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

Abstract

The present invention relates to a method for producing compounds that have at least one structure represented by formula (I) or (II), wherein the production method comprises a step for contacting a compound having at least one structure represented by formula (III) or (IV) with a prenyl transferase in order to introduce a prenyl group into said structure. This prenyl transferase is LimF or an enzyme exhibiting homology therewith. [Chem. 1]

Description

Compound manufacturing method, compound library manufacturing method, compound library and screening method

[0001] The present invention relates to a method for producing a compound, a method for producing a compound library, a compound library, and a screening method, and also to a method for producing a peptide or protein, a method for producing a peptide or protein library, a peptide or protein library, and a screening method.

[0002] Prenylation is a ubiquitous modification found in primary metabolism as well as in the biosynthetic pathways of diverse natural products, including terpenoids, polyketides, nonribosomal peptides, and ribosomal post-translationally modified peptides (RiPPs). Prenylation often plays an important role in the diverse biological activities of these natural products because the prenyl group generally enhances the molecule's lipophilicity and 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 for their 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. Therefore, developing new biocatalysts for peptide prenylation will not only expand the toolbox for synthetic biology but also facilitate the design of bioactive peptides for drug discovery.

[0003] Prenyltransferases (PTases), a class of RiPP involved in the biosynthesis of cyanobactin, are intriguing enzymes that can be used as prenylation biocatalysts for a variety of peptides. These prenyltransferases, so-called F family enzymes, adopt a truncated α / β PTase barrel-fold structure with a unique solvent-exposed cavity, enabling the binding of large peptide substrates. This unique structure confers strict selectivity for prenyl group donor and acceptor residues, but loose selectivity for the overall substrate peptide sequence, making them promising biocatalysts for facile and versatile peptide alkylation. Despite sharing a similar tertiary structure, F family prenyltransferases catalyze diverse prenylation reactions. To date, specific prenyltransferases have been reported in this family that have Tyr O-prenylation activity, Trp C- or N-prenylation activity, Arg N-prenylation activity, Ser / Thr O-prenylation activity, or terminal N- and C-prenylation activity toward peptide substrates (Patent Document 1, Non-Patent Documents 1 to 6).

[0004] International Publication No. 2020 / 080490

[0005] Hao, Y. et al. Molecular basis for the broad substrate selectivity of a peptide prenyltransferase. Proc. Natl. Acad. Sci. USA, 113, 14037-14042 (2016).Dalponte, L. et al. N-Prenylation of tryptophan by an aromatic prenyltransferase from the cyanobactin biosynthetic pathway. Biochemistry, 57, 6860-6867 (2018).Tianero, M.D. et al. Metabolic model for diversity-generating biosynthesis. Proc. Natl. Acad. Sci. USA, 113, 1772-1777 (2016).Phan, C.-S. et al. Argicyclamides A-C Unveil Enzymatic Basis for Guanidine Bis-prenylation. J. Am. Chem. Soc., 143, 10083-10087 (2021).Parajuli, A. et al. A Unique Tryptophan C-Prenyltransferase from the Kawaguchipeptin Biosynthetic Pathway. Angew. Chem., 55, 3596-3599 (2016).Okada, M. et al. Stereospecific prenylation of tryptophan by a cyanobacterial post-translational modification enzyme. Org. Biomol. Chem., 14, 9639-9644 (2016).

[0006] Among the reported prenylation modifications, C-prenylation is one of the most attractive because carbon-carbon bond formation is of central importance to both biological and organic chemistry for constructing the carbon backbone of molecules. However, most F-family prenyltransferases are known to catalyze the prenylation of electron-rich heteroatoms. The only known prenyltransferase with C-prenylation activity in the cyanobactin biosynthetic gene cluster (BGC) is KgpF from the Kawaguchipeptin BGC, which catalyzes the C-3 prenylation of Trp (Non-Patent Documents 5-6). Therefore, it is desirable to explore novel prenyltransferases that catalyze C-prenylation of unprecedented acceptors, not only to expand the chemical space of prenylated natural products but also to provide potential biocatalysts for the diverse alkylation of various bioactive peptides.

[0007] It is also desirable to search for prenyltransferases and prenylation methods that are capable of prenylation of a variety of substrates, not limited to C-prenylation.

[0008] An object of the present invention is to provide a novel method for producing a prenylated compound, a novel method for producing a prenylated compound library, and the like, which can solve any of the above problems.

[0009] As a result of extensive research to solve the above problems, the present inventors have found that certain prenyltransferases, including LimF, can not only catalyze novel C-prenylation but also catalyze the prenylation of various substrates, thereby completing the present invention.

[0010] That is, the present invention is as follows: [1] A method for producing a compound having at least one structure represented by the following formula (I) or (II), (In the above formula (I), R １ represents a hydrogen atom or an arbitrary substituent, and n represents an integer of 0 to 11. In the above formula (II), R ２are each independently an arbitrary substituent, p is an integer of 0 to 4, and n is an integer of 0 to 11. The method includes a step of contacting a compound having at least one structure represented by the following formula (III) or (IV) with a prenyltransferase to introduce a prenyl group into the structure, (In the above formula (III) and formula (IV), R １ , R ２and p have the same meanings as above.) A production method, wherein the prenyltransferase is LimF or an enzyme homologous thereto. [2] The production method according to [1], comprising contacting a peptide or protein having an amino acid sequence containing at least one His, Tyr, or a derivative thereof with a prenyltransferase to introduce a prenyl group into at least one His residue, Tyr residue, or derivative residue thereof. [3] A method for producing a compound library containing prenylated peptides or proteins, comprising a step of contacting a compound library containing peptides or proteins having an amino acid sequence containing at least one His, Tyr, or derivative thereof with a prenyltransferase to introduce a prenyl group into at least one His residue, Tyr residue, or derivative residue thereof, wherein the prenyltransferase is LimF or an enzyme homologous thereto. [4] The production method according to [3], further comprising the step of translating an mRNA library using a cell-free translation system to prepare the compound library containing peptides or proteins having an amino acid sequence containing at least one His, Tyr, or derivative thereof. [5] The production method according to [3] or [4], wherein the peptides or proteins in the compound library are peptides or proteins bound to genotypes. [6] The production method according to [5], further comprising the step of preparing the compound library containing peptides or proteins bound to genotypes by an mRNA display method, the step comprising: preparing an mRNA library in which each mRNA encodes a peptide or protein having an amino acid sequence containing at least one His, Tyr, or derivative thereof; binding puromycin to the 3' end of each mRNA in the mRNA library to produce a puromycin-bound mRNA library; and translating the puromycin-bound mRNA library using a cell-free translation system. [7] The prenyltransferase is selected from the group consisting of Limnothrix sp. or Symploca sp., or an enzyme having homology thereto.[8] The production method according to any one of [1] to [6], wherein the prenyltransferase comprises an enzyme comprising an amino acid sequence corresponding to the following (1), (2), or (3): In this embodiment, the prenyltransferase may comprise an enzyme comprising an amino acid sequence corresponding to the following (4): The prenyltransferase may comprise an enzyme having an amino acid sequence corresponding to the following (1), (2), (3), or (4): (1) An amino acid sequence represented by any one of SEQ ID NOs: 1 to 12. (2) An amino acid sequence represented by any one of SEQ ID NOs: 1 to 12, in which one or several amino acids are deleted, substituted, or added. (3) An amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 1. (4) An amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 2. [9] The production method according to any one of [1] to [6], wherein the prenyltransferase is at least one selected from the group consisting of enzymes having an amino acid sequence selected from the group consisting of the amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence represented by SEQ ID NO: 2, and these amino acid sequences in which one or several amino acids are deleted, substituted, or added.

[10] The production method according to any one of [1] to [9], wherein the prenylated compound is a peptide or protein having a cyclic structure formed by four or more amino acids.

[11] The production method according to any one of [1] to

[10] , wherein the compound to be contacted with the prenyltransferase is a peptide or a protein, wherein the peptide or protein has a partial structure represented by Xaa1-Xaa2 or an amino acid sequence having His, Tyr, or a derivative thereof at the N-terminus, wherein Xaa1 is a neutral amino acid and Xaa2 is His, Tyr, or a derivative thereof.

[12] The production method according to any one of [1] to

[11] , wherein the compound to be contacted with the prenyltransferase is a peptide or a protein, wherein the peptide or protein has a partial structure represented by Xaa2-Xaa3 or an amino acid sequence having His, Tyr, or a derivative thereof at the C-terminus, wherein Xaa2 is His, Tyr, or a derivative thereof, and Xaa3 is any amino acid other than Pro and derivatives thereof.

[13] A compound library produced by the production method according to any one of [1] to

[12] .

[14] A screening method for identifying a compound that binds to a target substance, comprising the steps of: contacting a compound library produced by the production method according to any one of [1] to

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

[0011] According to the present invention, it is possible to provide a novel method for producing a prenylated compound, a novel method for producing a prenylated compound library, and the like.

[0012] Figure 1 shows SSNs indicating PTase clusters obtained from Blastp analysis. (a) SSNs indicating PTase clusters obtained from Blastp analysis using KgpF as a query. (b) SSNs indicating PTase clusters obtained from BLASTp analysis using LimF as a query. Figure 2 shows SSNs indicating PTase clusters obtained from BLASTp analysis using LimF as a query. Figure 3 shows that the putative cyanobactin gene cluster (BGC) from Limnothrix sp. CACIAM 69d contains a unique PTase, LimF. (a) Phylogenetic tree analysis including LimF. For AgeMTPT, the PTase domain region (275-559) was used for alignment. LimF, Ser / Thr O-PTase, Tyr O-PTase, Trp N / C-PTase, and terminal N / O-PTase are highlighted in orange (LimF), blue (LynF, TolF, TruF1), light blue (PirF, PagF), green (KgpF, AcyF), and gray (MusF1, MusF2, AgeMTPT), respectively. (b) A cyanobactin-related BGC from the cyanobacterium Limnothrix sp. CACIAM 69d is shown. The limE3 gene is encoded by a distant locus. (c) A sequence alignment comparing several known cyanobactin precursors with the putative precursor peptides (LimE1-3) found in the limBGC is shown. The recognition sequences for A family proteases (RSII) and G family macrocyclases (RSII) are highlighted in blue. The core peptide (CP) region is highlighted in red. In vitro LimF treatment of bcLimE2 yielded the geranylated product, limnothamide. (a) LC-MS analysis of bcLimE2 before and after 12 hours of LimF treatment. The extracted ion chromatogram (EIC) shown includes all possible charge states of the substrate bcLimE2 and the geranylated product. The mass spectrum, integrating the peak areas observed in the EIC, is also shown. (b) The structure of limnothamide, as identified by NMR analysis. COSY and HMBC correlations are shown to confirm C-forward geranylation at the C2 position of His.Figure 1 shows MS / MS spectra and their assignments for (a) bcLimE2 and (b) LimF-modified bcLimE2. Fragmentation types (b-type and a-type) are indicated. The effects of (a) temperature, (b) pH, and (c) metals on the LimF-catalyzed bcLimE2 prenylation reaction are shown. (d) Time course analysis of LimF under standard conditions. (e) MgCl2 on LimF activity. ２ (f) GPP concentration dependence of LimF activity. (g) Reaction rate of LimF-catalyzed prenylation reaction at various bcLimE2 substrate concentrations. Various His-containing dipeptides (Ac-XH-NH ２ and Ac-HX-NH ２The results of His prenylation of teMP by LimF are shown. The prenylation reaction was carried out with 20 μM LimF for 16 hours. Figure 1 shows the one-pot in vitro biosynthesis of prenylated teMP using the FIT-LimF system. (a) Expression and modification of teLimE2 in the FIT-LimF system are shown. The EICs, which include all possible charge states of the substrate teLimE2 and its corresponding prenylated product, are shown. The modification efficiency, calculated based on the area of ​​the peak observed in the EIC, is shown on the chromatogram. (b) Mutation introduction at the −1 position of teLimE2 is shown. The bar graph shows the modification efficiency of the mutants in LimF-catalyzed prenylation. The results of three independent experiments are shown as black dots, the average is shown as a bar graph, and the standard deviation is shown as an error bar. (c) Mutation introduction at the +1 position of teLimE2 is shown. The asterisk indicates modification of His at the −1 position. (d) LimF modification of teMP with different ring sizes. (e) LimF modification of teMP (teR1-5) with randomly selected overall sequences and their -1 or +1 mutants. (f) Mutagenesis of -1 and +1 positions in various peptides. "y" in the cyclic peptide sequences represents D-Tyr. Figure 1 shows the results of one-pot in vitro biosynthesis of prenylated teMP using the FIT-LimF system. (a) LimF prenylation efficiency of cyclic peptides with various random overall sequences. (b) LimF prenylation of cyclic peptides with two or more His residues. (c) LimF modification of His derivatives incorporated by genetic code reprogramming. Structural changes in each derivative residue compared to the His residue are highlighted in red. Figure 2 shows Tyr O-prenylation by LimF. (a) LimF modification of teR6 results in the formation of two distinct prenylation products. EICs are shown that include all possible charge states of the substrate teR6 and its single prenylation product. His-prenylated and Tyr-prenylated products (teR6-H) are shown. Ｇｅｒ and teR6-Y Ｇｅｒ ) are highlighted with orange and light blue dotted lines. (b) teR6-H Ｇｅｒ and teR6-Y ＧｅｒThe mass spectrum of teR6-Y is shown. Ｇｅｒ In (a), the peak resulting from the loss of the prenyl moiety is shown. (c) Structure of the prenylated tripeptide identified by NMR analysis. 2D-NMR correlation for identifying Tyr O-prenylation is shown. (d) Changes in prenylation efficiency due to mutation at -1 position of teR6-H5A are shown. The bar graph shows the conversion efficiency by LimF. The asterisk indicates prenylation of His at -1 position. The effects of (a) temperature, (b) pH, and (c) metals on the prenylation reaction of teR6-H5A by LimF are shown. The effects of (d) GPP and (e) MgCl on the prenylation reaction of teR6-H5A by LimF are also shown. ２(f) The reaction rate of the prenylation reaction with varying concentrations of teR6-H5A is shown. Figure 1 shows the prenylation efficiency of LimF and its mutants for different reaction modes. The average modification efficiency for three independent reactions is shown. Results showing significant His-prenylation and Tyr-prenylation are highlighted in orange (row 2, columns 2 and 3; row 5, column 2) and light blue (row 2, columns 4 and 5; row 5, columns 4 and 5; row 7, column 5; row 8, columns 4 and 5), respectively. Figure 2 shows the prenylation of linear peptides of different lengths by LimF. The linear peptide with the longest sequence also yielded a product with two prenylated His residues. Figure 3 shows the prenylation of high-molecular-weight proteins (Ac-BSA and GST) by LimF. Mass spectra of (a) Ac-BSA and (b) GST before and after prenylation by LimF are shown.

[0033] Figure 1 shows a portion of the alignment results for SEQ ID NOS: 1 to 13.

[0034] Figure 1 shows a portion of the alignment results for SEQ ID NOS: 1 to 13.

[0035] Figure 1 shows the results of (a) serum stability test, (b) DPP-4 digestion test, and (c) cAMP production activation test for GLP-1 and its derivatives.

[0036] Figure 1 shows the relationship between the amino acid at position -1 of His and geranylation efficiency for LimF and its mutants.

[0037] The Q72A mutant, I52A mutant, and wild-type LimF are shown in the top, middle, and bottom rows, respectively.

[0038] Figure 1 shows the sequence alignment of multiple prenyltransferases.

[0039] Figure 2 shows the relationship between the type of prenyl donor and prenylation efficiency for each LimF mutant.

[0039] GPP (C10) and DMAPP (C5) are shown in the top and bottom rows, respectively.

[0039] Figure 2 shows the search for LimF mutants capable of selective farnesylation.

[0039] GPP (C10) and FPP (C15) are shown in the top and bottom rows, respectively. 1 shows the farnesylation efficiency of unnatural substrates by the LimF_H239G / W273T mutant.

[0013] 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.

[0014] [Method for Producing Prenylated Compound] The method for producing a compound of this embodiment is a method for producing a compound having at least one structure represented by the following formula (I) or (II), and includes a step of contacting a compound having at least one structure represented by the following formula (III) or (IV) with a prenyltransferase to introduce a prenyl group into the structure, wherein the prenyltransferase is LimF or an enzyme homologous thereto. In addition, in formula (I), R １ is a hydrogen atom or an arbitrary substituent, n is an integer of 0 to 11, and in formula (II), R ２ are each independently an arbitrary substituent, p represents an integer of 0 to 4, n represents an integer of 0 to 11, and in formula (III) and formula (IV), R １ , R ２ and p have the same definitions as in formulae (I) and (II).

[0015] According to the compound production method of this embodiment, a compound having at least one structure represented by the following formula (III) or (IV) is prenylated using a specific prenyltransferase, thereby producing a highly hydrophobic compound. Furthermore, since the compound production method of this embodiment uses LimF or an enzyme homologous thereto as the prenyltransferase, the substrate tolerance of the prenyltransferase is high, and a variety of compounds having a prenyl group can be produced. As described above, the compound production method of this embodiment can efficiently introduce a prenyl group into a variety of compounds, thereby potentially producing compounds with high cell membrane affinity and / or high cell membrane permeability.

[0016] Hereinafter, the process of contacting a compound having at least one structure represented by the above formula (III) or (IV) with LimF, which is a prenyltransferase, or an enzyme homologous thereto, to obtain a compound having at least one structure represented by the above formula (I) or (II) is referred to as the "prenylation process."

[0017] (Substrate) The substrate in the prenylation step (hereinafter also simply referred to as "substrate") is not particularly limited as long as it is a compound having at least one structure represented by the above formula (III) or (IV). In formula (III), R １ is a hydrogen atom or an arbitrary substituent, and in formula (IV), R ２ are each independently an arbitrary substituent, and p is an integer of 0 to 4.

[0018] The structure represented by the formula (III) above means a structure represented by either of the following formulas (III-1) or (III-2). The structure represented by the formula (IV) above means a structure represented by either of the following formulas (IV-1), (IV-2) or (IV-3). In formulas (III-1) and (III-2), R １ is defined as in formula (III), and in formulas (IV-1), (IV-2) and (IV-3), R ２ and p are as defined in formula (IV).

[0019] In the substrate, the structure represented by formula (III) may be a structure represented by any one of formulas (III-1) and (III-2). In addition, in the substrate, the structure represented by formula (IV) may be a structure represented by any one of formulas (IV-1), (IV-2), and (IV-3), but is preferably a structure represented by formula (IV-3).

