is a hydrogen atom or a hydrocarbon group, X represents a group represented by formula (1), Y represents a group represented by formula (2), Xa' is an amino acid, LP may or may not be present, and LP is a peptide including 1-100 amino acids and / or an analog thereof when LP is present. In formula (I), any of Xa, Xb, Xc, and Z includes at least one amino acid having a prenyl group.

Description

Method for producing cyclic compounds, method for producing compound libraries, screening method, and cyclic compounds

[0001] The present invention relates to a method for producing a cyclic compound, a method for producing a compound library, a screening method, and a cyclic compound, particularly a cyclic compound having a closed ring structure formed by a prenyl group and a nitrogen-containing 6-membered ring, a method for producing a cyclic compound, a method for producing a compound library, a screening method, and a cyclic compound.

[0002] With the recent advancement of genome sequencing technology, attention has been focused on a group of peptide compounds called Ribosomally synthesized and post-translationally modified peptides (RiPPs), which are biosynthesized through post-translational modifications after synthesis in the ribosome (Non-Patent Document 1). RiPPs are synthesized as precursor peptides through the ribosome translation of biosynthetic genes called structural genes present in the biosynthetic gene cluster. Subsequently, post-translational modification enzymes encoded by other biosynthetic genes apply multiple modifications to the precursor peptide, such as azole ring formation and dehydroamino acid formation, to synthesize the mature product.

[0003] Thiopeptides are a type of RiPPs and possess interesting physiological activities, such as potent translation inhibitory activity, antibacterial activity against multidrug-resistant bacteria, antiparasitic activity, antiviral activity, immunosuppressive activity, and anticancer activity. Known examples of thiopeptides include lactazols A, B, and C. It has also been reported that the lactazol biosynthetic gene cluster lazA-F was discovered in the genome of the actinomycete Streptomyces lactacystinaeus (Non-Patent Document 2). Patent Document 1 discloses a method for producing a peptide compound library using a thiopeptide biosynthetic system.

[0004] Prenylation is known as a post-translational modification. Prenylation is a ubiquitous modification found in primary metabolism as well as in the biosynthetic pathways of various natural products, including terpenoids, polyketides, nonribosomal peptides, and RiPPs. Prenylation often plays an important role in the diverse biological activities of these natural products. This is because the prenyl group generally enhances the lipophilicity of the molecule and its interaction with lipid membranes, thereby resulting in desirable bioavailability and membrane permeability. These properties of the prenyl group are of particular interest in the development of peptide pharmaceuticals, which are known to have poor membrane permeability. Alkylation is known as one of the most successful and important strategies for overcoming poor membrane permeability and has been used medicinally in the development of peptide pharmaceuticals such as liraglutide and insulin detemir.

[0005] Patent Documents 2 and 3 disclose methods for producing prenylated compounds.

[0006] International Publication No. 2020 / 067550 International Publication No. 2020 / 080490 International Publication No. 2023 / 068296

[0007] Arnison, PG et al. Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature. Nat. Prod. Rep. 30, 108-160, (2013). Hayashi, S. et al. Genome reveals mining a minimum gene set for the biosynthesis of 32-membered macrocyclic thiopeptides lactazoles. Chem. Biol. 21, 679-688, (2014).

[0008] Although the above-mentioned prior art documents disclose methods for producing compounds using a thiopeptide biosynthetic system and methods for producing prenylated compounds, a compound having a closed ring structure formed by an azole ring and a nitrogen-containing six-membered ring and a prenyl group in one molecule, and a method for producing the same, have not yet been discovered. Compounds having both a closed ring structure formed by an azole ring and a nitrogen-containing six-membered ring and a prenyl group could be promising candidates for pharmaceuticals.

[0009] An object of the present invention is to provide a novel compound having a closed ring structure formed by an azole ring and a nitrogen-containing six-membered ring, and a prenyl group, and a method for producing the same.

[0010] As a result of extensive research to solve the above problems, the present inventors have found that by applying a thiopeptide biosynthesis system and carrying out a prenylation reaction in a specific order, a general-purpose method for producing a cyclic compound having a closed ring structure formed by an azole ring and a nitrogen-containing six-membered ring and a prenyl group can be achieved, and have completed the present invention.

[0011] That is, the present disclosure includes the following embodiments: [1] Formula (I): [In formula (I), ａ , X ｂ , X ｃ , m X ｄ , and n X ｅ are each independently an amino acid or an analog thereof, Z may be present or absent, and when present, is a peptide consisting of 1 to 100 amino acids and / or analogs thereof, m is an integer selected from 0 to 39, n is an integer selected from 0 to 39, the sum of m and n is an integer selected from 1 to 39, ring A is a nitrogen-containing 6-membered ring which may have a substituent, and B １ is an oxygen atom, a sulfur atom, or an NH group, R １ is a hydrogen atom or a hydrocarbon group, and X ａ , X ｂ , X ｃ and Z each contain at least one amino acid having a prenyl group.] A method for producing a cyclic compound represented by formula (II): LP-X-(Xｄ ) ｍ -X ａ '-(X ｅ ) ｎ -X ｂ -Y-X ｃ -Z (II) [In formula (II), X is a group represented by formula (1): (R ２ represents a hydrogen atom or a hydrocarbon group; Y represents a group represented by formula (2): (R １ and B １ has the same meaning as above, and R ３ represents a hydrogen atom or a hydrocarbon group; ａ X' is an amino acid or an analog thereof; ｂ , X ｃ , m X ｄ , n X's ｅ , m, n, and Z are as defined above, and LP may or may not be present, and when present, it is a peptide consisting of 1 to 100 amino acids and / or analogs thereof. ] is reacted with a macrocyclase to form a nitrogen-containing six-membered ring A while eliminating LP, if present, to obtain a peptide represented by formula (I'): [In formula (I'), X ａ ', X ｂ , X ｃ , m X ｄ , n X's ｅ , m, n, Z, ring A, B １ , and R １ has the same meaning as defined above.]; and allowing a prenylation enzyme to act on the cyclic compound represented by formula (I') to form a cyclic compound represented by formula (I') containing at least X ａ ', X ｂ , X ｃ and Z, by prenylation of at least one amino acid contained in the cyclic compound represented by formula (I). [2] Formula (III): LP'-X'-(X ｄ ') ｍ -X ａ "-(X ｅ ') ｎ -X ｂ'-Y'-X ｃ '-Z' (III) [in formula (III), X' is serine or threonine, or an analog thereof, Y' is a peptide represented by -Y'(11)-Y'(12)-, Y'(11) is serine, cysteine, threonine, or diaminopropionic acid, or an analog thereof, Y'(12) is serine or threonine, or an analog thereof, m and n are as defined above, X ａ ", X ｂ ', X ｃ ', m X's ｄ ', n X's ｅ ', Z', and LP' are each the X in formula (II). ａ ', X ｂ , X ｃ , m X ｄ , n X's ｅ, Z, and LP.] and forming the peptide represented by formula (II) from the precursor peptide represented by formula (III) by the following reactions: a reaction of forming an azole ring at least on Y' (11) in the precursor peptide represented by formula (III) with an azole ring-forming enzyme; and a reaction of converting at least X' and Y' (12) in the precursor peptide represented by formula (III) into α,β-unsaturated amino acid residues with an α,β-unsaturated amino acid-forming enzyme in the presence of a cosubstrate for glutamylation and an aminoacylase. [3] The production method according to [1] or [2], wherein the at least one amino acid to be prenylated in formula (I') is tryptophan, arginine, histidine, tyrosine, serine, or threonine, or an analog thereof. [4] The method according to any one of [1] to [3], wherein the prenylation enzyme comprises at least one selected from KgpF, OltF, PT5, PT12, PT18, PT19, PT23, PT30, LimF, and PagF, and prenylation enzymes having an amino acid sequence that shares 80% or more sequence identity with the amino acid sequences of these enzymes. [5] The method according to any one of [1] to [4], wherein LP in formula (II) is a peptide consisting of 11 to 100 amino acids and / or analogs thereof. [6] The method according to any one of [1] to [5], wherein, in formula (I), m is an integer selected from 0 to 23, n is an integer selected from 0 to 23, and the sum of m and n is an integer selected from 1 to 23. [7] The method according to any one of [1] to [5], wherein -X in formula (II) ｂ -Y-X ｃ - has the following structure: The method according to any one of [1] to [6], wherein R １ , R ３ , R Ｘ１ , and R Ｃ are each independently a hydrogen atom or a hydrocarbon group, B １ and B Ｃare each independently an oxygen atom, a sulfur atom, or an NH group.) [8] The production method according to any one of [2] to [7], wherein the macrocyclization enzyme comprises LazC and / or an enzyme homologous to LazC, the azole ring-forming enzyme comprises at least one selected from LazD, LazE, and LazF, and enzymes homologous thereto, and the α,β-unsaturated amino acid-forming enzyme comprises at least one selected from LazB and LazF, and enzymes homologous thereto. [9] The production method according to any one of [2] to [7], wherein the cyclic compound represented by formula (I') is allowed to react with the prenylation enzyme to form at least X ａ

[10] The method according to any one of [1] to [8], wherein the amino acid sequence of formula (I): [In formula (I), ａ , X ｂ , X ｃ , m X ｄ , and n X ｅ are each independently an amino acid or an analog thereof, Z may be present or absent, and when present, is a peptide consisting of 1 to 100 amino acids and / or analogs thereof, m is an integer selected from 0 to 39, n is an integer selected from 0 to 39, the sum of m and n is an integer selected from 1 to 39, ring A is a nitrogen-containing 6-membered ring which may have a substituent, and B １ is an oxygen atom, a sulfur atom, or an NH group, R １ is a hydrogen atom or a hydrocarbon group, and X ａ , X ｂ , X ｃ and Z each contain at least one amino acid having a prenyl group.] ｄ ') ｍ -X ａ "-(X ｅ ') ｎ -X ｂ '-Y'-X ｃ'-Z' (III) [in formula (III), X' is serine or threonine, or an analog thereof, Y' is a peptide represented by -Y'(11)-Y'(12)-, Y'(11) is serine, cysteine, threonine, or diaminopropionic acid, or an analog thereof, Y'(12) is serine or threonine, or an analog thereof, m and n are as defined above, X ａ ", X ｂ ', X ｃ ', m X's ｄ ', n X's ｅ ', Z', and LP' are each the X in formula (II). ａ ', X ｂ , X ｃ , m X ｄ , n X's ｅ , Z, and LP. ] and preparing a precursor peptide library containing two or more precursor peptides represented by the formula (III) by the following reactions: a reaction of forming an azole ring at least on Y' (11) in the precursor peptide represented by the formula (III) using an azole ring-forming enzyme; and a reaction of converting at least X' and Y' (12) in the precursor peptide represented by the formula (III) into α,β-unsaturated amino acid residues using an α,β-unsaturated amino acid-forming enzyme in the presence of a cosubstrate for glutamylation and an aminoacylase. ｄ ) ｍ -X ａ '-(X ｅ ) ｎ -X ｂ -Y-X ｃ -Z (II) [In formula (II), X is a group represented by formula (1): (R ２ represents a hydrogen atom or a hydrocarbon group; Y represents a group represented by formula (2): (R １ and B １ has the same meaning as above, and R ３ represents a hydrogen atom or a hydrocarbon group; ａX' is an amino acid or an analog thereof; ｂ , X ｃ , m X ｄ , n X's ｅ , m, n, and Z are as defined above, and LP may or may not be present, and when present, it is a peptide consisting of 1 to 100 amino acids and / or analogs thereof.]; and allowing a macrocyclase to act on the peptide represented by formula (II), to form a nitrogen-containing six-membered ring A while eliminating LP, if present, to form a peptide represented by formula (I'): [In formula (I'), X ａ ', X ｂ , X ｃ , m X ｄ , n X's ｅ , m, n, Z, ring A, B １ , and R １ has the same meaning as defined above.]; and allowing a prenylation enzyme to act on the cyclic compound represented by formula (I') to form a cyclic compound represented by formula (I') containing at least X ａ ', X ｂ , X ｃand Z, prenylating at least one amino acid included in formula (I) to form a cyclic compound represented by formula (I), thereby forming a compound library containing two or more types of cyclic compounds represented by formula (I).

[11] The production method according to

[10] , wherein preparing the precursor peptide library comprises: preparing a library of nucleic acids encoding the precursor peptide represented by formula (III): and expressing the precursor peptide represented by formula (III) based on the library of nucleic acids.

[12] The production method according to

[11] , further comprising binding puromycin to the 3' end of each nucleic acid in the nucleic acid library, thereby forming a precursor peptide library containing two or more types of complexes between the precursor peptide represented by formula (III) and the nucleic acid encoding the precursor peptide by expression.

[13] A screening method for identifying a compound that binds to the target substance, comprising: contacting a compound library produced by the production method according to any one of

[10] to

[12] with a target substance; and selecting a compound that binds to the target substance.

[14] [In formula (I), ａ , X ｂ , X ｃ , m X ｄ , and n X ｅ are each independently an amino acid or an analog thereof, Z may be present or absent, and when present, is a peptide consisting of 1 to 100 amino acids and / or analogs thereof, m is an integer selected from 0 to 39, n is an integer selected from 0 to 39, the sum of m and n is an integer selected from 1 to 39, ring A is a nitrogen-containing 6-membered ring which may have a substituent, and B １ is an oxygen atom, a sulfur atom, or an NH group, R １ is a hydrogen atom or a hydrocarbon group, and X ａ , X ｂ , X ｃand Z each independently represent a cyclic compound represented by the formula (I), wherein the amino acid having a prenyl group is selected from tryptophan, arginine, histidine, tyrosine, serine, or threonine, or an analog thereof.

[16] The cyclic compound represented by the formula (I), wherein m is an integer selected from 0 to 23, n is an integer selected from 0 to 23, and the sum of m and n is an integer selected from 1 to 23.

[17] In the formula (I), X ａ

[18] The cyclic compound according to any one of

[14] to

[16] , wherein the formula (I): [In formula (I), ａ , X ｂ , X ｃ , m X ｄ , and n X ｅ are each independently an amino acid or an analog thereof, Z may be present or absent, and when present, is a peptide consisting of 1 to 100 amino acids and / or analogs thereof, m is an integer selected from 0 to 39, n is an integer selected from 0 to 39, the sum of m and n is an integer selected from 1 to 39, ring A is a nitrogen-containing 6-membered ring which may have a substituent, and B １ is an oxygen atom, a sulfur atom, or an NH group, R １ is a hydrogen atom or a hydrocarbon group, and X ａ , X ｂ , X ｃ and Z each contain at least one amino acid having a prenyl group. [In formula (I'), X ａ X' is an amino acid or an analog thereof; ｂ , X ｃ , m X ｄ , n X's ｅ , m, n, Z, ring A, B １ , and R １has the same meaning as above.], and a prenylation enzyme is allowed to act on the cyclic compound represented by the formula (I) to give a cyclic compound represented by the formula (I), ａ ', X ｂ , X ｃ and Z to form a cyclic compound represented by formula (I).

[19] The production method according to

[18] , wherein the at least one amino acid to be prenylated in formula (I') is tryptophan, arginine, histidine, tyrosine, serine, or threonine, or an analog thereof, and the prenylation enzyme is at least one selected from KgpF, OltF, PT5, PT12, PT18, PT19, PT23, PT30, LimF, and PagF, and prenylation enzymes having an amino acid sequence having 80% or more sequence identity with the amino acid sequences of these enzymes. [20 ... prenylation enzyme acts on the cyclic compound represented by formula (I') to form at least X ａ The method according to

[18] or

[19] , wherein ' is prenylated.

[0012] According to the present invention, it is possible to provide a novel compound having a closed ring structure formed by an azole ring and a nitrogen-containing six-membered ring, and a prenyl group, as well as a method for producing the same.

[0013] (A) Protocol overview and (B) extracted ion chromatogram (EIC) results for multiple setups are shown. (A) Synthetic method overview and (B) synthesis results (yields) for multiple prenylated thiopeptides are shown. LP and tail are the same as MS1 to 3. Sequences 1 to 5 are shown as SEQ ID NOS: 95 to 99. Temperature dependence of the prenylation reaction is shown. (A) Protocol overview and (B) experimental results. Enzyme concentration dependence of the prenylation reaction is shown. (A) Protocol overview and (B) experimental results. Results of an investigation into the concentration conditions of LazD, E, and F are shown. The volumes of the reaction solution in each reaction before and after adjusting the concentrations of LazD, E, and F are shown. (A) mRNA design and (B) conditions for the extension reaction and PCR reaction in the construction of a prenylated thiopeptide library are shown. The sequences of the designed peptides are shown as SEQ ID NOS: 101 to 109.

[0033] Figure 1 shows a scheme for selecting prenylated thiopeptides that specifically bind to WNK4. Figure 2 shows the results of selecting prenylated thiopeptides that specifically bind to WNK4. Figure 3 shows peptides selected as prenylated thiopeptides that specifically bind to WNK4. (A) UMAP results. (B) Sequences of selected peptides. The sequences of W4ip_1 to W4ip_14 are shown in SEQ ID NOS: 78 to 91. Figure 4 shows the analysis results of 11 selected peptides. (A) Measurement scheme and (B) results of WNK4 / WNK1 selectivity for hit peptides. Figure 5 shows confirmation of modified residues in the sequences of hit peptides. (A) Scheme for confirmation. (B) Confirmation results for W4-01. Confirmation results for modified residues in (A) W4-01 and (B) W4-03. Confirmation results for modified residues in (A) W4-06 and (B) W4-07. This shows that selective binding to WNK4 was maintained even when cysteine ​​was replaced with glutamine. (A) A scheme for confirmation. (B) Results of confirming the effect of cysteine ​​replacement in W4-06 and W4-07. Comparison of recovery rates with and without a prenyl group. (A) A scheme for comparison. (B) Results of confirming the effect of a prenyl group in W4-01, W4-06-C41Q, and W4-07-C51Q. (A) A scheme for investigation of prenylation enzymes other than KgpF, and (B) the results of investigation are shown.Figure 1 shows the results of a prenylation reaction by OltF. Figure 2 shows the results of a prenylation reaction by PT5. Figure 3 shows the results of a prenylation reaction by PT18. Figure 4 shows the results of a prenylation reaction by PT19. Figure 5 shows the results of a prenylation reaction by PT30. Figure 6 shows the results of a prenylation reaction by LimF. Figure 7 shows the results of a prenylation reaction by PagF 222G / 237G / 271N. Figure 8 shows the substrate tolerance for prenylation by (A) KgpF and OltF, and (B) OltF. Figure 9 shows the substrate tolerance for prenylation by PT5. Figure 10 shows the substrate tolerance for prenylation by PT18. Figure 11 shows the substrate tolerance for prenylation by PT19. Figure 12 shows the substrate tolerance for prenylation by PT12 and PT23. The upper row shows the prenylation efficiency of mutants in which the Pro adjacent to Trp was substituted with various amino acids, and the lower row shows the prenylation efficiency of mutants in which the Gly adjacent to Trp was substituted with various amino acids. The figures show the results of measuring the serum stability of prenylated thiopeptides in the examples. In the figure, PrPl+ represents the result for prenylated thiopeptides, and PrPl- represents the result for non-prenylated thiopeptides.

[0014] Hereinafter, an embodiment for carrying out the present invention (hereinafter referred to as "the present embodiment") will be described in detail, but the present invention is not limited to this embodiment and various modifications are possible within the scope of the gist thereof. In this specification, a wavy line in a chemical structural formula indicates a bonding site of a group represented by the structural formula.

[0015] [Cyclic Compound] The present embodiment provides a cyclic compound represented by formula (I): Formula (I):

[0016] In the formula (I), X ａ , X ｂ , X ｃ , m X ｄ , and n X ｅare each independently an amino acid or an analog thereof, Z may be present or absent, and when present, is a peptide consisting of 1 to 100 amino acids and / or analogs thereof, m is an integer selected from 0 to 39, n is an integer selected from 0 to 39, the sum of m and n is an integer selected from 1 to 39, ring A is a nitrogen-containing 6-membered ring which may have a substituent, and B １ is an oxygen atom, a sulfur atom, or an NH group, R １ is a hydrogen atom or a hydrocarbon group, and X ａ , X ｂ , X ｃ and Z each contain at least one amino acid having a prenyl group.

[0017] Thus, this embodiment provides a cyclic compound having a prenylated amino acid residue in addition to a closed ring structure formed by an azole ring and a nitrogen-containing six-membered ring. As mentioned above, Patent Document 1 discloses the formation of a closed ring structure formed by an azole ring and a nitrogen-containing six-membered ring using a thiopeptide biosynthesis system. Furthermore, Patent Documents 2 and 3 disclose the introduction of a prenyl group into an amino acid residue using a prenylation enzyme (synonymous with prenyltransferase). One aspect of the present disclosure is based on the discovery that the cyclic compound represented by formula (I) can be efficiently obtained only when the formation of a closed ring structure formed by an azole ring and a nitrogen-containing six-membered ring and prenylation by a prenylation enzyme are carried out in this order.

[0018] That is, while searching for a general method for producing a cyclic compound having a closed ring structure of an azole ring and a nitrogen-containing six-membered ring and a prenyl group, the inventors found that changing the order of the azole ring formation, the ring-closure reaction by a macrocyclase, and the prenylation reaction significantly changes the yield of the target compound. Furthermore, they found that a general method for producing the cyclic compound represented by formula (I) can be achieved only when the formation of the closed ring structure of an azole ring and a nitrogen-containing six-membered ring and the prenylation by a prenylation enzyme are carried out in this order. Furthermore, they found that the cyclic compound thus obtained, having a closed ring structure of an azole ring and a nitrogen-containing six-membered ring and a prenyl group, is a promising candidate for a pharmaceutical. The method for producing the cyclic compound of this embodiment will be described later.

[0019] In the above formula (I), X ａ , X ｂ , X ｃ and Z, at least one amino acid having a prenyl group is contained. The amino acid having a prenyl group is at least ａ , X ｂ , X ｃ At least one of X and Z may be included. ａ and Z. In this case, X ａ may be an amino acid having a prenyl group, or Z may comprise an amino acid having a prenyl group, or X ａ may be an amino acid having a prenyl group, and Z may contain an amino acid having a prenyl group. ａ is preferably an amino acid having a prenyl group. In the cyclic compound represented by formula (I), the number of amino acids having a prenyl group is not particularly limited, but may be, for example, 1 to 6, 1 to 4, or 1 to 3. In this specification, the term "prenyl group" refers to the following structure (also referred to as a forward prenyl group): or the following structure (also called a reverse prenyl group): In the above formula, k represents an integer of 0 to 11. When k is 0, 1, 2, 3, 4, 5, 7, or 9, the groups represented by the above formula are also referred to as dimethylallyl, geranyl, farnesyl, geranylgeranyl, geranylfarnesyl, hexaprenyl, octaprenyl, and decaprenyl groups, respectively. They may also be referred to as Cx prenyl groups depending on the number of carbon atoms x. For example, dimethylallyl, geranyl, and farnesyl groups are also referred to as C5 prenyl, C10 prenyl, and C15 prenyl groups, respectively. Therefore, in this specification, unless a specific group is particularly referred to, the term "prenyl group" is understood to include dimethylallyl, geranyl, farnesyl, geranylgeranyl, geranylfarnesyl, hexaprenyl, octaprenyl, and decaprenyl groups, etc. Which of the above structures is introduced depends on the type of prenylation enzyme described below, and the prenyl group may have any of the above structures, for example, a forward prenyl group.

[0020] As used herein, the term "amino acid" includes not only naturally occurring amino acids but also artificial amino acid variants. Examples of amino acids include proteinogenic amino acids and non-proteinogenic amino acids (natural or non-natural non-proteinogenic amino acids, and chemically synthesized compounds having properties known in the art as characteristic of amino acids). Proteinogenic amino acids are represented by the three-letter symbols commonly known in the art: Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gln, Cys, Gly, Pro, Ala, Ile, Leu, Met, Phe, Trp, Tyr, and Val. Proteinogenic amino acids are represented by the one-letter symbols commonly known in the art: R, H, K, D, E, S, T, N, Q, C, G, P, A, I, L, M, F, W, Y, and V. Proteinogenic amino acids are usually L-amino acids, but may have a structure as D-amino acids contained in non-proteinogenic amino acids. Non-proteinogenic amino acids include natural or unnatural amino acids other than proteinogenic amino acids.

[0021] Examples of non-proteinogenic amino acids include amino acids whose main chain structure differs from that of natural amino acids (α,α-disubstituted amino acids (α-methylalanine, cycloleucine, etc.), N-methyl amino acids, N-alkyl amino acids, D-amino acids, β-amino acids, γ-amino acids, δ-amino acids, long-chain amino acids, α-hydroxy acids, α-thio acids, and cyclic amino acids (cyclic α-amino acids, cyclic β-amino acids, aromatic amino acids, etc.)); amino acids whose side chain structure differs from that of natural amino acids (selenocysteine, norleucine, spinasine, nitrophenylalanine, tetrahydroisoquinolinecarboxylic acid, tetrahydroisoquinolinecarboxylic acid having a substituent (e.g., a hydroxy group, a C1-C3 alkyl group, a halogen group, etc.) such as hydroxytetrahydroisoquinolinecarboxylic acid, hydroxytryptophan, pentafluorophenylalanine, methoxyphenylalanine, γ-amino acids, γ-amino acids, γ-amino acids, α-hydroxy acids, α-thio acids, and cyclic amino acids (cyclic α-amino acids, cyclic β-amino acids, aromatic amino acids, etc.)); Ｓ，ＬExamples of unnatural amino acids include amino acids having a structure consisting of multiple rings, such as homoglutamine and bicycloamino acids, azide group-containing amino acids, alkyne group-containing amino acids, alkene group-containing amino acids, chloroacetamide group-containing amino acids, photoreactive group-containing amino acids, fluorescent amino acids, ε-alkylated lysine, biotin group-containing amino acids, citrulline, ester group-containing amino acids, amino acids having an additional methylene in the side chain ("homo" amino acids; e.g., homophenylalanine, homoglutamine, and homohistidine), and amino acids in which the carboxylic acid functional group in the side chain is replaced with a sulfonic acid group (e.g., cysteic acid); and combinations thereof. These combinations include amino acids whose main chain structure and side chain structure differ from those of natural amino acids, N-methylated versions of the above amino acids in which the main chain amino group is methylated, and D-amino acids of the above amino acids. Specific examples of other unnatural amino acids include the amino acids described in WO 2015 / 030014.

[0022] In this specification, unless an amino acid is specified as being in the L- or D-form, it may be in the L-form, the D-form, a racemic mixture, or a mixture of the L- and D-forms in any ratio. Unless otherwise specified, the term "amino acid" includes prenylated amino acids.