[0020] In the above formulas (III), (III-1) and (III-2), R １ is 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 in the above formulas (III), (III-1), and (III-2)１ The substituent in the above formula (III), (III-1) and (III-2) is preferably 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. The saturated or unsaturated hydrocarbon group is preferably an alkyl group. １ 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.

[0021] In the above formulas (IV), (IV-1), (IV-2) and (IV-3), R ２ 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 in the above formulas (IV), (IV-1), (IV-2), and (IV-3) ２ The substituent in 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. In the above formulae (IV), (IV-1), (IV-2), and (IV-3), R ２ If there are multiple R ２ may be the same or different.

[0022] In the above formulas (IV), (IV-1), (IV-2) and (IV-3), p represents an integer of 0 to 4 (both inclusive; the same applies to the ranges of values ​​expressed by "to" in this specification). When p is 0, the benzene ring in the above formulas (IV), (IV-1), (IV-2) and (IV-3) is a substituent R ２In the formulas (IV), (IV-1), (IV-2) and (IV-3), p is preferably 0 to 3, more preferably 0 to 2, even more preferably 0 or 1, and even more preferably 0.

[0023] The substrate is preferably a compound having at least one structure represented by the following formula (III') or (IV'). In the above formula (III′), R １ is R in the above formula (III) １ is synonymous with R ３ are each independently a hydrogen atom or an arbitrary substituent, and in the above formula (IV′), R ２ and p is R in the above formula (IV). ２ and p, and R ４ are each independently a hydrogen atom or an arbitrary substituent.

[0024] In the structure represented by the above formula (III′), R １ The bonding position on the imidazole ring is not particularly limited, that is, the structure represented by formula (III') above can have structures corresponding to formulas (III-1) and (III-2) in formula (III). In the structure represented by formula (IV') above, the bonding position of the hydroxy group on the benzene ring may be any of the ortho-position, meta-position, and para-position, that is, the structure represented by formula (IV') above can have structures corresponding to formulas (IV-1), (IV-2), and (IV-3) in the structure represented by formula (IV). In the structure represented by formula (IV'), the bonding position of the hydroxy group on the benzene ring is preferably the para-position.

[0025] R in the above formula (III′) １ Examples and preferred embodiments (embodiments described as preferred, more preferred, even more preferred, even more preferred, etc.; the same applies hereinafter in this specification) of １ In addition, R in the above formula (IV′) ２Examples and preferred embodiments of p are R in the above formula (IV). ２ and p are the same as those in the formula (III'). ３ and R in the above formula (IV′). ４ 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. ３ and two R ４ In each R ３ and R ４ may be the same or different. ３ and R in the above formula (IV′). ４ The substituent in the formula (III') 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. ３ and R in the above formula (IV′). ４ Preferred embodiments of the two R in the formula (III′) 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. ３ may be the same or different. ４ may be the same or different.

[0026] A preferred embodiment of the substrate is a compound represented by the formula (III′) above in which R １ is a hydrogen atom, a methyl group, or an ethyl group, and two R ３ In addition, a preferred embodiment of the substrate is a compound having at least one structure in which p is 0 and two R ４is a hydrogen atom and a hydroxy group is bonded at the para position.

[0027] The substrate in the prenylation step is not particularly limited as long as it is a compound having at least one structure represented by formula (III) or (IV) above, and may be a peptide or protein, or other compounds. The substrate is typically a peptide, protein, or low molecular weight compound (including amino acid monomer derivatives). In this specification, "low molecular weight compound" means a compound with a molecular weight of 1,000 or less. The substrate is preferably configured so that the surrounding environment of the structure represented by formula (III) or (IV) above is an environment that can interact with a prenyltransferase.

[0028] The substrate in the prenylation step is preferably a peptide or protein having at least one structure represented by formula (III) or (IV). As used herein, "peptide" refers to a compound in which 2 to 50 amino acids are bonded together via peptide bonds, and "protein" refers to a compound in which 51 or more amino acids are bonded together via peptide bonds. Furthermore, as used herein, "peptide or protein" refers to a compound in which two or more amino acids are bonded together via peptide bonds, and the number of bonded amino acids is not particularly limited.

[0029] As used herein, the term "amino acid" is used in its broadest sense and includes not only naturally occurring amino acids but also artificial amino acid variants and derivatives. Furthermore, as used herein, the term "amino acid" encompasses proteinogenic amino acids and non-proteinogenic amino acids. Proteinogenic amino acids, as represented by the three-letter abbreviations known in the art, are Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gln, Cys, Gly, Pro, Ala, Ile, Leu, Met, Phe, Trp, Tyr, and Val. Proteinogenic amino acids, as represented by the one-letter abbreviations known in the art, are R, H, K, D, E, S, T, N, Q, C, G, P, A, I, L, M, F, W, Y, and V. Non-proteinogenic amino acids refer to natural or unnatural amino acids other than proteinogenic amino acids.

[0030] Amino acids or derivatives thereof include naturally occurring proteinogenic L-amino acids; unnatural amino acids; and chemically synthesized compounds having properties known in the art as amino acid properties. Examples of unnatural amino acids include, but are not limited to, amino acids whose main chain structure differs from that of natural amino acids, such as α,α-disubstituted amino acids (such as α-methylalanine), N-alkyl-α-amino acids, D-amino acids, β-amino acids, and α-hydroxy acids; amino acids whose side chain structure differs from that of natural amino acids (such as norleucine and homohistidine); amino acids having an extra methylene in the side chain (such as "homo" amino acids, homophenylalanine and homohistidine); and amino acids in which the carboxylic acid functional group in the side chain is replaced with a sulfonic acid group (such as cysteic acid). Specific examples of unnatural amino acids include the amino acids described in WO 2015 / 030014. Furthermore, amino acid derivatives include forms protected with protecting groups, as described below.

[0031] When the substrate is a peptide or protein, the number of amino acids forming the substrate is not particularly limited and may be, for example, 2 to 2000. Within the above range, the number of amino acids forming the substrate may be 3 or more, 4 or more, 5 or more, 6 or more, or 10 or more, and may be 1500 or less, 1000 or less, 800 or less, 600 or less, 500 or less, 400 or less, 300 or less, 200 or less, 100 or less, 90 or less, 80 or less, 70 or less, 60 or less, 55 or less, 50 or less, 45 or less, 40 or less, 30 or less, or 20 or less. The number of amino acids forming the substrate may be, for example, 2 to 600 or less, 2 to 300 or less, 2 to 80 or less, or 2 to 60 or less. Within the above range, the number of amino acids forming the substrate may be within a range obtained by arbitrarily combining the above upper and lower limits.

[0032] When the substrate is a peptide or protein, its primary structure is not particularly limited, and may be, for example, linear or may contain a cyclic portion. The peptide or protein substrate may be a "lasso-shaped" substrate with a linear peptide attached like a tail to a cyclic structure. The substrate may also have a structure other than a peptide or protein. From the viewpoint of increasing target binding and / or cell membrane permeability and / or bioavailability, the substrate is preferably a cyclic peptide. As used herein, a peptide or protein having a "cyclic structure" or a "cyclic portion" means that it has a closed ring structure formed by two amino acids separated by one or more amino acid residues in the amino acid sequence, linked together directly or via a linker or the like.

[0033] When the substrate is a peptide or protein containing a cyclic structure, the closed ring structure in the cyclic structure is not particularly limited, and it is sufficient that two amino acids are covalently bonded via a linker or the like as necessary. Examples of the covalent bond between two amino acids include a disulfide bond, a peptide bond, an alkyl bond, an alkenyl bond, an ester bond, a thioester bond, an ether bond, a thioether bond, a phosphonate ether bond, an azo bond, a C-S-C bond, a C-N-C bond, a C=N-C bond, an amide bond, a lactam bridge, a carbamoyl bond, a urea bond, a thiourea bond, an amine bond, and a thioamide bond. In addition, the closed ring structure may be an N-CO-CH ２ The covalent bond between two amino acids may be an -S structure, or a structure formed by an amino acid having functional group 1 and an amino acid having the corresponding functional group 2 shown in Table 4 below. The covalent bond between two amino acids may be formed by bonding between the side chains of the two amino acids, between the main chains of the two amino acids, or between the side chains and the main chain of the two amino acids. When two amino acids are bonded at the main chain of the amino acids, a closed ring structure is typically formed by a peptide bond.

[0034] The cyclic structure that can be contained in a peptide or protein is not limited to a bond between the N-terminal amino acid and the C-terminal amino acid of a linear peptide, but may also be formed by a bond between a terminal amino acid and an amino acid other than the terminal amino acid, or by a bond between amino acids other than the terminal amino acid. When one of the amino acids bonded to form the cyclic structure is a terminal amino acid and the other is a non-terminal amino acid, the cyclic peptide has a lariat-shaped structure. When a peptide or protein has a lariat-shaped structure, it is preferable that a linear peptide is bonded to the C-terminus of the amino acid that constitutes the cyclic structure. In other words, it is preferable that the N-terminal amino acid in the linear peptide is bonded to the side chain of an amino acid other than the C-terminus to form a cyclic structure. The number of amino acid residues forming the cyclic structure of a peptide or protein is not particularly limited as long as it is 3 or more, but may be, for example, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more, or 60 or less, 50 or less, 40 or less, 30 or less, 25 or less, 20 or less, 18 or less, or 15 or less. When the substrate is a peptide containing a cyclic structure (also referred to as a "cyclic peptide" in this specification), the number of amino acids forming the cyclic structure is usually 3 to 40. The number of amino acids forming the cyclic structure may be 4 or more, 5 or more, 8 or more, or 10 or more, or 30 or less, 25 or less, 20 or less, or 18 or less, within the above-mentioned range. A preferred embodiment of the substrate in the prenylation step is, for example, a cyclic peptide having an amino acid sequence containing at least one His, Tyr, or a derivative thereof, and the number of amino acids forming the cyclic structure is preferably 3 to 30, more preferably 4 to 20.

[0035] Examples of peptides or proteins having at least one structure represented by the above formula (III) or (IV) include peptides or proteins having an amino acid sequence containing at least one His, Tyr, or derivatives thereof.

[0036] As used herein, derivatives of His include those having the following structure: and derivatives corresponding to the β-amino acids, γ-amino acids, and δ-amino acids of the derivatives and His; and derivatives corresponding to the α-alkyl-amino acids of these derivatives (for example, α-alkyl-amino acids having an alkyl group of 1 to 3 carbon atoms, typically α-methyl-amino acids). １ and R ３ is R in the above formula (III′). １ and R ３ and k represents an integer of 0 to 3. In the above formula (V), R １ is preferably a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, more preferably a hydrogen atom or a methyl group; R ３ are each independently preferably a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, more preferably a hydrogen atom or a methyl group, and even more preferably a hydrogen atom; and k is preferably 1 or 2. When k is 2, the above formula (V) corresponds to a homoamino acid. However, derivatives of His exclude those having the same structure as His.

[0037] As used herein, derivatives of Tyr include those having the following structure: and derivatives corresponding to the β-amino acid, γ-amino acid, and δ-amino acid of the derivatives and Tyr; and derivatives corresponding to the α-alkyl-amino acid of these derivatives (for example, an α-alkyl-amino acid having an alkyl group of 1 to 3 carbon atoms, typically an α-methyl-amino acid). ２ , R ４ and p is R in the above formula (IV′). ２ , R ４ and p have the same meanings as those of the formula (VI). ２ are each independently preferably an alkyl group having 1 to 3 carbon atoms, more preferably a methyl group; p is preferably 0 to 2, more preferably 0 or 1, and even more preferably 0; R ４are each independently preferably a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, more preferably a hydrogen atom or a methyl group, and even more preferably a hydrogen atom; the hydroxy group in the benzene ring is preferably located at the para position; and k preferably represents 1 or 2. When k is 2, the above formula (VI) corresponds to a homoamino acid. However, derivatives of Tyr exclude those having the same structure as Tyr.

[0038] In a peptide or protein, His, Tyr, or a derivative thereof may be in the L-form or the D-form. Furthermore, the derivatives of His and Tyr also include His and its derivatives, and Tyr and its derivatives, respectively, in forms protected by a protecting group described below.

[0039] In a peptide or protein having an amino acid sequence containing at least one His, Tyr, or derivative thereof as a substrate, the number of His, Tyr, or derivatives thereof is not particularly limited. The number of His, Tyr, or derivatives thereof may be 1 to 10, and within the above ranges, may be 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, or 3 or less, or may be 1 or more, 2 or more, 3 or more, or 4 or more. Of His, Tyr, or derivatives thereof, the peptide or protein may contain only His or a derivative thereof, only Tyr or a derivative thereof, or both His or a derivative thereof and Tyr or a derivative thereof.

[0040] When the substrate in the prenylation step is a peptide or protein having an amino acid sequence containing at least one His, Tyr, or a derivative thereof, the sequences preceding or following the His, Tyr, or derivative thereof are not particularly limited. In the substrate peptide or protein, the amino acid on the N-terminal side adjacent to His, Tyr, or a derivative thereof is preferably absent or an amino acid other than Lys, Arg, and derivatives thereof (limited to those having a positive charge in the side chain); more preferably absent, a neutral amino acid, or an amino acid with little steric hindrance; even more preferably absent or a neutral amino acid; and even more preferably absent, a neutral amino acid, or an amino acid with little steric hindrance. That is, the peptide or protein preferably (1) has a partial structure represented by Xaa1-Xaa2, or (2) has an amino acid sequence having His, Tyr, or a derivative thereof at the N-terminus. Here, Xaa1 is an amino acid other than Lys and Arg, preferably an amino acid other than Lys, Arg, and derivatives thereof (limited to those having a positive charge in the side chain), more preferably a neutral amino acid or an amino acid with little steric hindrance, even more preferably a neutral amino acid, and even more preferably a neutral amino acid and an amino acid with little steric hindrance. Furthermore, Xaa2 is His, Tyr, or a derivative thereof.

[0041] In a peptide or protein used as a substrate, the amino acid on the C-terminal side adjacent to His, Tyr, or a derivative thereof is preferably either absent or 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); more preferably either absent or 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. That is, the peptide or protein preferably (1) has a partial structure represented by Xaa2-Xaa3, or (2) has an amino acid sequence having His, Tyr, or a derivative thereof at the C-terminus. Here, Xaa2 is His, Tyr, or a derivative thereof. Furthermore, Xaa3 is an amino acid other than Pro, 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), 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.

[0042] As used herein, "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, 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 (R ５ and R ６are 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.

[0043] When an amino acid is present on the N-terminal side adjacent to His, Tyr, or a derivative thereof in a peptide or protein, preferred proteinogenic amino acids 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. When an amino acid is present C-terminally adjacent to His, Tyr, or a derivative thereof in a peptide or protein, preferred proteinogenic amino acids include Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gln, Cys, Gly, Ala, Ile, Leu, Met, Phe, Trp, Tyr, and Val.

[0044] The substrate in the prenylation step may be a peptide or protein modified by phosphorylation, methylation, acetylation, adenylylation, ADP-ribosylation, glycosylation, polyethylene glycol addition, or two or more types of peptides and / or proteins fused together directly or via a linker, etc. Furthermore, the peptide or protein may be biotinylated via a linker, etc.

[0045] The substrate may also be a compound in which a structure other than amino acids is bound to a peptide or protein by a covalent or non-covalent bond. Examples of compounds in which a structure other than amino acids is bound to a peptide or protein include compounds in which a nucleic acid molecule that is the genotype of the peptide or protein is bound to the peptide or protein, either directly or via a linker, and compounds in which a label for detecting the peptide or protein (such as a fluorescent substance, a radioactive substance, a metal nanoparticle, a quantum dot, or an enzyme) is bound to the peptide or protein, either directly or via a linker. The linker bound to the peptide or protein is not particularly limited, and any linker with a structure well known in the field of peptide synthesis as a linker for connecting peptides can be used.

[0046] The substrate in the prenylation step may be a compound that does not fall under the category of a peptide or a protein, and a preferred embodiment of such a compound includes a low-molecular-weight compound having at least one structure represented by formula (III) or (IV) above. Examples of such a low-molecular-weight compound include His, Tyr, or a derivative thereof as a simple substance. Examples of His derivatives and Tyr derivatives include those described above. His, Tyr, or a derivative thereof may be in the L-form or the D-form. Furthermore, His, Tyr, or a derivative thereof may be any of an α-amino acid, β-amino acid, γ-amino acid, or δ-amino acid, but is preferably an α-amino acid.

[0047] The substrate in the prenylation step may be His, Tyr, or a derivative thereof, in which at least one of the amino group, carboxy group, and side chain functional group is protected by a protecting group. The protecting group is not particularly limited as long as it is one commonly used in the art. Examples of protecting groups for amino groups include benzyloxycarbonyl (Cbz) group, tert-butoxycarbonyl (Boc) group, fluorenylmethoxycarbonyl (Fmoc) group, benzyl group, allyl group, and allyloxycarbonyl (Alloc) group. Examples of protecting groups for carboxy groups include methyl group, ethyl group, benzyl group, tert-butyl group, and cyclohexyl group. As for other functional groups of amino acids, for example, protecting groups for hydroxy groups in serine and threonine include, for example, benzyl and tert-butyl groups, and protecting groups for hydroxy groups in tyrosine include, for example, 2-bromobenzyloxycarbonyl and tert-butyl groups. Protecting groups can be protected and deprotected according to the method described in *Protective Groups in Organic Synthesis Second Edition* by T.W. Greene and P.G.M.Wuts, John Wiley & Sons, Inc., or methods equivalent thereto. His, Tyr, or a derivative thereof may be protected with a protecting group only at the amino group, only at the carboxy group, or only at other functional groups in the side chain, or may be protected with a protecting group at two or more or all of the amino group, carboxy group, and other functional groups in the side chain. His, Tyr, or a derivative thereof may be protected with a protecting group, for example, at the amino group, by Fmoc.

[0048] The low molecular weight compound other than His, Tyr, or a derivative thereof is not particularly limited as long as it has at least one structure represented by formula (III) or (IV) above, but is preferably, for example, a compound for which pharmacological activity has already been found. By using such a compound as a substrate to produce a prenylated compound by the method of this embodiment, it is possible to enhance the previously found pharmacological activity, or improve cell membrane affinity and / or cell membrane permeability and / or bioavailability. An example of such a low molecular weight compound is cimetidine, which has the structure shown below.

[0049] Other low molecular weight compounds that can be substrates include compounds having the following structures: In the above structure, R ８ is a divalent hydrocarbon group which may contain a heteroatom, and R ９ is a hydrogen atom or any substituent, and X is any atomic group. ８ R preferably has 1 to 15 carbon atoms, more preferably 1 to 10. ８ The heteroatom that may be contained in R is not particularly limited, and examples thereof include a nitrogen atom, a sulfur atom, and an oxygen atom. ９ is 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 ９ 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. ９Preferred 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. In the above structure, X is any atomic group. The number of atoms contained in X is not particularly limited, and may be, for example, 1 to 20, or 2 to 10. The type of atoms contained in X is also not particularly limited, and may include, for example, at least one of carbon atom, hydrogen atom, nitrogen atom, oxygen atom, sulfur atom, and phosphorus atom. The atomic group X may be a moiety that mainly contributes to the pharmacological activity of the compound represented by the above formula.

[0050] The above substrates may be subjected to the prenylation step singly or in combination of two or more. Furthermore, the substrate may be in the form of a pharmacologically acceptable salt of any of the above compounds. As used herein, the term "pharmacologically acceptable salt" includes, for example, salts with pharmaceutically acceptable bases or acids. 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.

[0051] (Prenyltransferase) The prenylation step is a step in which any of the above substrates is contacted with a prenyltransferase (PTase), and the prenyltransferase is LimF or an enzyme homologous thereto. As described in the Examples below, the present inventors have found that LimF and enzymes homologous thereto can prenylate any of the above substrates and can catalyze the prenylation of a variety of substrates. Furthermore, they have found that LimF and enzymes homologous thereto are novel prenyltransferases that catalyze a novel C-prenylation.

[0052] LimF is a prenyltransferase derived from Limnothrix sp. CACIAM 69d, and is an enzyme having the amino acid sequence of SEQ ID NO: 1 (described in Table 1 below). As used herein, "prenyltransferase" refers to an enzyme that can bind a prenyl group composed of a 5-carbon isoprene unit to a substrate. As used herein, "prenyl group" refers to an enzyme having the following structure: In the above formula, n represents an integer of 0 to 11. When n 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 Cn prenyl groups depending on the number of carbon atoms n. 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.

[0053] Enzymes homologous to LimF are not particularly limited, but can be suitably used, for example, proteins having an amino acid sequence that is found to be homologous to the amino acid sequence of LimF as a result of phylogenetic tree analysis performed by creating a sequence alignment using ClustalW or MEGA7 software. Proteins having an amino acid sequence that is found to be homologous as a result of the analysis also include proteins annotated as hypothetical proteins. Examples of enzymes homologous to LimF include enzymes having an amino acid sequence in which one or more amino acids are deleted, substituted, or added to the amino acid sequence of LimF (SEQ ID NO: 1), as well as enzymes having an amino acid sequence that is at least 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 homologous to the amino acid sequence of LimF (SEQ ID NO: 1) and that have prenyl transfer activity. Examples of such enzymes homologous to LimF include enzymes containing or having any of the amino acid sequences represented by SEQ ID NOS: 2 to 12 shown in Table 1 below (shown as Tables 1-1 and 1-2). The enzymes having the amino acid sequences of SEQ ID NOS: 2 to 12 are enzymes that are predicted to have particularly high homology to LimF and to have prenyl transfer activity similar to that of LimF, based on the results of the phylogenetic tree analysis and sequence similarity network analysis described in the Examples. In Table 1, the amino acid sequence represented by SEQ ID NOS: 1 is the amino acid sequence of LimF, and SEQ ID NOS: 13 is the amino acid sequence of LimF in which His at position 172 is replaced with Leu.

[0054]

[0055]

[0056] The prenyltransferase used in the prenylation step preferably includes an enzyme having an amino acid sequence corresponding to the following (1), (2), or (3), and more preferably includes an enzyme having an amino acid sequence corresponding to the following (1), (2), or (3). In this embodiment, these enzymes preferably have prenyltransferase activity. (1) An amino acid sequence represented by any one of SEQ ID NOS: 1 to 12 in Table 1 above. (2) An amino acid sequence in which one or several amino acids are deleted, substituted, or added in the amino acid sequence represented by any one of SEQ ID NOS: 1 to 12. (3) An amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NOS: 1.

[0057] As used herein, when a phrase "having an amino acid sequence in which one or several amino acids are deleted, substituted, or added" is used, the number of deleted, substituted, or added amino acids is not particularly limited, as long as the resulting prenyltransferase retains its function. Here, "several" refers to an integer of 2 or greater, preferably 2 to 15, more preferably 2 to 10, even more preferably 2 to 5, and even more preferably 2, 3, or 4. The position of the deletion, substitution, or addition in each prenyltransferase is not particularly limited, as long as the resulting prenyltransferase retains its function, and may be the N-terminus, C-terminus, or any intermediate position in each prenyltransferase. As used herein, the phrase "having Y% or more homology 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 shared amino acid residues to the total number of amino acids in SEQ ID NO: X is Y% or more. As used herein, "homology of Y% or more" can also be rephrased as "identity of Y% or more."

[0058] The amino acid sequence of (2) above is an amino acid sequence in which preferably 1 to 7, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 or 2 amino acids have been deleted, substituted, or added in the amino acid sequence represented by any of SEQ ID NOs: 1 to 12. The amino acid sequence of (3) above is an amino acid sequence that has a homology of preferably 85% or more, more preferably 90% or more, even more preferably 92% or more, even more preferably 95% or more, still more preferably 98% or more, and particularly preferably 99% or more to the amino acid sequence represented by SEQ ID NO: 1.

[0059] The prenyltransferase in the prenylation step preferably includes an enzyme containing an amino acid sequence corresponding to the following (4), and also preferably includes an enzyme having an amino acid sequence corresponding to the following (4). In this embodiment, these enzymes preferably have prenyltransferase activity. (4) An amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 2.

[0060] The amino acid sequence (4) above is an amino acid sequence having a homology of preferably 85% or more, more preferably 90% or more, even more preferably 92% or more, still more preferably 95% or more, even more preferably 98% or more, and particularly preferably 99% or more with the amino acid sequence represented by SEQ ID NO: 2.

[0061] The prenyltransferase preferably includes a prenyltransferase derived from Limnothrix sp. or Symploca sp., or an enzyme homologous thereto. The enzyme homologous to the prenyltransferase derived from Limnothrix sp. or Symploca sp. is not particularly limited, and examples include enzymes having an amino acid sequence that has at least 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 homology to the amino acid sequence of the prenyltransferase derived from Limnothrix sp. or Symploca sp. (e.g., the amino acid sequence represented by SEQ ID NO: 1 or 2).

[0062] The prenyltransferase preferably includes an enzyme having an amino acid sequence selected from the group consisting of the amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence represented by SEQ ID NO: 2, and these amino acid sequences in which one or several amino acids have been deleted, substituted, or added. Furthermore, the prenyltransferase is particularly preferably an enzyme having an amino acid sequence selected from the group consisting of the amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence represented by SEQ ID NO: 2, and these amino acid sequences in which one or several amino acids have been deleted, substituted, or added. The amino acid sequence represented by SEQ ID NO: 1 in which one or several amino acids have been deleted, substituted, or added may be the amino acid sequence represented by SEQ ID NO: 13.

[0063] When the substrate for the prenylation step does not have a structure represented by formula (IV) above but is a compound having at least one structure represented by formula (III) above, and the prenyltransferase includes an amino acid sequence corresponding to the following (2') or (3), or includes an enzyme having either of these amino acid sequences, in these enzymes, it is preferable that Glu at position 54 and His at position 172 in the amino acid sequence represented by SEQ ID NO: 1 are not deleted or substituted, and it is more preferable that Glu at position 54, His at position 172, Tyr at position 188, Tyr at position 237, and Tyr at position 292 are not deleted or substituted. Furthermore, Asp at position 70 in the amino acid sequence represented by SEQ ID NO: 1 may be deleted or substituted, and if substituted, it may be substituted with Ala. (2') An amino acid sequence in which one or several amino acids are deleted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 1. (3) An amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 1.

[0064] When the substrate for the prenylation step does not have a structure represented by formula (IV) above but is a compound having at least one structure represented by formula (III) above, and the prenyltransferase includes an amino acid sequence corresponding to (2) or (4) above, or includes an enzyme having an amino acid sequence corresponding to (2) or (4), when the amino acid sequences of such enzymes are aligned with the amino acid sequence represented by SEQ ID NO: 1, it is preferred that the amino acids corresponding to Glu at position 54 and His at position 172 of SEQ ID NO: 1 are not deleted or substituted, and it is more preferred that the amino acids corresponding to Glu at position 54, His at position 172, Tyr at position 188, Tyr at position 237, and Tyr at position 292 of SEQ ID NO: 1 are not deleted or substituted. Furthermore, the amino acid corresponding to Asp at position 70 of SEQ ID NO: 1 may be deleted or substituted, or if substituted, it may be substituted with Ala.

[0065] Furthermore, when the substrate for the prenylation step is a compound having at least one structure represented by (IV), and the prenyltransferase includes an amino acid sequence corresponding to (2') or (3) above, or an enzyme having either of these amino acid sequences, in such enzymes, it is preferable that Glu at position 54 in the amino acid sequence represented by SEQ ID NO: 1 is not deleted or substituted, and it is more preferable 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 represented by SEQ ID NO: 1 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 represented by SEQ ID NO: 1 may be deleted or substituted, and if substituted, it may be substituted with Ala.

[0066] Furthermore, when the substrate for the prenylation step is a compound having at least one structure represented by (IV), and the prenyltransferase contains an amino acid sequence corresponding to (2) or (4) above, or an enzyme having an amino acid sequence corresponding to (2) or (4), when the amino acid sequences of such enzymes are aligned with the amino acid sequence represented by SEQ ID NO: 1, it is preferable that the amino acid corresponding to Glu at position 54 of SEQ ID NO: 1 is not deleted or substituted, and it is more preferable that the amino acids corresponding to Glu at position 54, Tyr at position 188, Tyr at position 237, and Tyr at position 292 are not deleted or substituted. Furthermore, the amino acid corresponding to His at position 172 of SEQ ID NO: 1 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, the amino acid corresponding to Asp at position 70 of SEQ ID NO: 1 may be deleted or substituted, and if substituted, it may be substituted with Ala.

[0067] Furthermore, when a deletion or substitution has been made in the amino acid sequence represented by SEQ ID NO: 1, 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 the amino acid sequence represented by SEQ ID NO: 1, 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. Furthermore, when a deletion or substitution has occurred in the amino acid sequence represented by any one of SEQ ID NOs: 2 to 12, the deleted or substituted amino acid may be at least one of the amino acids corresponding to 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 of SEQ ID NO: 1 when the amino acid sequence represented by any one of SEQ ID NOs: 2 to 12 is aligned with the amino acid sequence represented by SEQ ID NO: 1. In the amino acid sequence represented by any one of SEQ ID NOs: 2 to 12, one or more amino acids may be deleted, substituted, or added to the amino acids other than the amino acids corresponding to E at position 54, Y at position 188, Y at position 237, and Y at position 292 when the amino acid sequence is aligned with the amino acid sequence represented by SEQ ID NO: 1.

[0068] The alignment results of SEQ ID NOs: 2 to 13 with SEQ ID NO: 1 are shown in Figures 14-1 and 14-2. In the figures, the amino acids corresponding to the sites where the prenyl group donor binds (is accommodated) in the enzyme LimF having the amino acid sequence represented by SEQ ID NO: 1 are indicated by arrows.

[0069] In the production method of this embodiment, the prenyltransferase may be selected appropriately depending on the type of substrate to be prenylated and the type of compound capable of donating a prenyl group. For example, when the substrate to be prenylated is a peptide or protein and His or a derivative thereof is to be prenylated, if the amino acid adjacent to the N-terminus of His or a derivative thereof has a positive charge or is highly sterically hindered, an enzyme may be used that contains or has an amino acid sequence in which I at position 52 in the amino acid sequence represented by SEQ ID NO: 1 has been substituted, or an amino acid sequence in which the amino acid corresponding to I at position 52 in SEQ ID NO: 1 has been substituted in an amino acid sequence represented by any of SEQ ID NOs: 2 to 12 when aligned with the amino acid sequence represented by SEQ ID NO: 1. Here, the substituted amino acid is not particularly limited, and may be substituted, for example, with an amino acid with little steric hindrance, preferably Ala.

[0070] Furthermore, when the prenyl group introduced into the substrate in the production method of this embodiment is a dimethylallyl group, an enzyme may be used that contains or has an amino acid sequence in which the G at position 224 in the amino acid sequence represented by SEQ ID NO: 1 is substituted, or an amino acid sequence in which the amino acid corresponding to the G at position 224 in SEQ ID NO: 1 is substituted in an amino acid sequence represented by any of SEQ ID NOs: 2 to 12 when aligned with the amino acid sequence represented by SEQ ID NO: 1. Here, the substituted amino acid may be, for example, an amino acid that is bulkier than Gly, and is preferably substituted with Cys, Met, Gln, Leu, Thr, Val, or His, more preferably substituted with Met or His, and more preferably substituted with Met.

[0071] Furthermore, in the production method of this embodiment, when the prenyl group introduced into the substrate is a relatively large group such as a farnesyl group, a geranylgeranyl group, a geranylfarnesyl group, a hexaprenyl group, an octaprenyl group, or a decaprenyl group, particularly when it is a farnesyl group, an enzyme may be used that contains or has an amino acid sequence in which the L at position 222 or the W at position 273 of SEQ ID NO: 1 in the amino acid sequence represented by SEQ ID NO: 1 is substituted, or an amino acid sequence in which the amino acid corresponding to the L at position 222 or the W at position 273 of SEQ ID NO: 1 when aligned with the amino acid sequence represented by SEQ ID NO: 1 is substituted. Here, the amino acid corresponding to the W at position 273 or the W at position 273 may be substituted, for example, with a neutral amino acid or an amino acid with little steric hindrance, preferably with a neutral amino acid with little steric hindrance, more preferably with Asn, Ser, Thr, Val, Ile, Cys, Gly, or Ala, and even more preferably with Asn or Thr. When an enzyme is used that contains or has an amino acid sequence in which the W at position 273 of SEQ ID NO: 1 is substituted in the amino acid sequence represented by SEQ ID NO: 1, or an amino acid sequence in which the amino acid corresponding to the W at position 273 of SEQ ID NO: 1 is substituted in an amino acid sequence represented by any of SEQ ID NOs: 2 to 12 when aligned with the amino acid sequence represented by SEQ ID NO: 1, the enzyme may further contain a substitution at H at position 239 of SEQ ID NO: 1, or at H at position 239 of SEQ ID NO: 1 in the amino acid sequence represented by any of SEQ ID NOs: 2 to 12 when aligned with the amino acid sequence represented by SEQ ID NO: 1. Here, the substituted amino acid may be substituted with, for example, Gln, Gly, Arg, Val, Cys, or Ala, and is preferably substituted with Gly or Ala.In the production method of this embodiment, when the prenyl group introduced into the substrate is a relatively large group such as a farnesyl group, a geranylgeranyl group, a geranylfarnesyl group, a hexaprenyl group, an octaprenyl group, or a decaprenyl group, any of the following may be used: an H239G / W237T mutant and an H239G / W237N mutant of an enzyme having the amino acid sequence represented by SEQ ID NO: 1; or an enzyme containing or having an amino acid sequence represented by any of SEQ ID NOs: 2 to 12, which has mutations corresponding to the H239G / W237T mutation and the H239G / W237N mutation of the amino acid sequence represented by SEQ ID NO: 1 when aligned with the amino acid sequence represented by SEQ ID NO: 1.

[0072] Furthermore, when the prenyl group introduced into the substrate in the production method of this embodiment is a geranyl group, the amino acid sequence may be: (i) an amino acid sequence represented by SEQ ID NO: 1; (ii) an amino acid sequence in which, in the amino acid sequence represented by SEQ ID NO: 1, one or several amino acids other than E at position 54, Y at position 188, Y at position 237, and Y at position 292 of SEQ ID NO: 1 are deleted or substituted; preferably one or several amino acids other than E at position 54, H at position 172, Y at position 188, Y at position 237, and Y at position 292; or at least one amino acid selected from 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; more preferably, at least one amino acid selected from I at position 52, D at position 70, Q at position 72, L at position 122, E at position 190, L at position 222, G at position 224, H at position 239, and W at position 273 is deleted or substituted; or (iii) an enzyme containing or having an amino acid sequence in which, when aligned with the amino acid sequence of SEQ ID NO: 1, one or several amino acids other than E at position 54, Y at position 188, Y at position 237, and Y at position 292 of SEQ ID NO: 1; preferably one or several amino acids other than E at position 54, H at position 172, Y at position 188, Y at position 237, and Y at position 292 of SEQ ID NO: 1; or at least one amino acid selected from 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; more preferably I at position 52, D at position 70, Q at position 72, L at position 122, E at position 190, L at position 222, G at position 224, H at position 239, and W at position 273 of SEQ ID NO: 1; Here, I at position 52, D at position 70, Q at position 72, L at position 122, E at position 190, L at position 222, and H at position 239 can be substituted with any amino acid, for example, Ala. H at position 172 can be substituted with any amino acid, for example, Leu, Phe, or Ala.G at position 224 can be substituted with any amino acid, for example, Phe or Tyr. W at position 273 can be substituted with any amino acid, for example, Asn, Ser, Thr, Val, Ile, Cys, Phe, Met, Gly, or Ala.

[0073] Among the enzymes described above as preferred enzymes for introducing relatively large groups such as dimethylallyl, geranyl, farnesyl, geranylgeranyl, geranylfarnesyl, hexaprenyl, octaprenyl, and decaprenyl groups (particularly farnesyl groups), the enzymes described above can be preferably used in both cases. For example, if an enzyme described above as a preferred enzyme for introducing a geranyl group is also described as a preferred enzyme for introducing relatively large groups such as farnesyl, geranylgeranyl, geranylfarnesyl, hexaprenyl, octaprenyl, and decaprenyl groups (particularly farnesyl groups), the enzyme is preferably used both for introducing a geranyl group and for introducing relatively large groups such as farnesyl, geranylgeranyl, geranylfarnesyl, hexaprenyl, octaprenyl, and decaprenyl groups (particularly farnesyl groups).