[0023] X in formula (I) ａ , X ｂ , X ｃ The amino acid having a prenyl group contained in any of (I), (II), and (II) is not particularly limited, and may be, for example, an amino acid residue obtained by prenylation of a proteinaceous amino acid. The amino acid having a prenyl group is preferably tryptophan, arginine, histidine, tyrosine, serine, or threonine, or an analog thereof, contained as the prenylated amino acid residue, and more preferably tryptophan, arginine, histidine, or tyrosine, or an analog thereof, contained as the prenylated amino acid residue.

[0024] Examples of tryptophan analogs include those represented by the following formulae (Trp-1), (Trp-2), (Trp-3), and (Trp-4). The chemical structural formulae in this specification encompass tautomers, geometric isomers, optical isomers, and the like. For example, the asymmetric carbons in formulae (Trp-1), (Trp-2), (Trp-3), and (Trp-4) may be in the R-configuration, the S-configuration, or a mixture thereof.

[0025] In formula (Trp-1), ring B is an aromatic six-membered ring which may contain a heteroatom, and R １＿Ｔｒｐ is an optional substituent on ring B, and R ２＿Ｔｒｐ and R ３＿Ｔｒｐ are each independently a hydrogen atom or an arbitrary substituent, m_Trp is an integer of 0 to 4, and l is an integer of 1 or 2. Examples of ring B include a benzene ring, a pyridine ring, a pyridazine ring, a pyrimidine ring, and a pyrazine ring, and a benzene ring is preferred. １＿Ｔｒｐ , R ２＿Ｔｒｐ and R ３＿Ｔｒｐ Examples of the alkyl group include, but are not limited to, a hydroxy group; a halogen group such as a fluorine group, a chloro group, a bromo group, or an iodo group; an alkyl group having 1 to 10 carbon atoms such as a methyl group, an ethyl group, or a propyl group; an aryl group such as a phenyl group or a naphthyl group; an aralkyl group such as a benzyl group; and an aralkyloxy group such as a benzyloxy group.

[0026] In formulas (Trp-2) to (Trp-4), ring B, R １＿Ｔｒｐ , R ２＿Ｔｒｐ , R ４＿Ｔｒｐ , m_Trp, and l represent ring B, R in formula (Trp-1). １＿Ｔｒｐ , R ２＿Ｔｒｐ , R ４＿Ｔｒｐ , m_Trp, and 1. Ring B in formulae (Trp-2) to (Trp-4) is preferably a benzene ring.

[0027] Taking the analog of formula (Trp-1) as an example, the following derivatives are examples of analogs of formula (Trp-1): Similar derivatives are also applicable to the analogs of formulas (Trp-2) to (Trp-4).

[0028] Among these derivatives, R １＿Ｔｒｐ , R ２＿Ｔｒｐ , R ３＿Ｔｒｐ , and m_Trp is R in formula (Trp-1). １＿Ｔｒｐ , R ２＿Ｔｒｐ , R ３＿Ｔｒｐ , and m_Trp.

[0029] Arginine analogs include analogs having any of the following structures: １＿Ａｒｇ and R ２＿Ａｒｇ are each independently a hydrogen atom or an arbitrary substituent; analogs thereof and analogs of arginine corresponding to β-amino acids, γ-amino acids, and δ-amino acids; analogs thereof and homoamino acids of arginine; and analogs thereof and analogs of arginine corresponding to α-alkyl-amino acids (for example, α-alkyl-amino acids having an alkyl group of 1 to 3 carbon atoms, typically α-methyl-amino acids). However, arginine analogs do not include those having the same structure as arginine.

[0030] In the above formula, R １＿Ａｒｇ and R ２＿Ａｒｇ are each independently a hydrogen atom or an arbitrary substituent. Examples of the arbitrary substituent include saturated or unsaturated hydrocarbon groups, hydroxy groups, alkoxy groups, carboxy groups, aldehyde groups, amino groups, amide groups, azide groups, mercapto groups (thiol groups), sulfo groups, halogen groups, and heteroaryl groups. The number of carbon atoms in the arbitrary substituent is not particularly limited, and is, for example, 0 to 20, preferably 0 to 10. R １＿Ａｒｇ and R ２＿ＡｒｇThe substituent in R is preferably a saturated or unsaturated hydrocarbon group, and the number of carbon atoms in the hydrocarbon group is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. The saturated or unsaturated hydrocarbon group is preferably an alkyl group. １＿Ａｒｇ and R ２＿Ａｒｇ Preferred embodiments of include a hydrogen atom and an alkyl group having 1 to 3 carbon atoms, and more preferred embodiments include a hydrogen atom and a methyl group.

[0031] Histidine analogs include, for example, any of the following structures: and analogs of these analogs and histidine corresponding to β-amino acids, γ-amino acids, and δ-amino acids; and analogs of these analogs and histidine corresponding to α-alkyl-amino acids (for example, α-alkyl-amino acids having an alkyl group of 1 to 3 carbon atoms, typically α-methyl-amino acids). However, histidine analogs do not include those having the same structure as histidine.

[0032] In the above formula (His-1), R １＿Ｈｉｓ and R ２＿Ｈｉｓ are each independently a hydrogen atom or an arbitrary substituent. Examples of the arbitrary substituent include saturated or unsaturated hydrocarbon groups, hydroxy groups, alkoxy groups, carboxy groups, aldehyde groups, amino groups, amide groups, azide groups, mercapto groups (thiol groups), sulfo groups, halogen groups, and heteroaryl groups. The number of carbon atoms in the arbitrary substituent is not particularly limited, and is, for example, 0 to 20, preferably 0 to 10. R １＿Ｈｉｓ and R ２＿Ｈｉｓ The substituent in R is preferably a saturated or unsaturated hydrocarbon group, and the number of carbon atoms in the hydrocarbon group is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. The saturated or unsaturated hydrocarbon group is preferably an alkyl group. １＿Ｈｉｓ and R ２＿ＨｉｓPreferred embodiments of R are each independently a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, and more preferred embodiments are a hydrogen atom or a methyl group. ２＿Ｈｉｓ If there are multiple R ２＿Ｈｉｓ may be the same or different.

[0033] In the above formula (His-1), l is an integer of 1 or 2. When l is 2, the above formula (His-1) corresponds to a homoamino acid.

[0034] Examples of tyrosine analogs include those with the following structures: and analogs of these analogs and tyrosine corresponding to β-amino acids, γ-amino acids, and δ-amino acids; and analogs of these analogs and tyrosine corresponding to α-alkyl-amino acids (for example, α-alkyl-amino acids having an alkyl group of 1 to 3 carbon atoms, typically α-methyl-amino acids). However, tyrosine analogs do not include those having the same structure as tyrosine.

[0035] In the above formulas (Tyr-1) and (Tyr-2), R １＿Ｔｙｒ are each independently an arbitrary substituent. Examples of the arbitrary substituent include saturated or unsaturated hydrocarbon groups, hydroxy groups, alkoxy groups, carboxy groups, aldehyde groups, amino groups, amide groups, azide groups, mercapto groups (thiol groups), sulfo groups, halogen groups, and heteroaryl groups. The number of carbon atoms in the arbitrary substituent is not particularly limited, and is, for example, 0 to 20, preferably 0 to 10. R １＿Ｔｙｒ The substituent in R is preferably a saturated or unsaturated hydrocarbon group, and the number of carbon atoms in the hydrocarbon group is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. The saturated or unsaturated hydrocarbon group is preferably an alkyl group. １＿Ｔｙｒ If there are multiple R １＿Ｔｙｒ may be the same or different.

[0036] In the above formula (Tyr-1), R ２＿Ｔｙｒare each independently a hydrogen atom or an arbitrary substituent. Examples of the arbitrary substituent include saturated or unsaturated hydrocarbon groups, hydroxy groups, alkoxy groups, carboxy groups, aldehyde groups, amino groups, amide groups, azide groups, mercapto groups (thiol groups), sulfo groups, halogen groups, and heteroaryl groups. The number of carbon atoms in the arbitrary substituent is not particularly limited, and is, for example, 0 to 20, preferably 0 to 10. ２＿Ｔｙｒ In each R ２＿Ｔｙｒ may be the same or different. ２＿Ｔｙｒ The substituent in R is preferably a saturated or unsaturated hydrocarbon group, and the number of carbon atoms in the hydrocarbon group is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. The saturated or unsaturated hydrocarbon group is preferably an alkyl group. ２＿Ｔｙｒ Preferred embodiments of include a hydrogen atom and an alkyl group having 1 to 3 carbon atoms, and more preferred embodiments include a hydrogen atom and a methyl group.

[0037] In the above formula (Tyr-1), m_Tyr represents an integer of 0 to 4, preferably 0 to 3, more preferably 0 to 2, even more preferably 0 or 1, and even more preferably 0. In the above formula (Tyr-1), l is an integer of 1 or 2. When l is 2, the above formula (Tyr-1) corresponds to a homoamino acid. In the above formula (Tyr-1), the OH group bonded to the benzene ring is preferably bonded at the para position relative to the part to which the amino acid main chain is bonded.

[0038] In the above formula (Tyr-2), m_Tyr represents an integer of 0 to 3, preferably 0 to 2, more preferably 0 to 1, and even more preferably 0.

[0039] Examples of serine analogs include those with the following structure: and analogs of the analogs and serine corresponding to β-amino acids, γ-amino acids, and δ-amino acids; and analogs of the analogs and serine corresponding to α-alkyl-amino acids (for example, α-alkyl-amino acids having an alkyl group of 1 to 3 carbon atoms, typically α-methyl-amino acids). However, serine analogs do not include those having the same structure as serine. In addition, in formula (Ser-1), analogs of the analogs having one R １＿Ｓｅｒ is a hydrogen atom, and one R １＿Ｓｅｒ When is a methyl group, the structure of formula (Ser-1) corresponds to threonine, and therefore such a structure is also excluded from analogs of serine.

[0040] In the above formula (Ser-1), R １＿Ｓｅｒ are each independently a hydrogen atom or an arbitrary substituent. Examples of the arbitrary substituent include saturated or unsaturated hydrocarbon groups, hydroxy groups, alkoxy groups, carboxy groups, aldehyde groups, amino groups, amide groups, azide groups, mercapto groups (thiol groups), sulfo groups, halogen groups, and heteroaryl groups. The number of carbon atoms in the arbitrary substituent is not particularly limited, and is, for example, 0 to 20, preferably 0 to 10. １＿Ｓｅｒ In each R １＿Ｓｅｒ may be the same or different. １＿Ｓｅｒ The substituent in R is preferably a saturated or unsaturated hydrocarbon group, and the number of carbon atoms in the hydrocarbon group is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. The saturated or unsaturated hydrocarbon group is preferably an alkyl group. １＿Ｓｅｒ Preferred embodiments of include a hydrogen atom and an alkyl group having 1 to 3 carbon atoms, and more preferred embodiments include a hydrogen atom and a methyl group.

[0041] In the above formula (Ser-1), l is an integer of 1 to 4, and may be 1 or 2. When l is 2, the above formula (Ser-1) corresponds to a homoamino acid.

[0042] Examples of threonine analogs include those having the following structure: and analogs of these analogs and threonine corresponding to β-amino acids, γ-amino acids, and δ-amino acids; and analogs of these analogs and threonine corresponding to α-alkyl-amino acids (for example, α-alkyl-amino acids having an alkyl group of 1 to 3 carbon atoms, typically α-methyl-amino acids). However, threonine analogs do not include those having the same structure as threonine.

[0043] In the above formula (Thr-1), R １＿Ｔｈｒ are each independently a hydrogen atom or an arbitrary substituent. Examples of the arbitrary substituent include saturated or unsaturated hydrocarbon groups, hydroxy groups, alkoxy groups, carboxy groups, aldehyde groups, amino groups, amide groups, azide groups, mercapto groups (thiol groups), sulfo groups, halogen groups, and heteroaryl groups. The number of carbon atoms in the arbitrary substituent is not particularly limited, and is, for example, 0 to 20, preferably 0 to 10. １＿Ｔｈｒ In each R １＿Ｔｈｒ may be the same or different. １＿Ｔｈｒ The substituent in R is preferably a saturated or unsaturated hydrocarbon group, and the number of carbon atoms in the hydrocarbon group is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. The saturated or unsaturated hydrocarbon group is preferably an alkyl group. １＿Ｔｈｒ Preferred embodiments of include a hydrogen atom and an alkyl group having 1 to 3 carbon atoms, and more preferred embodiments include a hydrogen atom and a methyl group.

[0044] In the above formula (Thr-1), l_Thr is an integer of 0 or 1. When l is 1, the above formula (Thr-1) corresponds to a homoamino acid.

[0045] Prenylation of a tryptophan residue occurs, for example, on the indole ring of the side chain. Modes of prenylation include C3 prenylation, C6 prenylation, C7 prenylation, and N1 prenylation. The mode of prenylation can be selected depending on the prenylation enzyme used for prenylation. C6 prenylation, C7 prenylation, and N1 prenylation of tryptophan or its analogs respectively produce the following structures. Note that the group represented by Prenyl is a prenyl group.

[0046] Furthermore, C3 prenylation forms the following structure in which the main chain nitrogen is alkylated: In the following structure, the prenyl group may be a reverse prenyl group instead of a forward prenyl group, but a forward prenyl group is preferred.

[0047] The configuration of the structure is not particularly limited, but may be, for example, the configurations represented by the following formulas (T1-1) and (T1-2), and preferably the configuration represented by the following formula (T1-1).

[0048] Furthermore, when tryptophan analogs represented by formula (Trp-1), formula (Trp-2), and formula (Trp-3) are C3 prenylated, structures represented by the following formulas (Trp-1-Prenyl), (Trp-2-Prenyl), and (Trp-3-Prenyl), respectively, are obtained, taking the forward prenyl group as an example.

[0049] In the formula (Trp-1-Prenyl), the formula (Trp-2-Prenyl), and the formula (Trp-3-Prenyl), ring B, R １＿Ｔｒｐ , R ２＿Ｔｒｐ , R ３＿Ｔｒｐ , R ４＿Ｔｒｐ , m_Trp, l, and k are as defined above.

[0050] Prenylation of tyrosine, serine, and threonine residues occurs, for example, at the oxygen atoms of the side chains, and prenylation of arginine residues occurs, for example, at the imino and primary amino groups (NH ２Prenylation of arginine residues occurs at least on one of the imino and primary amino groups (NH ２ The aryl group may be substituted for both the aryl and aryl groups.

[0051] Prenylation of a histidine residue may occur, for example, at the carbon of the imidazole ring of the side chain. Prenylation of a histidine residue may occur at the C2 carbon of the imidazole ring of the side chain. When a histidine analog represented by formula (His-1) or formula (His-2) is C2-forward prenylated, a histidine analog having a structure represented by the following formula (His-Prenyl) is obtained.

[0052] As will be described later, the carboxy group of the C-terminal amino acid of Z may have a prenyl group. The prenylation of the carboxy group may occur in the form of —C(═O)—O-Prenyl.

[0053] In formula (I), the amino acid having a prenyl group is C3-prenylated, C6-prenylated, C7-prenylated, or N1-prenylated tryptophan and its analogs, an imino group in the side chain, and a primary amino group (NH ２ Preferably, the prenyl group-containing amino acid is a residue of an amino acid selected from the group consisting of arginine and analogs thereof in which at least one of the oxygen atoms in the side chain is prenylated, C2-prenylated histidine and analogs thereof, tyrosine and analogs thereof in which the oxygen atom in the side chain is prenylated, serine and analogs thereof in which the oxygen atom in the side chain is prenylated, and threonine and analogs thereof in which the oxygen atom in the side chain is prenylated. The amino acid having a prenyl group is preferably a residue of an amino acid selected from the group consisting of C3-prenylated, C6-prenylated, C7-prenylated, or N1-prenylated tryptophan and analogs thereof, and a residue of an amino acid selected from the group consisting of C3-prenylated, C6-prenylated, C7-prenylated, or N1-prenylated tryptophan and analogs thereof, a residue of an imino group and a primary amino group (NH ２ Preferably, at least one of the amino acid residues (amino acid residues) is a residue of an amino acid selected from prenylated arginine and analogs thereof, C2-prenylated histidine and analogs thereof, and tyrosine and analogs thereof in which the oxygen atom of the side chain is prenylated.

[0054] In formula (I), X ａ , X ｂ , X ｃ, m X ｄ , and n X ｅ are each independently an amino acid or an analog thereof. ｄ , and n X ｅ In formula (I), X may be the same or different and independently represent an amino acid residue selected from the group consisting of any amino acid and its analog. ａ , X ｂ , X ｃ and Z each contain at least one amino acid having a prenyl group, but m X ｄ , and n X ｅ In the above, an amino acid having a prenyl group may be included. ｄ ) ｍ -X ａ -(X ｅ ) ｎ When one amino acid having a prenyl group is contained in -, the amino acid having a prenyl group is ａ Also, it corresponds to -(X ｄ ) ｍ -X ａ -(X ｅ ) ｎ When two or more amino acids having a prenyl group are contained in -, one amino acid having a prenyl group is X ａ The remaining amino acids having prenyl groups correspond to m X ｄ , and n X ｅ will be included in the

[0055] As used herein, the term "amino acid or analog thereof" means that in addition to the amino acids defined above, it may also include, for example, α,β-unsaturated amino acids, azolines, and azoles derived from amino acids.

[0056] An α,β-unsaturated amino acid refers to an amino acid in which an unsaturated double bond is formed between the α-carbon and the β-carbon adjacent to the α-carbon, as shown in the structure below. The α,β-unsaturated amino acid may be a single isomer of the E or Z isomer, or a mixture of the E and Z isomers. (In the formula, R ａ is any organic group.

[0057] When an α,β-unsaturated amino acid as an amino acid analog is contained in a peptide, it is specifically included as the following structure, analog (I). Analog (I) may be a single isomer of E or Z isomer, or a mixture of E and Z isomers, but is preferably an isomer having NH and R Ｘ１ is a single isomer in the cis configuration. (R Ｘ１ is any organic group, preferably a hydrogen atom or a hydrocarbon group.) In this specification, unless otherwise defined, the "hydrocarbon group" may be a saturated or unsaturated hydrocarbon group, and the number of carbon atoms in the hydrocarbon group is preferably 1 or more and 10 or less, more preferably 1 or more and 5 or less, and even more preferably 1 or more and 3 or less.

[0058] When an azole is included as an amino acid analog in a peptide, it is specifically included as the following structure analog (II). When an azoline is included as an amino acid analog in a peptide, it is specifically included as the following structure analog (III). (In analog (II) and analog (III), B Ｘ is an oxygen atom, a sulfur atom, or an NH group, and R Ｘ２ is a hydrogen atom or a hydrocarbon group.

[0059] R of analog (I), analog (II), and analog (III) Ｘ１ and R Ｘ２ The hydrocarbon group in R is preferably an alkyl group having 1 to 10 carbon atoms which may have a substituent, or an aromatic group which may have a substituent, and is preferably an alkyl group having 1 to 6 carbon atoms or a phenyl group which may have a substituent, more preferably an alkyl group having 1 to 4 carbon atoms or a phenyl group, and even more preferably a methyl group. Ｘ１ and R Ｘ２ Examples of the substituent in the hydrocarbon group of R include an alkyl group, an aromatic group, a halogen group, an azide group, and an amino group, and preferably an alkyl group having 1 to 4 carbon atoms, and more preferably a methyl group. Ｘ１ and RＸ２ Preferred embodiments of include a hydrogen atom and an alkyl group having 1 to 3 carbon atoms, and more preferred embodiments include a hydrogen atom and a methyl group.

[0060] In formula (I), X ａ , X ｂ , X ｃ , m X ｄ , and n X ｅ are preferably each independently selected from the 20 proteinogenic amino acids and D-amino acids of the 19 proteinogenic amino acids excluding glycine, cysteine ​​analogs, serine or threonine analogs, diaminopropionic acid (Dap) and analogs thereof, α,β-unsaturated amino acids and prenylated analogs thereof, and azoles. Azolines may be contained instead of or in addition to azoles. Prenylated amino acid analogs include C3-prenylated, C6-prenylated, C7-prenylated, or N1-prenylated tryptophan and analogs thereof, and analogs of tryptophan with a side chain imino group and a primary amino group (NH ２ Preferably, the amino acid sequence is selected from C3-, C6-, C7-, or N1-prenylated tryptophan and analogs thereof, at least one of an imino group and a primary amino group (NH) in the side chain is prenylated, C2-prenylated histidine and analogs thereof, tyrosine and analogs thereof, serine and analogs thereof, and threonine and analogs thereof, at least one of an imino group and a primary amino group (NH) in the side chain is prenylated. ２ It is more preferred that at least one of the C1, C2, and C3 groups is selected from arginine and analogs thereof in which the C2 and C3 groups are prenylated, histidine and analogs thereof in which the C2 and C3 groups are prenylated, and tyrosine and analogs thereof in which the oxygen atom of the side chain is prenylated.

[0061] In formula (I), X ａ , X ｂ , X ｃ , m X ｄ , and n X ｅExamples of cysteine ​​analogs in the above formula include, but are not limited to, those represented by the following formula: (In the formula, R is a hydrogen atom or a hydrocarbon group.)

[0062] In formula (I), X ａ , X ｂ , X ｃ , m X ｄ , and n X ｅ Examples of serine or threonine analogs in the above formula include, but are not limited to, those represented by the following formula: (In the formula, R is a hydrogen atom or a hydrocarbon group.)

[0063] In formula (I), X ａ , X ｂ , X ｃ , m X ｄ , and n X ｅ Examples of analogs of diaminopropionic acid (Dap) in the above formula include, but are not limited to, those represented by the following formula: (In the formula, R is a hydrogen atom or a hydrocarbon group.)

[0064] As will be described later, cysteine, serine, threonine, diaminopropionic acid (Dap) and analogs thereof can be derivatized into azolines and azoles.

[0065] In formula (I), X ａ , X ｂ , X ｃ , m X ｄ , and n X ｅIn the above structure, the hydrocarbon group represented by R as an analog of cysteine, serine, threonine, and diaminopropionic acid (Dap) is preferably an alkyl group having 1 to 10 carbon atoms which may have a substituent, or an aromatic group which may have a substituent, preferably an alkyl group having 1 to 6 carbon atoms, or a phenyl group which may have a substituent, more preferably an alkyl group having 1 to 4 carbon atoms, or a phenyl group, and even more preferably a methyl group. Examples of the substituent on the hydrocarbon group include an alkyl group, an aromatic group, a halogen group, an azide group, an amino group, etc., preferably an alkyl group having 1 to 4 carbon atoms, more preferably a methyl group.

[0066] In formula (I), X ａ is preferably an amino acid having a prenyl group. ｂ is preferably the above analog (I), and more preferably R Ｘ１ is a hydrogen atom. ｃ is preferably an amino acid other than an acidic amino acid or an analog thereof, and more preferably an azole. ｃ is an azole, the cyclic compound of formula (I) is Ｃ In the following structure, X ｃ Except for this, it is the same as formula (I).

[0067] Formula (IX Ｃ ) Medium, B Ｃ is an oxygen atom, a sulfur atom, or an NH group, and R Ｃ is a hydrogen atom or a hydrocarbon group. Ｃ ) in R Ｃ Examples and preferred embodiments (embodiments described as preferred, more preferred, even more preferred, even more preferred, etc.; the same applies hereinafter in this specification) are R １ The same as Formula (I-X Ｃ ) in B Ｃ is preferably a sulfur atom.

[0068] In formula (I), Z may or may not be present, and when present, it is a peptide consisting of 1 to 100 amino acids and / or analogs thereof. The presence or absence and sequence of Z do not significantly affect the activity of prenylation enzymes or macrocyclases when producing the cyclic compound of this embodiment, as will be described later. In formula (I), when Z is present, its sequence is not particularly limited, but is preferably a peptide consisting of 1 to 100 amino acids and / or analogs thereof, more preferably a peptide consisting of 1 to 50 amino acids and / or analogs thereof, even more preferably a peptide consisting of 1 to 40 amino acids and / or analogs thereof, and even more preferably a peptide consisting of 3 to 30 amino acids and / or analogs thereof. The amino acid contained in Z may have a prenyl group. An amino acid having a prenyl group is an amino acid that is present at the N-terminus (i.e., X ｃ It may be present either at the N-terminus (i.e., the amino acid adjacent to ring A) or at the C-terminus (i.e., the amino acid most distant from ring A), or it may be present in the peptide chain between the N-terminus and C-terminus. When the C-terminus of Z is prenylated, the C-terminal carboxylic acid may be prenylated.

[0069] In formula (I), when Z is present, the amino acids and / or analogs thereof forming Z are preferably each independently selected from the 20 proteinogenic amino acids and D-amino acids of the 19 proteinogenic amino acids excluding glycine, cysteine ​​analogs, serine or threonine analogs, diaminopropionic acid (Dap) and analogs thereof, α,β-unsaturated amino acids and prenylated analogs thereof, and azoles. Azoline may be contained instead of or in addition to azoles. Prenylated amino acid analogs include C3-prenylated, C6-prenylated, C7-prenylated, or N1-prenylated tryptophan and analogs thereof, and analogs of tryptophan having a side chain imino group and a primary amino group (NH ２Preferably, the amino acid sequence is selected from C3-, C6-, C7-, or N1-prenylated tryptophan and analogs thereof, at least one of an imino group and a primary amino group (NH) in the side chain is prenylated, C2-prenylated histidine and analogs thereof, tyrosine and analogs thereof, serine and analogs thereof, and threonine and analogs thereof, at least one of an imino group and a primary amino group (NH) in the side chain is prenylated. ２ It is more preferred that at least one of the C1, C2, and C3 groups is selected from arginine and analogs thereof in which the C2 and C3 groups are prenylated, histidine and analogs thereof in which the C2 and C3 groups are prenylated, and tyrosine and analogs thereof in which the oxygen atom of the side chain is prenylated.

[0070] In formula (I), m and n each independently represent an integer selected from 0 to 39, and preferably an integer of 1 to 23, 1 to 19, 2 to 19, 1 to 15, or 2 to 15. Furthermore, in formula (I), the sum of m and n is an integer selected from 1 to 39, and preferably an integer of 1 to 23, 1 to 19, 2 to 19, 3 to 19, 4 to 19, 1 to 15, 2 to 15, 3 to 15, or 4 to 15. When m and n are within the above ranges, cyclization tends to proceed smoothly when a macrocyclase is allowed to act, and prenylation tends to proceed smoothly when a prenylation enzyme is allowed to act.

[0071] In formula (I), ring A is a nitrogen-containing 6-membered ring which may have a substituent, and the nitrogen-containing 6-membered ring which may have a substituent is not particularly limited as long as it is a nitrogen-containing 6-membered ring which is generated by the reaction of formula (1) and formula (2) described below, i.e., 2+4 cycloaddition. Examples of such a nitrogen-containing 6-membered ring include the following structures. (In the formula, R Ａ１ and R Ａ２ are each independently a hydrogen atom or a hydrocarbon group.

[0072] R in the above structure given as the nitrogen-containing 6-membered ring structure Ａ１ and R Ａ２The hydrocarbon group in R is preferably an alkyl group having 1 to 10 carbon atoms which may have a substituent, or an aromatic group which may have a substituent, and is preferably an alkyl group having 1 to 6 carbon atoms or a phenyl group which may have a substituent, more preferably an alkyl group having 1 to 4 carbon atoms or a phenyl group, and even more preferably a methyl group. Ａ１ and R Ａ２ Examples of the substituent in the hydrocarbon group of R include an alkyl group, an aromatic group, a halogen group, an azide group, and an amino group, and preferably an alkyl group having 1 to 4 carbon atoms, and more preferably a methyl group. Ａ１ and R Ａ２ is preferably a hydrogen atom.

[0073] In formula (I), B １ is an oxygen atom, a sulfur atom, or an NH group, and is preferably an oxygen atom.

[0074] In formula (I), R １ is a hydrogen atom or a hydrocarbon group. １ The hydrocarbon group in R is preferably an alkyl group having 1 to 10 carbon atoms which may have a substituent, or an aromatic group which may have a substituent, and is preferably an alkyl group having 1 to 6 carbon atoms or a phenyl group which may have a substituent, more preferably an alkyl group having 1 to 4 carbon atoms or a phenyl group, and even more preferably a methyl group. １ Examples of the substituent in the hydrocarbon group of R include an alkyl group, an aromatic group, a halogen group, an azide group, and an amino group, and preferably an alkyl group having 1 to 4 carbon atoms, and more preferably a methyl group. １ is preferably a methyl group or a hydrogen atom.

[0075] In formula (I), the peptide bond between amino acids is omitted. ｄ ) ｍ and ring A are the N-terminal X ｄ and a carboxy group bonded to ring A, or ｄ and a carboxy group bonded to ring A to form an azoline or azole, the azoline or azole and ring A are linked by a single bond.ｂ and the azoline or azole bonded to ring A are X ｂ The carboxyl group of X is bonded to the azoline or azole so as to form a part of the azoline or azole. ｂ For example, when X is an α-amino acid, the α-carbon and the azoline or azole bonded to ring A are connected by a single bond. ｃ That is, X ｃ and a carboxy group bonded to ring A, or ｃ and a carboxy group bonded to ring A to form an azoline or azole, the azoline or azole and ring A are linked by a single bond.

[0076] As described below, the cyclic compound of formula (I) can be produced, for example, by applying multiple enzymatic reactions to a precursor peptide. Examples of enzymatic reactions include an azole ring formation reaction, an α,β-unsaturated amino acid formation reaction, a macrocyclization reaction, and a prenylation reaction. The amino acid sequence of the precursor peptide may be designed to increase the yield of these enzymatic reactions. As a result, the cyclic compound of formula (I) preferably has the following characteristics. For such design, reference may be made to, for example, J. Am. Chem. Soc. 2020, 142, 20329-20334.

[0077] In formula (I), m X ｄ Among them, the N-terminal X ｄ , that is, X adjacent to ring A ｄ is preferably an amino acid other than an acidic amino acid, more preferably an aromatic amino acid, and even more preferably Trp or an analog thereof. ｄ may be prenylated, an α,β-unsaturated bond may be formed, and an azoline or an azole may be formed. ｄ When X adjacent to ring A is an amino acid other than an acidic amino acid, the macrocyclization reaction tends to proceed favorably. ｄ In the formulas (II) and (III) described below, X and X′ are adjacent to each other. ｄ and X ｄ' corresponds to '.

[0078] As used herein, an acidic amino acid refers to an amino acid that exhibits acidity, for example, an amino acid having an acidic functional group such as a carboxyl group in its side chain. Taking proteinogenic amino acids as an example, acidic amino acids include Asp and Glu. Examples of amino acids other than acidic amino acids include proteinogenic amino acids such as Arg, His, Lys, Ser, Thr, Asn, Gln, Cys, Gly, Pro, Ala, Ile, Leu, Met, Phe, Trp, Tyr, and Val.

[0079] As used herein, the term "aromatic amino acid" refers to an amino acid having an aromatic ring in the side chain. Taking proteinogenic amino acids as examples, aromatic amino acids include Phe, Trp, His, and Tyr.

[0080] In formula (I), m X ｄ The second X from the N-terminus ｄ , that is, the two C-terminal Xs of ring A ｄ is preferably an amino acid with little steric hindrance. ｄ may be prenylated, an α,β-unsaturated bond may be formed, and an azoline or an azole may be formed. ｄ However, if the amino acid has little steric hindrance, the macrocyclization reaction tends to proceed favorably. ｄ In the formulas (II) and (III) described below, the two C-terminal Xs of X and X' are ｄ and X ｄ ' corresponds to '.

[0081] In this specification, an amino acid with little steric hindrance means an amino acid with little steric hindrance in its side chain, and examples thereof include amino acids whose side chains have four or fewer atoms other than hydrogen atoms. Examples of proteinogenic amino acids with little steric hindrance include Gly, Ala, Ser, Pro, Val, Thr, Cys, Leu, Ile, Asn, Asp, and Met.

[0082] In formula (I), n X ｅ Among them, the C-terminal Xｅ , i.e., X ｂ X adjacent to ｅ is preferably an amino acid other than an acidic amino acid. ｂ X adjacent to ｅ may be prenylated, an α,β-unsaturated bond may be formed, or an azoline or an azole may be formed. ｂ X adjacent to ｅ When is an amino acid other than an acidic amino acid, the macrocyclization reaction tends to proceed favorably.

[0083] In formula (I), n X ｅ The second X from the C-terminus ｅ , i.e., X ｂ The two N-terminal X ｅ is preferably an amino acid other than an acidic amino acid or a basic amino acid, i.e., a neutral amino acid. ｂ The two N-terminal X ｅ may be prenylated, an α,β-unsaturated bond may be formed, or an azoline or an azole may be formed. ｂ The two N-terminal X ｅ When is a neutral amino acid, the macrocyclization reaction tends to proceed favorably.

[0084] As used herein, a basic amino acid refers to an amino acid that exhibits basicity, for example, an amino acid having a basic functional group such as an amino group in its side chain. Examples of proteinogenic amino acids include Arg, Lys, and His. Examples of neutral amino acids include proteinogenic amino acids such as Gly, Ala, Phe, Leu, Ile, Cyc, Met, Tyr, Val, Thr, Ser, Pro, Trp, Asn, and Gln.

[0085] In formula (I), when Z is present, the C-terminal amino acid, i.e., X ｃThe amino acid adjacent to is preferably an amino acid other than an acidic amino acid. The amino acid or analog thereof constituting Z may be prenylated, may have an α,β-unsaturated bond formed therein, or may form an azoline or an azole. When the C-terminal amino acid of the amino acids or analogs thereof constituting Z is an amino acid other than an acidic amino acid, the macrocyclization reaction tends to proceed favorably.

[0086] In formula (I), X ａ and the amino acid adjacent to X ａ The amino acid adjacent to the amino acid (X ａ The amino acid next to it is X ａ and X ａ The prenylation enzyme for prenylation of X can be appropriately selected. ａ and the amino acid adjacent to X ａ The design of these four amino acids and the prenylation enzyme, which are adjacent to each other via one amino acid, will be described later. These four amino acids may be prenylated, may form an α,β-unsaturated bond, or may form an azoline or azole.

[0087] [Method for Producing Cyclic Compound] The method for producing the cyclic compound represented by formula (I) of this embodiment comprises the steps of: ｄ ) ｍ -X ａ '-(X ｅ ) ｎ -X ｂ -Y-X ｃ -Z (II) is treated with a macrocyclase to form a nitrogen-containing six-membered ring A while eliminating LP if present, to obtain a peptide represented by formula (I'): and forming a cyclic compound represented by formula (I') with a prenylation enzyme to form at least X ａ ', X ｂ , X ｃ and Z.

[0088] In formula (II), X represents a group represented by formula (1): (R ２ represents a hydrogen atom or a hydrocarbon group; Y represents a group represented by formula (2): (R １ and B １ has the same meaning as above, and R ３ represents a hydrogen atom or a hydrocarbon group; ａ X' is an amino acid or an analog thereof; ｂ , X ｃ , m X ｄ , n X's ｅ , m, n, and Z are X in the above formula (I). ｂ , X ｃ , m X ｄ , n X's ｅ , m, n, and Z; LP may be present or absent, and if present, is a peptide consisting of 1-100 amino acids and / or analogs thereof.

[0089] In addition, in formula (I'), X ａ ', X ｂ , X ｃ , m X ｄ , n X's ｅ , m, n, Z, ring A, B １ , and R １ represents X in the above formulas (I) and (II). ａ ', X ｂ , X ｃ , m X ｄ , n X's ｅ , m, n, Z, ring A, B １ , and R １ is synonymous with.

[0090] As described above, the production method of this embodiment can be used as a general-purpose production method for the cyclic compound represented by formula (I) above by carrying out the formation of the nitrogen-containing six-membered ring A by a macrocyclization enzyme and prenylation by a prenylation enzyme in this order. This is thought to be because macrocyclization enzymes have low tolerance for substrates containing prenyl groups, whereas prenylation enzymes have high tolerance for the cyclic compound of formula (I') above. Therefore, this embodiment also demonstrates the high substrate tolerance of prenylation enzymes.

[0091] (Macrocyclization Reaction) The production method of this embodiment involves allowing a macrocyclization enzyme to act on the peptide represented by formula (II) above, and forming a nitrogen-containing six-membered ring A while eliminating LP, if present, to form a cyclic compound represented by formula (I') above (hereinafter referred to as "macrocyclization reaction"). In the macrocyclization reaction, X and Y in formula (II) undergo 2+4 cycloaddition to generate a nitrogen-containing six-membered ring.

[0092] In formula (II), X is not particularly limited as long as it is a group represented by formula (1) above, and may be a single isomer of E or Z isomer, or a mixture of E and Z isomers. In formula (II), X is a group having the following structure: It is preferable that:

[0093] In formula (1), R ２ is a hydrogen atom or a hydrocarbon group. ２ The hydrocarbon group in R is preferably an alkyl group having 1 to 10 carbon atoms which may have a substituent, or an aromatic group which may have a substituent, preferably an alkyl group having 1 to 6 carbon atoms, or a phenyl group which may have a substituent, more preferably an alkyl group having 1 to 4 carbon atoms, or a phenyl group, and even more preferably a methyl group. Examples of the substituent in the hydrocarbon group include an alkyl group, an aromatic group, a halogen group, an azide group, an amino group, etc., preferably an alkyl group having 1 to 4 carbon atoms, more preferably a methyl group. ２ Preferred embodiments of include a hydrogen atom and an alkyl group having 1 to 3 carbon atoms, and more preferred embodiments include a hydrogen atom and a methyl group.

[0094] In formula (II), Y is not particularly limited as long as it is a group represented by formula (2), and R ３ In formula (II), Y may be a single isomer of E or Z isomer, or a mixture of E and Z isomers. It is preferable that:

[0095] B in formula (2) １ and R １ Examples and preferred embodiments of the compound are B in formula (I). １ and R １ is the same as:

[0096] In formula (2), R ３ is a hydrogen atom or a hydrocarbon group. ３ The hydrocarbon group in R is preferably an alkyl group having 1 to 10 carbon atoms which may have a substituent, or an aromatic group which may have a substituent, preferably an alkyl group having 1 to 6 carbon atoms, or a phenyl group which may have a substituent, more preferably an alkyl group having 1 to 4 carbon atoms, or a phenyl group, and even more preferably a methyl group. Examples of the substituent in the hydrocarbon group include an alkyl group, an aromatic group, a halogen group, an azide group, an amino group, etc., preferably an alkyl group having 1 to 4 carbon atoms, more preferably a methyl group. ３ Preferred embodiments of include a hydrogen atom and an alkyl group having 1 to 3 carbon atoms, and more preferred embodiments include a hydrogen atom and a methyl group.

[0097] In formula (II), X ａ X' is not particularly limited as long as it is an amino acid or an analog thereof. ａ X' can be prenylated by the prenylation reaction described below. ａ If X' is not prenylated, ｂ , X ｃ At least one amino acid contained in any of X, Z, and Z is prenylated. ａ') is preferably tryptophan, arginine, histidine, tyrosine, serine, or threonine, or an analog thereof, more preferably tryptophan, arginine, histidine, or tyrosine, or an analog thereof. The analog of each amino acid has the same meaning as defined above.

[0098] X in formula (II) ｂ , X ｃ , m X ｄ , n X's ｅ , m, n, and Z are exemplified and preferred embodiments of X in formula (I) except for the following points. ｂ , X ｃ , m X ｄ , n X's ｅ , m, n, and Z. In the peptide represented by formula (II) used in the production of the cyclic compound represented by formula (I), X ｂ , X ｃ , m X ｄ , n X's ｅ , m, n, and Z may be the same as or different from those of the cyclic compound represented by formula (I). ａ ', X ｂ , X ｃ , m X ｄ , n X's ｅ In the above, and Z, the amino acid or analogue thereof is preferably selected from non-prenylated amino acids or analogues thereof.

[0099] In formula (II), -X ｂ -Y- preferably has the following structure: R １ , B １ , R ３ and R Ｘ１ has the same meaning as above. １ , B １ , R ３ and R Ｘ１ Examples and preferred embodiments of the formula (I) are １ and B １ , R in formula (2) ３ , and R in analog(II) Ｘ１ is the same as:

[0100] In formula (II), -Y-X ｃ - preferably has the following structure: R １ , R ３ , R Ｃ , B １ and B Ｃ has the same meaning as above. １ and R ３ Examples and preferred embodiments of the formula (I) are １ and B １ is the same as R ３ Examples and preferred embodiments of the formula (2) are ３ is the same as R Ｃ and B Ｃ Examples and preferred embodiments of the compound of formula (I-X) Ｃ ) in R Ｃ and B Ｃ is the same as:

[0101] In formula (II), -X ｂ -Y-X ｃ - preferably has the following structure: R １ , R ３ , R Ｘ１ , R Ｃ , B １ and B Ｃ has the same meaning as above. １ and R ３ Examples and preferred embodiments of the formula (I) are １ and B １ is the same as R ３ Examples and preferred embodiments of the formula (2) are ３ is the same as R Ｘ１ Examples and preferred embodiments of the compound are R in analog (II). Ｘ１ is the same as R Ｃ and B Ｃ Examples and preferred embodiments of the compound of formula (I-X) Ｃ ) in R Ｃ and B Ｃ is the same as:

[0102] In formula (II), -Xｂ -Y-X ｃ - has the following structure: or the following structure: or more preferably has the following structure: Even more preferably, it has the following structure: In addition, R １ , R ３ , R Ｘ１ , R Ｃ , B １ and B Ｃ has the same meaning as above.

[0103] In formula (II), LP is a leader peptide sequence and represents a peptide consisting of 1 to 100 amino acids and / or analogs thereof. LP may or may not be present. The presence of LP makes it easier for enzymes such as macrocyclases, azole ring-forming enzymes, and α,β-unsaturated amino acid-forming enzymes to recognize it, and the reaction tends to proceed smoothly. The number of amino acids and / or analogs thereof constituting LP is preferably 11 to 100, 11 to 60, or 16 to 45 in order to further enhance substrate recognition by modifying enzymes. For details of LP, see International Publication No. WO 2020 / 080490.

[0104] The sequence of LP is not particularly limited, but it is preferably a peptide in which the amino acids -20 to -1 (-1 is adjacent to X) in formula (II), where X is the first amino acid, are -(-20)LSELTVTSLRDTVALPENGA(-1)- (SEQ ID NO: 74) in order from the N-terminus, or a peptide having homology to this peptide. The sequence identity of LP to the peptide -LSELTVTSLRDTVALPENGA- is preferably 50% or more, more preferably 70% or more, and even more preferably 90% or more. The sequence of LP is more preferably a peptide in which the amino acids -30 to -1, with X as the 1st position, are -(-30)VESLDLQDLDLSELTVTSLRDTVALPENGA(-1)- (SEQ ID NO: 75), in order from the N-terminus, and even more preferably a peptide in which the amino acids -38 to -1, with X as the 1st position, are -(-38)MSDITASRVESLDLQDLDLSELTVTSLRDTVALPENGA(-1)- (SEQ ID NO: 76), in order from the N-terminus, or a peptide having homology to this peptide. The sequence identity to these peptides is preferably 50% or more, more preferably 70% or more, and even more preferably 90% or more.

[0105] In LP, the amino acid at position -17, with X at the first position in formula (II), is preferably a hydrophobic amino acid, more preferably leucine or an analog thereof, and even more preferably leucine. In LP, the amino acid at position -20, with X at the first position in formula (II), is preferably a hydrophobic amino acid, more preferably leucine or an analog thereof, and even more preferably leucine. In LP, the amino acid at position -21, with X at the first position in formula (II), is preferably an acidic amino acid, more preferably aspartic acid or an analog thereof, and even more preferably aspartic acid. In LP, the amino acids at positions -17 and -20, with X at the first position in formula (II), are preferably hydrophobic amino acids, more preferably leucine or an analog thereof, and even more preferably leucine. In LP, it is preferred that, with X in formula (II) as the 1st position, the amino acids at positions −17 and −20 are hydrophobic amino acids and the amino acid at position −21 is an acidic amino acid, it is more preferred that the amino acids at positions −17 and −20 are leucine or an analog thereof and the amino acid at position −21 is aspartic acid or an analog thereof, it is even more preferred that the amino acids at positions −17 and −20 are leucine and the amino acid at position −21 is aspartic acid.

[0106] Leucine analogs include β-amino acid, γ-amino acid, and δ-amino acid analogs of leucine; homoleucine; and analogs of these analogs and α-alkyl-amino acids of leucine (e.g., α-alkyl-amino acids having an alkyl group of 1 to 3 carbon atoms, typically α-methyl-amino acids). Aspartic acid analogs include β-amino acid, γ-amino acid, and δ-amino acid analogs of aspartic acid; homoaspartic acid; and analogs of these analogs and α-alkyl-amino acids of aspartic acid (e.g., α-alkyl-amino acids having an alkyl group of 1 to 3 carbon atoms, typically α-methyl-amino acids).

[0107] X in formula (I') formed by macrocyclization reaction ａExamples and preferred embodiments of X′ in formula (II) are ａ The same applies to X in formula (I'). ｂ , X ｃ , m X ｄ , n X's ｅ , m, n, Z, ring A, B １ , and R １ The examples and preferred embodiments of the formula (I) are the same as those of the formula (I) except for the following points: ｂ , X ｃ , m X ｄ , n X's ｅ , m, n, Z, ring A, B １ , and R １ In the production of the cyclic compound represented by formula (I), in the peptide represented by formula (I′) obtained by macrocyclization reaction, X ｂ , X ｃ , m X ｄ , n X's ｅ , m, n, Z, ring A, B １ , and R １ may be the same as or different from the cyclic compound represented by formula (I). ａ ', X ｂ , X ｃ , m X ｄ , n X's ｅ In the above, and Z, the amino acid or analogue thereof is preferably selected from non-prenylated amino acids or analogues thereof.

[0108] The macrocyclization enzyme in the macrocyclization reaction preferably includes LazC. As the macrocyclization enzyme, an enzyme having homology to LazC can also be used. As the macrocyclization enzyme, the macrocyclization enzymes shown in the following table may be used. LazC is shown, for example, in Table 7-1 disclosed in WO 2020 / 067550.

[0109]

[0110] Examples of enzymes homologous to LazC include those shown in Tables 7-1 to 7-6 disclosed in WO 2020 / 067550, and two or more of these enzymes may be used in combination. Enzymes homologous to LazC are also called LazC homologs, and "homolog" refers to an enzyme other than LazC that can be used to detect a peptide in which a macrocyclization reaction similar to that described in the Examples below is performed on a peptide at the same concentration as LazC, thereby detecting a peptide in which a macrocycle has been formed. The sequence identity of a LazC homolog may be 20% or more, 40% or more, 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 92% or more, 95% or more, 98% or more, or 99% or more.

[0111] The macrocyclization enzyme is not particularly limited as long as it contains at least a domain involved in macrocyclization, and a homologue of LazC may be an enzyme containing both a domain involved in macrocyclization and a domain involved in another function (a bifunctional enzyme), or may contain two or more separate domains involved in macrocyclization. A homologue of LazC preferably contains a domain with 80% or more sequence identity, more preferably 85% or more sequence identity, and even more preferably 90% or more sequence identity to the macrocyclization domain in LazC.

[0112] As used herein, "having Y% or more sequence identity with the amino acid sequence represented by SEQ ID NO: X" means that when the amino acid sequences of the two polypeptides are aligned to maximize identity, the ratio of the number of common amino acid residues to the total number of amino acids represented by SEQ ID NO: X is Y% or more.

[0113] In the macrocyclization reaction, the concentration of the macrocyclization enzyme may be adjusted appropriately depending on the types of macrocyclization enzyme and the peptide of formula (II), and may be, for example, 0.1 to 100 μM or 0.5 to 50 μM. The reaction temperature in the macrocyclization reaction is not particularly limited, but is preferably within the range of the optimal temperature of the macrocyclization enzyme used ±20°C, and may be, for example, 0 to 50°C, or 0 to 35°C. In the production method of this embodiment, an azole ring formation reaction and / or an α,β-unsaturated amino acid formation reaction, which will be described later, may be carried out simultaneously with or sequentially to the macrocyclization reaction.

[0114] (Prenylation Reaction) In the production method of this embodiment, a prenylation enzyme is allowed to act on the cyclic compound represented by the above formula (I′) to form a cyclic compound represented by at least X ａ ', X ｂ , X ｃ and Z to form a cyclic compound represented by the above formula (I) (hereinafter referred to as "prenylation reaction").

[0115] In the prenylation reaction, a prenylation enzyme reacts with at least X of formula (I'). ａ ', X ｂ , X ｃ At least one amino acid in any of X, Z, and Z is prenylated. ａ ', X ｂ , X ｃ Prenylation may occur at either X or Z, but preferably at ａ At least one amino acid in either X' or Z is prenylated. ａ X' may be prenylated, the amino acid contained in Z may be prenylated, ａ Furthermore, other m X' of formula (I') may be prenylated, and the amino acid contained in Z may be prenylated. ｄ , and n X ｅ At least one amino acid or analog thereof selected from X may be prenylated. ａ When X' is prenylated, -(X ｄ )ｍ -X ａ '-(X ｅ ) ｎ - corresponds to the prenylation of two or more amino acids. The number of prenyl groups introduced by the prenylation reaction is not particularly limited, and may be, for example, 1 to 6, 1 to 4, or 1 to 3.

[0116] As used herein, the term "prenylation enzyme" refers to an enzyme capable of attaching a prenyl group composed of a five-carbon isoprene unit to at least some of the amino acids or analogs thereof contained in a peptide. In the prenylation reaction, it is preferable to contact the cyclic compound represented by formula (I') with the prenylation enzyme in the presence of a compound capable of donating a prenyl group (hereinafter referred to as a "prenyl group donor"). The prenyl group donor is not particularly limited as long as it is a compound having a prenyl group or a functional group that can be converted into a prenyl group. Examples of prenyl group donors include prenyl diphosphates such as dimethylallyl diphosphate (DMAPP) (also referred to as dimethylallyl pyrophosphate; the same applies hereinafter), isoprenyl diphosphate (IPP), geranyl diphosphate (GPP), farnesyl diphosphate (FPP), geranylgeranyl diphosphate (GGPP), and phytyl diphosphate (PDP). These prenyl group donors may be used singly or in combination of two or more. The prenyl group donor may be changed appropriately depending on the type of prenylation enzyme.

[0117] The prenylation enzyme used in the prenylation reaction is not particularly limited as long as it exhibits prenylation activity toward an amino acid or its analog. Examples of prenylation enzymes include those disclosed in WO 2020 / 080490, WO 2023 / 068296, and Trends Biochem. Sci., 48, 360-374 (2023). Specific examples of the enzymes disclosed in WO 2020 / 080490 and WO 2023 / 068296 include the enzymes disclosed in the following table.

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132] In addition to the enzymes listed in the table above, known prenylation enzymes (enzymes obtained as a result of BLAST search analysis) can be selected as prenylation enzymes. Sequence alignments can be created using, for example, ClustalW or MEGA7 software, and enzymes with amino acid sequences that are found to be homologous to the amino acid sequences of the known prenylation enzymes as a result of phylogenetic tree analysis can be suitably used. Enzymes with amino acid sequences that are found to be homologous as a result of analysis also include proteins annotated as hypothetical proteins. Prenylation enzymes can be used singly or in combination of two or more types.

[0133] The prenylation enzyme may be a homolog of an enzyme listed in the table above. Homologs of the enzymes listed in the table above are not particularly limited as long as they are enzymes that have the function of prenylating an amino acid or an analog thereof, and examples include (1) enzymes that have an amino acid sequence in which one or several amino acids are deleted, substituted, and / or added to the enzymes listed in the table above and have prenylation activity, as well as (2) enzymes that have an amino acid sequence that shares at least 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 92% or more, 95% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence of the enzymes listed in the table above and have prenylation activity.

[0134] Furthermore, the prenylation enzyme in this specification is not particularly limited as long as it contains at least a domain involved in the prenylation reaction. Homologs of the enzymes listed in the above table may each be in the form of an enzyme containing both a domain involved in the prenylation reaction and a domain involved in another function, or may be in the form of two or more enzymes each containing a separate domain involved in prenylation.

[0135] Preferable prenylation enzymes include KgpF, OltF, PT5, PT12, PT18, PT19, PT23, PT30, LimF, TruF1, TruF2, PatF, LynF, AcyF, OscF, and PagF listed in the above table, as well as prenylation enzymes having amino acid sequences that share 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more sequence identity with the amino acid sequences of these enzymes. The prenylation enzyme may be a prenylation enzyme having an amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in KgpF, OltF, PT5, PT12, PT18, PT19, PT23, PT30, LimF, TruF1, TruF2, PatF, LynF, AcyF, OscF, and PagF.

[0136] The prenylation enzyme more preferably comprises at least one selected from KgpF, OltF, PT5, PT12, PT18, PT19, PT23, PT30, LimF, and PagF, and prenylation enzymes having an amino acid sequence that has 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more sequence identity with the amino acid sequences of these enzymes. It is particularly preferable to comprise at least one selected from KgpF, OltF, PT5, PT18, PT19, PT30, LimF, and PagF, and prenylation enzymes having an amino acid sequence that has 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more sequence identity with the amino acid sequences of these enzymes. The prenylation enzyme may be a prenylation enzyme having an amino acid sequence in which one or several amino acids are deleted, substituted and / or added in KgpF, OltF, PT5, PT18, PT19, PT30, LimF, and PagF.