[0074] Furthermore, in the case where the substrate for the prenylation step does not have a structure represented by formula (IV) above but is a compound having at least one structure represented by formula (III) above; in the case where the substrate for the prenylation step is a compound having at least one structure represented by (IV); and in the case where the substrate to be prenylated is a peptide or protein and His or a derivative thereof is prenylated when the amino acid adjacent to the N-terminus of His or a derivative thereof has a positive charge or is significantly sterically hindered, the enzymes described above as usable or preferably used enzymes may be introduced with the above-mentioned mutations introduced in the enzymes described above as enzymes used to introduce relatively large groups (particularly farnesyl groups) such as a dimethylallyl group, a geranyl group, and a farnesyl group, a geranylgeranyl group, a geranylfarnesyl group, a hexaprenyl group, an octaprenyl group, and a decaprenyl group, depending on the type of prenyl group to be introduced into the substrate. In addition to the above-mentioned mutations, one or several amino acids may be deleted, substituted, or added. The prenyltransferase may include an enzyme that contains or has an amino acid sequence that is at least 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 homologous to the above-mentioned enzymes and mutants thereof. In the production method of this embodiment, before the prenylation step, a prenyltransferase may be selected depending on the types of substrate to be prenylated and compound capable of donating a prenyl group.

[0075] Any of the above prenyltransferases may be used in a state where they are immobilized on a solid support such as magnetic beads. Therefore, the prenyltransferases used in the prenylation step include enzymes immobilized on a solid support. Furthermore, the prenyltransferases may have additional sequences such as His tags, glutathione S-transferase (GST), and maltose-binding protein (MBP). The above prenyltransferases may be used alone or in combination of two or more types in the prenylation step.

[0076] (Prenylation Step) The prenylation step is a step of contacting any one of the above substrates or a combination thereof with any one of the above prenyltransferases or a combination thereof to obtain a compound having at least one structure represented by the following formula (I) or (II): In addition, in formula (I), R １ is a hydrogen atom or an arbitrary substituent, n is an integer of 0 to 11, and in formula (II), R ２ are each independently an arbitrary substituent, p is an integer of 0 to 4, and n is an integer of 0 to 11.

[0077] In the structure represented by the above formula (I), R １ The bonding position in the imidazole ring is not particularly limited, that is, the structure represented by the formula (I) above can have structures corresponding to the formulas (III-1) and (III-2) in the formula (III). In the structure represented by the formula (II) above, the bonding position of the oxygen atom in the benzene ring may be any of the ortho-position, meta-position, and para-position, that is, the structure represented by the formula (II) above can have structures corresponding to the formulas (IV-1), (IV-2), and (IV-3) in the structure represented by the formula (IV). In the structure represented by the formula (II) above, the bonding position of the oxygen atom in the benzene ring is preferably the para-position.

[0078] R in the above formula (I) １ Examples and preferred embodiments of the formula (III) are １ In addition, R in the above formula (II) is the same as ２ Examples and preferred embodiments of p are R in the above formula (IV). ２ and p are the same as those in Formulas (I) and (II). n in the above formulae (I) and (II) represents an integer of 0 to 11, and depends on the type of prenyl group donor used, which will be described later, and other factors. n in the above formulae (I) and (II) is, for example, 0 to 8, and may be 0 to 6, 0 to 5, 0 to 3, 0 to 2, or 0, 1, or 2.

[0079] The compound obtained by the prenylation step corresponds to the substrate used in the prenylation step. Any of the above substrates can be used to obtain a compound having the structure of formula (I) or (II). １ , R in the above formula ２ and p, as well as other structures, a structure corresponding to the substrate used is obtained. Note that, when the substrate has two or more structures represented by the above formula (III) or (IV), it is sufficient that at least one or more of the multiple moieties having the structures are prenylated, and only one moiety may be prenylated, or all moieties may be prenylated. Similarly, when the substrate has both a structure represented by the above formula (III) and a structure represented by the above formula (IV), it is sufficient that either the structure represented by the above formula (III) or the structure represented by the above formula (IV) is prenylated, and only the structure represented by the above formula (III) may be prenylated, or only the structure represented by the above formula (IV) may be prenylated, or both the structure represented by the above formula (III) and the structure represented by the above formula (IV) may be prenylated.

[0080] The prenylation step may be, for example, a step of reacting any one or a combination of the above-mentioned substrates, any one or a combination of the above-mentioned prenyltransferases, and any one or a combination of compounds capable of donating a prenyl group (hereinafter referred to as "prenyl group donors") at an appropriate reaction temperature and reaction time. 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, hereinafter), isoprenyl diphosphate (IPP), geranyl diphosphate (GPP), farnesyl diphosphate (FPP), geranylgeranyl diphosphate (GGPP), and phytyl diphosphate (PDP). These prenyl group donors may be used alone or in combination of two or more in the prenylation step. The prenyl group donor may be appropriately selected depending on the type of prenyltransferase.

[0081] The prenylation step may be carried out in a reaction system in which all of the substrate, prenyltransferase, and prenyl group donor are added to a solvent, or may be carried out by contacting a reaction system in which any of the substrate, prenyltransferase, and prenyl group donor is supported on a solid phase support such as magnetic beads with a solution containing the remaining components. When the prenyltransferase 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 prenyltransferase concentration may be, for example, 0.050 μM to 1.0 mM, or 0.10 to 500 μM. When the substrate 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 100 times or 1.0 to 50 times the concentration of the substrate, specifically 0.010 μM to 100 mM or 0.10 to 50 mM.

[0082] The reaction temperature (incubation temperature) in the prenylation step is not particularly limited, but is preferably within a range of the optimal temperature of the prenyltransferase 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 step may be adjusted appropriately depending on the reaction temperature and the concentrations of the substrate, prenyltransferase, 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.

[0083] The prenylation step may contain components in the reaction system other than the substrate, prenyltransferase, and prenyl group donor. Such components are not particularly limited and include, for example, components used in preparing any of the substrate, prenyltransferase, and prenyl group donor, as well as cofactors that enhance the catalytic activity of the prenyltransferase. 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, magnesium ions are preferably present. Whether or not a cofactor is used in the prenylation step can be appropriately selected depending on various conditions, such as the type of prenyltransferase used.

[0084] A preferred embodiment of the prenylation step is a step of contacting a peptide or protein having an amino acid sequence containing at least one His, Tyr, or derivative thereof with any one of the above-mentioned prenyltransferases or a combination thereof to introduce a prenyl group into at least one His residue, Tyr residue, or derivative residue thereof. According to this embodiment, a prenyl group can be introduced into a peptide or protein that generally tends to have low cell membrane permeability and / or low bioavailability, thereby obtaining a potentially promising drug.

[0085] (Other Steps) The method for producing a compound of this embodiment may include steps other than the above-mentioned prenylation step. Such steps include, for example, a step of preparing at least one of a substrate, a prenyltransferase, and a prenyl group donor to be subjected to the prenylation step, a step of separating and purifying a compound prenylated in the prenylation step, and a step of analyzing the physical properties of the purified prenylated compound.

[0086] The step of preparing a substrate to be subjected to the prenylation step (hereinafter referred to as the "substrate preparation step") is a step of preparing a compound having at least one structure represented by the above formula (III) or (IV). The substrate preparation step may be a step of producing such a compound by a conventionally known production method, or may be a step of purchasing a commercially available compound and optionally subjecting it to purification or the like.

[0087] In the substrate preparation step, one or more types of substrates as described above may be prepared. When two or more types of substrates are prepared in the substrate preparation step, the substrate preparation step can be said to be a step of preparing a library of compounds having at least one structure represented by the above formula (III) or (IV). By carrying out the compound production method of this embodiment using such a compound library, it is possible to produce a library of compounds having at least one structure represented by the above formula (I) or (II). That is, one aspect of the production method of this embodiment is a method for producing a compound library having at least one structure represented by the above formula (I) or (II).

[0088] Furthermore, when two or more compounds containing peptides or proteins having an amino acid sequence containing at least one His, Tyr, or derivative thereof are prepared in the substrate preparation step, the substrate preparation step can be said to be a step of preparing a library of such peptides or proteins. By carrying out the compound production method of this embodiment using such a peptide or protein library, it is possible to produce a library of peptides or proteins having a prenyl group on at least one His residue, Tyr residue, or derivative residue thereof. That is, one aspect of the production method of this embodiment is a production method of a peptide or protein library having a prenyl group on at least one His residue, Tyr residue, or derivative residue thereof. For the production method of a peptide or protein library, reference can be made to the production method of a prenylated compound library of this embodiment described below.

[0089] The substrate preparation step may be produced by a conventionally known chemical synthesis method. In particular, when the substrate is a peptide or protein, a synthesis method using a translation synthesis system may be used. Such translation synthesis systems include cellular translation systems and cell-free translation systems. When producing a peptide or protein 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. The method for producing a peptide or protein using a cell-free translation system will be described later.

[0090] The step of preparing a prenyltransferase (hereinafter referred to as the "prenyltransferase preparation step") is a step of preparing the above-mentioned LimF or an enzyme homologous thereto. In the prenyltransferase preparation step, the enzyme may be isolated from a naturally occurring organism (e.g., Limnothrix sp., Symploca sp., etc.), or may be obtained by heterologously expressing a gene encoding the target enzyme in an appropriate host (e.g., Escherichia coli). Alternatively, the enzyme may be obtained by a cell-free translation system or chemical synthesis. In the prenyltransferase preparation step, a prenyltransferase may be selected depending on the type of substrate to be prenylated and the type of compound capable of donating a prenyl group.

[0091] The step of separating and / or purifying the prenylated compound (hereinafter referred to as the "separation / purification step") is a step of separating the compound having at least one structure represented by formula (I) or (II) obtained by the production method of this embodiment from raw materials and / or by-products, and / or purifying each of the target compounds when multiple target compounds are obtained. A known separation method such as liquid chromatography may be used for the separation / purification step.

[0092] The step of analyzing the physical properties of the purified prenylated compound (hereinafter referred to as the "analysis step") is a step of analyzing the target compound obtained by the prenylation step or the target compound purified by the separation / purification step. The analysis step may include identification of the obtained compound, calculation of the prenylation efficiency, measurement of the dissociation constant for the desired target substance, etc. Specifically, the analysis step may include the compound analysis method described in the Examples.

[0093] [Method for Producing a Prenylated Compound Library] In the compound production method of this embodiment, a library of compounds containing prenylated peptides or proteins can be produced by using, as substrates, a library containing two or more compounds containing peptides or proteins having an amino acid sequence containing at least one His, Tyr, or a derivative thereof. That is, the method for producing a compound library of this embodiment is a method for producing a compound library containing prenylated peptides or proteins, and includes the step of contacting a compound library containing peptides or proteins having an amino acid sequence containing at least one His, Tyr, or a derivative thereof with a prenyltransferase to introduce a prenyl group into at least one His residue, Tyr residue, or derivative residue thereof, wherein the prenyltransferase is LimF or an enzyme homologous thereto.

[0094] According to the method for producing a compound library of this embodiment, a library of compounds containing highly hydrophobic peptides or proteins can be produced by prenylating at least one His, Tyr, or a portion of a derivative thereof using a predetermined prenyltransferase. Furthermore, since the method for producing a compound library of this embodiment uses LimF or an enzyme homologous thereto as the prenyltransferase, the substrate tolerance of the prenyltransferase is high, making it possible to produce a library containing a variety of compounds having a prenyl group. As described above, the method for producing a compound library of this embodiment can efficiently introduce a prenyl group into a variety of compounds, potentially producing compounds with high cell membrane affinity and / or high cell membrane permeability.

[0095] 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 library of compounds containing peptides or proteins in which at least one His residue, Tyr residue, or derivative residue thereof is prenylated. The compounds contained in the library may be peptides or proteins, or may be compounds in which a structure other than an amino acid is bound to a peptide or protein by a covalent or non-covalent bond. Examples of the moiety other than a peptide or protein in a compound in which a structure other than an amino acid is bound to a peptide or protein include linkers, nucleic acid molecules such as DNA and RNA, and labels for detecting peptides or proteins (fluorescent substances, radioactive substances, metal nanoparticles, quantum dots, enzymes, etc.).

[0096] The method for producing a compound library of this embodiment can be carried out in the same manner as the method for producing a compound of this embodiment described in detail above, except that a library containing two or more compounds containing a peptide or protein having an amino acid sequence containing at least one His, Tyr, or a derivative thereof is used as a substrate. The process for producing a library used as a substrate (hereinafter referred to as the "library preparation process") will be described in detail below. The compound library may be prepared by purchasing a commercially available product.

[0097] (Library Preparation Step) The library preparation step is not particularly limited as long as it is a step capable of preparing a library containing two or more compounds including peptides or proteins having an amino acid sequence containing at least one His, Tyr, or a derivative thereof. The compounds 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.

[0098] The library preparation step is preferably a step of translating an mRNA library using a cell-free translation system to prepare a compound library containing peptides or proteins having an amino acid sequence containing at least one His, Tyr, or a derivative thereof.

[0099] 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.

[0100] For convenience, the following describes a step of preparing a compound library containing peptides using an mRNA library, but a step of preparing a compound library containing proteins using an mRNA library can also be carried out in a similar manner by adjusting the number of bases in the 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 peptides having an amino acid sequence containing at least one His, Tyr, or a derivative thereof.

[0101] In one embodiment of the library preparation step, first, an mRNA library containing mRNA encoding peptides contained in each compound of the compound library is prepared (hereinafter referred to as the "mRNA preparation step"). The sequence of the mRNA encoding the peptide contained in each compound of the compound library 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.

[0102] 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:

[0103]

[0104] 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.

[0105] 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.

[0106] 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).

[0107] 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. However, the process can be carried out in the same manner when other mRNA libraries are used, as long as the peptides contained in the translated compound library are prenylated. In the natural genetic code, N1N2K represents 20 amino acids whose right column in Table 2 above is G or U.

[0108] 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, using amino acids containing a cyclic structure and / or N-alkylamino acids as non-proteinogenic amino acids tends to result in a compound library with increased resistance to proteolysis, cell membrane permeability, and / or conformational rigidity. Such a compound library is useful for screening compounds that target intracellular disease-related molecules and / or molecules with protease activity. 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.

[0109] Preferably, each mRNA in the mRNA library contains a start codon, multiple N1N2K sequences, and a stop codon, in this order. Here, at least one set of multiple N1N2K sequences is a codon assigned to His or Tyr. When using the natural genetic code, each mRNA contains, for example, a sequence of AUG-(N1N2K)n-UAG. Here, N1 and N2 are each independently selected from adenine (A), guanine (G), cytosine (C), and uracil (U), and K is each independently either uracil (U) or guanine (G). Furthermore, n is an integer between 1 and 49. Within the above range, n may be 2 or more, 3 or more, 4 or more, 5 or more, or 9 or more, and may be 44 or less, 39 or less, 29 or less, or 19 or less.

[0110] In the library preparation step, when an mRNA library encoding peptides having an amino acid sequence containing at least one His, Tyr, or derivative thereof is prepared using the natural genetic code, each mRNA preferably contains a start codon, multiple N1N2Ks, and a stop codon, in this order, and at least one set of the multiple N1N2Ks is any of CAU, CAC, UAU, and UAC (i.e., any codon assigned to His or Tyr). In this case, for example, taking an example containing CAU, each mRNA contains the sequence AUG-(N1N2K)n1-CAU-(N1N2K)n2-UAG. Here, N1, N2, and K are as defined above, and n1 and n2 are integers of 0 to 48, inclusive. The sum of n1 and n2 may be determined to be n-1 (n is as defined above).

[0111] In the description of the substrate for the compound production method of this embodiment, it was explained that, in the amino acid sequence of the peptide or protein serving as the substrate, particularly preferred amino acids on the N-terminal side adjacent to His, Tyr, or a derivative thereof are Thr, Ala, Gly, Ser, Val, Leu, Asn, Met, Pro, and Ile. From this perspective, it is preferable that each mRNA in the mRNA library contains, for example, a sequence shown in Table 3 below (in the sequences shown in Table 3, RBU is an example and may be replaced with a codon encoding any of Thr, Ala, Gly, Ser, Val, Leu, Asn, Met, Pro, and Ile). In Table 3, N1, N2, and K are as defined above, R is either adenine (A) or guanine (G), B is either guanine (G), cytosine (C), or uracil (U), and m1 and m2 are each an integer of 0 to 47. m1 and m2 may be selected in the range such that the sum of m1 and m2 is 0 or more and 47 or less. The sum of m1 and m2 may be 1 or more, 2 or more, 3 or more, or 7 or more, and may be 42 or less, 37 or less, 27 or less, or 17 or less. In Table 3, the CAU codon may be replaced with other codons encoding His or Tyr (e.g., CAC, UAU, UAC, etc.).

[0112]

[0113] When the library preparation step is a step of preparing a library of compounds containing cyclic peptides, for example, an mRNA library containing mRNAs encoding peptides containing an amino acid having functional group 1 and an amino acid having the corresponding functional group 2 shown in Table 4 below may be used.

[0114] In the chemical structural formula of Table 4 above, X １ is a leaving group, and Ar is an aromatic ring which may have a substituent. Examples of the leaving group include halogen atoms such as Cl, Br, and I.

[0115] When such an mRNA library is used, it is possible to obtain a library of compounds containing cyclic peptides cyclized from an amino acid having functional group 1 and an amino acid having the corresponding functional group 2. For example, when the library preparation step is carried out using an mRNA library encoding peptides containing an amino acid having a functional group (A-1) and an amino acid having a functional group (A-2), N-CO-CH ２ Cyclic peptides can be prepared that contain a ring structure formed by an -S structure.

[0116] Either functional group 1 or 2 may be located on the N-terminus, or may be located at the N-terminus or C-terminus, or one may be a terminal amino acid and the other a non-terminal amino acid, or both may be non-terminal amino acids. The bond formed by functional group 1 and functional group 2 can be said to be a chemical crosslinking structure for forming a molecular cyclic structure in a cyclic peptide.

[0117] Examples of the amino acid having the functional group (A-1) include chloroacetylated amino acids, such as N-chloroacetyl-L-alanine, N-chloroacetyl-L-phenylalanine, N-chloroacetyl-L-tyrosine, N-chloroacetyl-L-tryptophan, N-3-(2-chloroacetamido)benzoyl-L-phenylalanine, N-3-(2-chloroacetamido)benzoyl-L-tyrosine, N-3-(2-chloroacetamido)benzoyl-L-tryptophan, β-N-chloroacetyl-L-diaminopropanoic acid, γ-N-chloroacetyl-L-diaminobutyric acid, δ-N-chloroacetyl-L-ornithine, ε-N-chloroacetyl-L-lysine, and their corresponding D-amino acid derivatives. As the amino acid having a functional group (A-1), N-chloroacetyl-L-tyrosine and N-chloroacetyl-D-tyrosine are preferably used.

[0118] Examples of the amino acid having the functional group (A-2) include cysteine, homocysteine, mercaptonorvaline, mercaptonorleucine, 2-amino-7-mercaptoheptanoic acid, and 2-amino-8-mercaptooctanoic acid, etc. Cysteine ​​is preferably used as the amino acid having the functional group (A-2).