[0137] As used herein, when a phrase "having one or several amino acid deletions, substitutions, and / or additions" is used, the number of amino acids to be deleted, substituted, etc. is not particularly limited, as long as the resulting prenylation enzyme retains the function of prenylating amino acid residues. Furthermore, "multiple" here means an integer of 2 or more, and preferably several, for example, 2 to 15, more preferably 2 to 10, even more preferably 2 to 5, and still more preferably 2, 3, or 4. The position of the deletion, substitution, or addition in each prenylation enzyme may be the N-terminus, C-terminus, or any intermediate position in each prenylation enzyme, as long as the resulting prenylation enzyme retains the function of prenylating amino acid residues.

[0138] When the prenylation enzyme is LimF and the amino acid to be prenylated is histidine or an analog thereof, LimF preferably has neither a deletion nor a substitution of Glu at position 54 and His at position 172 in the amino acid sequence of LimF listed in the above table, and more preferably has neither a deletion nor a substitution of Glu at position 54, His at position 172, Tyr at position 188, Tyr at position 237, and Tyr at position 292. Furthermore, Asp at position 70 in the amino acid sequence of LimF listed in the above table may be deleted or substituted, and if substituted, may be substituted with Ala.

[0139] When the prenylation enzyme is LimF and the amino acid to be prenylated is tyrosine or an analog thereof, it is preferred that Glu at position 54 in the amino acid sequence of LimF listed in the table above is not deleted or substituted, and it is more preferred that Glu at position 54, Tyr at position 188, Tyr at position 237, and Tyr at position 292 are not deleted or substituted. In this embodiment, His at position 172 in the amino acid sequence of LimF listed in the table above may be deleted or substituted, and if substituted, it is preferably substituted with an amino acid having an aliphatic residue (e.g., Leu, Met, Ile, or Val), more preferably with Leu. Furthermore, Asp at position 70 in the amino acid sequence of LimF listed in the table above may be deleted or substituted, and if substituted, it may be substituted with Ala.

[0140] Furthermore, when LimF is deleted or substituted, the deleted or substituted amino acid may be at least one of I at position 52, D at position 70, Q at position 72, L at position 122, H at position 172, E at position 190, L at position 222, G at position 224, H at position 239, and W at position 273. In LimF, one or more amino acids may be further deleted, substituted, or added to the amino acids other than E at position 54, Y at position 188, Y at position 237, and Y at position 292.

[0141] When the prenylation enzyme is a LimF homolog, the LimF homolog may have the same amino acids as above at the same positions as above when aligned with LimF. When the prenylation enzyme is LimF or a homolog thereof, preferred prenylation enzymes depending on the type of substrate to be prenylated and prenyl group donor are disclosed in WO 2023 / 068296.

[0142] Before carrying out the prenylation reaction in the production method of this embodiment, a prenylation enzyme may be selected depending on the type of substrate to be prenylated, the amino acid to be prenylated, and the prenyl group donor.

[0143] Any of the above prenylation enzymes may be used in a state where they are immobilized on a solid support such as magnetic beads. Therefore, the prenylation enzymes used in the prenylation reaction include enzymes immobilized on a solid support. Furthermore, the prenylation enzymes may have additional sequences such as a His tag, glutathione S-transferase (GST), and maltose-binding protein (MBP).

[0144] As verified in the Examples described below, these prenylation enzymes are capable of prenylating a wide range of amino acids and are capable of prenylating the cyclic compound of formula (I') regardless of the sequence preceding or following the amino acid to be prenylated.

[0145] In the prenylation reaction, the amino acid residues adjacent to the amino acid residue to be prenylated are not particularly limited, but depending on the prenylation enzyme used, it is possible to further increase the prenylation efficiency by designing a total of four amino acid residues, including the amino acid residue adjacent to the amino acid residue and the adjacent amino acid, or a total of two amino acid residues, including the amino acid residue adjacent to the amino acid residue. Examples of such amino acid residue designs include those disclosed in WO 2020 / 080490 and WO 2023 / 068296.

[0146] When LimF or a homolog thereof is used as the prenylation enzyme, and the amino acid residue to be prenylated is designated Xaa2, and the substrate to be prenylated has a partial structure represented by Xaa1-Xaa2-Xaa3, Xaa1 may be any amino acid, preferably an amino acid other than Lys or Arg, more preferably an amino acid other than Lys, Arg, or their analogs (limited to those having a positive charge in the side chain), even more preferably a neutral amino acid or an amino acid with little steric hindrance, even more preferably a neutral amino acid, and particularly preferably a neutral amino acid and an amino acid with little steric hindrance. Xaa3 may be any amino acid, preferably an amino acid other than Pro, more preferably an amino acid other than Pro and its derivatives (limited to those whose side chain is bonded to the nitrogen atom of the amino group to form a ring), and even more preferably an amino acid other than an amino acid whose side chain is bonded to the nitrogen atom of the amino group to form a ring. Xaa2 may be His, Tyr, or an analog thereof.

[0147] "Neutral amino acid" refers to an amino acid that does not have a basic or acidic side chain in the molecule, and examples of proteinogenic amino acids include Gly, Ala, Phe, Leu, Ile, Cyc, Met, Tyr, Val, Thr, Ser, Pro, Trp, Asn, and Gln. Furthermore, "amino acid with little steric hindrance" refers to an amino acid with little steric hindrance in the side chain, as described above, and examples include amino acids whose side chains contain four or fewer atoms other than hydrogen atoms. Examples of proteinogenic amino acids include Gly, Ala, Ser, Pro, Val, Thr, Cys, Leu, Ile, Asn, Asp, and Met. Furthermore, "neutral amino acids with little steric hindrance" refers to amino acids that fall under the neutral amino acids and amino acids with little steric hindrance in the above definitions, and examples of proteinogenic amino acids include Gly, Ala, Ser, Pro, Val, Thr, Cys, Leu, Ile, Asn, and Met. Furthermore, "amino acids whose side chains are bonded to the nitrogen atom of the amino group to form a ring" refer to, for example, amino acids with the following structure: Amino acids having the formula (R５ and R ６ are each independently a hydrogen atom or an arbitrary substituent, and R ７ are each independently any divalent group (typically a methylene group), m1 represents an integer of 0 or more, and m2 represents an integer of 1 or more. m1 is typically 0 or 1.), and an example of a proteinogenic amino acid is Pro.

[0148] Preferred amino acids for Xaa1 include Thr, Ala, Met, Pro, Ser, Val, Ile, Leu, Gly, Asn, Asp, His, Gln, Glu, Phe, Cys, Trp, and Tyr, more preferably Thr, Ala, Met, Pro, Ser, Val, Ile, Leu, Gly, Glu, Asp, His, Gln, and Asn, and even more preferably Thr, Ala, Gly, Ser, Val, Leu, Asn, Met, Pro, and Ile. Preferred amino acids at Xaa3 include Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gln, Cys, Gly, Ala, Ile, Leu, Met, Phe, Trp, Tyr, and Val.

[0149] When KgpF or a homolog thereof is used as the prenylation enzyme, and the amino acid residue to be prenylated is Xaa2, and the substrate to be prenylated has a partial structure represented by Xaa4-Xaa1-Xaa2-Xaa3-Xaa5, Xaa4 is any amino acid, and is preferably G, N, Y, I, R, E, F, S, W, H, D, T, P, A, V, L, Q, or K, or an analog thereof, and is preferably G, N, Y, I, R, E, F, S, W, H, D, T, P, A, V, L, Q, or K. Xaa1 is any amino acid, preferably an amino acid with little steric hindrance as defined above, and is preferably V, T, I, A, P, Q, N, S, F, H, W, L, or G, or an analog thereof, and is preferably V, T, I, A, P, Q, or N, or an analog thereof, and is preferably V, T, I, A, or N, or an analog thereof, and is more preferably V, T, I, A, P, Q, N, S, F, H, W, L, or G, or V, T, I, A, P, Q, or N, or V, T, I, A, or N. Xaa3 is any amino acid, preferably G, W, S, A, L, F, T, R, N, Y, I, V, H, Q, K, D or E, or an analog thereof, more preferably G, W, S, A, L, F, T, R, N, or Y, or an analog thereof, and more preferably G, W or S, A, L, F, T, R, N, Y, I, V, H, Q, K, D or E, or G, W, S, A, L, F, T, R, N, or Y, or G, W or S. Xaa5 may be any amino acid, preferably G, N, Y, I, R, E, F, S, W, H, D, T, P, A, V, L, Q, or K, or an analog thereof, and more preferably G, N, Y, I, R, E, F, S, W, H, D, T, P, A, V, L, Q, or K. Xaa2 may be Trp or an analog thereof.

[0150] Preferred motifs when KgpF or a homolog thereof is used as the prenylation enzyme are disclosed in WO 2020 / 080490.

[0151] When OltF or a homolog thereof is used as the prenylation enzyme, and the amino acid residue to be prenylated is Xaa2, and the substrate to be prenylated has a partial structure represented by Xaa4-Xaa1-Xaa2-Xaa3-Xaa5, Xaa4 may be any amino acid, preferably an amino acid other than an acidic amino acid, and preferably R, H, K, S, T, N, Q, C, G, P, A, I, L, M, F, W, Y, or V or an analog thereof, and preferably V, G, A, S, P, L, F, N, Y, or K or an analog thereof, and preferably R, H, K, S, T, N, Q, C, G, P, A, I, L, M, F, W, Y, or V, or V, G, A, S, P, L, F, N, Y, or K. Xaa1 is any amino acid, preferably an amino acid other than an acidic amino acid, and is preferably R, H, K, S, T, N, Q, C, G, P, A, I, L, M, F, W, Y, or V or an analog thereof, and is preferably R, H, K, S, T, N, Q, G, P, A, I, L, F, W, Y, or V or an analog thereof, and is preferably R, H, Q, G, P, I, L, F, W, Y, or V or an analog thereof. Preferably, it is I, L, F, W, Y, or V or an analog thereof, and more preferably, it is R, H, K, S, T, N, Q, C, G, P, A, I, L, M, F, W, Y, or V, or R, H, K, S, T, N, Q, G, P, A, I, L, F, W, Y, or V, or R, H, Q, G, P, I, L, F, W, Y, or V, or I, L, F, W, Y, or V.Xaa3 is any amino acid, preferably an amino acid other than acidic amino acids and amino acids whose side chains are bonded to the nitrogen atom of the amino group to form a ring, and is preferably R, H, K, S, T, N, Q, C, G, A, I, L, M, F, W, Y, or V or an analog thereof, and is preferably R, H, K, S, T, N, Q, C, G, A, I, M, F, W, Y, or V or an analog thereof, and is preferably V, G, A, S, F, N, Y, or K or an analog thereof, and is preferably R, H, K, S, T, N, Q, C, G, A, I, L, M, F, W, Y, or V, or R, H, K, S, T, N, Q, C, G, A, I, M, F, W, Y, or V, or V, G, A, S, F, N, Y, or K. Xaa5 is any amino acid, preferably an amino acid other than an amino acid whose side chain is bonded to the nitrogen atom of the amino group to form a ring, and is preferably R, H, K, D, E, S, T, N, Q, C, G, A, I, L, M, F, W, Y, or V, or an analog thereof, and is preferably R, H, K, D, E, S, T, N, Q, C, G, A, I, L, M, F, W, or V, or an analog thereof, and is preferably V, G, A, S, L, F, N, D, or K, or an analog thereof, and is preferably R, H, K, D, E, S, T, N, Q, C, G, A, I, L, M, F, W, Y, or V, or R, H, K, D, E, S, T, N, Q, C, G, A, I, L, M, F, W, or V, or V, G, A, S, L, F, N, D, or K. Xaa2 may be Trp or an analog thereof.

[0152] Preferred motifs when OltF or a homolog thereof is used as the prenylation enzyme are disclosed in WO 2020 / 080490.

[0153] When PT5 or a homolog thereof is used as the prenylation enzyme, and the amino acid residue to be prenylated is Xaa2 and the substrate to be prenylated has a partial structure represented by Xaa1-Xaa2-Xaa3, Xaa1 is any amino acid, preferably an amino acid other than glutamine and its homologs, and is preferably R, H, K, D, E, S, T, N, C, G, P, A, I, L, M, F, W, Y, or V or analogs thereof, and is preferably R, H, D, E, S, T, N, C, G, P, A, I, L, M, F, W, Y, or V or analogs thereof, and is preferably H, D, E, S , T, N, G, P, A, I, L, F, W, Y, or V or analogs thereof, and preferably R, H, K, D, E, S, T, N, C, G, P, A, I, L, M, F, W, Y, or V, or R, H, D, E, S, T, N, C, G, P, A, I, L, M, F, W, Y, or V, or H, D, E, S, T, N, G, P, A, I, L, F, W, Y, or V. Xaa3 is any amino acid, preferably an amino acid other than acidic amino acids and amino acids whose side chains are bonded to the nitrogen atom of the amino group to form a ring, and is preferably H, K, S, T, N, G, A, I, L, F, W, Y, or V or an analog thereof, and is preferably R, H, K, S, T, N, C, G, A, I, L, M, F, W, Y, or V or an analog thereof, and is preferably H, K, S, T, N, G, A, I, L, F, W, Y, or V, or R, H, K, S, T, N, C, G, A, I, L, M, F, W, Y, or V. Xaa2 may be Arg or an analog thereof.

[0154] When PT12 or a homolog thereof is used as the prenylation enzyme, and the amino acid residue to be prenylated is Xaa2, and the substrate to be prenylated has a partial structure represented by Xaa1-Xaa2-Xaa3, Xaa1 is any amino acid, and is preferably H, K, D, E, T, N, Q, C, G, P, A, I, L, M, F, W, or Y or an analog thereof, and more preferably H, K, D, E, T, N, Q, G, P, A, I, L, F, or Y or an analog thereof, and is preferably H, K, D, E, T, N, Q, C, G, P, A, I, L, M, F, W, or Y, or H, K, D, E, T, N, Q, G, P, A, I, L, F, or Y. Xaa3 may be any amino acid, preferably A, D, E, N, Q, or T or an analog thereof, and more preferably A, D, E, N, Q, or T. Xaa2 may be Trp or an analog thereof.

[0155] When PT18 or a homolog thereof is used as the prenylation enzyme, and the amino acid residue to be prenylated is Xaa2 and the substrate to be prenylated has a partial structure represented by Xaa1-Xaa2-Xaa3, Xaa1 may be any amino acid, preferably an amino acid other than an acidic amino acid or an amino acid whose side chain is bonded to the nitrogen atom of an amino group to form a ring, and is preferably R, H, K, S, T, N, Q, C, G, A, I, L, M, F, W, Y, or V or an analog thereof, and is preferably R, H, K, S, T, N, Q, G, A, I, L, F, Y, or V or an analog thereof, and is preferably R, H, K, S, T, N, Q, C, G, A, I, L, M, F, W, Y, or V, or R, H, K, S, T, N, Q, G, A, I, L, F, Y, or V. Xaa3 is any amino acid, preferably an amino acid other than an amino acid whose side chain is bonded to the nitrogen atom of the amino group to form a ring, and is preferably R, H, K, D, E, S, T, N, Q, C, G, A, I, L, M, F, W, Y, or V, or an analog thereof, and is preferably R, H, K, D, E, S, T, N, Q, G, A, I, L, M, F, Y, or V, or an analog thereof, and is preferably R, H, K, D, E, S, T, N, Q, C, G, A, I, L, M, F, W, Y, or V, or R, H, K, D, E, S, T, N, Q, G, A, I, L, M, F, Y, or V. Xaa2 may be Trp or an analog thereof.

[0156] When PT19 or a homolog thereof is used as the prenylation enzyme, and the amino acid residue to be prenylated is Xaa2 and the substrate to be prenylated has a partial structure represented by Xaa1-Xaa2-Xaa3, Xaa1 may be any amino acid, preferably an amino acid other than a basic amino acid, and preferably a neutral amino acid, and is preferably H, S, T, N, Q, C, G, P, A, I, L, M, F, W, Y, or V, or an analog thereof, and is preferably H, S, T, N, Q, C, G, P, A, I, L, M, F, W, Y, or V, or H, S, T, N, Q, G, P, A, I, L, F, Y, or V. Xaa3 is any amino acid, preferably an amino acid other than an amino acid whose side chain is bonded to the nitrogen atom of the amino group to form a ring, and is preferably R, H, K, D, E, S, T, N, Q, C, G, A, I, L, M, F, W, Y, or V, or an analog thereof, and more preferably H, D, E, S, T, N, Q, C, G, A, I, L, M, F, W, Y, or V, or an analog thereof. Xaa2 is preferably H, D, E, S, T, N, Q, G, A, I, L, F, Y, or V, or an analog thereof, and more preferably R, H, K, D, E, S, T, N, Q, C, G, A, I, L, M, F, W, Y, or V, or H, D, E, S, T, N, Q, C, G, A, I, L, M, F, W, Y, or V, or H, D, E, S, T, N, Q, G, A, I, L, F, Y, or V. Xaa2 may be Trp or an analog thereof.

[0157] When PT23 or a homolog thereof is used as the prenylation enzyme, and the amino acid residue to be prenylated is Xaa2, and the substrate to be prenylated has a partial structure represented by Xaa1-Xaa2-Xaa3, Xaa1 is any amino acid and is preferably D, E, T, N, Q, C, G, P, I, L, M, W, or Y or an analog thereof, and is preferably D, E, T, N, Q, G, P, I, L, M, W, or Y, or D, E, T, N, Q, G, P, I, L, or Y. Xaa3 is any amino acid and is preferably A, D, E, F, H, N, Q, or T or an analog thereof, and is preferably A, D, E, F, H, N, Q, or T. Xaa2 may be Trp or an analog thereof.

[0158] Preferred motifs when PT30 and PagF or any of their homologs (particularly PagF 222G / 237G / 271N) are used as the prenylation enzyme are disclosed in the Examples.

[0159] In addition, when not defined above, the analogs of each of the amino acids R, H, K, D, E, S, T, N, Q, C, G, P, A, I, L, M, F, W, Y, and V in Xaa1 to Xaa5 above may be analogs corresponding to β-amino acids, γ-amino acids, and δ-amino acids of those amino acids; analogs corresponding to homoamino acids; and analogs corresponding to α-alkyl-amino acids (for example, α-alkyl-amino acids having an alkyl group of 1 to 3 carbon atoms, typically α-methyl-amino acids) of those analogs and amino acids. Furthermore, the amino acids in Xaa1 to Xaa5 above may be L- or D-isomers, or a mixture thereof.

[0160] Here, Xaa1 is the amino acid adjacent to the amino acid to be prenylated on the N-terminal side. ａ For example, when prenylation of m X' is performed, ｄ Among them, the C-terminal X ｄ , i.e., X ａX adjacent to ｄ , m X's in formula (I') ｄ Among them, the C-terminal X ｄ , i.e., X ａ X adjacent to ' ｄ , m X's in formula (II) ｄ Among them, the C-terminal X ｄ , i.e., X ａ X adjacent to ' ｄ and / or m X's of formula (III) below ｄ ', the C-terminal X ｄ ', i.e., X ａ X adjacent to " ｄ Xaa2 is an amino acid to be prenylated. X in formula (I') may be ａ For example, when X′ in formula (I) is prenylated, ａ , X in formula (I') ａ ', X in formula (II) ａ ', and / or X in formula (III) below ａ Xaa3 is the amino acid adjacent to the amino acid to be prenylated on the C-terminal side. X in formula (I') ａ For example, when prenylation of n X' is performed, ｅ Among them, the N-terminal X ｅ , i.e., X ａ X adjacent to ｅ , n X in formula (I') ｅ Among them, the N-terminal X ｅ , i.e., X ａ X adjacent to ' ｅ , n X in formula (II) ｅ Among them, the N-terminal X ｅ , i.e., X ａ X adjacent to ' ｅ and / or n X in formula (III) below ｅ ', the N-terminal X ｅ ', i.e., X ａ X adjacent to " ｅ Xaa4 is the second amino acid on the N-terminal side of the amino acid to be prenylated. ａ For example, when prenylation of m X' is performed, ｄThe second X from the C-terminus ｄ , m X's in formula (I') ｄ The second X from the C-terminus ｄ , m X's in formula (II) ｄ The second X from the C-terminus ｄ and / or m X's of formula (III) below ｄ ', the second X from the C-terminus ｄ Xaa5 is the second amino acid on the C-terminal side of the amino acid to be prenylated. ａ For example, when prenylation of n X' is performed, ｅ The second X from the N-terminus ｅ , n X in formula (I') ｅ The second X from the N-terminus ｅ , n X in formula (II) ｅ The second X from the N-terminus ｅ and / or n X in formula (III) below ｅ ', the second X from the N-terminus ｅ ' may be.

[0161] In formula (I), Xaa1, Xaa2, Xaa4, and Xaa5 may be prenylated, may form an α,β-unsaturated bond, or may form an azoline or azole. In formula (I'), Xaa1, Xaa2, Xaa4, and Xaa5 are preferably not prenylated, may form an α,β-unsaturated bond, or may form an azoline or azole. In formula (II), Xaa1, Xaa2, Xaa4, and Xaa5 are preferably not prenylated, may form an α,β-unsaturated bond, or may form an azoline or azole. In formula (III), Xaa1, Xaa2, Xaa4, and Xaa5 are preferably not prenylated, preferably do not form an α,β-unsaturated bond, and preferably do not form an azoline or azole. Xaa3 is preferably prenylated in formula (I) and is preferably not prenylated in formula (I'), formula (II) and formula (III).

[0162] The prenylation reaction may be carried out in a reaction system in which all of the cyclic compound of formula (I') (substrate), prenylation enzyme, and prenyl group donor are added to a solvent, or may be carried out by contacting a reaction system in which any of these is supported on a solid phase support such as magnetic beads with a solution containing the remaining components. When the prenylation enzyme is present in a solution, its concentration may be appropriately adjusted depending on the expression and purification conditions of the enzyme, and is not particularly limited. The concentration of the prenylation enzyme may be, for example, 0.050 μM to 1.0 mM, or 0.10 to 500 μM. When the cyclic compound of formula (I') is present in a solution, its concentration is not particularly limited, but may be, for example, 0.010 μM to 50 mM, or 0.10 μM to 10 mM. When the prenyl group donor is present in a solution, its concentration is not particularly limited, and may be, for example, 0.20 to 10,000 times or 1.0 to 5,000 times the concentration of the substrate, specifically 0.010 μM to 100 mM or 0.10 to 50 mM.

[0163] The reaction temperature (incubation temperature) in the prenylation reaction is not particularly limited, but is preferably within a range of the optimal temperature of the prenylation enzyme used ±20°C. The reaction temperature may be, for example, 0 to 50°C, and preferably 0 to 40°C. The reaction time in the prenylation reaction may be adjusted appropriately depending on the reaction temperature and the concentrations of the substrate, prenylation enzyme, prenyl group donor, etc. The reaction time is, for example, 30 minutes to 100 hours, 1.0 to 80 hours, 3.0 to 50 hours, or 5.0 to 40 hours.

[0164] The prenylation reaction may contain components in the reaction system other than the substrate, prenylation enzyme, and prenyl group donor. Such components are not particularly limited and include, for example, components used in preparing any of the substrate, prenylation enzyme, and prenyl group donor, as well as cofactors that enhance the catalytic activity of the prenylation enzyme. Cofactors are not particularly limited and include, for example, magnesium ions, copper ions, iron ions, manganese ions, molybdenum ions, nickel ions, selenium ions, and zinc ions. These cofactors may be present alone or in combination of two or more. Among these, the presence of magnesium ions is preferred. Whether or not a cofactor is used in the prenylation reaction can be appropriately selected depending on various conditions, such as the type of prenylation enzyme used.

[0165] (Preparation of precursor peptide) The production method of this embodiment comprises preparing a precursor peptide of formula (III): LP'-X'-(X ｄ ') ｍ -X ａ "-(X ｅ ') ｎ -X ｂ '-Y'-X ｃ The method may further include preparing a precursor peptide represented by formula (III), and forming a peptide represented by formula (II) from the precursor peptide represented by formula (III) by the following reactions: a reaction of forming an azole ring at at least Y' (11) in the precursor peptide represented by formula (III) using an azole ring-forming enzyme (hereinafter referred to as "azole ring-forming reaction"); and a reaction of converting at least X' and Y' (12) in the precursor peptide represented by formula (III) into α,β-unsaturated amino acid residues using an α,β-unsaturated amino acid-forming enzyme in the presence of a cosubstrate for glutamylation and an aminoacylase (hereinafter referred to as "α,β-unsaturated amino acid-forming reaction").

[0166] In formula (III), X' is serine or threonine or an analog thereof, Y' is a peptide represented by -Y'(11)-Y'(12)-, Y'(11) is serine, cysteine, threonine, or diaminopropionic acid or an analog thereof, and Y'(12) is serine or threonine or an analog thereof, m and n are as defined above, and X ａ ", X ｂ ', X ｃ ', m X's ｄ ', n X's ｅ ', Z', and LP' are each X in formula (II). ａ ', X ｂ , X ｃ , m X ｄ , n X's ｅ , Z, and LP.

[0167] The precursor peptides may be produced by a conventionally known production method, or may be purchased commercially and optionally prepared after purification, etc. The precursor peptides may be prepared using a synthesis method using a translation synthesis system. Examples of such translation synthesis systems include a cellular translation system and a cell-free translation system. When producing a peptide library, it is preferable to use a synthesis method using a cell-free translation system from the viewpoint of improving the diversity of the library, etc. The method for producing peptides using a cell-free translation system will be described later. When producing precursor peptides on a large scale, synthesis methods using a cellular translation system or solid-phase synthesis methods using beads or columns are preferred. Solid-phase synthesis methods can be performed according to known methods.

[0168] When a cell translation system is used, cells genetically modified to produce a precursor peptide may be used. Nucleic acids encoding precursor peptides can be prepared by known methods or methods equivalent thereto. For example, they can be synthesized using an automated synthesizer. Restriction enzyme recognition sites may be added to insert the resulting nucleic acid into a vector, or a base sequence encoding an amino acid sequence for excising the produced peptide chain with an enzyme or the like may be incorporated.

[0169] The nucleic acid encoding the precursor peptide can be expressed by inserting it downstream of a promoter in an expression vector. Examples of the vector that can be used include Escherichia coli-derived plasmids (pBR322, pBR325, pUC12, pUC13, pUC18, pUC19, pUC118, pBluescript II, etc.), Bacillus subtilis-derived plasmids (pUB110, pTP5, pC1912, pTP4, pE194, pC194, etc.), yeast-derived plasmids (pSH19, pSH15, YEp, YRp, YIp, YAC, etc.), bacteriophages (λ phage, M13 phage, etc.), viruses (retrovirus, vaccinia virus, adenovirus, adeno-associated virus (AAV), cauliflower mosaic virus, tobacco mosaic virus, baculovirus, etc.), and cosmids.

[0170] The promoter can be selected appropriately depending on the type of host. When the host is an animal cell, for example, a promoter derived from SV40 (simian virus 40) or a promoter derived from CMV (cytomegalovirus) can be used. When the host is Escherichia coli, a trp promoter, a T7 promoter, a lac promoter, etc. can be used. The expression vector can also incorporate nucleic acids encoding a DNA replication origin (ori), a selection marker (antibiotic resistance, auxotrophy, etc.), an enhancer, a splicing signal, a poly(A) addition signal, a tag (FLAG, HA, GST, GFP, etc.), etc.

[0171] The host used for transformation can be selected appropriately in relation to the vector, and examples thereof include Escherichia coli, Bacillus subtilis, Bacillus species, yeast, insects or insect cells, and animal cells. Examples of animal cells that can be used include HEK293T cells, CHO cells, COS cells, myeloma cells, HeLa cells, and Vero cells. Transformation can be carried out according to known methods, such as lipofection, calcium phosphate, electroporation, microinjection, and particle gun technology, depending on the type of host. The transformant is cultured according to standard methods to express the desired peptide. The medium is selected appropriately depending on the type of host, etc.

[0172] Peptides can be purified from a transformant culture by recovering the cultured cells, suspending them in an appropriate buffer, disrupting the cells by ultrasonication, freeze-thawing, or other methods, and then obtaining a crude extract by centrifugation or filtration. If the peptide is secreted into the culture medium, the supernatant is recovered. Purification from the crude extract or culture supernatant can also be carried out by known methods or methods equivalent thereto (e.g., salting out, dialysis, ultrafiltration, gel filtration, SDS-PAGE, ion exchange chromatography, affinity chromatography, reversed-phase high performance liquid chromatography, etc.). The obtained peptide can be converted from a free form to a salt, or from a salt to a free form, by known methods or methods equivalent thereto.

[0173] When preparing precursor peptides, one or more types of precursor peptides represented by formula (III) may be prepared. When there are two or more types of precursor peptides represented by formula (III), this can be said to be a step of preparing a library containing precursor peptides represented by formula (III). By carrying out the production method of this embodiment using such a compound library, a library of cyclic compounds of formula (I) can be produced. That is, one aspect of the production method of this embodiment is a method for producing a library containing the cyclic compounds of formula (I) above. For the method for producing the library, please refer to the method for producing the compound library of this embodiment described below.

[0174] In formula (III), X' is serine or threonine or an analog thereof, preferably serine or threonine, more preferably serine. The serine or threonine analogs are as described above.

[0175] In formula (III), Y' is a peptide represented by -Y'(11)-Y'(12)-. Y'(11) is serine, cysteine, threonine, or diaminopropionic acid, or an analog thereof, preferably serine, cysteine, or threonine, or an analog thereof, more preferably serine, cysteine, or threonine, and even more preferably serine. Y'(12) is serine or threonine, or an analog thereof, preferably serine or an analog thereof, more preferably serine. The serine, cysteine, threonine, and diaminopropionic acid analogs are as described above.

[0176] Examples and preferred embodiments of m and n in formula (III) are the same as those of m and n in formula (I). ａ ", X ｂ ', X ｃ ', m X's ｄ ', n X's ｅ ', Z', and LP' are each the same as X in formula (II), except as follows: ａ ', X ｂ , X ｃ , m X ｄ , n X's ｅ In the precursor peptide of formula (III) used to obtain the peptide of formula (II), X ａ ", X ｂ ', X ｃ ', m X's ｄ ', n X's ｅ X′, Z′, and LP′ may be the same as or different from the peptide of formula (II). X′, Z′, and LP′ may be the same as or different from the peptide of formula (III). ａ ", X ｂ ', X ｃ ', m X's ｄ ', n X's ｅ In LP′, Z′, and LP′, the amino acid or analog thereof is preferably selected from amino acids which are not prenylated and which do not have an azoline, an azole ring, or an α,β-unsaturated bond.

[0177] In formula (III), X ｂX' is not particularly limited as long as it is an amino acid or an analog thereof, but is preferably serine or threonine, or an analog thereof, more preferably serine or threonine, and even more preferably serine. ｂ When ' is one of the above amino acids, the efficiency of the macrocyclization reaction tends to be even higher.

[0178] In formula (III), X ｃ is not particularly limited as long as it is an amino acid or an analog thereof, but is preferably serine, cysteine, threonine, or diaminopropionic acid or an analog thereof, more preferably serine, cysteine, or threonine, and even more preferably cysteine.

[0179] (Azole ring formation reaction / α,β-unsaturated amino acid formation reaction) In the azole ring formation reaction, an azole ring is formed at least at Y′(11) in the precursor peptide represented by formula (III) by an azole ring-forming enzyme. ａ ", X ｂ ', X ｃ ', m X's ｄ ', n X's ｅ In the azole ring-forming reaction, an azoline or azole ring may be formed in at least one amino acid or analog thereof selected from X′, Z′, and LP′. ｃ ', m X's ｄ ', n X's ｅ An azole ring may be formed in at least one amino acid or analog thereof selected from LP', Z', and LP'. For the azole ring formation reaction, see International Publication No. WO 2020 / 067550.

[0180] The azole ring-forming enzyme in the azole ring-forming reaction preferably includes LazD, LazE, and LazF. Enzymes having homology to each of LazD, LazE, and LazF can also be used as the azole ring-forming enzyme. The azole ring-forming enzymes shown together with the macrocyclases in Table 1 above may also be used as the azole ring-forming enzyme.

[0181] Examples of enzymes homologous to LazD, LazE, and LazF include those shown in Tables 3-1 and 3-2 (for LazD), Tables 4-1 to 4-81 (for LazE), and Tables 5A-1 to 5A-51 (for LazF) disclosed in WO 2020 / 067550. These may be used in combination with two or more enzymes. LazD, LazE, and LazF are shown in Tables 3-1, 4-1, and 5A-1, respectively, of WO 2020 / 067550, for example. Enzymes that are homologous to LazD, LazE, and LazF, respectively, are also referred to as homologs of LazD, LazE, and LazF, respectively. A "homolog" refers to an enzyme other than LazD, LazE, or LazF that is capable of detecting an azole-cyclized peptide by performing an azole ring-forming reaction similar to that described in the Examples below using a peptide at the same concentration as LazD, LazE, or LazF. The sequence identity of a homolog of LazD, LazE, or LazF to those of LazD, LazE, or LazF may be 20% or more.

[0182] When LazD, LazE, and LazF are used in an azole ring-forming reaction, LazD and LazE form an azoline, followed by the formation of an azole by LazF (the C-terminal domain involved in azole ring formation). In this embodiment, the azole ring-forming enzyme refers to a group of enzymes involved in azoline formation and azole formation. Here, the homologs of LazD and LazE may each be enzymes containing both a domain involved in azoline formation and a domain involved in other functions, or may be two or more enzymes containing separate domains involved in azoline formation. Furthermore, LazF contains a domain involved in azole formation at the C-terminus and a domain involved in α,β-unsaturated amino acid formation at the N-terminus. Therefore, a homolog of LazF as an azole ring-forming enzyme may be any enzyme containing at least a domain involved in azole formation, or may be two or more enzymes containing separate domains involved in azole formation. As described above, the azole ring-forming enzyme is not particularly limited as long as it contains at least the domains involved in azoline formation and azole formation. Homologs of LazD, LazE, and LazF preferably contain domains with 80% or more sequence identity, more preferably 85% or more sequence identity, and even more preferably 90% or more sequence identity to the azoline- and azole-forming domains in LazD, LazE, and LazF, respectively.

[0183] In the azole ring-forming reaction, the concentration of the azole ring-forming enzyme may be adjusted appropriately depending on the types of the azole ring-forming enzyme and the precursor peptide of formula (III), and may be, for example, 0.1 to 100 μM or 0.5 to 50 μM. The reaction temperature in the azole ring-forming reaction is not particularly limited, but is preferably within the range of the optimal temperature of the azole ring-forming enzyme used ±20°C, and may be, for example, 0 to 50°C, or 0 to 35°C.

[0184] In the α,β-unsaturated amino acid forming reaction, at least X′ and Y′(12) in the precursor peptide represented by formula (III) are converted to α,β-unsaturated amino acid residues by the α,β-unsaturated amino acid forming enzyme. ａ ", X ｂ ', X ｃ ', m X's ｄ ', n X's ｅ At least one amino acid or analog thereof selected from X′, Z′, and LP′ may be converted into an α,β-unsaturated amino acid residue. ｂ ', m X's ｄ ', n X's ｅ At least one amino acid or analog thereof selected from LP′, Z′, and LP′ may be converted into an α,β-unsaturated amino acid residue. For the α,β-unsaturated amino acid forming reaction, see International Publication No. WO 2020 / 067550.

[0185] The α,β-unsaturated amino acid forming enzyme in the α,β-unsaturated amino acid forming reaction preferably includes LazB and LazF. As the α,β-unsaturated amino acid forming enzyme, an enzyme having homology to each of LazB and LazF can also be used. As the α,β-unsaturated amino acid forming enzyme, an α,β-unsaturated amino acid forming enzyme shown together with the macrocyclase in Table 1 above may also be used.

[0186] Examples of enzymes homologous to LazB and LazF include those shown in Tables 6-1 to 6-13 (for LazB) and Tables 5B-1 to 5B-32 (for LazF) disclosed in WO 2020 / 067550. Two or more of these enzymes may be used in combination. LazB and LazF are shown in Tables 6-1 and 5B-1, respectively, of WO 2020 / 067550.

[0187] Enzymes that are homologous to LazB and LazF are also referred to as homologs of LazB and LazF, respectively. A "homolog" refers to an enzyme other than LazB and LazF that is capable of detecting a peptide in which an α,β-unsaturated amino acid is formed when an α,β-unsaturated amino acid-forming enzyme similar to that described in the Examples below is used on a peptide at the same concentration as LazB and LazF. The sequence identity of a homolog of LazB or LazF may be 20% or more with respect to LazB or LazF.

[0188] When LazB and LazF are used in the α,β-unsaturated amino acid formation reaction, LazB performs glutamylation, followed by deglutamylation by LazF (the domain involved in α,β-unsaturated amino acid formation on the N-terminal side of LazF), thereby forming α,β-unsaturated amino acid residues. In this embodiment, the α,β-unsaturated amino acid forming enzyme refers to a group of enzymes involved in glutamylation and deglutamylation. Here, homologs of LazB and LazF may be enzymes that contain both domains involved in glutamylation and deglutamylation and domains involved in other functions, or may be two or more enzymes that contain separate domains involved in glutamylation and deglutamylation. As described above, the α,β-unsaturated amino acid forming enzyme is not particularly limited as long as it contains at least domains involved in glutamylation and deglutamylation. Homologs of LazB and LazF preferably contain domains with 80% or more sequence identity to the domains involved in glutamylation and deglutamylation in LazB and LazF, more preferably with 85% or more sequence identity, and even more preferably with 90% or more sequence identity.

[0189] The α,β-unsaturated amino acid forming reaction is carried out in the presence of a cosubstrate and an aminoacylase for the glutamylation reaction. When synthesis of the precursor peptide is carried out in a cell-free translation system, the cosubstrate and aminoacylase for the glutamylation reaction may be derived from the cell-free translation system, or may be derived from a different enzyme than those contained in the cell-free translation system. That is, when the cell-free translation system contains a cosubstrate and an aminoacylase for the glutamylation reaction, the α,β-unsaturated amino acid forming reaction may be carried out using those present in the cell-free translation system. Alternatively, the α,β-unsaturated amino acid forming reaction may be carried out in the presence of a cosubstrate and / or aminoacylase for the glutamylation reaction that is not contained in the cell-free translation system, which are separately added. Whether a cosubstrate and aminoacylase contained in the cell-free translation system or a cosubstrate and aminoacylase other than those contained in the cell-free translation system are used may be appropriately selected depending on the type of α,β-unsaturated amino acid forming enzyme to be used, etc.

[0190] The cosubstrate in the glutamylation reaction is tRNA. Ｇｌｕ (Glutamyl tRNA Ｇｌｕ When LazB and LazF, and / or their homologs, are used as the α,β-unsaturated amino acid forming enzyme, tRNA Ｇｌｕ is not particularly limited as long as it is acceptable to at least the α,β-unsaturated amino acid forming enzyme. From the viewpoint of efficiently proceeding with the reaction, tRNA derived from actinomycetes is preferred. Ｇｌｕ and / or tRNA derived from Escherichia coli Ｇｌｕ It is preferable to use tRNA derived from Streptomyces lactacystinaeus, a lactazole-producing actinomycete. Ｇｌｕ and / or tRNA from Streptomyces lividans used as a heterologous expression host for lactazol. Ｇｌｕ It is more preferable to use tRNA. Ｇｌｕ Glutamyl tRNA Ｇｌｕ The aminoacyltransferase GluRS is used to form the tRNA GluR. GluRS is used when LazB and LazF, and / or their homologs, are used as the α,β-unsaturated amino acid forming enzymes. Ｇｌｕis not particularly limited as long as it is glutamylated, but is preferably GluRS derived from actinomycetes and / or GluRS derived from Escherichia coli, and more preferably GluRS derived from Streptomyces lactacystinaeus, a lactazole-producing actinomycete, and / or GluRS derived from Streptomyces lividans, which is used as a heterologous expression host for lactazole. The GluRS derived from actinomycetes may be GluRS extracted from actinomycetes, or may be GluRS obtained as a recombinant protein from GluRS in the actinomycete genome. The GluRS derived from Escherichia coli may be GluRS extracted from Escherichia coli, or may be GluRS obtained as a recombinant protein from GluRS in the Escherichia coli genome.

[0191] In the α,β-unsaturated amino acid forming reaction, the concentration of the α,β-unsaturated amino acid forming enzyme may be adjusted appropriately depending on the types of the α,β-unsaturated amino acid forming enzyme and the precursor peptide of formula (III), and may be, for example, 0.1 to 100 μM or 0.5 to 50 μM. The reaction temperature in the α,β-unsaturated amino acid forming reaction is not particularly limited, but is preferably within the range of the optimal temperature of the α,β-unsaturated amino acid forming enzyme used ±20°C, and may be, for example, 0 to 50°C, or 0 to 35°C.

[0192] In the production method of this embodiment, the azole ring-forming reaction and the α,β-unsaturated amino acid-forming reaction may proceed simultaneously with the macrocyclization reaction or sequentially. For example, in the production method of this embodiment, the macrocyclization reaction, the azole ring-forming reaction, and the α,β-unsaturated amino acid-forming reaction may be carried out in any of the following modes: 1) a mode in which the azole ring-forming reaction, the α,β-unsaturated amino acid-forming reaction, and the macrocyclization reaction are carried out step by step; 2) a mode in which the azole ring-forming reaction, the α,β-unsaturated amino acid-forming reaction, and the macrocyclization reaction are carried out simultaneously; 3) a mode in which the azole ring-forming reaction and the α,β-unsaturated amino acid-forming reaction are carried out before the macrocyclization reaction; and 4) a mode in which the azole ring-forming reaction is carried out before the α,β-unsaturated amino acid-forming reaction and the macrocyclization reaction are carried out.

[0193] When the production method of this embodiment includes the preparation of a precursor peptide, an azole ring formation reaction, and an α,β-unsaturated amino acid formation reaction, the reactions may be allowed to proceed by sequentially adding an azole ring-forming enzyme, an α,β-unsaturated amino acid-forming enzyme, a macrocyclization enzyme, and a prenylation enzyme to a precursor peptide synthesis system. In this case, two or more types of each enzyme may be added simultaneously, but the macrocyclization reaction and the prenylation reaction must occur stepwise in this order.

[0194] It is preferable that the synthesis of the precursor peptide, the azole ring formation reaction, the α,β-unsaturated amino acid formation reaction, the macrocyclization reaction, and the prenylation reaction be performed in such a way that the final volume of the reaction system is reduced while the yield of each reaction is increased. For example, the production method of this embodiment includes a step of preparing a precursor peptide, a step of allowing the azole ring formation reaction and the α,β-unsaturated amino acid formation reaction to proceed, a step of allowing the macrocyclization reaction to proceed, and a step of allowing the prenylation reaction to proceed, and it is preferable that the step of allowing the azole ring formation reaction and the α,β-unsaturated amino acid formation reaction to proceed be performed under concentration conditions for the azole ring-forming enzyme and the α,β-unsaturated amino acid-forming enzyme that achieve both high azole ring formation efficiency and a small volume when the enzyme solution is added.

[0195] Such conditions may be, for example, conditions in which the factor defined in the Examples for v.207, a model peptide described below, is 2.3 or more, 2.5 or more, or 2.6 or more, and the enzyme solution used in the step of promoting the azole ring-forming reaction and the α,β-unsaturated amino acid-forming reaction is 0.7 equivalents or less, 0.5 equivalents or less, or 0.3 equivalents or less of the solution used in the step of preparing the precursor peptide. Such concentration conditions for the azole ring-forming enzyme and the α,β-unsaturated amino acid-forming enzyme can be achieved by appropriately adjusting the concentration of each enzyme.

[0196] (Other Steps) The production method of this embodiment may include steps other than the steps described above. Such steps include, for example, a step of preparing substrates for the enzymatic reactions, a step of preparing each enzyme, a step of separating and purifying the products obtained by each reaction, and a step of analyzing the physical properties of the purified compounds.

[0197] The step of preparing an enzyme is a step of preparing an enzyme as described above. In this step, the enzyme may be isolated from a naturally occurring organism, or may be obtained by heterologously expressing a gene encoding the target enzyme in a suitable host (e.g., Escherichia coli), or may be obtained by a cell-free translation system or chemical synthesis.

[0198] The step of separating and / or purifying the product is a step of separating the product obtained in each step of the production method of this embodiment, particularly the cyclic compound of formula (I), from raw materials and / or by-products, and / or purifying each of the target compounds when multiple target compounds are obtained. For such a step, a known separation method such as liquid chromatography may be used.

[0199] [Method for Producing a Compound Library] The method for producing a compound library containing two or more types of cyclic compounds represented by formula (I) of this embodiment includes: preparing a precursor peptide library containing two or more types of precursor peptides represented by formula (III); forming a peptide represented by formula (II) from a precursor peptide represented by formula (III) by the following reactions: a reaction of forming an azole ring at at least Y' (11) in the precursor peptide represented by formula (III) using an azole ring-forming enzyme; and a reaction of converting at least X' and Y' (12) in the precursor peptide represented by formula (III) into α,β-unsaturated amino acid residues using an α,β-unsaturated amino acid-forming enzyme in the presence of a cosubstrate for glutamylation and an aminoacylase; allowing a macrocyclase to act on the peptide represented by formula (II) to form a nitrogen-containing six-membered ring A while eliminating LP, if present, to form a cyclic compound represented by formula (I'); and allowing a prenylation enzyme to act on the cyclic compound represented by formula (I') to convert at least X ａ ', X ｂ , X ｃ and Z, to form a cyclic compound represented by formula (I), thereby forming a compound library containing two or more cyclic compounds represented by formula (I).

[0200] As used herein, the term "compound library" refers to a group of compounds containing at least two compounds. The compound library obtained by the compound library production method of this embodiment is a compound library containing two or more types of cyclic compounds of formula (I) above. The compounds contained in the library may be compounds in which another structure is bound to the cyclic compound of formula (I) above by a covalent or non-covalent bond. Examples of moieties having such another structure include linkers, nucleic acid molecules such as DNA and RNA, and labels for detecting the cyclic compound of formula (I) (fluorescent substances, radioactive substances, metal nanoparticles, quantum dots, enzymes, etc.).

[0201] The method for producing a compound library of this embodiment can be carried out in the same manner as the method for producing a cyclic compound of this embodiment described in detail above, with the exception that a precursor peptide library containing two or more precursor peptides represented by formula (III) is used as a substrate. Hereinafter, as an example of a process for preparing a precursor peptide library to be used as a substrate, a process for adjusting a precursor peptide library (hereinafter referred to as a "library preparation process") will be described in detail. The precursor peptide library may also be prepared by purchasing a commercially available product.

[0202] (Library Preparation Step) The library preparation step is not particularly limited as long as it is a step that can prepare a library containing two or more precursor peptides represented by formula (III). The precursor peptides in the library may be synthesized by solid-phase or liquid-phase chemical synthesis, or may be prepared by a method using a translation synthesis system, but are preferably prepared by a method using a cell-free translation system.

[0203] The library preparation step is preferably a step of expressing a precursor peptide represented by formula (III) based on a library of nucleic acids. The nucleic acids may be DNA, cDNA, or mRNA. More preferably, the library preparation step is a step of translating an mRNA library using a cell-free translation system to prepare a compound library containing the precursor peptide represented by formula (III).

[0204] Hereinafter, a method using an mRNA library as a nucleic acid library will be described in detail, but the nucleic acid is not limited to mRNA.

[0205] As used herein, the term "mRNA library" refers to a group of mRNAs containing two or more types of mRNAs each having a plurality of N1N2N3 codons. Furthermore, as used herein, "N1N2N3" refers to a codon that specifies any amino acid, where N1, N2, and N3 are each independently selected from adenine (A), guanine (G), cytosine (C), and uracil (U). A single mRNA contains multiple N1N2N3s, each of which is independently selected. Therefore, for example, when an mRNA contains -N1N2N3-N1N2N3-, each of the two N1s, N2s, and N3s may be the same or different.

[0206] In one embodiment of the library preparation step, an mRNA library containing mRNA encoding peptides for each compound in the compound library is first prepared (hereinafter referred to as the "mRNA preparation step"). The sequence of the mRNA encoding the peptide is determined according to the amino acid sequence of the desired peptide. Such an mRNA library may be prepared, for example, by synthesizing a DNA library encoding the peptide and transcribing it.

[0207] In the mRNA preparation step, any amino acid may be reassigned to the N1N2N3 codon. The reassignment may be different from the relationship between codons and amino acids in the natural genetic code, or the same relationship may be assigned. As used herein, the term "natural genetic code" refers to a table showing amino acids represented by genetic codes consisting of triplets of mRNA in living organisms. In the natural genetic code, N1N2N3 encodes the following amino acids:

[0208]

[0209] More specifically, in the mRNA preparation step, for example, Leu may be assigned to the UUG codon according to the above-mentioned natural genetic code, or an amino acid other than Leu may be assigned by amino acid reassignment. "Assigning an amino acid to a codon" means rewriting the genetic code so that a certain codon encodes that amino acid. In this specification, "assigning an amino acid to a codon" and "reassigning a codon" are used synonymously.

[0210] Assignment of an amino acid to each codon that differs from that in the natural genetic code can be achieved, for example, by codon reassignment using the artificial aminoacylation RNA catalyst Flexizyme. Flexizyme allows a desired amino acid to be attached to a tRNA having any anticodon, thereby making it possible to assign any amino acid to any codon. Known Flexizymes, such as those disclosed in H. Murakami, H. Saito, and H. Suga, (2003), Chem. Biol., Vol. 10, 655-662, may be used. Herein, attaching an amino acid to a tRNA is referred to as charging an amino acid to the tRNA, aminoacylating the tRNA, or acylating the tRNA with an amino acid.

[0211] The multiple N1N2N3 in each mRNA are not particularly limited and may be completely random, or may have some bases fixed to specific bases, such as multiple N1N2K, multiple N1N2S, multiple N1N2M, multiple N1N2W, multiple N1N2A, multiple N1N2U, multiple N1N2C, or multiple N1N2G. In the above, N1 and N2 are synonymous with N1 and N2 in N1N2N3, K is independently either uracil (U) or guanine (G), S is independently either cytosine (C) or guanine (G), M is independently either adenine (A) or cytosine (C), and W is independently either adenine (A) or uracil (U).

[0212] For convenience, the mRNA preparation process will be described below using an example in which the mRNA library contains mRNAs containing multiple N1N2K codons, i.e., each mRNA in the mRNA library contains multiple N1N2K codons, but the process can be carried out in the same manner when other mRNA libraries are used, as long as the translated compound library contains the precursor peptide represented by formula (III). In the natural genetic code, N1N2K represents 20 amino acids whose right column in Table 2 above is G or U.

[0213] In the mRNA preparation step, any amino acid can be assigned to the "N1N2K" codon, and not only proteinogenic amino acids but also, for example, non-proteinogenic amino acids may be assigned. For example, amino acids having a cyclic structure and / or N-alkyl amino acids may be used as non-proteinogenic amino acids. In the mRNA preparation step, all of the multiple N1N2Ks may be assigned to non-proteinogenic amino acids, or some of them may be assigned to non-proteinogenic amino acids.

[0214] Preferably, each mRNA in the mRNA library contains, in this order, a start codon, multiple N1N2N3 sequences including multiple N1N2K sequences, and a stop codon. When using the natural genetic code, each mRNA contains, for example, a sequence of AUG-(N1N2N3)s-UAG, where s corresponds to the number of amino acids contained in LP' to Z' in formula (III).

[0215] The plurality of N1N2N3 includes X', X in formula (III). ｂ The codons to which X′, X′, and Y′ are assigned are ｂ In addition, when a specific prenylation enzyme is used, it is preferable that the X ａ ", and a codon encoding an amino acid that can be prenylated by the prenylation enzyme is X in formula (III). ａ The codon encoding an amino acid that can be prenylated by the prenylation enzyme is preferably contained at a position corresponding to X in formula (III). ｂ ', X ｃ ', and / or Z' may be included at positions corresponding to Z'.

[0216] Furthermore, in formula (III), where preferred amino acids or analogs thereof are described above, it is preferable to design multiple N1N2N3 so that the translated peptide contains the preferred amino acids or analogs thereof.

[0217] An example of the design of a precursor peptide and the corresponding mRNA is shown in Figure 7(A). In Figure 7(A), in formula (III), X' is serine, and the N-terminal X adjacent to X' is ｄ X' is tryptophan, and X' is adjacent to X' with one tryptophan in between. ｄ ' is an amino acid encoded by DSK, and X ａ " is tryptophan, and X ａ The amino acid adjacent to the N-terminal side of X is an amino acid encoded by RYU, ｂ '-Y'-X ｃ ' is SSSC, and X ｂThe amino acids adjacent to the N-terminal of X' are encoded by NNU, and the other random sequences are encoded by NNK. ａ RYU, which corresponds to the amino acid adjacent to the N-terminal side of ", is ａ " is selected to have high prenylation efficiency. RYU may be, for example, CMG.

[0218] Here, R each independently represents G or A, Y each independently represents U or C, M each independently represents A or C, K each independently represents G or U, D each independently represents A, G or U, and S each independently represents G or C.