[0119] Examples of the cyclization method using an amino acid having a functional group (A-1) and an amino acid having a functional group (A-2) include those described in Kawakami, T. et al., Nat. Chem. Biol. 5, 888-890 (2009); Yamagishi, Y. et al., ChemBioChem 10, 1469-1472 (2009); Sako, Y. et al., J. Am. Chem. Soc. 130, 7932-7934 (2008); Goto, Y. et al., ACS Chem. Biol. 3, 120-129 (2008); Kawakami T. et al., Chem. Biol. 15, 32-42 (2008), and WO 2008 / 117833.

[0120] Examples of amino acids having the functional group (B-1) include propargylglycine, homopropargylglycine, 2-amino-6-heptynoic acid, 2-amino-7-octynoic acid, and 2-amino-8-nonynoic acid. 4-pentynoylated or 5-hexynoylated amino acids may also be used. Examples of 4-pentenoylated amino acids include N-(4-pentenoyl)-L-alanine, N-(4-pentenoyl)-L-phenylalanine, N-(4-pentenoyl)-L-tyrosine, N-(4-pentenoyl)-L-tryptophan, N-3-(4-pentynoylamido)benzoyl-L-phenylalanine, N-3-(4-pentynoylamido)benzoyl-L-tyrosine, N-3-(4-pentynoylamido)benzoyl-L-tryptophan, β-N-(4-pentenoyl)-L-diaminopropanoic acid, γ-N-(4-pentenoyl)-L-diaminobutyric acid, σ-N-(4-pentenoyl)-L-ornithine, ε-N-(4-pentenoyl)-L-lysine, and the corresponding D-amino acid derivatives thereof. Examples of 5-hexynoyl amino acids include amino acids in which the 4-pentynoyl group in the compounds exemplified as 4-pentynoyl amino acids has been substituted with a 5-hexynoyl group.

[0121] Examples of amino acids having the functional group (B-2) include azidoalanine, 2-amino-4-azidobutanoic acid, azidoptonorvaline, azidonorleucine, 2-amino-7-azidoheptanoic acid, and 2-amino-8-azidooctanoic acid. Azidoacetylated or 3-azidopentanoylated amino acids can also be used. Examples of azidoacetylated amino acids include N-azidoacetyl-L-alanine, N-azidoacetyl-L-phenylalanine, N-azidoacetyl-L-tyrosine, N-azidoacetyl-L-tryptophan, N-3-(4-pentynoylamido)benzoyl-L-phenylalanine, N-3-(4-pentynoylamido)benzoyl-L-tyrosine, N-3-(4-pentynoylamido)benzoyl-L-tryptophan, β-N-azidoacetyl-L-diaminopropanoic acid, γ-N-azidoacetyl-L-diaminobutyric acid, σ-N-azidoacetyl-L-ornithine, ε-N-azidoacetyl-L-lysine, and their corresponding D-amino acid derivatives. Examples of 3-azidopentanoylated amino acids include amino acids in which the azidoacetyl group in the compounds exemplified as azidoacetylated amino acids has been substituted with a 3-azidopentanoyl group.

[0122] Examples of the cyclization method using an amino acid having a functional group (B-1) and an amino acid having a functional group (B-2) include the methods described in Sako, Y. et al., J. Am. Chem. Soc. 130, 7932-7934 (2008) and WO 2008 / 117833.

[0123] Examples of amino acids having a functional group (C-1) include N-(4-aminomethyl-benzoyl)-phenylalanine (AMBF) and 3-aminomethyltyrosine. Examples of amino acids having a functional group (C-2) include 5-hydroxytryptophan (WOH). Examples of cyclization methods using amino acids having a functional group (C-1) and amino acids having a functional group (C-2) include those described in Yamagishi, Y. et al., Chembiochem 10, 1469-1472 (2009) and WO 2008 / 117833.

[0124] Examples of amino acids having a functional group (D-1) include 2-amino-6-chloro-hexynoic acid, 2-amino-7-chloro-heptynoic acid, and 2-amino-8-chloro-octynoic acid. Examples of amino acids having a functional group (D-2) include cysteine, homocysteine, mercaptonorvaline, mercaptonorleucine, 2-amino-7-mercaptoheptanoic acid, and 2-amino-8-mercaptooctanoic acid. Examples of methods for cyclization using amino acids having a functional group (D-1) and amino acids having a functional group (D-2) include the methods described in WO 2012 / 074129.

[0125] Examples of the amino acid (E-1) include N-3-chloromethylbenzoyl-L-phenylalanine, N-3-chloromethylbenzoyl-L-tyrosine, N-3-chloromethylbenzoyl-L-tryptophan, and their corresponding D-amino acid derivatives. Examples of the amino acid (E-2) include cysteine, homocysteine, mercaptonorvaline, mercaptonorleucine, 2-amino-7-mercaptoheptanoic acid, and 2-amino-8-mercaptooctanoic acid. The cyclization method using the amino acid having a functional group (E-1) and the amino acid having a functional group (E-2) can be carried out with reference to, for example, the cyclization method between (A-1) and (A-2) or the cyclization method between (D-1) and (D-2).

[0126] When the library preparation step is a step of preparing a library of compounds containing cyclic peptides, each mRNA in the mRNA library preferably contains, for example, a sequence shown in Table 5 below. In Table 5 below, N1, N2, K, R, and B are defined as in Table 3 above, m3 and m4 are each an integer of 0 to 46, and (linker) is an arbitrary sequence. m3 and m4 may be selected in the range such that the sum of m3 and m4 is 0 to 46. The sum of m1 and m2 may be 1 or more, 2 or more, 3 or more, or 6 or more, and may be 41 or less, 36 or less, 26 or less, or 16 or less.

[0127]

[0128] In the above table, an N-chloroacetyl amino acid is assigned to the AUG codon, and a cyclic peptide is formed by the chloroacetyl group of the amino acid and the mercapto group of the downstream Cys. In the above table, D- or L-Tyr, Trp, or Phe is assigned to the AUG codon, but the amino acids that can be assigned are not limited to Tyr, Trp, and Phe. To assign an N-chloroacetyl amino acid to an AUG codon, for example, the above-mentioned Flexizyme may be used.

[0129] 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.

[0130] 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.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] 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 １３ It is possible to produce libraries containing more than one different peptide.

[0137] The library preparation step may be a step of preparing a compound library containing genotype-bound peptides or proteins by a display method, particularly an mRNA display method.

[0138] A display method refers to a system that displays a phenotype on a genotype by covalently or non-covalently linking the phenotype to the genotype encoding its sequence, and enables the enrichment and amplification (i.e., selection) of active species using a replication system reconstructed in a test tube. Examples of display methods include phage display, which uses E. 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.

[0139] The step of preparing a compound library containing genotype-bound peptides or proteins by the mRNA display method preferably includes the steps of: preparing an mRNA library in which each mRNA encodes a peptide or protein having an amino acid sequence containing at least one His, Tyr, or a derivative thereof; binding puromycin to the 3' end of each mRNA in the mRNA library to produce a puromycin-bound mRNA library; and translating the puromycin-bound mRNA library using a cell-free translation system.

[0140] The step of preparing an mRNA library and the step of translating the puromycin-bound mRNA library using a cell-free translation system can be carried out in the same manner as the above-described mRNA preparation step and translation step, respectively. The step of producing a puromycin-bound mRNA library can be carried out by binding puromycin to the downstream region of the ORF (open reading frame) of each mRNA when preparing an mRNA library in the above-described method of producing a peptide library. 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. Such peptide-mRNA complexes can correlate genotypes with phenotypes and can be applied to in vitro display.

[0141] (Compound Library) One aspect of this embodiment is a compound library produced by the above-described production method. The compound library of this embodiment includes a library of compounds containing the above-described peptides or proteins, and a library of complexes of compounds containing peptides or proteins with mRNA.

[0142] [Screening Method] One aspect of this embodiment is a screening method for identifying compounds that bind to a target substance, comprising the steps of contacting a compound library produced by any of the production methods described above, preferably a library of complexes of compounds containing peptides or proteins and mRNA, with a target substance, and selecting compounds that bind to the target substance.

[0143] 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.

[0144] 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, etc. In particular, the above-mentioned compound library can be used when the target substance has protease activity or is an intracellular molecule.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] (Screening Kit) Another aspect of this embodiment is a compound screening kit. One aspect of the screening kit of this embodiment includes a compound library produced by the above-described production method. In addition to the compound library, the screening kit may include reagents and devices necessary for detecting binding between a target substance and a compound. Examples of such reagents and devices include, but are not limited to, a solid phase support, a buffer solution, a labeling reagent, an enzyme, an enzyme reaction stop solution, and a microplate reader.

[0151] 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.

[0152] 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.

[0153] [Materials] Unless otherwise noted, reagents used in the examples were purchased from Nacalai Tesque, Wako Pure Chemical Industries, Sigma-Aldrich Japan, Kanto Chemical, or Watanabe Chemical Industry and used without purification. Geranyl pyrophosphate was purchased from Isopronoid, LC. Farnesyl pyrophosphate was purchased from Sigma-Aldrich Japan. All oligo primers were purchased from Eurofins Genomics (OPC purified grade).

[0154] General Experimental Procedures Liquid chromatography-mass spectrometry (LC-MS) experiments for the analysis of synthetic substrates and enzymatic reaction products were performed using a Waters Xevo G2-XS QToF instrument equipped with an Acquity I-Class UPLC system. Injected samples were separated on an Acquity UPLC Petide BEH C18 column (300 Å, 1.7 μm, 2.1 mm × 150 mm) using the following gradient: 1% B × 2 min; 1–61% B × 6 min; 95% B × 1 min; 1% B × 3 min (total run time: 12 min), or 1% B × 2 min; 1–81% B × 16 min; 95% B × 1 min; 1% B × 3 min (total run time: 22 min) (Mobile phase A: 0.1% (v / v) formic acid in water; and B: 0.1% (v / v) formic acid in acetonitrile) and then directly injected onto the Xevo QTof MS.

[0155] All MS analyses were performed in positive mode with a capillary voltage of 0.7 kV, using nitrogen as the cone gas (50 L / min) and desolvation gas (1000 L / min). The ionization source temperature was 120°C, and the desolvation gas was 400°C. For MS / MS analysis, spectra were acquired with a scan time of 0.3 seconds, and selected ions were subjected to a collision energy ramp of 15-30 or 25-40 eV. LC-MS data were analyzed using MassLynx 4.1, and MS / MS spectral assignments were performed manually. Extracted ion chromatograms (EICs) with selected ions were generated with an m / z tolerance of 0.10. For the purification of synthetic substrates and enzymatic reaction products, an LC-20AP system (Shimadzu) equipped with a Chromolith Prep column (100 × 25 mm, C18 phase, Merck) was used with mobile phase A: 0.1% (v / v) trifluoroacetic acid (TFA) in water, and B: 0.1% (v / v) TFA in acetonitrile.

[0156] [Method for Expression and Purification of Prenyltransferase] A codon-optimized LimF synthetic gene was purchased from Eurofins Genomics, and the cDNA encoding SUMOstar-LimF was introduced into the pET32 vector (Novagen) using the In-Fusion HD Cloning kit (TaKaRa Clontech) to generate the pET32-LimF-SMS plasmid. For expression of the LimF-SUMO fusion protein in E. coli, the pET32-LimF-SMS plasmid was transformed into E. coli BL21 Gold (DE3) using 100 mg / L sodium ampicillin as a selection marker. The resulting transformants were cultured in 2xYT medium containing 100 mg / L sodium ampicillin. The culture was incubated at 37°C with shaking at 160 rpm until the OD600 reached 0.8. The culture was then cooled on ice, and isopropyl-β-D-1-thiogalactopyranoside (IPTG) and ethanol were immediately added to final concentrations of 0.2 mM and 3% (v / v), respectively. After IPTG induction, the culture was incubated at 20°C and 180 rpm for 20 hours, and then the cell pellet was collected by centrifugation (6000 x g, 15 minutes) at 4°C. The pellet was dissolved in 100 mL of lysis buffer (40 mM K ２ HPO ４ , 10 mM KH ２ P.O. ４ The cells were resuspended in 5% CO₂Cl, 500 mM NaCl, 20 mM imidazole, 1 mM DTT, 0.1 mM PMSF, 0.5% Triton X-100, pH 7.5, disrupted by sonication on ice, centrifuged at 4°C (20,000 × g, 30 min), and then filtered to recover the soluble fraction.

[0157] Buffer A (40mM K ２ HPO ４ , 10 mM KH ２ P.O. ４The collected soluble fraction was loaded onto a Histrp HP 5 mL column (Cytiva) pre-equilibrated with Buffer A (20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 20 mM imidazole, 1 mM DTT, pH 7.5). The His-tagged LimF-SUMO fusion protein was eluted with Buffer A containing 200 mM imidazole. The protein was then dialyzed against Buffer B (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM DTT) at 4°C for 6 hours. SUMOstar protease was then added to the dialysis membrane, and the SUMO tag was removed by further dialysis at 4°C for 6 hours. The tag-free LimF was then loaded onto a Hitrap SP 5 mL column (Cytiva) pre-equilibrated with Buffer C (10 mM Hepes pH 7.5, 150 mM NaCl, 1 mM DTT) and eluted with Buffer C containing 250 mM NaCl. The purified fraction was then loaded onto a HiLoad 16 / 60 Superdex 200 pre-packed gel filtration column (Cytiva) at 4°C and eluted with Buffer C. The tag-free LimF fraction was then concentrated using an Amicon centrifugal filter (Merck). Protein purity was confirmed by SDS-PAGE analysis, and protein concentration was measured using absorbance at 280 nm calculated using the ExPASy ProtParam tool. The protein was then flash-frozen in liquid nitrogen and stored at -80°C.

[0158] [Chemical Synthesis of Peptides] The cyclic peptides bcLimE1, E2, and E3 used in the following examples were synthesized using a standard Fmoc SPPS protocol on a Syro I automated synthesizer (Biotage). 2-Chlorotrityl chloride resin was precharged with Fmoc-Tyr(tBu)-OH (0.877 mmol / g loading), and peptides were synthesized as C-terminal acids using 0.025 mmol of resin. The side chain protecting groups were the following combinations: Asp(OtBu), Glu(OtBu), His(Boc), Lys(Boc), Asn(Trt), Gln(Trt), Arg(Pbf), Ser(tBu), Thr(tBu), and Tyr(tBu). Peptide coupling reactions were carried out using 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) as the activator and diisopropylethylamine (DIPEA) as the base. Fmoc-deprotection was carried out using 20% ​​(v / v) piperidine in DMF. Unless otherwise noted, all reagents were dissolved in dimethylformamide (DMF) and reactions were carried out in DMF.

[0159] After SPPS, the resin was washed four times with dichloromethane (DCM) and dried under vacuum. The fully protected linear precursor peptide was cleaved from the resin by incubating it in a 20% solution of 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) in DCM with 5 mL of 2.5% triisopropylsilane (TIS) for 3 hours at room temperature. The peptide was precipitated by adding 40 mL of ice-cold diethyl ether to the solution containing the cleaved peptide and then leaving it at -20°C for 30 minutes. The peptide pellet was collected by manual centrifugation and washed four times with 10 mL of ice-cold diethyl ether. The washed pellet was dried overnight in a fume hood to remove residual diethyl ether. Each linear precursor peptide pellet was dissolved in 10 mL of DMF. 1.1 equivalents of benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) and 2.2 equivalents of DIPEA were added, and the cyclization reaction was carried out at room temperature for 6 hours. After cyclization, 10 mL of DCM was added, and the mixture was extracted twice with 100 mL of water. The organic phase containing the peptide was collected, and the solvent was removed using a rotary evaporator.

[0160] The side chain protecting groups were then deprotected by reaction with 5 mL of TFA / water / TIS (95:2.5:2.5, v / v) solution at room temperature for 3 hours. The deprotected peptide was precipitated with diethyl ether as described above and then washed with additional diethyl ether. The dried pellet was dissolved in 5 mL of dimethyl sulfoxide (DMSO) containing 0.1% TFA, filtered, and purified by preparative reverse-phase HPLC. Fractions containing the desired product were collected, lyophilized, and redissolved in DMSO to obtain the peptide solution.

[0161] For the synthesis of short peptides, the same SPPS method as above was carried out using NovaPEG Rink Amide resin (0.44 mmol / g loading) to synthesize peptides with amidated C-terminus. After peptide chain elongation, 0.5 M Ac ２The N-terminus was acetylated by incubating the resin in 2 mL of NMP containing 0 and 0.25 M DIPEA at room temperature for 60 minutes. After washing the resin three times with DMF and four times with DCM, the peptide was cleaved from the resin by incubating it in a TFA / water / TIS solution at room temperature for 3 hours. The purified peptide was obtained by the same workup and purification procedure as described above. The synthesis of thioether cyclic peptides was performed using a previously published method (Passioura, T.; Liu, W.; Dunkelmann, D.; Higuchi, T.; Suga, H. Display selection of exotic macrocyclic peptides expressed under a radically reprogrammed 23 amino acid genetic code. J. Am. Chem. Soc., 2018, 140 (37), 11551-11555.).

[0162] [Method for preparing Flexizyme and tRNA] dFx, eFx, tRNA ＧｌｕＥ２ ＧＵＧ , and tRNA ｆＭｅｔ ＣＡＵ was prepared by in vitro transcription using T7 RNA polymerase as previously reported (Goto, Y.; Katoh, T.; Suga, Nat. Protoc., Flexizymes for genetic code reprogramming. 2011, 6 (6), 779-790.).