[0219] In the library preparation step, the mRNA library prepared in the mRNA preparation step is then translated (hereinafter referred to as the "translation step"). The translation step may be carried out, for example, in a cell-free translation system having an anticodon corresponding to one of the N1N2N3 codons and containing a tRNA charged with the amino acid assigned to that codon. The N1N2N3 codon and tRNA contained in the cell-free translation system may be, for example, 20 types.

[0220] The cell-free translation system may be a translation system obtained by freely removing components of an existing translation system according to the purpose, or freely adding other components, and reconstructing only the necessary components. For example, when a translation system from which a specific amino acid has been removed is reconstructed, the codon corresponding to that amino acid becomes an empty codon that does not encode any amino acid. Therefore, by using flexizyme or the like to charge a tRNA having an anticodon complementary to that empty codon with an arbitrary amino acid and then adding this to perform translation, the arbitrary amino acid will be encoded by that codon. This allows the preparation of a peptide in which the arbitrary amino acid has been introduced in place of the removed amino acid.

[0221] The tRNA in the cell-free translation system may be a wild-type tRNA derived from an organism (e.g., Escherichia coli) or an artificial tRNA prepared by in vitro transcription. The 20 types of tRNA corresponding to the 20 types of N1N2N3 codons used in the cell-free translation system may have the same sequence except for the anticodon loop portion. In this embodiment, the reactivity of a particular tRNA is not higher or lower, and each tRNA has uniform reactivity, making it possible to express a desired peptide with good reproducibility.

[0222] As used herein, the term "cell-free translation system" refers to a translation system that does not contain cells. Examples of cell-free translation systems that can be used include Escherichia coli extract, wheat germ extract, rabbit erythrocyte extract, and insect cell extract. Alternatively, a reconstructed cell-free translation system constructed by reconstructing purified ribosomal proteins, aminoacyl-tRNA synthetases (aaRS), ribosomal RNA, amino acids, rRNA, GTP, ATP, translation initiation factors (IF), elongation factors (EF), release factors (RF), and ribosome recycling factors (RRF), as well as other factors necessary for translation, may also be used. From the viewpoint of simultaneously performing transcription from DNA, a cell-free translation system containing RNA polymerase may also be used.

[0223] Commercially available cell-free translation systems include RTS-100 (registered trademark) from Roche Diagnostics, which is a system derived from Escherichia coli; PURESYSTEM (registered trademark) from PGI, PUREfrex from GeneFrontier, and PURExpress In Vitro Protein Synthesis Kit from New England BioLabs, which are reconstructed translation systems; and systems using wheat germ extract from Zoigene or CellFree Sciences. Furthermore, as a system using Escherichia coli ribosomes, the techniques described in the following documents are known, for example: H. F. Kung et al., 1977, The J. Biol. Chem. Vol. 252, No. 19, 6889-6894; M. C. Gonza et al., 1985, Proc. Natl. Acad. Sci. USA Vol. 82, 1648-1652; M. Y. Pavlov and M. Ehrenberg, 1996, Arch. Biochem. Biophys. Vol. 328, No. 1, 9-16; Y. Shimizu et al., 2001, Nat. Biotechnol. Vol. 19, No. 8, 751-755; H. Ohashi et al., 2007, Biochem. Biophys. Res. Commun. Vol. 352, No. 1, 270-276.

[0224] The cell-free translation system includes an elongation tRNA and may further include an initiator tRNA. The present inventors have previously constructed a translation system in which N1N2N3 encodes any amino acid by codon reassignment using flexizyme. In natural translation systems, tRNAs exist with anticodons corresponding to each amino acid, and each tRNA has a unique sequence even in regions other than the anticodon loop. However, by using flexizyme to reassign any amino acid to all N1N2N3, all tRNAs can be artificial. In this case, the elongation tRNAs corresponding to each N1N2N3 contained in the translation system may have an identical base sequence for 80% or more, 85% or more, 88% or more, or 90% or more of their total length. In other words, a group of elongation tRNAs with almost the same sequence, excluding the anticodon, can be used. The group of elongation tRNAs may have the same base sequence except for the anticodon loop. The extension tRNAs corresponding to each N1N2N3 added to the translation system may have sequences other than the anticodon loop that are 85% or more, 88% or more, 90% or more, 93% or more, 95% or more, 98% or more, or 99% or more identical.

[0225] As used herein, the term "anticodon loop" refers to a single-stranded loop portion of a tRNA that contains an anticodon. The sequence of the anticodon loop can be appropriately determined by those skilled in the art so as to complement the codon-anticodon interaction.

[0226] According to the method using the cell-free translation system as described above, it is possible to obtain an expression product in a highly pure form without purification. Furthermore, based on the diversity of N1N2N3 in mRNA, １２ ~1 x 10 １４ It is possible to produce libraries containing more than one different peptide.

[0227] The library preparation step may be a step of preparing a precursor peptide library containing genotype-linked precursor peptides by a display method.

[0228] A display method refers to a system in which a phenotype is displayed on a genotype by covalently or non-covalently linking the phenotype to a genotype encoding the sequence, and active species can be enriched and amplified (i.e., selected) using a replication system reconstructed in a test tube. Examples of display methods include phage display, which uses Escherichia coli as a replication medium, and yeast display. Display methods also include in vitro display, which does not use prokaryotic or eukaryotic organisms as a medium. In particular, in vitro display enables the exploration of more diverse libraries than phage display. Examples of in vitro display include ribosome display, cDNA display, and mRNA display. The library preparation step may be a step of preparing a precursor peptide library containing precursor peptides bound to genotypes using an mRNA display method.

[0229] The library preparation step may be a step of forming a precursor peptide library containing two or more complexes of precursor peptides represented by formula (III) and nucleic acids encoding the precursor peptides by expression, further comprising binding puromycin to the 3' end of each nucleic acid in the nucleic acid library. The nucleic acid may be mRNA, and in this case, the step is a step of preparing a precursor peptide library containing precursor peptides to which genotypes are bound by an mRNA display method.

[0230] The step of binding puromycin to the 3' end of a nucleic acid can be carried out by a conventionally known method. For example, when the nucleic acid is mRNA, this can be carried out by binding puromycin to the downstream region of the ORF (open reading frame) of each mRNA in the above-mentioned mRNA preparation step. Puromycin may be bound to the mRNA via a linker composed of a peptide or nucleic acid. By binding puromycin to the downstream region of the ORF of the mRNA, the ribosome that translated the ORF of the mRNA takes up puromycin, forming a complex between the mRNA and the peptide.

[0231] [Screening Method] One aspect of this embodiment is a screening method for identifying a compound that binds to a target substance, comprising contacting a compound library containing two or more types of cyclic compounds of formula (I) produced by the above-described production method with a target substance, and selecting a compound that binds to the target substance.

[0232] The step of contacting a compound with a target substance may be, for example, a method of incubating a system in which the compound and the target substance coexist, or a method of immobilizing the target substance on a solid phase carrier and contacting a liquid phase containing the compound with the solid phase carrier. The contact may be carried out in an appropriately selected buffer solution, and the pH, temperature, contact time, etc. may be adjusted to allow interaction. As used herein, the term "solid phase carrier" is not particularly limited as long as it is a carrier capable of immobilizing a target substance, and examples thereof include glass, metal, and resin microtiter plates, substrates, beads, nitrocellulose membranes, nylon membranes, and PVDF membranes. The target substance can be immobilized on these solid phase carriers according to known methods.

[0233] In this specification, the "target substance" is not particularly limited, and may be a low molecular weight compound, a high molecular weight compound, a nucleic acid, a peptide, a protein, a sugar, a lipid, or the like.

[0234] The step of selecting a compound that binds to a target substance can be carried out, for example, by detectably labeling the compound according to a known method, contacting the compound with the target substance, washing the surface of the solid support with a buffer solution, and detecting the compound that binds to the target substance. Detectable labels include enzymes such as peroxidase and alkaline phosphatase; １３１ I, ３５ S. １８ F and ３ Examples of the label include radioactive substances such as H; fluorescent substances such as fluorescein isothiocyanate, rhodamine, dansyl chloride, phycoerythrin, tetramethylrhodamine isothiocyanate, and near-infrared fluorescent materials; luminescent substances such as luciferase, luciferin, and aequorin; and nanoparticles such as gold colloids and quantum dots. When an enzyme is used as a label, the enzyme substrate can be brought into contact with the enzyme to develop color and then detected. Alternatively, biotin can be bound to the compound, and avidin or streptavidin labeled with an enzyme or the like can be bound to the compound for detection.

[0235] Furthermore, it is possible to not only detect and measure the presence or absence or the degree of binding, but also to measure the enhancement or inhibition of the activity of the target substance and identify compounds having such enhancing or inhibiting activity. By using such a method, it is also possible to identify compounds that have physiological activity and are useful as pharmaceuticals.

[0236] In the screening method of this embodiment, a display method may be applied to the step of selecting a compound that binds to a target substance. In the screening method of this embodiment, when a library of compounds containing mRNA is used, an mRNA display method can be applied to the step of selecting a compound that binds to a target substance.

[0237] In the mRNA display method, a cDNA library is obtained by performing a reverse transcription reaction on a library of compounds containing mRNA. Each cDNA in this cDNA library encodes a peptide or protein that binds to a target molecule. The cDNA library is amplified and transcribed to obtain a new mRNA library. The obtained mRNA library has a higher concentration of molecules that bind to the target molecule compared to the original mRNA library. Therefore, by repeating the above process multiple times, molecules that bind to the target molecule can be gradually enriched. The amino acid sequences of the enriched peptides or proteins can be identified by analyzing the cDNA sequence, making it easy to select compounds with high affinity for the target molecule based on the sequence information.

[0238] The RaPID system (Yamagishi, Y. et al., Chem. Biol., 2011, 18(12), 1562-70) is an example of a screening system that combines the FIT system used in the Examples described below with the mRNA display method.

[0239] [Cyclic compounds that specifically bind to WNK4] As described below, in this embodiment, it has been discovered that there are multiple cyclic compounds of formula (I) that selectively bind to WNK4 (see Am. J. Physiol. Renal Physiol. 320, F378-F403 (2021)).

[0240] WNK4 is a kinase present in cells and is involved in ion homeostasis, so inhibitors of WNK4 are promising drugs for treating hypertension. Furthermore, WNK1, a kinase in the same family as WNK4, is more widely distributed than WNK4, so it is desirable to selectively inhibit only WNK4 without inhibiting WNK1. Therefore, compounds that bind to WNK4 but not WNK1 are particularly promising drugs.

[0241] From the viewpoint of increasing the binding strength to WNK4 and decreasing the binding strength to WNK1, the cyclic compound represented by formula (I) preferably has the following characteristics.

[0242] X ａ is preferably a prenylated tryptophan, more preferably a C3 prenylated tryptophan.

[0243] X ｂ is preferably the above analog (I), and more preferably R Ｘ１ is a group in which the atom is a hydrogen atom.

[0244] X ｃ is preferably the azole of the above analog (II). In this case, the cyclic compound represented by formula (I) is preferably the azole of the above formula (I-X Ｃ ) structure. ｃ is more preferably R in the azole of analog (II) above. Ｃ is a methyl group or a hydrogen atom, and B Ｃ is a group in which the sulfur atom is

[0245] R １ is preferably a methyl group or a hydrogen atom.

[0246] B １ is preferably an oxygen atom.

[0247] Ring A preferably has the following structure: (In the formula, R Ａ１ and R Ａ２ is preferably a hydrogen atom or a methyl group. More preferably, it is a pyridine ring.

[0248] Z is preferably a peptide consisting of 1 to 50 amino acids and / or analogs thereof, more preferably a peptide consisting of 1 to 40 amino acids and / or analogs thereof, even more preferably a peptide consisting of 3 to 30 amino acids and / or analogs thereof, and even more preferably AQPYPYDVPDYAVIQPLE (SEQ ID NO: 77) or an amino acid sequence in which one or several amino acids have been deleted, substituted and / or added in this sequence. The amino acid in Z may form an α,β-unsaturated bond, may form an azoline or azole, or may have a prenyl group.

[0249] The sum of m and n is preferably an integer of 4 or more and 19 or less, 5 or more and 18 or less, 6 or more and 17 or less, 8 or more and 15 or less, or 9 or more and 14 or less.

[0250] -(X ｄ ) ｍ -X ａ -(X ｅ ) ｎ - is one of the following: WX １０ VX １１ WX １２ (X １３ ) ３ GWX １４ X １５ (WNK4-1) WX １６ KQHWMWNPY (WNK4-2) WSNX １６ AWX １７ HNPGX １８ P (WNK4-3) , or a sequence in which C is replaced with Q in the above formulas (WNK4-1), (WNK4-2), and (WNK4-3), or an amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in these sequences.

[0251] X １０ is R, S, or T, or analogs thereof. １１ is V or T, or an analog thereof. １２ is L or M, or an analog thereof. １３ are each independently an amino acid or an analog thereof. １４is Y, F, or W, or an analog thereof. １５ is G or A, or an analog thereof. １６ is D or A, or an analog thereof. １７ is W or L, or an analog thereof. １８ is C, or Q, or an analog thereof.

[0252] In formulae (WNK4-1), (WNK4-2), and (WNK4-3), each amino acid may have an α,β-unsaturated bond, W may be prenylated, S and T may have an azoline or azole formed therein, and C may have an azoline or azole formed therein, and iodoacetamide may be bonded to the thiol group.

[0253] In formula (WNK4-1), the fourth W from the N-terminus is preferably prenylated. The third W from the C-terminus may be prenylated, but is preferably not prenylated.

[0254] In formula (WNK4-2), it is preferred that the sixth W from the N-terminus is prenylated.

[0255] In formula (WNK4-3), it is preferred that the sixth W from the N-terminus is prenylated.

[0256] -(X ｄ ) ｍ -X ａ -(X ｅ ) ｎ It is more preferable that - is -W-(amino seq)-, where -(amino seq)- is any of the following amino acid sequences: The following are SEQ ID NOS: 78 to 91, respectively. In the following amino acid sequences, one or several amino acids may be deleted, substituted, and / or added.

[0257] In the above amino acid sequences, each amino acid may have an α,β-unsaturated bond, W may be prenylated, S and T may form an azoline or azole, and C may form an azoline or azole, and iodoacetamide may be bonded to the thiol group. In addition, in each sequence corresponding to formula (WNK4-1), the fourth W from the N-terminus is preferably prenylated, and the third W from the C-terminus is preferably not prenylated. In each sequence corresponding to formula (WNK4-2), the sixth W from the N-terminus is preferably prenylated. In each sequence corresponding to formula (WNK4-3), the sixth W from the N-terminus is preferably prenylated.

[0258] The cyclic compounds of formula (I) may be antagonists of WNK4.

[0259] This embodiment may further be embodied in the following aspects: a pharmaceutical composition comprising a cyclic compound of formula (I); a cyclic compound of formula (I) for use in the treatment or prevention of a disease; a pharmaceutical composition comprising a cyclic compound of formula (I) for use in the treatment or prevention of a disease; use of a cyclic compound of formula (I) for the treatment or prevention of a disease; use of a cyclic compound of formula (I) in the manufacture of a medicament for the treatment or prevention of a disease; or a cyclic compound of formula (I) for use in the manufacture of a medicament for the treatment or prevention of a disease.

[0260] The disease may be a disease associated with WNK4, or a disease that is treated or prevented by inhibiting WNK4, such as hypertension.

[0261] In each of the above aspects, the cyclic compound of formula (I) may be in the form of a pharmacologically acceptable salt. Pharmacologically acceptable salts include, for example, salts with pharmaceutically acceptable bases or acids. Specific, non-limiting examples of pharmacologically acceptable salts include addition salts with inorganic acids (hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, etc.), addition salts with organic acids (p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carboxylic acids, succinic acid, citric acid, benzoic acid, acetic acid, etc.), addition salts with inorganic bases (ammonium hydroxide or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, etc.), and addition salts of amino acids.

[0262] In each of the above aspects, the pharmaceutical composition may further contain a pharmaceutically acceptable carrier and / or additive in addition to the cyclic compound of formula (I) and may be formulated. Examples of formulations include oral preparations such as tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions, and emulsions; and parenteral preparations such as injections, infusions, suppositories, ointments, and patches. The content of the carrier or additive may be appropriately set based on the range commonly used in the pharmaceutical field. Examples of the carrier include, but are not limited to, water, physiological saline, other aqueous solvents, and aqueous or oily bases. Examples of the additive include, but are not limited to, excipients, binders, pH adjusters, disintegrants, absorption enhancers, lubricants, colorants, flavoring agents, and fragrances. The above carriers and / or additives may be used alone or in combination of two or more.

[0263] In each of the above aspects, the pharmaceutical composition may be administered parenterally (e.g., intravenously, intramuscularly, intradermally, intraperitoneally, subcutaneously, or topically) as an injection or infusion formulated with a pharmaceutically acceptable carrier.

[0264] Furthermore, the injection or infusion solution may contain additives such as stabilizers, solubilizing agents, suspending agents, emulsifying agents, soothing agents, buffers, preservatives, antiseptics, and pH adjusters.

[0265] The subject to which the above-mentioned method for treating or preventing a disease is applied may be a human or a non-human mammal (e.g., rat, mouse, rabbit, sheep, pig, cow, cat, dog, monkey, etc.). The dose of the cyclic compound of formula (I) may be appropriately determined taking into consideration the purpose, severity of the disease, age, weight, sex, medical history, type of active ingredient, etc. of the patient.

[0266] In any of the above embodiments described in this specification, the aspects described in this specification and the aspects described as preferred aspects, etc. in this specification may be arbitrarily combined.

[0267] The present invention will be described in more detail below using examples and comparative examples, but the present invention is not limited to the following examples.

[0268] [Exploring the biosynthetic pathway of prenylated thiopeptides] In order to explore the biosynthetic pathway of molecules having a prenyl group and a dehydroamino acid, an azole, and a pyridine ring-closed skeleton, which are characteristic of thiopeptides, in the same molecule, peptide synthesis was carried out using three setups.

[0269] (Preparation of DNA templates) DNA templates were prepared to synthesize the following model peptides, MS1, MS2, and MS3 (SEQ ID NOs: 92 to 94). Note that "LP" is the leader peptide sequence MSDITASRVESLDLQDLDLSELTVTSLRDTVALPENGA (SEQ ID NO: 76), and "tail" is the sequence AQPYPYDVPDYAVIQPLE (SEQ ID NO: 77). The same applies hereinafter unless otherwise specified.

[0270] To prepare the DNA templates for MS1 and MS2, 0.5 μM of the appropriate forward and reverse primers (see the table below) were added to 1x Taq PCR master mix (composition described below) and denatured at 95°C for 1 minute. Five cycles of annealing (50°C for 1 minute) and extension (72°C for 1 minute) were then performed, and 1 μL of the resulting product was used as the template for the next step. A second PCR was performed with 15 cycles of denaturation (95°C for 40 seconds), annealing (50°C for 40 seconds), and extension (72°C for 40 seconds). Amplification of the PCR products was confirmed by 3% agarose gel electrophoresis. To extract the DNA, an equal volume of a 25 / 24 / 1 mixture of phenol / chloroform / isoamyl alcohol was added to the PCR mixture and centrifuged at 15,300 g for 2 minutes. The supernatant was collected, and an equal volume of a 24:1 mixture of chloroform and isoamyl alcohol was added. The mixture was centrifuged at 15,300 g for 2 minutes to obtain the supernatant. Ethanol and aqueous NaCl solution were then added to the sample mixture to make 70% ethanol and 0.1 M NaCl. The mixture was centrifuged at 15,300 g for 15 minutes, the supernatant was removed, and the precipitated DNA or RNA was resuspended in 70% ethanol. The resulting mixture was centrifuged at 15,300 g for 5 minutes. The supernatant was removed, and the precipitated DNA was dried at room temperature for 10 minutes.

[0271] To prepare the DNA template for MS3, 1x Taq PCR master mix and 0.5 μM of the appropriate forward and reverse primers (see the table below) were prepared. The primers and 0.4 nM template DNA LP-SW were added to the 1x Taq PCR master mix, and five cycles of denaturation (95°C for 40 seconds), annealing (50°C for 40 seconds), and extension (72°C for 40 seconds) were performed. 1 μL of the product was used as the template for the next step. A second PCR was performed for nine cycles under the same conditions as the first PCR. After confirming the amplification of the PCR product by 3% agarose gel electrophoresis, the DNA was extracted and precipitated in the same manner as for MS1 and MS2. To prepare DNA LP-SW and the LP-S described below, 1x Taq PCR master mix was added with 0.5 μM of the appropriate forward and reverse primers (see the table below) and 0.4 nM template DNA v. 151 was added. The first PCR consisted of 13 cycles of denaturation (95°C for 40 seconds), annealing (50°C for 40 seconds), and extension (72°C for 40 seconds). DNA was extracted and precipitated in the same manner as above.

[0272] The composition of the 1x Taq PCR master mix is ​​as follows: 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% (v / v) Triton X-100, 2.5 mM MgCl ２ , 250 μM each dNTP, 0.75% DNA polymerase solution. For electrophoresis, 2x DNA loading buffer was used, prepared as follows: 968 mg Tris, 228.4 μL acetic acid, 400 μL 0.5 M EDTA, 16.7 mL glycerol, and 1 μL 2% BPB solution were dissolved in 50 mL distilled water. 3% agarose gel was prepared as follows: 3 g agarose was added to 40 mM Tris-acetate and 1 mM EDTA solution to make a 100 mL solution. After heating, 5 μL of ethidium bromide was added.

[0273] (Setup 1) An in vitro translation mixture (composition described below) containing 0.08 μM DNA template was incubated at 37°C for 30 minutes and then used in the following multi-step enzymatic reaction. An equal volume of 2x Laz DEF mixture (composition described below) was added to the translation mixture and incubated at 25°C for 2 hours. Next, iodoacetamide (IAA) was added to a final concentration of 5 mM and incubated at 4°C for 2 minutes. Then, an equal volume of 2x Laz BFC mixture (composition described below) was added to the reaction mixture and incubated at 25°C for 6 hours. Next, KgpF and DMAPP (dimethylallyl diphosphate) were added to a final concentration of 72 μM for KgpF and 1 mM for DMAPP, and the mixture was incubated at 37°C for 18 hours. Next, an equal volume of MeOH was added and the mixture was incubated on ice for at least 10 minutes. After centrifugation, the supernatant was used for LC-MS analysis.

[0274] The composition of the in vitro translation mixture is as follows: a solution containing the following final concentrations of each component: 50 mM HEPES-K (pH 7.6), 100 mM KOAc, 2 mM GTP, 2 mM ATP, 1 mM CTP, 1 mM UTP, 20 mM creatine phosphate, and 12 mM Mg(OAc). ２, 2 mM spermidine, 2 mM DTT, 1.5 mg / mL E. coli total tRNA, 1.2 μM ribosome, 0.6 μM MTF, 2.7 μM IF1, 0.4 μM IF2, 1.5 μM IF3, 10 μM EF-Tu, 10 μM EF-Ts, 0.26 μM EF-G, 1 μM RF1, 0.25 μM RF2, 0.17 μM RF3, 0.5 μM RRF, 4 μg / mL creatine kinase, 3 μg / mL myokinase, 0.1 μM pyrophosphatase, 0.1 μM nucleotide-diphosphatase kinase, 0.1 μM T7 RNA polymerase, 0.73 μM AlaRS, 0.03 μM ArgRS, 0.38 μM AsnRS, 0.13 μM AspRS, 0.02 μM CysRS, 0.06 μM GlnRS, 0.23 μM GluRS, 0.09 μM GlyRS, 0.02 μM HisRS, 0.4 μM IleRS, 0.04 μM LeuRS, 0.1 1 μM LysRS, 0.03 μM MetRS, 0.68 μM PheRS, 0.16 μM ProRS, 0.04 μM SerRS, 0.09 μM ThrRS, 0.03 μM TrpRS, 0.02 μM TyrRS, 0.02 μM ValRS, 500 μM each proteinogenic amino acid, and 100 μM 10-HCO-H4folate.

[0275] The composition of the 2x LazDEF mixture was as follows: 100 mM Tris-HCl (pH 8.0), 9 mM ATP (pH 7.0), 2 mM DTT, 20 mM MgCl ２ , 4 μM lazD, 2 μM lazE, and 4 μM lazF. The composition of the 2x LazBFC mixture was as follows: 50 mM Tris-HCl (pH 8.0), 4.5 mM ATP (pH 7.0), 0.5 mM glucose, and 10 mM MgCl. ２ , 4 μM lazB, 8 μM lazC, 3.1 μM lazF, 2 μM GluRS S. lividans, 40 μM tRNAGlu S. lactacystinaeus.

[0276] LC-MS analysis was performed using a Waters Xevo G2-XS QTof equipped with an Acquity I-Class UPLC system. The UPLC method was performed using an Acquity UPLC Peptide BEH C18 column (dimensions: 150 x 1 mm; pore size: 300 Å; particle size: 1.7 μm) with 0.1% (v / v) acetic acid in water (solvent A) or acetonitrile (solvent B) as the mobile phase. The analysis was performed at 60°C with a flow rate of 250 μL / min, running the following gradient: 1% B for 2 min; 1% to 60% B over 7.5 min; 95% B for 2 min; and 1% B for 8.5 min (total run time: 20 min). MS analysis was performed in positive polarity / high sensitivity mode with a scan time of 0.3 s. The capillary voltage was set to 700 V, and the ESI source temperature and desolvation temperature were 120°C and 400°C, respectively. Leucine Enkephalin, supplied by the manufacturer, was used as a lockspray standard for mass calibration during analysis, and the recommended lockspray setup procedure was performed before each measurement. Prenylation efficiency was calculated using the following formula: Unless otherwise specified, XIC peak areas were used for calculations. The XIC absolute window was always m / z = calculated value ± 0.05.

[0277] Like, D, Z, Z, GluRS, and Streptomyces lactacystinaeus Streptomyces lactacystinaeus Streptomyces lactacystinaeus tRNAGlu is a strong mechanism by tRNAGlu (Vinogradov, AA; Shimomura , M. ; Goto, Y.; Sugai, Y.; Suga, H.; Onaka, H. Minimal Lactazole Scaffold for In Vitro Thiopeptide Bioengineering. Nat. Commun. 2020, 11, 2272. Vinogradov, AA; Nagai, E.; Chang , JS ; Narumi , K. ; Onaka, H.; Goto, Y.; Suga, H. Accurate Broadcasting of Substrate Fitness for Lactazole Biosynthetic Pathway from Reactivity- Profiling MRNA Display. J. Am. Chem. Soc. Rev. 2020, 142, 20329–2 K Vinogradov, AA; Nagano, M.; Goto, Y.; Suga, H. Site-Specific Nonenzymatic Peptide S / O- Glutamylation Reveals the Extent of Substrate Promiscuity in Glutamate Elimination Domains. J. Am. Chem. Soc. 2021, 143, 13358-13369.

[0278] KgpF was expressed in E. coli BL21(DE3) pLysS using the appropriate pET32 plasmid. An overnight LB culture (10 mL) was inoculated into 1 L of SB medium containing 100 μg / mL ampicillin and 100 μg / mL chloramphenicol. The cells were grown at 30°C for 5 hours with shaking (180 rpm) and then induced with a final concentration of 100 μM isopropyl β-D-1-thiogalactopyranoside (IPTG). After 16 hours of KgpF expression at 16°C with shaking at 180 rpm, the cells were harvested by centrifugation (5000 × g, 10 min). The pellet was resuspended in 30 mL of KgpF disruption buffer and disrupted by sonication on ice. The supernatant was filtered through a 0.44 mm filter (Millipore). The filtrate was applied to a 5 mL His-trap HP column (GE Healthcare) using an AKTA Avant 25 (GE Healthcare). KgpF was eluted with imidazole using a gradient method using KgpF buffer A and KgpF buffer B. Fractions were analyzed by 10% SDS-PAGE, and fractions containing the corresponding band were pooled. 20% SUMO protease (volume %) was added to the collected solution and dialyzed against 2 L of KgpF buffer C for 20 hours. The collected solution was directly applied to a His-trap HP column (GE Healthcare) connected to a His-trap Q HP column (GE Healthcare). KgpF was eluted with KgpF buffer D. Fractions were analyzed by absorbance at 280 nm using a Nanodrop (Thermo). The collected solution was concentrated to a total volume of 2.5 mL using an Amicon® Ultra 50 kDa (Millipore). The solution was then subjected to size-exclusion chromatography using a HiLoad 16 / 200 Superdex 200pg column (GE Healthcare) with AKTA and KgpF storage buffer. Fractions were analyzed by 10% SDS-PAGE, and fractions containing the corresponding bands were pooled. The pooled solution was concentrated using an Amicon® Ultra 50 kDa column (Millipore). The concentration was confirmed by measuring the absorbance at 280 nm using a Nanodrop (Thermo).

[0279] The composition of the reagents used was as follows: Buffer A for KgpF: 40 mM K ２ HPO ４ , 10 mM KH ２ P.O. ４ (pH 7.8), 500 mM NaCl, 20 mM imidazole (pH 7.8), 0.5% Triton-X-100, 1 mM DTT. Buffer B for KgpF: 40 mM K ２ HPO ４ , 10 mM KH ２ P.O. ４ (pH 7.8), 500 mM NaCl, 500 mM imidazole (pH 7.8), 0.5% Triton-X-100, 1 mM DTT. Buffer C for KgpF: 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM DTT. Buffer D for KgpF: 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 20 mM imidazole (pH 7.8), 1 mM DTT. KgpF storage buffer: 10 mM HEPES (pH 7.5), 150 mM NaCl. 10% Glycine SDS-PAGE Gel: Separating gel: A solution containing 10% (19:1) acrylamide, 375 mM Tris-HCl (pH 8.8), 0.1% sodium lauryl sulfate was prepared and mixed with 0.1% ammonium persulfate and 0.05% tetramethylethylenediamine (TEMED) immediately before adding to the glass plate. Stacking gel: A solution containing 4% (29:1) acrylamide, 62.5 mM Tris-HCl (pH 6.8), 0.1% sodium lauryl sulfate was prepared and mixed with 0.1% ammonium persulfate and 0.05% tetramethylethylenediamine (TEMED) immediately before adding to the glass plate. 2x glycine-SDS loading buffer: 1 mL of 500 mM Tris-HCl (pH 6.8), 1 mL of 1 M DTT, 0.2 g of SDS, and 1 mL of glycerol were mixed, and distilled water was added to prepare a 5 mL solution.

[0280] (Setup 2) As in Setup 1, an in vitro translation mixture containing 0.08 μM DNA template was incubated at 37°C for 30 minutes and then used in the following multi-step enzymatic reaction. An equal volume of 2x Laz DEF mixture was added to the translation mixture and incubated at 25°C for 2 hours. IAA was then added to a final concentration of 5 mM and incubated at 4°C for 2 minutes. KgpF and DMAPP (dimethylallyl diphosphate) were then added to final concentrations of 72 μM and 1 mM, respectively, and the mixture was incubated at 37°C for 18 hours. An equal volume of 2x Laz BFC mixture was then added to the reaction mixture and incubated at 25°C for 6 hours. An equal volume of MeOH was then added and the mixture was incubated on ice for at least 10 minutes. The mixture was then centrifuged, and the supernatant was used for LC-MS analysis.

[0281] (Setup 3) As in Setup 1, an in vitro translation mixture containing 0.08 μM DNA template was incubated at 37°C for 30 minutes and then used in the following multi-step enzymatic reaction. KgpF and DMAPP (dimethylallyl diphosphate) were added to the translation mixture to final concentrations of 72 μM and 1 mM, respectively, and incubated at 37°C for 18 hours. An equal volume of 2x Laz DEF mixture was added and incubated at 25°C for 2 hours. IAA was then added to a final concentration of 5 mM and incubated at 4°C for 2 minutes. An equal volume of 2x Laz BFC mixture was then added to the reaction mixture and incubated at 25°C for 6 hours. Next, an equal volume of MeOH was added and incubated on ice for at least 10 minutes. The mixture was then centrifuged, and the supernatant was used for LC-MS analysis.

[0282] (Results) Figure 1 shows (A) an overview of the protocol for each setup and (B) the extracted ion chromatogram (EIC) results. In Setup 1, the synthesis of the target prenylated thiopeptide was confirmed in all model sequences MS1, 2, and 3. In Setup 2, the synthesis of prenylated thiopeptide was confirmed in MS1 ​​and 2, but not in MS3. In MS3, the product confirmed was an over-prenylated product containing two prenylated pyrroloindoline moieties and incorporating a thiopeptide moiety other than the pyridine ring. This is thought to be due to prenylation of the tryptophan one residue downstream of the dehydroalanine immediately following the leader peptide sequence LP, which inhibited peptide recognition by LazC, which catalyzes the pyridine ring closure reaction, preventing the reaction from proceeding. In Setup 3, the synthesis of prenylated thiopeptide was confirmed in MS1 ​​and 2, but it was not the major product. In MS3, the prenylated thiopeptide was not confirmed.

[0283] To demonstrate that the established biosynthetic pathway can produce prenylated thiopeptides with various sequences, five sequences containing random regions were designed. These sequences were translated in vitro and subjected to enzymatic reactions similar to those described in Setup 1 above.

[0284] (DNA Template Preparation) To prepare DNA templates for Sequences 1, 2, 3, and 4 shown in Figure 2(B), 0.5 μM of the appropriate forward and reverse primers (see the table below) were added to 1x Taq PCR master mix and denatured at 95°C for 1 minute. The DNA templates were then prepared in the same manner as for the MS1 and MS2 DNA templates. To prepare DNA template for Sequence 5 shown in Figure 2(B), 1x Taq PCR master mix and 0.5 μM of the appropriate forward and reverse primers (see the table below) were prepared. The initial PCR consisted of 13 cycles of denaturation (95°C for 40 seconds), annealing (50°C for 40 seconds), and extension (72°C for 40 seconds). After DNA extraction and precipitation as described above, 1x Taq PCR master mix, 0.5 μM of the appropriate forward and reverse primers (see table below), and 0.4 nM of the extracted DNA were heated for 5 cycles under the same conditions as the first PCR. 1 μL of the product was used as template for the next step. A third PCR was performed for 9 cycles under the same conditions as the first PCR. After PCR product amplification was confirmed by 3% agarose gel electrophoresis, the DNA was extracted and precipitated in the same manner as described above.

[0285] (Results) Figure 2 shows (A) an outline of the protocol and (B) the yield of peptides having each sequence. As shown in Figure 2(B), it was confirmed that the biosynthetic pathway constructed above can synthesize multiple prenylated thiopeptides.

[0286] [Investigation of Prenylation Reaction Conditions] To investigate the temperature dependency of the prenylation reaction, the modification reaction of prenylated thiopeptides was carried out for the three sequences MS1 to 3 under multiple temperature conditions, and the results were analyzed by LC-MS analysis.

[0287] In Setup 1 above, the prenylation reaction was allowed to proceed at incubation temperatures of 0°C, 16°C, 25°C, or 37°C after the addition of KgpF. Figure 3(B) shows the prenylation efficiency at each temperature for MS1 to 3. It was found that the modification efficiency hardly changed between 0 and 37°C.

[0288] Next, to examine the enzyme concentration dependency in the prenylation reaction, the modification reaction of prenylated thiopeptides was carried out for the three sequences MS1 to 3 under multiple enzyme concentration conditions, and the results were analyzed by LC-MS analysis.

[0289] In Setup 1 described above, the prenylation reaction was allowed to proceed with the addition of KgpF at concentrations of 0 μM, 20 μM, 50 μM, or 72 μM, and the incubation temperature after the addition of KgpF was set to 0°C. Figure 4(B) shows the prenylation efficiency at each concentration for MS1 to 3. It was found that there was almost no change in the modification efficiency within the concentration range of 20 to 72 μM.

[0290] [Consideration of Enzyme Reaction Conditions] When the reaction proceeds by sequentially adding enzyme solutions as described above, if the volume of the solution being handled becomes too large, the required amounts of reagents such as target protein and beads will become very large. Therefore, the conditions for an enzyme solution that shows sufficiently high reaction efficiency while reducing the total solution volume were investigated for the LazDEF mixture as follows.

[0291] The following v.207 (SEQ ID NO: 100) was used as a model peptide. To prepare a DNA template for v.207, 0.5 μM of the appropriate forward and reverse primers (see the table below) were added to 1× Taq PCR master mix and denatured at 95° C. for 1 minute. Thereafter, the DNA template was prepared in the same manner as for the DNA templates for MS1 and MS2.

[0292] The in vitro translation mixture containing 0.08 μM of DNA template was incubated at 37°C for 30 minutes and then used in the following multi-step enzymatic reaction: 60 mM Tris-HCl (pH 8.0), 5.4 mM ATP (pH 7.0), 1.2 mM DTT, 12 mM MgCl２ A LazDEF mixture containing 1.2, 2.4, or 3.6 μM lazD, 1.2, 2.4, or 3.6 μM lazE, and 2.4 μM lazF was added to the translation mixture at a volume five times that of the translation mixture and incubated at 25°C for 30 minutes. An equal volume of MeOH was then added, and the mixture was incubated on ice for at least 10 minutes, centrifuged, and the supernatant was used for LC-MS analysis. In LC-MS analysis, the factor was calculated according to the following formula: Thzx0, Thzx1, Thzx2, and Thzx3 correspond to the peaks shown in Figure 5.

[0293] From Figure 5, we determined that the optimal enzyme concentrations for reducing the reaction solution volume as much as possible while maintaining sufficiently high efficiency of the modification reaction were LazD = 2 μM, LazE = 1 μM, and LazF = 2 μM. As a result, as shown in Figure 6, the volume of the reaction solution in the final reaction was successfully reduced to approximately 65%.

[0294] [Construction of a prenylated thiopeptide library] The biosynthetic pathway of prenylated thiopeptides described above was applied to the mRNA display method to construct a prenylated thiopeptide library. 9.02 × 10 mRNAs encoding the precursor sequences of prenylated thiopeptides containing random regions (sequences from the leader peptide sequence to the tail sequence) were used. １３ The mRNA was synthesized with a diversity of 100%. As shown in Figure 7(A), the mRNA was designed so that at least one tryptophan to be prenylated always appears in the random region. The codon (RYU) immediately upstream of the tryptophan, the codon (DSK) immediately downstream of SW downstream of the leader peptide sequence, and the codon (NNT) immediately upstream of SSCS were set to increase the reaction efficiency of the enzyme used under the conditions described here, but they do not necessarily have to be RYU, DSK, and NNT.

[0295] DNA libraries were constructed de novo using PCR with oligonucleotide primers. Libraries encoding the UGG codon at different positions were constructed separately. The construction scheme and primer sequences are summarized in the table below. First, overlapping forward and reverse primers were annealed and extended. 100 μL of Platinum SuperFi DNA Polymerase solution [PCR buffer (1x), SuperFi Pol (1x), 250 μM each dNTP; SF PCR mix; buffer and enzyme (Thermo Fisher)] containing 300 nM forward primer and 250 nM reverse primer was denatured by heating at 95°C for 60 seconds. Three cycles of annealing (53.1°C for 30 seconds) and extension (72°C for 30 seconds) were then performed. In the next step, the extension product (100 μL) was added to 1900 μL of SF PCR mix containing 250 nM of forward and reverse primers, and six cycles of amplification were performed (20 s denaturation step at 95°C, 20 s annealing at 60°C, and 30 s extension at 72°C). The mixture was then further incubated at 72°C for 60 s and cooled to 10°C. The products were evaluated using a TapeStation (Agilent Technologies). DNA was extracted with phenol / chloroform / isoamyl alcohol (25:24:1, saturated with 10 mM Tris (pH 8.0), 1 mM EDTA) and then extracted with chloroform / isoamyl alcohol (24:1).

[0296] The extracted DNA was precipitated with ethanol, washed with 70% aqueous ethanol (v / v), dissolved in water, and added to the transcription reaction mixture [40 mM Tris buffer (pH 8.0), 22 mM MgCl ２The following buffer was added to the 500-kDa ... RNA extracted from the gel with 300 mM NaCl was recovered by ethanol precipitation, dissolved in water, and stored at −80° C. From round 2 of selection onwards, transcription reactions were scaled down to 25 μL reactions, and PAGE purification was only performed in rounds 5 and 6.

[0297] [Selection of prenylated thiopeptides that specifically bind to WNK4] To obtain prenylated thiopeptides that specifically bind to WNK4 (see Am J Physiol Renal Physiol. 320, F378-F403 (2021)), selection was performed using the scheme shown in Figure 8. WNK4 is an intracellular kinase involved in ion homeostasis, and therefore WNK4 inhibitors are promising drugs for treating hypertension. Furthermore, WNK1, a kinase in the same family as WNK4, is more widely distributed than WNK4, so it is desirable to selectively inhibit only WNK4 without inhibiting WNK1. Therefore, peptides that bind to WNK4 but not WNK1 were selected.

[0298] The selection scale was 150 μL in the first round and 2.5 μL in the other rounds. Unless otherwise specified, the following procedure will be performed at a 2.5 μL scale.

[0299] (Puromycin Ligation) Each mRNA library containing a UGG codon (corresponding to tryptophan) at a different position was mixed in an equimolar ratio. The resulting mRNA library was ligated to puromycin by Y-ligation using T4 ligase. The ligation buffer (40 mM Tris (pH 7.8), 10 mM MgCl ２ A reaction mixture containing 1 μM mRNA, 1.5 μM puromycin linker, and 1 μM T4 ligase in a 5% PBS solution (10 mM DTT, 0.5 mM ATP, water / DMSO mixture [8:2, v / v]) was incubated at 25°C for 45 minutes. The ligated mRNA was extracted with phenol / chloroform as described above, precipitated with ethanol, washed with 70% ethanol, and redissolved in water. The results of the puromycin ligation were confirmed by PAGE (6%, 6 M urea). The resulting product was diluted to 6 μM in water and frozen at -20°C. This product was used for in vitro translation during selection.

[0300] (Translation and reverse transcription) The in vitro translation system was reconstituted by mixing purified ribosomes, enzymes, and translation factors. The final reaction mixture contained: 50 mM HEPES-KOH (pH 7.6), 100 mM KOAc, 2 mM GTP, 2 mM ATP, 1 mM CTP, 1 mM UTP, 20 mM creatine phosphate, and 12 mM Mg(OAc). ２, 2 mM spermidine, 2 mM DTT, 1.5 mg / mL E. coli total tRNA (Roche), 1.2 μM ribosome, 0.6 μM MTF, 2.7 μM IF1, 0.4 μM IF2, 1.5 μM IF3, 10 μM EF-Tu, 10 μM EF-Ts, 0.26 μM EF-G, 0.25 μM RF2, 0.17 μM RF3, 0.5 μM RRF, 4 μg / mL creatine kinase, 3 μg / mL myokinase, 0.1 μM pyrophosphatase, 0.1 μM nucleotide-diphosphatase kinase, 0.1 μM T7 RNA. polymerase, 0.73 μM AlaRS, 0.03 μM ArgRS, 0.38 μM AsnRS, 0.13 μM AspRS, 0.02 μM CysRS, 0.06 μM GlnRS, 0.23 μM GluRS, 0.09 μM GlyRS, 0.02 μM HisRS, 0.4 μM IleRS, 0.04 μM LeuRS, 0.11 μM 0.03 μM LysRS, 0.03 μM MetRS, 0.68 μM PheRS, 0.16 μM ProRS, 0.04 μM SerRS, 0.09 μM ThrRS, 0.03 μM TrpRS, 0.02 μM TyrRS, 0.02 μM ValRS, 500 μM of each proteinogenic amino acid, and 1.2 μM puromycin-bound mRNA library.

[0301] The reaction mixture was first incubated at 37°C for 30 minutes, then at 25°C for 12 minutes, after which EDTA (pH 8.0) was added to a final concentration of 17 mM. The quenched translation reaction was incubated for an additional 10 minutes at 37°C. Reverse transcription was carried out with MMLV(H-) reverse transcriptase (Promega) using the Laz-HA-l4.R44 primer (see table below) at 42°C for 60 minutes. The final reaction mixture contained 0.84 μL of 150 mM Tris-HCl (pH 8.3), 1.5 mM dNTPs, 10 μM Laz-HA-l4.R44 primer, 150 mM Mg(OAc). ２0.5 μL, 0.25 μL of 0.2 M KOH, and 0.125 μL of M-MLV RTase(H-)Point Mutant (Promega) were added to 3 μL of the translation product (total volume 4.72 μL).

[0302] (Enzyme Reaction) The ice-cold reverse transcription product was diluted with 100 mM Tris buffer (pH 8.0) (20 mM MgCl ２ A 0.31-fold volume of LazDEF mixture containing 4 μM LazD, 2 μM LazE, and 4 μM LazF in 50 mM Tris buffer (pH 8.0) (containing 9 mM ATP and 2 mM DTT) was added on ice. The mixture was transferred to a 25°C thermostatic bath and incubated for 2 hours. Next, an IAA solution (final concentration: 5 mM) was added on ice. After 5 minutes, the mixture was diluted with 50 mM Tris buffer (pH 8.0) (10 mM MgCl ２ To the resulting mixture (containing 4 μM LazB, 8 μM LazC, 2.1 μM LazF, 2 μM S. lividans GluRS, and 40 μM S. lactacystinaeus tRNAGlu in a 1x volume of LazBCF mixture (containing 4.5 mM ATP and 0.5 mM glutamate), was added. The mixture was further incubated at 25°C for 6 hours. KgpF and DMAPP (dimethylallyl diphosphate) were then added to a final concentration of 72 μM KgpF and 1 mM DMAPP, and the mixture was incubated on ice for 18 hours.

[0303] (WNK4 affinity pull-down) The reaction mixture was returned to ice, and 0.11 volume of 10x blocking buffer [TBS-T (50 mM Tris, 150 mM NaCl, 0.2% (v / v) Tween-20 (pH 7.6)) containing 20 mg / mL bovine serum albumin and 20 mg / mL yeast total RNA] was added. The resulting sample was then subjected to counterselection (not performed in the first round). A mixture containing M280 streptavidin Dynabeads, the same beads conjugated with D-biotin (prepared by incubating the beads with a saturating concentration of D-biotin at 4°C for 10 minutes), and WNK1-immobilized M280 streptavidin Dynabeads (0.97 pmol / μL bead slurry; prepared by shaking in TBS-T containing 1 μM WNK1 at 4°C for 10 minutes and washing three times with TBS-T) in a ratio of 1:1:2.8 was washed three times with TBS-T and added to the library. After three 10-minute incubations at 4°C, the supernatant was collected and added to WNK4-immobilized M280 streptavidin Dynabeads (1.92 pmol / μL bead slurry; prepared by shaking 1 μM WNK4 in TBS-T at 4°C for 10 minutes and washing three times with TBS-T). The mixture was shaken at 4°C for 30 minutes, and the beads were washed three times with TBS-T. The selected cDNA was eluted by heating the beads suspended in elution buffer [SuperFi PCR buffer (1x), 250 μM each dNTP, T7g10M.F46, and Laz-HA-l4.R44 primers at 95°C for 5 minutes].

[0304] (PCR amplification) The concentration of the recovered cDNA and the cDNA recovery rate were measured by qPCR. A portion of the recovered sample was amplified in 10 mM Tris (pH 8.4), 50 mM KCl, 0.1% (v / v) Triton X-100, 2.5 mM MgCl ２The elution product was eluted with 250 μM of each dNTP, 250 nM of T7g10M.F46 and Laz-HA-l4.R44 primers, Taq DNA polymerase (1x), and Sybr Green I (1x; Thermo Fisher Scientific). The results were analyzed by comparing with a calibration curve generated with a library cDNA sample of known concentration. Thermal cycling for library DNA recovery was performed based on the qPCR results to avoid over-amplification of the cDNA. SuperFi DNA polymerase (final concentration: 1x) was added to the eluted product, followed by three thermal cycling steps (denaturation at 95°C for 15 seconds, annealing at 57.2°C for 15 seconds, and extension at 72°C for 15 seconds). DNA isolation, transcription, and puromycin ligation steps were then performed.

[0305] (Results) The results of selection against WNK4 are shown in Figure 9. At the seventh round, the recovery rate and P / N ratio increased significantly. The recovery rate indicates the proportion of peptides bound to each bead relative to the total. The P / N ratio is the ratio of the number of peptides bound to the positive beads to the number of peptides bound to the negative beads. As the rounds increased, the recovery rate and P / N ratio increased, indicating that sequences that selectively bind to WNK4 carried on the positive beads but do not bind to WNK1 carried on the negative beads or the beads themselves were enriched.

[0306] To elucidate the sequences of the peptides obtained in the fifth round, DNA obtained using a next-generation sequencer was analyzed, and 14 hit peptides were selected based on the number of reads and other factors. The amplified cDNA was diluted with a PCR mix containing 1 μM Miseq_f_150cyc.F50 and Jun-forward-lib.Rd2SP.R56. The product was subjected to a second PCR step using SuperFi Polymerase and Nextera XT v3 Set primers (Illumina) to introduce sequence barcodes. All PCR products were mixed and purified with NucleoSpin® Gel and PCR cleanup kit (Takara Bio). The purified PCR product was then diluted to 4 nM with 10 mM Tris-HCl (pH 8.5) / 0.1% Tween 20. 5 μL of 0.2 M NaOH was added to 4 μL of the cDNA library and left at room temperature for 5 minutes to denature it into ssDNA. In parallel, the same procedure was performed on 4 nM PhiX Control v3 (Illumina). The cDNA library and PhiX Control v3 were diluted to a final concentration of 20 pM in Hyb buffer (Illumina). Next-generation sequencing was then performed using Miseq (Illumina). MiSeq Reagent Kit v3 (150 cycles) was used.

[0307] The fasta-formatted data were analyzed using an NGS analysis script developed by Dr. Alexander Vinogradov (https: / / github.com / avngrdv / FastQProcessor). First, all DNA sequences were translated and sequences with ambiguous base calls were removed. Next, sequences with a Q score below 20 were removed. Finally, sequences that did not match the constructed library configuration were removed. The remaining sequences after each data processing step are listed below.

[0308] The 14 peptides selected according to the number of reads and UMAP are shown in Figure 10 .

[0309] To confirm that prenylated thiopeptides were formed for all hit peptides, DNA encoding the hit peptide sequences was prepared (see the table below), and after in vitro translation, an enzymatic modification reaction was carried out, and the reaction products were analyzed by LC-MS. The analysis results are shown in Figure 11. The center of Figure 11 shows the results of LC-MS analysis. It was confirmed that the desired prenylated thiopeptides were formed for all sequences.

[0310] To confirm the WNK4 / WNK1 selectivity of the hit peptides, a pull-down assay was performed using a mixture of clone DNAs of the hit peptides. Equal amounts of DNA for the peptides shown in Figure 11 (W4-01, W4-02, W4-03, W4-04, W4-05, W4-06, W4-07, W4-08, W4-11, W4-013, and W4-14) were mixed and transcribed as described above. Puromycin ligation, translation, reverse transcription, enzymatic reaction with Laz enzyme and KgpF, and WNK4 affinity pull-down were performed as described above. For the WNK4 affinity pull-down assay, the captured cDNA was incubated on ice for 30 minutes three times before washing. PCR amplification and next-generation sequencing were then performed as described above. The estimated recovery rate was calculated using the following formula: The number of recovered molecules and the number of input molecules were calculated from the qPCR results, and the frequency was determined from the number of reads in next-generation sequencing.

[0311] As shown in Figure 12, all peptides showed relatively high estimated recovery rates. A higher WNK4 / WNK1 ratio was obtained for peptides containing the SN-AW-HNPG-P motif.

[0312] [Identification of modified residues] To identify the residues in the hit peptide sequences that had been modified by the enzyme, several mutants were synthesized, subjected to modification reactions, and analyzed by LC-MS (Figure 13(A)). As shown in Figure 13(B), mutants were synthesized in which one of the two tryptophans in W4-01 was replaced with phenylalanine, and the results of LC-MS analysis were compared with those of W4-01. Figure 13(B) revealed that the tryptophan on the C-terminal side of the threonine in W4-01 was prenylated.

[0313] Similarly, as shown in Figure 14(A), consecutive serine residues in W4-01 were found to be dehydroalaninated, and as shown in Figure 14(B), the tryptophan immediately C-terminal to the valine in W4-03 was found to be prenylated.

[0314] Similarly, as shown in Figure 15(A), the tryptophan on the C-terminal side of one histidine in W4-06 was found to be prenylated, and as shown in Figure 15(B), the tryptophan on the N-terminal side of consecutive tryptophans in W4-07 was found to be prenylated.

[0315] [Mutant Clonal Assay] In the selection, the hit peptides described above had iodoacetamide (IAA) bound to the cysteine. Therefore, we examined whether mutants in which Cys in the hit peptides was replaced with Gln would selectively bind to WNK4. W4-06 and W4-07, as well as W4-06-C41Q and W4-07-C51Q, in which Cys in the respective sequences was replaced with Gln, were synthesized as described above, and WNK4 affinity pull-down assays were performed. As shown in Figure 16, in both W4-06 and W4-07, the mutants in which Cys was replaced with Gln similarly exhibited selective binding to WNK4.