[0163] [Method for preparing aminoacylated tRNA using Flexizyme] ClAc-D-Phe and ClAc-D-Tyr were prepared by tRNA synthesis using eFx. ｆＭｅｔ ＣＡＵ 3-Me-L-His, 1-Me-L-His, L-Ala(2-Thi), L-Ala(3-Thi), and α-Me-D / L-His were synthesized using eFx with tRNA ＧｌｕＥ２ ＧＵＧ L-His, D-His, and L-Ala (4-Thz) were synthesized using dFx as tRNA. ＧｌｕＥ２ ＧＵＧThe aminoacylation reaction was carried out using 25 μM Flexizyme (eFx or dFx), 25 μM tRNA, 5 mM activated amino acid donor (eFx reaction: cyanomethyl ester, dFx reaction: 3,5-dinitrobenzyl ester), 100 mM HEPES-KOH pH 7.5, 600 mM MgCl ２ The aminoacylation reaction was carried out in a solution containing 0.1% DMSO and 20% DMSO. Activated amino acids were prepared as previously described (Goto, Y.; Katoh, T.; Suga, Nat. Protoc., Flexizymes for genetic code reprogramming. 2011, 6 (6), 779-790; Passioura, T.; Liu, W.; Dunkelmann, D.; Higuchi, T.; Suga, HJ Am. Chem. Soc., Display selection of exotic macrocyclic peptides expressed under a radically reprogrammed 23 amino acid genetic code. 2018, 140 (37), 11551-11555). Each aminoacylation reaction mixture was incubated on ice for 2 hours (ClAc-D-Phe and ClAc-D-Tyr) or 6 hours (other amino acids), followed by the addition of 4 volumes of 0.3 M sodium acetate (pH 5.2) and 10 volumes of EtOH. The mixture was then centrifuged (15,300 × g) at room temperature for 15 minutes, and the supernatant was discarded. The aminoacyl-tRNA pellet was washed twice with 70% EtOH containing 0.1 M sodium acetate (pH 5.2) and once with 70% EtOH. The resulting aminoacyl-tRNA was dried for 10 minutes. The aminoacyl-tRNA was dissolved in 1 mM sodium acetate (pH 5.2) to a concentration of 250 μM immediately before addition to the translation mixture.

[0164] [Method for preparing DNA templates for substrates] Template DNA used in the following examples was prepared using the primers shown in Tables 6 (shown as Tables 6-1 and 6-2) and 7 (shown as Tables 7-1 to 7-4). Linear double-stranded DNA containing a T7 promoter upstream of a sequence encoding a thioether ring-closed cyclic peptide (hereinafter referred to as "teMP") was prepared using Taq DNA polymerase in a similar manner to a previous report (Vinogradov, A.A.; Shimomura, M.; Goto, Y.; Ozaki, T.; Asamizu, S.; Sugai, Y.; Suga, H.; Onaka, Nat. Commun., Minimal lactazole scaffold for in vitro thiopeptide bioengineering. 2020, 11 (1), 1-13.).

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171] [1. Search for prenyltransferases] (Method for generating sequence similarity networks (SSN)) The SSN was generated by using the hit sequences obtained by Blastp analysis and the Enzyme Function Initiative-Enzyme Similarity Tool (EFI-ESI) (Zallot, R.; Oberg, N.; Gerlt, The EFI web resource for genomic enzymology tools: leveraging protein, genome, and metagenome databases to discover novel enzymes and metabolic pathways. Biochemistry, 2019, 58 (41), 4169-4182.; Gerlt, JA; Bouvier, JT; Davidson, DB; Imker, HJ; Sadkhin, B.; Slater, DR; Whalen, KL. Enzyme function initiative-enzyme similarity tool (EFI-EST): a web tool for generating protein sequence similarity networks. BBA. PROTEINS AND PROTEOMICS, 2015, 1854). (8), 1019-1037. Nodes were created by appropriately setting alignment scores, and the resulting network was visualized using Cytoscape 3.8.2 to cluster PTases with different functions. KgpF or LimF was used as the query, as described below.

[0172] (Phylogenetic tree analysis method) The phylogenetic tree was constructed as follows. First, known cyanobactin PTase sequences were downloaded from the NCBI database. These sequences were aligned using MEGA 7.0 (Kumar, S.; Stecher, G.; Tamura, MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol., 2016, 33 (7), 1870-1874.), and then a maximum likelihood phylogenetic tree based on this alignment was constructed using 10,000 bootstrap replicates.

[0173] (Discussion) To search for potential peptide PTases with C-prenylation activity, we used the Basic Local Alignment Search Tool (BLAST) to search for proteins homologous to KgpF, the only known cyanobactin C-PTase. For the hits obtained, we set the alignment score to 10 to predict sequence-function relationships. －８６ A sequence similarity network (SSN) was generated by setting the seq. The resulting SSN revealed clusters with predicted functions, including Tyr O-prenylation, terminal N- / O-prenylation, Arg N-prenylation, and Trp N- / C-prenylation, as well as clusters with unspecified functions (Fig. 1a).

[0174] Among the unidentified proteins, we focused on a protein from Limnothrix sp. CACIAM 69d that did not belong to any cluster. Phylogenetic analysis of this protein, comparing it with other known cyanobactin PTases, revealed that it formed an independent clade between Tyr O-PTases and Trp C- / N-PTases (Fig. 2a). This protein was previously referred to as a PirF (Tyr O-PTase) homolog (Lima, ARJ et al. Insights Into Limnothrix sp. Metabolism Based on Comparative Genomics. Front. Microbiol., 9(2018).), but both the SSN and phylogenetic analysis suggested that this protein exhibited unique activity. Therefore, we designated this protein LimF for further study.

[0175] The limF gene was contained in a typical cyanobactin BGC, which also contained the cyanobactin protease (limA) and macrocyclase (limG), as well as putative precursor peptides (limE1 and limE2) (Fig. 2b and Table 8). Manual gene screening revealed another precursor candidate (limE3) encoded at a distant locus. The LimE1-3 peptide shared significant similarity with a previously identified cyanobactin precursor (Donia, MS, Schmidt, Linking chemistry and genetics in the growing cyanobactin natural products family. Chem. Biol., 18, 508-519 (2011)). Sequence alignment analysis revealed the recognition sequences (RSII and RSIII) of LimA and G (Fig. 2c).

[0176]

[0177] In the general biosynthetic pathway of cyanobactins, a family A protease cleaves the N-terminal RSII, followed by a family G macrocyclase cleaving the C-terminal RSII while cyclizing the core peptide (CP) region to produce a main chain cyclized peptide (Sarkar, S., Gu, W. & Schmidt, Expanding the chemical space of synthetic cyclic peptides using a promiscuous macrocyclase from prenylagaramide biosynthesis. ACS Catal., 10, 7146-7153 (2020).). The resulting cyclic core peptide is generally further modified by other tailoring enzymes, including PTases, to give the final cyanobactin product. Based on similarities to other cyanobactin biosynthetic gene clusters and the conserved RS motif, we identified the CP region of the LimE1-3 peptide and hypothesized that their main-chain macrocyclized peptides (bcLimE1, bcLimE2, and bcLimE3, the structures of which are shown in Table 9 below) could serve as substrates for LimF.

[0178]

[0179] In addition, for protein sequences hit in the BLASTp analysis using LimF as a query, the alignment score was set to 10 －７０ SSN analysis was performed with the following setting. As a result, 11 new proteins were found to form the same cluster as LimF (Fig. 1b). Their sequences are shown in Table 1 as SEQ ID NOS: 2 to 12. From the above, it was strongly suggested that these 11 proteins have the same function as LimF. Among them, the protein of SEQ ID NOS: 2 derived from Symploca sp. SIO1B1 was estimated to have the highest probability of having the same function as LimF.

[0180] [2. Evaluation of LimF Enzyme Activity] To examine the enzymatic activity of LimF, LimF was heterologously expressed in Escherichia coli by the method described above, and the enzyme was incubated with MgCl ２Chemically synthesized bcLimE1, bcLimE2, or bcLimE3 was incubated with LimF in the presence of LimF and a prenyl group donor (geranyl pyrophosphate (GPP)). The reaction conditions were as described below. LC-MS analysis of the reaction mixture showed that bcLimE2 was quantitatively converted to a product with an increased molecular weight (+136 Da) corresponding to geranylation (Fig. 3a). MS / MS analysis of geranylated bcLimE2 suggested that the His residue was geranylated by LimF treatment (Fig. 4). In contrast, bcLimE1 and bcLimE3 were not geranylated by LimF under these conditions.

[0181] LimF-modified bcLimE2 was purified by HPLC and subjected to 1D and 2D NMR analyses, including COSY and HMBC. The HMBC correlation observed between the geranyl group and the imidazole C2 carbon indicated a structure in which the terminal methylene of geranylation was attached to the C2 position of the His side chain (forward geranylation) (Figure 3b). From these results, the structure of limnothamide, the predicted product of limBGC, was identified (Figure 3b). Furthermore, LimF was identified as a unique PTase that forward geranylates the C2 carbon of the His side chain.

[0182] To investigate the biochemical properties of LimF, we performed in vitro modification of bcLimE2 with LimF under various reaction conditions. First, we performed the same experiment as above using farnesyl pyrophosphate instead of geranyl pyrophosphate as the prenyl group donor. As a result, a small amount of farnesylated bcLimE2 was produced, although the efficiency was lower than when GPP was used.

[0183] In addition, when the reaction was carried out under various conditions described below, LimF was found to be ２＋ The enzyme showed a clear dependence on ions and a relatively wide temperature and pH tolerance (Fig. 5a-c). ２＋ Based on the experiments using the concentrations (Fig. 5d-f), the LimF reaction conditions were optimized as follows: 1 mM GPP, 40 mM MgCl ２, 1 mM dithiothreitol (DTT), pH 7.2, reaction temperature 25° C. Hereinafter, the above reaction conditions were used unless otherwise specified.

[0184] Under optimal conditions, the steady-state kinetic parameters of the prenylation reaction of bcLimE2 by LimF were measured using the method described below. The results were as follows: k = 13.8 ± 0.6 min －１ , Km = 0.313 ± 0.039 mM (Table 10, Figure 5g), indicating that LimF is one of the most efficient cyanobactin PTases in terms of catalytic turnover.

[0185] Interestingly, LimF not only catalyzes its native substrate bcLimE2 but also His monomers (H-His-OH and Fmoc-His-OH) and His-containing dipeptides (Ac-AH-NH ２ ) were shown to prenylate with moderate and high efficiency, respectively, as shown in the table below.

[0186]

[0187] In order to verify that the geranylated structure of the compounds in Table 10 obtained by geranylation of the substrates is the same as that of limnothamide, Ac-AH-NH geranylated by LimF was used. ２ The purified LimF was analyzed by NMR. The COSY and HMBC correlations observed supported forward geranylation of the His C2 position, similar to that observed in bcLimE2. These results suggest that LimF has extremely high substrate sequence tolerance while exhibiting strict regioselectivity.

[0188] Furthermore, Ac-AH-NH ２ When the prenylation reaction with 20 μM LimF was applied to not only the above but also various His-containing dipeptides, it was revealed that the His-prenylation reaction with LimF proceeded for various His-containing dipeptides (FIG. 6).

[0189] (Method for Reaction of Synthetic Substrates with LimF) Initial evaluation of LimF using putative substrates (bcLimE1, bcLimE2, and bcLimE3) was performed as follows: The enzymatic reaction was carried out in a solution of LimF (20 μM), peptide substrate (200 μM), prenyl group donor (geranyl pyrophosphate or farnesyl pyrophosphate) (2 mM), HEPES (50 mM, pH 7.5), MgCl ２ The reaction was carried out at 37°C for 12 hours under conditions of 20 mM ATP and 1 mM DTT. After the substrates bcLimE1, bcLimE2, and bcLimE3 were reacted at 37°C for 12 hours, a 9-fold volume of 1% TFA was added, and the mixture was centrifuged at 4°C (15,300 x g, 10 minutes) to recover the supernatant. 10 μL of the resulting supernatant was subjected to LC-MS analysis.

[0190] (Large-scale LimF reaction method) Analytes for NMR analysis were prepared as follows. Geranylated bcLimE2 was prepared by performing 100 parallel 20 μL-scale reactions at 25° C. for 30 hours. The reaction mixture contained LimF (50 μM), bcLimE2 (2 mM), GPP (2.5 mM), HEPES (50 mM, pH 7.5), MgCl ２ The reaction was carried out under conditions of 100 mM (100%), 1 mM DTT, and DMSO (4%, v / v). After the completion of the reaction, each reaction solution was combined and 2 mL of methanol (MeOH) was added. The mixture was incubated on ice for 30 minutes, and then the supernatant was collected by centrifugation (15,300 x g, 10 minutes). The supernatant was then diluted with 9 volumes of water, filtered, and injected into a preparative reverse-phase HPLC for purification. A linear gradient of 1 to 60% buffer B over 60 minutes was used for separation. Fractions containing geranylated bcLimE2 (~2.0 mg) were combined, and their purity was confirmed by LC-MS. They were then dried and analyzed by NMR spectroscopy using deuterated methanol (CD). ３ The geranylated AH dipeptide and ATY tripeptide described above and below were also obtained by the enzymatic reaction and purification in the same manner as above.

[0191] (Method for evaluating biochemical properties of LimF) The effects of temperature, pH, and metal ions on the LimF-mediated bcLimE2 prenylation reaction were investigated as follows. The standard reaction conditions were as follows: 1 μM LimF, 1 mM GPP, 40 mM MgCl ２ The reaction mixture was 50 mM HEPES pH 7.5, DMSO (4%, v / v), 1 mM DTT, 0.1 mM substrate, at a reaction temperature of 30°C, and for 60 minutes. The reaction temperature, pH, and the type and concentration of metal ions were varied as appropriate. After the reaction, 9 volumes of 1% TFA were added, and the supernatant was subjected to LC-MS analysis. In the time course experiment for LimF, the reaction was incubated at 25°C and stopped by adding 9 volumes of 1% TFA at each time point. The prenylation efficiency was calculated as (product peak area / (product peak area + substrate peak area)) × 100 (%), unless otherwise specified.

[0192] GPP and MgCl in the bcLimE2 prenylation reaction by LimF ２ The concentration dependence of the enzyme activity was measured using 1 μM LimF, 1 mM or an appropriate concentration of GPP, 50 mM HEPES pH 7.2, 0.4 mM substrate, 1 mM DTT, and 40 mM or an appropriate concentration of MgCl ２ The reaction was measured in a reaction system containing GPP. A reaction solution containing all components except GPP was prepared and preheated to 25°C, and the reaction was initiated by adding GPP. After incubation at 25°C, the reaction was stopped at each time point and analyzed by LC-MS. The initial velocity was then calculated as the slope of the increase in the amount of prenylated product produced in the linear region. Three separate reactions were performed under each condition, and the mean and standard deviation of the results were used for analysis. Regarding the GPP concentration dependence and peptide substrate concentration dependence, the initial velocity and the GPP substrate concentration or peptide substrate concentration were fitted to the Michaelis-Menten model in Graphpad Prism 8.4.2 (GraphPad Prism Software Inc., San Diego, CA) software to estimate the kinetic parameters. ２ Concentration dependence (K Ｍｇ ), the initial rate, as well as MgCl ２The concentration was calculated using the following formula: To evaluate the Tyr prenylation activity of LimF, which will be described later, a similar reaction was carried out with 5 μM LimF using teR6-H5A as a substrate.

[0193] (Method for measuring steady-state kinetic parameters of LimF) The kinetic parameters of the His-prenylation reaction by LimF were determined from reactions under optimal conditions using different concentrations of substrate. For Tyr-prenylation described below, the kinetic parameters were measured under the same conditions except that LimF was used at 5 μM and the reaction temperature was 16°C. To estimate the kinetic parameters, the initial velocity and substrate concentration were fitted to the Michaelis-Menten model in Graphpad Prism 8.4.2 software.

[0194] 3. In Vitro Biosynthesis of Diverse Prenylated teMPs Using the FIT-LimF System Thioether-closed cyclic peptides (teMPs) have been used to develop novel peptide ligands for desired target proteins, and their usefulness has been demonstrated by notable achievements (Huang, Y., Wiedmann, M. M., & Suga, RNA display methods for the discovery of bioactive macrocycles. Chem. Rev., 119, 10360-10391 (2018).; Goto, Y. et al. Reprogramming the translation initiation for the synthesis of physiologically stable cyclic peptides. ACS Chem. Biol., 3, 120-129 (2008).). Therefore, we next examined whether LimF could modify the His residues on the unnatural teMP backbone.

[0195] First, we designed teMP (teLimE2) that mimics the sequence of bcLimE2. We then expressed it by genetic code reprogramming using a customized cell-free translation system, the so-called flexible in vitro translation (FIT) system (Goto, Y., Katoh, T. & Suga, Flexizymes for genetic code reprogramming. Nat. Protoc., 6, 779-790 (2011)). Specifically, the AUG start codon was substituted with N-chloroacetyl-D-phenylalanine ( ＣｌＡｃ D-F), a linear precursor with a chloroacetyl group (ClAc group) at the N-terminus was expressed, which spontaneously cyclized with the downstream Cys thiol. Ｎ A thioether bond was formed by the reaction (Fig. 7a). The FIT system had the composition described below. In vitro expressed teLimE2 was incubated in the presence of 100 μM LimF at 25°C for 16 hours and analyzed by LC-MS. The chromatogram shows the geranylated product (teLimE2-H). Ｇｅｒ ), indicating successful prenylation of teLimE2 by LimF (Fig. 7a). Hereinafter, the in vitro translation system coupled with LimF is referred to as the FIT-LimF system.

[0196] The FIT-LimF system allows for the easy preparation of various substrates, followed by LimF-mediated prenylation in a one-pot manner. Therefore, the FIT-LimF system facilitates the investigation of LimF's broad substrate tolerance. Because RiPP enzymes can be affected by the local sequence environment near the modification site, we investigated the prenylation efficiency of LimF for various substrates with altered residues near the prenylation site. The FIT-LimF system was configured as described below.

[0197] Thirty-eight teLimE2 mutants were designed, in which the His-1 and His+1 positions were substituted with 19 proteinogenic amino acids other than Cys. These mutants were expressed and modified using the FIT-LimF system, and the reaction results were analyzed by LC-MS. In this experiment, the prenylation step was intentionally performed under conditions with low modification efficiency by using a low LimF concentration (10 μM). This allowed for a more accurate investigation of the effects of the His-1 and His+1 amino acids.

[0198] The His-1 mutant series suggested that LimF tends to have weak preference for this position. Specifically, substrates with relatively small or hydrophobic residues at the -1 position, such as Thr, Ala, Met, Pro, Ser, or Val, were prenylated very efficiently by LimF, whereas substrates with bulky or charged residues at the -1 position were prenylated relatively inefficiently by LimF (Fig. 7b). On the other hand, only Pro exhibited low prenylation efficiency at the +1 position, and the amino acid at the +1 position was highly variable (Fig. 7c). A similar trend was confirmed for cyclic peptides with different sequences (Fig. 7f). This provided insight into the differences in LimF modification efficiency for substrates with different His sequences.

[0199] In the next series of experiments, we investigated whether teMP with various ring sizes could be tolerated by LimF. Thirteen teMP mutants with different ring sizes (4 to 18 residues) and a preferred local sequence motif (Ser-His-Ala / Cys) were designed. One-pot expression and modification of these teMPs using the FIT-LimF system (LimF concentration: 10 μM) demonstrated that all of them underwent quantitative prenylation (Fig. 7d). These results demonstrate that LimF can prenylate teMP regardless of ring size.

[0200] Next, we demonstrated that LimF can modify various random-sequence teMPs. Five random-sequence teMPs were expressed and modified using the FIT-LimF system (LimF concentration: 10 μM) (Fig. 7e). Of the five teMPs tested, two (teR1 and teR2) possessing the -1 and +1 residues favorable for LimF prenylation underwent quantitative prenylation. Mutation of the adjacent residues (-1 or +1 residue) of these teMPs to unfavorable amino acids suppressed LimF modification efficiency, as expected (teR1 (P-1K) and teR2 (W+1P)). On the other hand, the prenylation efficiency of the teR3, teR4, and teR5 peptides was moderate or low, but substitution of the residue adjacent to His with a preferred side chain significantly improved the modification efficiency with LimF (teR3(E-1S), teR4(W-1A), and teR5(P+1A)).

[0201] These results indicate that LimF can prenylate a variety of teMPs, regardless of their length or overall sequence composition, depending primarily on the local sequence environment around the His residue. A similar trend was observed in the prenylation of cyclic peptides with different sequences using the FIT-LimF system (LimF concentration: 20 μM) (Fig. 8a).

[0202] Furthermore, we demonstrated that multiple prenyl groups could be introduced when the substrate contained multiple prenylation-accepting moieties. Five teMPs with random sequences were expressed and modified using the FIT-LimF system (LimF concentration: 20 μM) (Figure 8b). Cyclic peptides with two His residues yielded a major monoprenylated product and a minor diprenylated product. Cyclic peptides with three His residues yielded a major diprenylated product and a minor monoprenylated product. These results demonstrate that the FIT-LimF system can produce cyclic peptides with multiple prenyl groups.

[0203] (Configuration of FIT-LimF system) The transcription-coupled in vitro translation system was configured to contain the following components: 500 μM of each proteinogenic amino acid (except methionine), 50 mM HEPES-KOH (pH 7.6), 12 mM Mg(OAc). ２ , 100 mM potassium acetate, 2 mM spermidine, 1 mM DTT, 20 mM creatine phosphate (Roche), 2 mM ATP, 2 mM GTP, 1 mM CTP, 1 mM UTP, 0.1 mM 10-formyl-5,6,7,8-tetrahydrofolate, 1.5 mg / ml Escherichia coli total tRNA (Roche), 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 His RS, 0.4 μM IleRS, 0.04 μM LeuRS, 0.11 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 S, 0.02 μM ValRS, 0.6 μM MTF, 2.7 μM IF1, 0.4 μM IF2, 1.5 μM IF3, 0.26 μM EF-G, 10 μM EF-Tu, 0.66 μM EF-Ts, 0.25 μM RF2, 0.17 μM RF3, 0.5 μM RRF, 0.1 μM T7 RNA polymerase, 0.1 μM nucleotide diphosphate kinase, 4 μg / ml creatine kinase, 3 μg / ml myokinase, 0.1 μM pyrophosphatase, and 1.2 μM ribosomes.

[0204] The composition is then mixed with a DNA template and a tRNA charged with a chloroacetyl amino acid. ｆＭｅｔ ＣＡＵ (ClAc-D-Phe-tRNA ｆＭｅｔ ＣＡＵ or ClAc-D-Tyr-tRNA ｆＭｅｔ ＣＡＵ) was added to final concentrations of 0.04 μM and 50 μM, respectively. The mixture was then incubated at 37°C for 1 hour to allow translation and spontaneous thioether ring formation. For substrates incorporating non-proteinogenic amino acid derivatives of histidine, as described below, similar in vitro translation reactions were carried out without His and with tRNA charged with a His analog. ＧｌｕＥ２ ＧＵＧ (50 μM) was added.

[0205] In the FIT-LimF system of this example, the LimF reaction was performed as follows, unless otherwise noted. After 1 hour of reaction at 37°C, the translation mixture was mixed with the LimF enzyme mixture to prepare a reaction mixture containing 10 μM LimF and 1 mM GPP. After 16 hours of incubation at 25°C, the reaction was stopped by adding 45 μL of a 1% TFA aqueous solution, and the mixture was then incubated on ice for 10 minutes. The precipitated fraction was separated by centrifugation (15,200 × g, 10 minutes) at 4°C, and 10 μL of the supernatant was subjected to LC-MS analysis.

[0206] [4. Prenylation of peptides containing His derivatives by the FIT-LimF system] Next, we investigated whether prenylation by LimF would proceed if the His residue to be prenylated was replaced with a similar side chain (His derivative). Using genetic code reprogramming with the FIT system, we expressed teLimE2 derivatives in which the His residue was replaced with various non-proteinogenic amino acids (Fig. 8c). Specifically, we replaced the proteinogenic amino acid L-His with Nτ- and Nπ-methylated His analogs ( Ｌ His(1-Me) and Ｌ His(3-Me)), amino acids with thiophene or thiazole side chains ( Ｌ Ala(2-Thi), Ｌ Ala(3-Thi), and Ｌ Cyclic peptides were synthesized in which the cyclic amino acid sequence was substituted with Ala(4-Thz), D-His, or α-methyl-His. Each of the synthesized teLimE2 derivatives was modified using the FIT-LimF system (LimF concentration: 100 μM).

[0207] As a control, teLimE2 expressed by genetic code reprogramming using L-His was used. This teLimE2 was prenylated by LimF with an efficiency equivalent to that of teLimE2 translated using the standard genetic code, confirming that the reprogrammed FIT-LimF system functions well.

[0208] As shown in Figure 8c, the teLimE2 derivative containing D-His and α-methyl-His ( Ｄ His and α(Me) Ｄ／Ｌ His) was a good substrate for LimF. However, the Nτ- and Nπ-methylated His analogs ( Ｌ His(1-Me) and Ｌ His(3-Me)) was not prenylated at all by LimF. Ｌ Ala(2-Thi), Ｌ Ala(3-Thi), and Ｌ Ala(4-Thz) was also not prenylated by LimF. This suggests that the NH of the imidazole may be involved in LimF recognition. These results suggest that the imidazole moiety of His is involved in LimF recognition, and that the main-chain structure, including α-substitution and configuration, is not an important determinant. Thus, the utility of the FIT-LimF system, which can produce teMP with various non-natural prenylated residues, was demonstrated.

[0209] [5. Tyrosine O-prenylation by LimF] During the evaluation of teMPs with various random sequences, we accidentally discovered a teMP (teR6) that gave two prenylated products after LimF treatment (Fig. 9a). A mutant (teR6-H5A) in which His of this peptide was replaced with Ala also gave prenylated products. This indicates that LimF adds a prenyl group to residues other than His. Mass spectrometry analysis of this peptide prenylated at sites other than His revealed that the prenyl moiety (C １０ H １７) was observed (Figure 9b), suggesting that the prenyl group is located on the heteroatom (McIntosh, JA, Donia, MS, Nair, SK & Schmidt, Enzymatic basis of ribosomal peptide prenylation in cyanobacteria. J. Am. Chem. Soc., 133, 13698-13705 (2011).; Donia, MS, Schmidt, Linking chemistry and genetics in the growing cyanobactin natural products family. Chem. Biol., 18, 508-519 (2011).).

[0210] MS / MS analysis of the prenylated peptide identified the Tyr at position 7 as being prenylated by LimF (Fig. 9a). For simple NMR analysis, the model tripeptide Ac-ATY-NH ２ was synthesized and modified with LimF to give the corresponding Tyr-prenylated product. COSY and HMBC correlations of the product confirmed that the prenylation site was the phenolic OH group (Fig. 9c). These results demonstrate that LimF can catalyze two distinct prenylation modes: His C-prenylation and Tyr O-prenylation.

[0211] To examine the local sequence selectivity of LimF-mediated Tyr-prenylation, we tested a series of Tyr-1 mutants of teR6-H5A in the presence of 10 μM LimF at 25°C for 16 hours, and found that they showed a preference for the -1 position, similar to His-prenylation (Fig. 9d).

[0212] To further evaluate the two distinct prenylation modes catalyzed by LimF, two teMPs (teR6-H5A / Y7H and teR6-H5A) that undergo efficient His-prenylation and Tyr-prenylation, respectively, were designed and chemically synthesized. Using these peptides, kinetic analysis was performed using the method described above. The Km and kcat values ​​for His-prenylation of teR6-H5A / Y7H obtained were comparable to those of bcLimE2, suggesting that the sequence of the closed ring backbone does not significantly affect catalytic efficiency (Table 11, entries 1 and 2).

[0213] Furthermore, by the above method, GPP or Mg ２＋ By varying the concentration of LimF modification of teR6-H5A, the amount of GPP and Mg required to exert half-maximal activity was ２＋ The concentrations of Tyr-prenylation and His-prenylation were found to be similar (Fig. 10a-e). This suggested that the two prenylation modes may be catalyzed by a similar binding site. However, the kcat and Km values ​​for Tyr-prenylation were lower than those for His-prenylation (Table 11, entries 2 and 3, Fig. 10f), suggesting that His-prenylation is the primary function of LimF. However, the catalytic turnover rate of Tyr-prenylation by LimF (kcat = 1.0 min －１ ) is comparable to or better than some of the previously reported cyanobactin PTases, indicating that Tyr-prenylation is a secondary but significant catalytic activity of LimF (Table 12).

[0214]

[0215]

[0216] References12 and 8 Grundmann, A.; Kuznetsova, T.; Afiyatullov, SS; Li, S.-M., FtmPT2, an N-Prenyltransferase from Aspergillus fumigatus, Catalyzes the Last Step in the Biosynthesis of Fumitremorgin B. Chembiochem, 2008, 9(13), 2059-2063. Li, G.-H.; Luo, S.-L.; Du, L.; Hu, Q.-Y.; Xu, H.-K.; Zhang, K.-Q.; Zhao, P.-J., Vib-PT, an aromatic prenyltransferase involved in the biosynthesis of vibralactone from Stereum vibrans. Appl. Approx. Microbiol., 2020, 86(10), e02687-19.11Haagen, Y.; Unsold, I.; Westrich , L. ; Gust , B. ; Richard, SB; Noel , JP ; Heide, L., A soluble, magnesium-independent prenyltransferase catalyzes reverse and regular C-prenylations and O-prenylations of aromatic substrates. FEBS Lett., 2007, 581(16), 2889-2893.

[0217] Furthermore, we confirmed that LimF can O-prenylate Tyr contained in teMP with various random sequences. When a peptide with a sequence different from teR6-H5A / Y7H was expressed in the FIT-LimF system and reacted with 20 μM LimF for 16 hours, Tyr-prenylation was confirmed. Furthermore, when the amino acid residue at position -1 of Tyr in this peptide was replaced with Thr, which is favorable for prenylation by LimF, Tyr-prenylation was confirmed to be more efficient. Table 13 shows the sequences of prenylated teMP and their prenylation efficiencies. In the table, "y" stands for D-Tyr, and "tecyclo" indicates a cyclic structure formed by a thioether bond between the chloroacetyl group of the N-terminal amino acid (D-Tyr) and the thiol group of the downstream Cys. Furthermore, the substrates shown in Table 13 were prenylated at the underlined Tyr.

[0218]

[0219] 6. Study of LimF Mutants Based on the crystal structure and sequence alignment of PagF, a cyanobactin PTase, we performed site-directed mutagenesis experiments to verify the importance of residues (Glu54, Asp70, and His172) predicted to be located near the substrate imidazole ring. Sequence alignment analysis of cyanobactin PTase revealed that Glu54 is highly conserved among F family PTases. While Asp70 and His172 are unique to LimF, other reported PTases that modify aromatic side chains have conserved aliphatic residues (Leu, Met, or Val) at His172. Two Glu54 mutants (LimF-E54A and LimF-E54Q), one Asp70 mutant (LimF-D70A), and three His172 mutants (LimF-H172A, LimF-H172F, and LimF-H172L) were prepared by the method described below and reacted with the above-mentioned bcLimE2 to confirm their His-prenylation ability. Specifically, 100 μM bcLimE2 was reacted with 1 μM wt-LimF or each LimF mutant for 2 hours under the previously optimized standard conditions.

[0220] As a result, LimF mutants other than LimF-D70A did not produce any or very little prenylated bcLimE2 product (Figure 11). This demonstrated that Glu54 and His172 play essential roles in His-prenylation by LimF. On the other hand, LimF-D70A showed His-prenylation ability equivalent to that of the wild-type, indicating that these residues do not play an important role in His-prenylation by LimF.

[0221] Similarly, we also evaluated the Tyr-prenylation ability of wild-type LimF and each LimF mutant to modify teR6 and teR6-H5A substrates. The reaction was performed in the same manner as for bcLimE2 prenylation, except that teR6 or teR6-H5A was used as the substrate and the reaction was performed in the presence of 5 μM LimF for 24 hours. The Glu54 mutation resulted in a loss of Tyr-prenylation. This result is reasonable, given that Glu54 is highly conserved among F-family PTases. In contrast, LimF-H172L, in which the unique His residue of LimF is replaced by a Leu residue conserved in Tyr-O-PTases, showed better modification efficiency for Tyr-containing substrates than wild-type LimF. Further steady-state kinetic assays of LimF-H172L confirmed a 5-fold increase in kcat / Km over the wild type, confirming its enhanced Tyr-prenylation activity (Table 11, entries 3 and 5). In contrast to His-prenylation, in which both Glu54 and His172 are essential, His172 does not play a critical role in Tyr-prenylation; rather, mutation to Leu enhanced Tyr-prenylation activity.

[0222] (Method for constructing plasmids for expression of LimF mutants) The pET32-LimF-SMS plasmid was used as a template for PCR-based site-directed mutagenesis. PCR reactions were performed using the In-Fusion® HD Cloning Kit (TaKaRa Clontech) according to the manufacturer's protocol. The sequence of the plasmid was confirmed by Sanger sequencing. The LimF mutants were purified using the same protocol as that for wild-type (wt) LimF. The primers used are shown in Table 14 below.

[0223]

[0224] [7. Prenylation of Various Compounds by LimF] Prenylation by LimF was performed using various low molecular weight compounds, peptides, and proteins containing an imidazole ring as substrates. First, we demonstrated that LimF prenylates not only cyclic peptides but also linear peptides consisting of several to several dozen amino acids. One cyclic peptide and three linear peptides of different lengths that preserve the core portion of the cyclic peptide were prepared. Each of the prepared peptides was prenylated by LimF (20 μM) with approximately the same efficiency (Figure 12). This suggests that prenylation by LimF is not limited to cyclic peptides but can be applied to a wide range of peptides or proteins.

[0225] Next, His-prenylation by LimF was performed on various small molecules, peptides, and proteins, including various compounds already approved as pharmaceuticals. Specifically, 500 μM substrate (except for Aviptadil, Pramlintide, GLP-1, Teduglutide, and Elcatonin, where 125 μM substrate was used) was reacted for 20 hours under the previously optimized standard conditions in the presence of 100 μM LimF and 2 mM GPP. Table 15 below (shown as Tables 15-1 and 15-2) shows the substrates used, their characteristics, target molecules, applications, and His-prenylation efficiency.

[0226]

[0227]

[0228] From Table 15 above, it can be seen that all imidazole-containing compounds and His-containing compounds were prenylated by LimF. Interestingly, the N-terminal His of Aviptadil, a synthetic vasoactive intestinal polypeptide recently approved for the treatment of COVID-19, which causes respiratory failure, was successfully prenylated by LimF with a conversion efficiency of approximately 90%. ２ The His residue or the C2 carbon of the imidazole moiety was prenylated with high efficiency in the receptor antagonist cimetidine, the anticancer drug leoprorelin, histrelin, which recognizes the gonadotropin-releasing hormone receptor (GNRH), the amylin analog pramlintide, the GLP-1 and GLP-2 analog teduglutide, gonadotropin-releasing hormone (GnRH), the thioether cyclic peptide PBm1, which inhibits the Plexin B1-semaphorin 4D interaction discovered through the Random Peptide Integrated Discovery (RaPID) system, and Lar-D5, which binds to receptor protein tyrosine phosphatases. In particular, the prenylation of cimetidine by LimF suggests that LimF can tolerate and prenylate imidazole rings bearing substituents at positions 4 and 5. These results demonstrate that LimF and its homologous enzymes are highly promising biocatalysts for the chemoselective and site-specific prenylation of His in a variety of compounds, including small molecules, peptides, and proteins.

[0229] Furthermore, His-prenylation of two high-molecular-weight proteins (acetylated bovine albumin (Ac-BSA, Wako Pure Chemical Industries, Ltd.) and equine glutathione S-transferase (GST, Sigma-Aldrich Japan)) with LimF was also performed. Specifically, 100 μM Ac-BSA or 100 μM GST was reacted for 20 hours in the presence of 100 μM LimF and 2 mM GPP under the previously optimized standard conditions. The reaction solution was desalted by solid-phase extraction (SPEC C-TIP, Nikkyo Technos Co., Ltd.) and then analyzed by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS, Bruker Daltonics Ultraflex Xtreme) using sinapinic acid as a matrix. The resulting mass spectrum is shown in Figure 13.

[0230] 13 shows that Ac-BSA was prenylated by LimF at two sites. Furthermore, GST was prenylated by LimF at one site. These findings demonstrate that LimF and enzymes homologous to it are highly promising biocatalysts for the prenylation of high-molecular-weight proteins.

[0231] Furthermore, Tyr-prenylation using the LimF-H172L mutant was carried out on various peptides known as bioactive peptide hormones and artificial cyclic peptide drugs discovered through the RaPID system. Specifically, 500 μM of substrate was reacted for 20 hours in the presence of 40 μM LimF-H172L and 2 mM GPP under the previously optimized standard conditions. Table 16 below (shown as Tables 16-1 and 16-2) shows the substrates used, their characteristics, target molecules and applications, and Tyr-prenylation efficiency.

[0232]

[0233]

[0234] As can be seen from Table 16 above, all Tyr-containing compounds were Tyr-prenylated by the LimF-H172L mutant. In particular, the Tyr residues in the endogenous opioid peptide neurotransmitters Leu-Enkephalin, angiotensin II, and gastrin I were prenylated with high efficiency. These results demonstrate that LimF and enzymes having homology therewith are highly promising biocatalysts for the chemoselective and regiospecific prenylation of Tyr in a variety of compounds.

[0235] [8. Introduction of a Prenyl Group into GLP-1] (8.1. Introduction of a Prenyl Group) Next, large-scale prenylation was carried out on GLP-1 and its derivatives, which are known as motifs for diabetes treatment drugs. Although GLP-1 itself has low stability in the blood, making it difficult to use as a diabetes treatment drug, it is known that modification with a fatty acid functional group improves stability in the blood. Fatty acid-modified GLP-1 derivatives are already known to be used as diabetes treatment drugs. GLP-1 is a compound that activates intracellular cAMP production through binding to a receptor.