[0316] [Comparison of recovery rates between the presence and absence of prenyl groups] To confirm the importance of the prenyl group for selective binding to WNK4, prenylated and non-prenylated peptides were prepared, and a clonal assay was performed to compare the recovery rates. For W4-01, W4-06-C41Q, and W4-07-C51Q, samples with and without prenylation were prepared, and a WNK4 affinity pull-down assay was performed ( Figure 17(A) ). As shown in Figure 17(B) , the prenylated samples showed higher recovery rates for all sequences.

[0317] [Study of prenylation enzymes other than KgpF] To confirm that prenylated thiopeptides can be synthesized using prenylation enzymes other than KgpF, prenylation reactions were carried out using seven prenylation enzymes: OltF, PT5, PT18, PT19, PT30, LimF, and PagF 222G / 237G / 271N.

[0318] DNA templates were prepared using the appropriate forward and reverse primers (see the table below) as described above. An in vitro translation mixture containing 0.08 μM DNA template was incubated at 37°C for 30 minutes and then used in the following multi-step enzymatic reaction. An equal volume of 2x Laz DEF mixture was added to the translation mixture and incubated at 25°C for 2 hours. Next, iodoacetamide (IAA) was added to a final concentration of 5 mM and incubated at 4°C for 2 minutes. An equal volume of 2x Laz BFC mixture was then added to the reaction mixture and incubated at 25°C for 6 hours. Then, a prenylation enzyme (OltF, PT5, PT18, PT19, PT30, LimF, or PagF 222G / 237G / 271N) and a prenyl group donor (DMAPP (dimethylallyl diphosphate), GPP (geranyl diphosphate), or FPP (farnesyl pyrophosphate)) were added to a final concentration of 72 μM for the prenylation enzyme and 1 mM for the prenyl group donor, and the mixture was incubated at 37°C for 18 hours. Next, an equal volume of MeOH was added, and the mixture was incubated on ice for 10 minutes or more. The mixture was then centrifuged, and the supernatant was used for LC-MS analysis.

[0319] The reaction schemes and analysis results of the reaction products when each prenylation enzyme was used are shown in Figures 19 to 25. Figures 19 to 25 demonstrate that prenylated thiopeptides can be synthesized even when prenylation enzymes other than KgpF are used. The study scheme and results are summarized in Figure 18. Although the synthesis of prenylated thiopeptides was not confirmed for PT12 and PT23, it is expected that prenylated thiopeptides can be synthesized by appropriately adjusting the reaction conditions. Therefore, in Figure 18(B), PT12 and PT23 are also shown, along with the prenylation enzymes for which the synthesis of prenylated thiopeptides was confirmed.

[0320] [Study on substrate tolerance of prenylation enzymes] WO 2020 / 080490 discloses the tolerance of KgpF and its homologs to the sequences before and after the amino acid residue to be prenylated in the substrate peptide. WO 2023 / 068296 also discloses the tolerance of LimF and its homologs to the sequences before and after the amino acid to be prenylated in the substrate peptide. Similarly, the substrate tolerance of other prenylation enzymes was examined below.

[0321] Multiple DNA templates were prepared using the appropriate forward and reverse primers as described above. An in vitro translation mixture containing 0.08 μM DNA template was incubated at 37°C for 30 minutes. A prenylation enzyme (KgpF, OltF, PT5, PT18, PT19, PT30, or LimF) and a prenyl group donor (DMAPP (dimethylallyl diphosphate) or GPP (geranyl diphosphate)) were then added to the respective prenylation enzyme concentrations shown in Figures 26-30 and the prenyl group donor concentration to 1 mM (2 mM when OltF was used). The mixture was then incubated at 37°C for 20 hours (18 hours when OltF was used). An equal volume of MeOH was then added, and the mixture was incubated on ice for at least 10 minutes. The mixture was then centrifuged, and the supernatant was used for analysis. P12 and PT23 were analyzed by MALDI-MS analysis, and the other prenylation enzymes were analyzed by LC-MS analysis.

[0322] For MALDI-MS analysis, peptides were desalted after translation and analyzed by MALDI-TOF MS. Peptides were loaded onto an SPE C-TIP (Nikkyo Technos) and desalted with a 4% acetonitrile, 0.5% acetic acid solution. Elution was performed with 1.2 μL of a 50% saturated α-cyano-4-hydroxycinnamic acid (HCCA) solution containing 80% acetonitrile and 0.5% acetic acid. MALDI-TOF MS analysis of peptides was performed in reflector / positive mode using an ultrafleXtreme (Bruker Daltonics). External mass calibration was performed using Peptide Calibration Standard II (Bruker Daltonics).

[0323] The peptide sequences used in each experiment, as well as the sequences preceding and following the amino acid residues to be prenylated and the prenylation efficiency, are shown in Figures 26 to 30. When PT30 was used, geranylation was confirmed at the Trp of the following peptide: Ac-TWH-NH ２ Ac-TWHA-NH ２ Ac-DWH-NH ２ Ac-DWHA-NH ２ Ac-VDWHA-NH ２

[0324] Also, 20 μM PagF 222G / 237G / 271N, 1 mM C １０ Az-OPP (a compound in which the tip of geranyl diphosphate (the end opposite the pyrophosphate group in the carbon chain) is azide-modified) and 150 mM MgCl ２ The following peptides (N-terminal Ｄ Y and the C-terminal C are connected via the S atom of Cys (NH—CO—CH ２ ) and the modification was confirmed at Tyr. The following sequences are SEQ ID NOs: 213-216 from left to right on the top row, and SEQ ID NOs: 217-220 from left to right on the bottom row. Ｄ Y means D-Tyr.

[0325] [Primers] The primers used in the examples are shown in the table below.

[0326] The amino acid sequences in the table below are SEQ ID NOs: 101 to 123, in order.

[0327] The amino acid sequences in the table below are SEQ ID NOs: 124-147 in order.

[0328] The base sequences in the table below are SEQ ID NOs: 148-149.

[0329] The base sequences in the table below are SEQ ID NOs: 150 to 184 in order.

[0330] The base sequences in the table below are SEQ ID NOs: 185 to 204, in order.

[0331] [Large-scale synthesis of prenylated thiopeptides] (Production of precursor peptides using Escherichia coli) To demonstrate large-scale synthesis of prenylated thiopeptides, precursor peptides were expressed in Escherichia coli. Two types of precursor peptide constructs were prepared: a His-tagged precursor peptide and a maltose binding protein (MBP)-tagged precursor peptide.

[0332] The His-tagged precursor peptide was produced using the following reagents: Buffer A: 50 mM Tris-HCl (pH 8), 300 mM NaCl, 10 mM imidazole (pH 8.0), 10 mM DTT. Buffer B: 50 mM Tris-HCl (pH 8), 300 mM NaCl, 500 mM imidazole (pH 8.0), 10 mM DTT.

[0333] His-tagged precursor peptides were expressed in E. coli BL21(DE3) gold using the appropriate pET-15b plasmid. One liter of LB medium containing 100 μg / mL ampicillin was inoculated with 10 mL of an overnight culture grown in LB medium. The culture was incubated at 37°C for 3 hours with shaking (180 rpm). Four peptides (W4-01, W4-02, W4-06-C41Q, and W4-07-C51Q in the table below) were induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) at a final concentration of 100 μM, and two peptides (W4-03 and W4-04 in the table below) were induced with IPTG at a final concentration of 1 mM. The incubation was continued at 37°C for 3 hours with shaking (180 rpm). After expressing the His-tagged precursor peptide with shaking at 5000 × g, the cells were collected by centrifugation (5,000 × g, 10 min). The precipitate was resuspended in 10 mL of buffer A and disrupted by sonication on ice. The supernatant was filtered through a 0.44 mm filter (Millipore). The filtrate was applied to 3 mL of Ni Sepharose 6 Fasr Flow (Cytiva) equilibrated with buffer A and loaded onto an Econopack column (Bio-Rad). After washing the Ni Sepharose 6 Fasr Flow with 40 mL of buffer A, the His-tagged precursor peptide was eluted with imidazole by adding 3 mL of buffer B. The eluate was purified using a gradient method with an HPLC LC-20AP preparative system (Shimadzu) using water containing 0.1% TFA and acetonitrile containing 0.1% TFA. Fractions were identified using an Autoflex (Bruker), and fractions containing the corresponding mass were collected. The collected solution was frozen in liquid nitrogen, and the solvent was removed using a freeze dryer (Tokyo Rikakikai). The final powder was dissolved in DMSO (dimethyl sulfoxide), and the concentration was confirmed by light absorption at 280 nm using a Nanodrop (Thermo).

[0334] Additionally, MBP-tagged precursor peptides were produced using the following reagents: Buffer A2: 20 mM Tris-HCl (pH 7.5), 200 mM NaCl, 1 mM EDTA (pH 8.0), 1 mM DTT. Buffer B2: 20 mM Tris-HCl (pH 7.5), 200 mM NaCl, 1 mM EDTA (pH 8.0), 10 mM maltose, 1 mM DTT. Storage buffer: 25 mM HEPES (pH 7.5), 500 mM NaCl, 1 mM DTT.

[0335] MBP-tagged precursor peptides were expressed in E. coli BL21(DE3) gold using the appropriate pMAL-c5e plasmid. An overnight LB culture (10 mL) was inoculated into 1 L of LB medium containing 100 μg / mL ampicillin and grown at 30°C for 2.5 hours with shaking (180 rpm). The cells were then induced with a final concentration of 100 μM isopropyl β-D-1-thiogalactopyranoside (IPTG). After 22 hours of MBP-tagged precursor peptide expression at 16°C with shaking at 180 rpm, the cells were harvested by centrifugation (5000 × g, 10 minutes). The pellet was resuspended in 30 mL of MBP-tagged buffer A2 and disrupted by sonication on ice. The supernatant was filtered through a 0.44 mm filter (Millipore). The filtrate was applied to a 5 mL MBPtrap HP column (Cytiva) using an AKTA Avant 25 (GE Healthcare). The MBP-tagged precursor peptide was eluted with maltose using a gradient method using buffer A2 and buffer B2. Fractions were analyzed by 10% SDS-PAGE, and fractions containing the corresponding band were collected. The collected solution was concentrated using an Amicon® Ultra 10 kDa (Millipore). The concentrated solution was then loaded onto a PD10 (Cytiva) and exchanged into storage buffer. The concentration was confirmed by measuring light absorbance at 280 nm using a Nanodrop (Thermo).

[0336] The sequences shown in the table below were used as precursor peptides. The yields for each sequence are shown in the table below. Note that the LP sequence (MSDITASRVESLDLQDLDLSELTVTSLRDTVALPENGA (SEQ ID NO: 76)) is bound to the N-terminus of each sequence shown in the table below, and the tail sequence (AQPQDM (SEQ ID NO: 212)) is bound to the C-terminus. The His tag is MGSSHHHHHHSSGLVPRGSH (SEQ ID NO: 205), and the MBP tag is MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENG KYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSSNNNNNNNNNNLGDDDDDK (SEQ ID NO: 221). The amino acid sequences in the table below are SEQ ID NOs: 206-211, in order. In addition, it is presumed that the underlined tryptophan in the sequence has been prenylated, and the serine and threonine marked with "*" in the upper right corner have been converted to α,β-unsaturated amino acid residues.

[0337]

[0338] (Enzymatic Reaction of Precursor Peptides) The obtained precursor peptides were subjected to an enzymatic reaction to synthesize prenylated thiopeptides for each sequence.

[0339] 100 μM of ice-cold His-tagged precursor peptide was diluted in 100 mM Tris buffer (pH 8.0) (20 mM MgCl ２A 1x volume of LazDEF mixture containing 3 μM LazD, 4 μM LazE, and 4 μM LazF in 50 mM Tris buffer (pH 8.0) (containing 9 mM ATP and 2 mM DTT) was added on ice. The mixture was transferred to a 25°C thermostatic bath and incubated for 2 hours. Next, an IAA solution (final concentration: 5 mM) was added on ice. After 5 minutes, the mixture was diluted with 50 mM Tris buffer (pH 8.0) (10 mM MgCl ２ To the resulting mixture was added a single volume of LazBCF mixture containing 4 μM LazB, 8 μM LazC, 2.1 μM LazF, 2 μM S. lividans GluRS, and 40 μM S. lactacystinaeus tRNAGlu in a buffer containing 4.5 mM ATP and 1 mM glutamate. The mixture was further incubated at 25°C for 6 hours. KgpF and DMAPP (dimethylallyl diphosphate) were then added to the mixture to a final concentration of 72 μM KgpF and 1 mM DMAPP, and the mixture was incubated on ice for 18 hours.

[0340] In addition, 100 μM of ice-cold MBP-tagged precursor peptide was diluted with 100 mM Tris buffer (pH 8.0) (20 mM MgCl ２ A 1x volume of LazDEF mixture containing 3 μM LazD, 4 μM LazE, and 4 μM LazF in 50 mM Tris buffer (pH 8.0) (containing 9 mM ATP and 2 mM DTT) was added on ice. The mixture was transferred to a 25°C thermostatic bath and incubated for 2 hours. Next, an IAA solution (final concentration: 5 mM) was added on ice. After 5 minutes, the mixture was diluted with 50 mM Tris buffer (pH 8.0) (10 mM MgCl ２ To the resulting mixture was added a single volume of LazBCF mixture containing 4 μM LazB, 8 μM LazC, 2.1 μM LazF, 2 μM S. lividans GluRS, and 40 μM S. lactacystinaeus tRNAGlu in a buffer containing 4.5 mM ATP and 1 mM glutamate. The mixture was further incubated at 25°C for 6 hours. KgpF and DMAPP (dimethylallyl diphosphate) were then added to the mixture to a final concentration of 72 μM KgpF and 1 mM DMAPP, and the mixture was incubated on ice for 18 hours.

[0341] The yield of prenylated thiopeptide for each sequence is shown in the table below, where "-" indicates that no measurement was performed.

[0342]

[0343] [Measurement of serum stability of prenylated thiopeptides] The serum stability of the three prenylated thiopeptides (W4-01, W4-07-C51Q, and W4-06-C41Q) synthesized in the above [Large-scale synthesis of prenylated thiopeptides] was measured.

[0344] On ice, an internal standard (int std) peptide (NH ２ -PEG ５ - Ｄ W Ｄ S Ｄ T Ｄ N Ｄ D Ｄ W Ｄ S Ｄ T Ｄ N Ｄ D-PEG ５ -CONH ２ (SEQ ID NO: 222)) and the prenylated thiopeptide synthesized in the above "Large-scale synthesis of prenylated thiopeptides," or a non-prenylated thiopeptide with the same sequence, were added to an internal standard (int std) peptide at a final concentration of 1 μM, and the prenylated thiopeptide or thiopeptide at a final concentration of 14 μM, followed by incubation at 37°C. After 0, 1, 2, 4, 8, and 24 hours of incubation, 4 μL of the reaction solution was taken, and 2.3 volumes of methanol were added on ice. The mixture was mixed well and allowed to stand for 5 minutes. The solution was then centrifuged at 153,000 × g for 5 minutes, and the supernatant was analyzed by LC-MS (waters). During analysis, the percentage of remaining peptide was calculated using the following formula:

[0345] % remaining = {Sample(t) / Standard(t)} / {Sample(0) / Standard(0)} × 100 Here, Sample(t) and Sample(0) mean the peak area of ​​the prenylated thiopeptide or thiopeptide at incubation times of t and 0 hours, respectively, and Standard(t) and Standard(0) mean the peak area of ​​the internal standard at incubation times of t and 0 hours, respectively.

[0346] The measurement results for each thiopeptide are shown in Figure 31. It can be seen that for all prenylated thiopeptides, 20% or more remained even after 8 hours of incubation in human serum. It can also be seen that the serum stability of W4-01 and W4-06-C41Q was improved by prenylation.

Claims

1. Formula (I): [In formula (I), ａ , X ｂ , X ｃ , m X's ｄ , and n X's ｅ are each independently an amino acid or an analogue thereof, Z may or may not be present, and when present, is a peptide consisting of 1 to 100 amino acids and / or analogues thereof, m is an integer selected from 0 to 39, n is an integer selected from 0 to 39, the sum of m and n is an integer selected from 1 to 39, ring A is a nitrogen-containing 6-membered ring which may have a substituent, B １ is an oxygen atom, a sulfur atom, or an NH group; R １ is a hydrogen atom or a hydrocarbon group; X ａ , X ｂ , X ｃ and Z each contain at least one amino acid having a prenyl group. ｄ ) ｍ -X ａ '-(X ｅ ) ｎ -X ｂ -Y-X ｃ -Z (II) [In the formula (II), X is a group represented by the formula (1): (R ２ is a hydrogen atom or a hydrocarbon group; Y is a group represented by formula (2): (R １ and B １ is as defined above, R ３ is a hydrogen atom or a hydrocarbon group; ａ X' is an amino acid or an analog thereof; ｂ , X ｃ , m X's ｄ , n X's ｅ , m, n, and Z are as defined above, and LP may or may not be present, and when present, it is a peptide consisting of 1 to 100 amino acids and / or analogs thereof.] is subjected to the action of a macrocyclase to form a nitrogen-containing six-membered ring A while eliminating LP, if present, to obtain a peptide represented by formula (I'): [In formula (I'), X ａ ', X ｂ , X ｃ , m X's ｄ , n X's ｅ , m, n, Z, ring A, B １ , and R １ has the same meaning as above.]; and reacting a prenylation enzyme with the cyclic compound represented by formula (I') to form a cyclic compound represented by formula (I') containing at least X ａ ', X ｂ , X ｃ and prenylating at least one amino acid contained in any one of Z and Z to form a cyclic compound represented by formula (I).

2. Formula (III): LP'-X'-(X ｄ ') ｍ -X ａ "-(X ｅ ') ｎ -X ｂ '-Y'-X ｃ In the formula (III), X' is serine or threonine, or an analogue thereof, Y' is a peptide represented by -Y'(11)-Y'(12)-, Y'(11) is serine, cysteine, threonine, diaminopropionic acid, or an analogue thereof, Y'(12) is serine or threonine, or an analogue thereof, m and n are as defined above, and X ａ ", X ｂ ', X ｃ ', m X's ｄ ', n X's ｅ ', Z', and LP' are each the X in formula (II). ａ ', X ｂ , X ｃ , m X's ｄ , n X's ｅ , Z, and LP are synonymous with each other, and forming the peptide represented by formula (II) from the precursor peptide represented by formula (III) by the following reactions: a reaction of forming an azole ring at least on Y' (11) in the precursor peptide represented by formula (III) with an azole ring-forming enzyme; and a reaction of converting at least X' and Y' (12) in the precursor peptide represented by formula (III) into α,β-unsaturated amino acid residues with an α,β-unsaturated amino acid forming enzyme in the presence of a cosubstrate for glutamylation and an aminoacylating enzyme.

3. The method of claim 1, wherein the at least one amino acid in formula (I') to be prenylated is tryptophan, arginine, histidine, tyrosine, serine, or threonine, or an analog thereof.

4. The production method according to any one of claims 1 to 3, wherein the prenylation enzyme comprises at least one selected from KgpF, OltF, PT5, PT12, PT18, PT19, PT23, PT30, LimF, and PagF, and prenylation enzymes having an amino acid sequence that has 80% or more sequence identity with the amino acid sequences of these enzymes.

5. The method according to any one of claims 1 to 3, wherein LP in formula (II) is a peptide consisting of 11 to 100 amino acids and / or analogs thereof.

6. The method according to any one of claims 1 to 3, wherein, in formula (I), m is an integer selected from 0 to 23, n is an integer selected from 0 to 23, and the sum of m and n is an integer selected from 1 to 23.

7. -X in the formula (II) ｂ -Y-X ｃ -has the following structure: The method according to any one of claims 1 to 3, wherein R １ , R ３ , R Ｘ１ , and R Ｃ are each independently a hydrogen atom or a hydrocarbon group; B １ and B Ｃ are each independently an oxygen atom, a sulfur atom, or an NH group.

8. The production method described in claim 2, wherein the macrocyclization enzyme comprises LazC and / or an enzyme having homology to LazC, the azole ring forming enzyme comprises at least one selected from LazD, LazE, LazF, and enzymes having homology thereto, and the α,β-unsaturated amino acid forming enzyme comprises at least one selected from LazB, LazF, and enzymes having homology thereto.

9. By allowing the prenylation enzyme to act on the cyclic compound represented by the formula (I'), at least X ａ The method according to any one of claims 1 to 3, wherein the amino acid sequence of the amino acid sequence of the present invention is prenylated.

10. Formula (I): [In formula (I), ａ , X ｂ , X ｃ , m X's ｄ , and n X's ｅ are each independently an amino acid or an analogue thereof, Z may or may not be present, and when present, is a peptide consisting of 1 to 100 amino acids and / or analogues thereof, m is an integer selected from 0 to 39, n is an integer selected from 0 to 39, the sum of m and n is an integer selected from 1 to 39, ring A is a nitrogen-containing 6-membered ring which may have a substituent, B １ is an oxygen atom, a sulfur atom, or an NH group; R １ is a hydrogen atom or a hydrocarbon group; X ａ , X ｂ , X ｃ and Z each independently represent an amino acid having a prenyl group. ｄ ') ｍ -X ａ "-(X ｅ ') ｎ -X ｂ '-Y'-X ｃ In the formula (III), X' is serine or threonine, or an analogue thereof, Y' is a peptide represented by -Y'(11)-Y'(12)-, Y'(11) is serine, cysteine, threonine, diaminopropionic acid, or an analogue thereof, Y'(12) is serine or threonine, or an analogue thereof, m and n are as defined above, and X ａ ", X ｂ ', X ｃ ', m X's ｄ ', n X's ｅ ', Z', and LP' are each the X in formula (II). ａ ', X ｂ , X ｃ , m X's ｄ , n X's ｅ and preparing a precursor peptide library containing two or more precursor peptides represented by the formula (III): LP-X-(X 11 ), and a precursor peptide library containing two or more precursor peptides represented by the formula (III): ｄ ) ｍ -X ａ '-(X ｅ ) ｎ -X ｂ -Y-X ｃ -Z (II) [In the formula (II), X is a group represented by the formula (1): (R ２ is a hydrogen atom or a hydrocarbon group; Y is a group represented by formula (2): (R １ and B １ is as defined above, R ３ is a hydrogen atom or a hydrocarbon group; ａ X' is an amino acid or an analog thereof; ｂ , X ｃ , m X's ｄ , n X's ｅ , m, n, and Z are as defined above, and LP may or may not be present, and when present, it is a peptide consisting of 1 to 100 amino acids and / or analogs thereof.]; and forming a peptide represented by formula (II) above with a macrocyclase to form a nitrogen-containing six-membered ring A while eliminating LP, if present, to obtain a peptide represented by formula (I'): [In formula (I'), X ａ ', X ｂ , X ｃ , m X's ｄ , n X's ｅ , m, n, Z, ring A, B １ , and R １ has the same meaning as above.]; and reacting a prenylation enzyme with the cyclic compound represented by formula (I') to form a cyclic compound represented by formula (I') containing at least X ａ ', X ｂ , X ｃ and Z, thereby forming a cyclic compound represented by formula (I), thereby forming a compound library containing two or more kinds of cyclic compounds represented by formula (I).

11. The method according to claim 10, wherein preparing the precursor peptide library comprises: preparing a library of nucleic acids encoding the precursor peptides represented by formula (III); and expressing the precursor peptides represented by formula (III) based on the library of nucleic acids.

12. The method according to claim 11, further comprising binding puromycin to the 3' end of each nucleic acid in the nucleic acid library, and forming a precursor peptide library containing two or more complexes between the precursor peptide represented by formula (III) and the nucleic acid encoding the precursor peptide by expression of the precursor peptide represented by formula (III).

13. A screening method for identifying a compound that binds to a target substance, comprising: contacting a compound library produced by the production method according to any one of claims 10 to 12 with a target substance; and selecting a compound that binds to the target substance.

14. Formula (I): [In formula (I), ａ , X ｂ , X ｃ , m X's ｄ , and n X's ｅ are each independently an amino acid or an analogue thereof, Z may or may not be present, and when present, is a peptide consisting of 1 to 100 amino acids and / or analogues thereof, m is an integer selected from 0 to 39, n is an integer selected from 0 to 39, the sum of m and n is an integer selected from 1 to 39, ring A is a nitrogen-containing 6-membered ring which may have a substituent, B １ is an oxygen atom, a sulfur atom, or an NH group; R １ is a hydrogen atom or a hydrocarbon group; X ａ , X ｂ , X ｃ and Z each independently represent an amino acid having a prenyl group.

15. The cyclic compound of claim 14, wherein the amino acid having a prenyl group in formula (I) is selected from tryptophan, arginine, histidine, tyrosine, serine, or threonine, or analogs thereof.

16. The cyclic compound according to claim 14 or 15, wherein, in formula (I), m is an integer selected from 0 to 23, n is an integer selected from 0 to 23, and the sum of m and n is an integer selected from 1 to 23.

17. In the formula (I), X ａ The cyclic compound according to claim 14 or 15, wherein is an amino acid having a prenyl group.

18. Formula (I): [In formula (I), ａ , X ｂ , X ｃ , m X's ｄ , and n X's ｅ are each independently an amino acid or an analogue thereof, Z may or may not be present, and when present, is a peptide consisting of 1 to 100 amino acids and / or analogues thereof, m is an integer selected from 0 to 39, n is an integer selected from 0 to 39, the sum of m and n is an integer selected from 1 to 39, ring A is a nitrogen-containing 6-membered ring which may have a substituent, B １ is an oxygen atom, a sulfur atom, or an NH group; R １ is a hydrogen atom or a hydrocarbon group; X ａ , X ｂ , X ｃ and Z each contain at least one amino acid having a prenyl group. [In formula (I'), X ａ X' is an amino acid or an analog thereof; ｂ , X ｃ , m X's ｄ , n X's ｅ , m, n, Z, ring A, B １ , and R １ is as defined above.], a prenylation enzyme is allowed to act on the cyclic compound represented by the formula (I) to obtain a cyclic compound represented by the formula (I), ａ ', X ｂ , X ｃ and Z, by prenylation of at least one amino acid contained in the cyclic compound represented by formula (I).

19. The method according to claim 18, wherein the at least one amino acid to be prenylated in formula (I') is tryptophan, arginine, histidine, tyrosine, serine, or threonine, or an analog thereof, and the prenylation enzyme comprises at least one selected from KgpF, OltF, PT5, PT12, PT18, PT19, PT23, PT30, LimF, and PagF, and prenylation enzymes having amino acid sequences having 80% or more sequence identity with the amino acid sequences of these enzymes.

20. By allowing the prenylation enzyme to act on the cyclic compound represented by the formula (I'), at least X ａ 20. The method of claim 18 or 19, wherein the ' is prenylated.

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