[0236] To obtain a sufficient amount of prenylated GLP-1 derivative for various downstream assays, the prenylation reaction was carried out on a 3 mL scale (at 25°C for 72 hours). The following two compounds were used as substrates. In the table below, "GLP-1" refers to the portion of GLP-1 excluding the portion from the first His to the sixth Arg, and "GLP-1_H7F / K34H" refers to the portion in which the seventh His has been replaced with Phe and the 34th Lys with His.

[0237]

[0238] The reaction solution contained LimF (50 μM), GLP-1 or GLP-1_H7F / K34H (1 mM), GPP (2.5 mM), HEPES (50 mM, pH 7.2), MgCl ２The reaction mixture was adjusted to contain 50 mM HCl, 1 mM DTT, and 4% DMSO (v / v). After the reaction was completed, the reaction mixtures were combined and mixed with 2 mL of methanol (MeOH). After incubation on ice for 30 minutes, the supernatant was separated by centrifugation (15,300 × g, 10 minutes) and diluted with a 9-fold volume of 0.1% TFA-water solution. The resulting solution was filtered and subjected to preparative reverse-phase HPLC. A linear gradient of 1 to 60% buffer B over 60 minutes was used for separation. Fractions containing prenylated GLP-1 were combined and lyophilized. Similarly, fractions containing prenylated GLP-1_H7F / K34H were combined and lyophilized. The purified peptide was dissolved in DMSO for further assays.

[0239] Native GLP-1 could be efficiently prenylated by LimF. Ultimately, 0.58 mg of prenylated GLP-1 was successfully obtained from 3.2 mg of GLP-1. Furthermore, GLP-1_H7F / K34H, in which the His position was modified, could also be efficiently prenylated by LimF. Ultimately, 0.42 mg of prenylated GLP-1_H7F / K34H was successfully obtained from 3.5 mg of GLP-1_H7F / K34H.

[0240] (8.2. Evaluation of Compounds) Using GLP-1 and GLP-1_H7F / K34H, and the obtained prenylated GLP-1 and GLP-1_H7F / K34H, a serum stability test, a DPP-4 digestion test, and a cAMP production activation test were carried out.

[0241] Serum stability tests were performed by incubating 50 μM solutions of each peptide (GLP-1 and GLP-1_H7F / K34H, and prenylated GLP-1 and GLP-1_H7F / K34H) in human serum at 37°C (100 μL scale). After various incubation times, 4 μL aliquots were sampled from the reaction system and mixed with an equal volume (4 μL) of methanol to terminate the reaction. After centrifugation at 13,000 rpm for 5 minutes, the supernatant was collected, and four volumes of 1% TFA-water solution were added, followed by further centrifugation for 5 minutes. 1 μL of the resulting supernatant was analyzed by LC / MS to quantify the amount of each remaining peptide.

[0242] DPP-4 digestion tests were performed by incubating 50 μM Tris-Cl solutions (pH 8.0) of each peptide (GLP-1 and GLP-1_H7F / K34H, and prenylated GLP-1 and GLP-1_H7F / K34H) in the presence of 2.5 ng / μL of DPP-4 at 37°C (100 μL scale). After various incubation times, 4 μL aliquots were taken from the reaction mixture, and the reaction was stopped by adding four volumes (16 μL) of a 1% TFA-water solution. After centrifugation at 13,000 rpm for 5 minutes, 1 μL of the supernatant was analyzed by LC / MS to quantify the amount of each remaining peptide.

[0243] cAMP production activation assays were performed for each peptide (GLP-1 and GLP-1_H7F / K34H, and prenylated GLP-1 and GLP-1_H7F / K34H) using the cAMP Hunter™ eXpress GLP1R CHO-K1 GPCR Assay Kit purchased from DiscoveRx (Fremont, CA, USA) according to the manufacturer's protocol. The peptide concentrations used ranged from 0.33 pM to 20 nM.

[0244] The results of each test are shown in Figure 15. As shown in Figures 15(a) and (b), unmodified GLP-1 and GLP-1_H7F / K34H exhibited rapid degradation, while their prenylated forms exhibited higher stability. Furthermore, as shown in Figure 15(c), native GLP-1 potently activated cAMP production, but the His7-prenylated peptide showed a significantly reduced ability to activate cAMP production. This is consistent with the known importance of His7 in GLP-1 receptor binding. Furthermore, GLP-1_H7F / K34H, in which the His residue was replaced, was found to maintain activity comparable to that of native GLP-1. Furthermore, the prenylated form of GLP-1_H7F / K34H also exhibited a similarly high ability to activate cAMP production.

[0245] These results suggest that prenylated GLP-1_H7F / K34H produced by the above method has higher stability in blood than native GLP-1 and has receptor activation ability equivalent to that of native GLP-1.

[0246] [9. Further Study of LimF Mutants] (9.1. Changes in Substrate Tolerance Due to LimF Mutations) We attempted to change the substrate tolerance of LimF shown in Figure 7 by introducing mutations into LimF. The substrate used was teLimE2, which had a mutation introduced at the -1 position of His, as shown in Figure 7(b). The transcription-coupled in vitro translation system contained the following components: 500 µM of each proteinogenic amino acid (except methionine), 50 mM HEPES-KOH (pH 7.6), and 12 mM Mg(OAc). ２, 100 mM potassium acetate, 2 mM spermidine, 1 mM DTT, 20 mM creatine phosphate (Roche), 2 mM ATP, 2 mM GTP, 1 mM CTP, 1 mM UTP, 0.1 mM 10-formyl-5,6,7,8-tetrahydrofolate, 1.5 mg / ml Escherichia coli total tRNA (Roche), 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 His RS, 0.4 μM IleRS, 0.04 μM LeuRS, 0.11 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 S, 0.02 μM ValRS, 0.6 μM MTF, 2.7 μM IF1, 0.4 μM IF2, 1.5 μM IF3, 0.26 μM EF-G, 10 μM EF-Tu, 0.66 μM EF-Ts, 0.25 μM RF2, 0.17 μM RF3, 0.5 μM RRF, 0.1 μM T7 RNA polymerase, 0.1 μM nucleotide diphosphate kinase, 4 μg / ml creatine kinase, 3 μg / ml myokinase, 0.1 μM pyrophosphatase, 1.2 μM ribosomes, 0.04 μM mDNA template.

[0247] The composition includes a DNA template, and ＣｌＡｃ D Phe-charged tRNA ｆＭｅｔ ＣＡＵ were added to final concentrations of 0.04 μM and 50 μM, respectively. The mixture was then incubated at 37° C. for 1 hour to promote translation and spontaneous thioether ring formation.

[0248] After 1 hour of translation, 3 μL of the translation mixture was individually mixed with 2 μL of the enzyme / GPP mixture to form reaction mixtures containing 20-100 μM LimF or mutants and 1 mM GPP. For reactions with in vitro-translated teMP to examine the substrate tolerance of LimF, the same conditions were used, except for 10 μM LimF. After incubation at 25°C for 16 hours, the reaction was stopped by adding 45 μL of 1% aqueous TFA solution. The mixture was then incubated on ice for 10 minutes, and the precipitate was separated by centrifugation at 4°C (15,300 × g, 10 minutes). 10 μL of the supernatant was subjected to LC-MS analysis.

[0249] For the site of mutation in LimF, amino acids that form a small pocket that accommodates the side chain at position -1 of the substrate in the crystal structure were selected. Specifically, Ile at position 52 and Gln at position 72 were selected, and mutants were prepared in which these were substituted with Ala. Figure 16 shows the relationship between the amino acid at position -1 and the prenylation efficiency for wild-type LimF and each mutant. Figure 16 demonstrates that the I52A mutant, in particular, can efficiently prenylate substrates containing bulky amino acids such as Leu and Phe at position -1.

[0250] (9.2. Changes in prenyl group donor tolerance by LimF mutation) We attempted to change the prenyl group donor selectivity of LimF examined in "2. Evaluation of LimF enzymatic activity" by introducing mutations into LimF. teLimE2, shown in Figure 7(a), was used as the substrate. For the assay of LimF mutants, 100 μM bcLimE2 was mixed with 1 mM GPP or DMAPP, 50 mM HEPES (pH 7.2), 40 mM MgCl ２ The mixture was incubated individually in 5 μL with 20-100 μM of LimF mutants in a 1 mM DTT DMSO (4%, v / v) solution. After 16 hours of incubation at 25°C, the reaction was stopped by adding 9 volumes (45 μL) of a 1% TFA aqueous solution. The mixture was then incubated on ice for 10 minutes, and the precipitate was separated by centrifugation at 4°C (15,300 × g, 10 minutes). 1 μL of the supernatant was subjected to LC-MS analysis.

[0251] The site of mutation in LimF was selected to be an amino acid that forms a pocket for storing a prenyl group in the crystal structure. Specifically, Gly at position 224 was selected and replaced with a larger amino acid. As shown in Figure 17, alignment of multiple prenyltransferases revealed that C10 prenyl (geranyl)transferases have a characteristic Gly at position 224 corresponding to that of LimF. This sequence alignment supported the hypothesis that Gly at position 224 contributes to prenyl group selectivity.

[0252] The relationship between the prenyl group donor and the prenylation efficiency for each mutant is shown in Figure 18. Figure 18 shows that C5 prenylation (dimethylallylation) occurs selectively, particularly in the G224M mutant.

[0253] Similarly, LimF mutants with high selectivity for C15 prenylation (farnesylation) were also investigated. For the LimF mutant assay, 100 μM bcLimE2 was incubated in 1 mM GPP or FPP, 50 mM HEPES (pH 7.2), 5 mM MgCl , and 100 μM bcLimE2. ２ The mixture was incubated individually in 5 μL with 20-100 μM of LimF mutants in a 1 mM DTT DMSO (4%, v / v) solution. After 16 hours of incubation at 25°C, the reaction was stopped by adding 9 volumes (45 μL) of a 1% TFA aqueous solution. The mixture was then incubated on ice for 10 minutes, and the precipitate was separated by centrifugation at 4°C (15,300 × g, 10 minutes). 1 μL of the supernatant was subjected to LC-MS analysis.

[0254] The site of mutation in LimF was selected to be an amino acid that forms a pocket for storing a prenyl group in the crystal structure. Specifically, the amino acid at the position shown in the left column of Figure 19 was substituted with a relatively small Ala. As a result, substitution of Trp at position 273 increased farnesylation activity (19%; Figure 19).

[0255] Therefore, we comprehensively tested substitution of amino acids other than Ala at position W273 (middle column in Figure 19). As a result, we focused on the W273N and W273T mutations, and in addition to these, we also simultaneously tried substituting His at position 239, the amino acid adjacent to position 273, with various amino acids (right column in Figure 19). As a result, we found that C15 prenylation (farnesylation) occurred highly efficiently and selectively in the H239G / W237T and H239G / W237N mutants. These results confirmed the production of peptides with a wider range of prenyl group backbones.

[0256] Next, kinetic parameters were measured for C10 prenylation of wild-type LimF, C5 prenylation of the G224M mutant, and C15 prenylation of the H239G / W237T mutant in a 10 μM LimF or LimF mutant, 1 mM GPP, DMAPP, FPP, or GGPP, 50 mM HEPES (pH 7.2), 5 mM MgCl ２ The modification reaction was carried out at 25°C with 1 mM DTT, DMSO (4%, v / v), and various concentrations of bcLimE2. The initial velocity and substrate concentration were fitted to the Michaelis-Menten model in Graphpad Prism 8.4.2 software to estimate kinetic parameters. To examine the concentration dependence of the prenyl group donor, the reaction was performed in 1 μM LimF or LimF mutants, 1 mM or various concentrations of GPP, DMAPP, FPP, or GGPP, 50 mM HEPES (pH 7.2), 0.1 mM bcLimE2, 1 mM DTT, and 5 mM MgCl. ２The modification reaction was carried out at 25°C without a prenyl group donor. After preheating to 25°C without a prenyl group donor, the reaction was initiated by the addition of the prenyl group donor. After incubation at 25°C, the reaction was stopped at each time point and analyzed by LC-MS. The initial rate was then calculated as the slope of the linear region. Each experiment was repeated three times. For the prenyl group donor concentration dependence, the initial rate was fitted to the Michaelis-Menten model using nonlinear regression analysis in Graphpad Prism 8.4.2 software. The results are shown in the table below. In Table 18, "nr" indicates that the reaction did not proceed.

[0257]

[0258] Next, we confirmed that the H239G / W237T mutant also has high substrate tolerance, similar to that of LimF. Three unnatural substrates (cimetidine, GLP-1, and GLP-1_H7F / K34H) were used as substrates. Specifically, for the farnesylation of cimetidine, GLP-1, or GLP-1_H7F / K34H, 0.2 mM of each compound was incubated with 50 μM LimF_H239G / W273T mutant and 1 mM FPP for 20 hours under the previously optimized standard conditions.

[0259] The farnesylation efficiency for each compound is shown in Figure 20. It was confirmed that farnesylation of unnatural substrates can be achieved by using the H239G / W273T mutant.

[0260] Finally, we confirmed that the regioselectivity of prenylation was unchanged for the G224M mutant (C5 prenylation) and the H239G / W237T mutant (C15 prenylation) compared with wild-type LimF. MS / MS analysis, similar to the method described in [2. Evaluation of LimF enzymatic activity], confirmed that the G224M and H239G / W237T mutants dimethylallylated and farnesylated the imidazole C2 carbon, respectively, just as wild-type LimF geranylated the C2 carbon on the imidazole ring of the His side chain.

[0261] [10. Supplementary Note] His is one of the four proteinogenic amino acids with an aromatic side chain and is characterized by an electron-deficient heteroaromatic imidazole side chain that plays essential roles in protein / peptide functions such as metal binding, hydrogen bonding, proton transport, and nucleophilicity (Liao, S.-M., Du, Q.-S., Meng, J.-Z., Pang, Z.-W. & Huang, The multiple roles of histidine in protein interactions. Chem. Cent. J., 7, 1-12 (2013).). However, modifications of His occur rarely. Among these, nucleophilic heteroatom modifications such as N-phosphorylation and alkylation are common, both enzymatically and chemically (Jia, S., He, D. & Chang, C.J. Bioinspired Thiophosphorodichloridate Reagents for Chemoselective Histidine Bioconjugation. Journal of the American Chemical Society 141, 7294-7301 (2019).; Kee, J.-M. & Muir, T.W. Chasing Phosphohistidine, an Elusive Sibling in the Phosphoamino Acid Family. ACS Chem. Biol. 7, 44-51 (2012).).

[0262] A series of aromatic PTases are characterized by diverse prenylation reactions of aromatic compounds, mainly small molecules. However, prenylation of aromatic residues in peptides is limited to the indole and phenol groups of Trp and Tyr residues, respectively. Because the carbon atom on the imidazole ring is electron-deficient and difficult to functionalize, few small natural molecules containing prenylated imidazole structures have been reported (Larsen, TO, Frisvad, JC, & Jensen, Aurantiamine, a diketopiperazine from two varieties of Penicillium aurantiogriseum. Phytochemistry, 31, 1613-1615 (1992)); Kanoh, K. et al. (-)-Phenylahistin: a new mammalian cell cycle inhibitor produced by Aspergillus ustus. Bioorg. Med. Chem. Lett., 7, 2847-2852 (1997)).

[0263] Natural products modified at the most electron-deficient C-2 position of imidazole are even rarer; only one compound with a C-2 dimethylallylated imidazole structure has been reported to date (Jan, C., Dippenaar, A. & Holzapfel, Crystal structure of the metal complexes of viridamine. S. Afr. J. Chem., 30, 161-168 (1977)). Enzymes catalyzing the C-alkylation of His have remained unexplored due to the rarity of such structures in nature. To our knowledge, LimF is the first structurally characterized PTase catalyzing the C-2 alkylation of His.

[0264] LimF showed the ability to prenylate Tyr in addition to His. Most cyanobactin PTases tolerate a wide variety of sequences, but are selective in the site of modification. It is unprecedented for a cyanobactin PTase, like LimF, to catalyze the C- and O-prenylation of two different amino acids.

[0265] On the other hand, peptide-based molecules have become promising therapeutic approaches, particularly due to their ability to target proteins of interest (POIs) within cells. Regarding peptide ligand discovery, the well-established RaPID system using thioether cyclic peptide libraries has proven to be a powerful workflow for screening novel peptide ligands for most types of POIs (Taguchi, S. & Suga, Targeting of extracellular protein-protein interactions with macrocyclic peptides. Curr. Opin. Chem. Biol. 62, 82-89 (2021).; Peacock, H. & Suga, Discovery of De Novo Macrocyclic Peptides by Messenger RNA Display. Trends Pharmacol. Sci., 42, 385-397 (2021).). Given that LimF has been demonstrated to be capable of prenylation of such unnatural macrocyclic scaffolds, the LimF-integrated RaPID system may enable the identification of diverse prenylated peptide ligands targeting desired POIs with improved membrane permeability.

[0266] Furthermore, the discovery of LimF is expected to contribute to the expansion of peptide modification methods. Despite considerable progress, previously reported peptide modification methods largely rely on nucleophilic residues such as Cys and Lys. Although two recent reports have demonstrated His C-2 alkylation mediated by a radical-mediated approach, direct late-stage functionalization of His is still in its infancy due to the presence of various hindering functional groups on peptides (Noisier, A. F. et al. Late-Stage Functionalization of Histidine in Unprotected Peptides. Angew. Chem., 58, 19096-19102 (2019).; Chen, X. et al. Histidine-Specific Peptide Modification via Visible-Light-Promoted CH Alkylation. J. Am. Chem. Soc. 141, 18230-18237 (2019).). Herein, we demonstrated that various compounds (small molecules, peptides, and proteins) can be selectively prenylated by LimF. Structural analysis of LimF enabled the design of LimF with enhanced versatility, such as by altering substrate-interacting residues to accommodate substrates with low prenylation efficiency. Due to its remarkable chemoselectivity and regioselectivity, high substrate tolerance, and mild reaction conditions, LimF is expected to serve as a powerful biocatalyst for the chemically challenging regioselective His C-2 functionalization.

[0267] Furthermore, phylogenetic tree analysis and SSN analysis suggested that 11 proteins derived from organisms other than LimF exhibit His C-prenylation activity and / or Tyr O-prenylation activity similar to LimF.

Claims

1. A method for producing a compound having at least one structure represented by the following formula (I) or (II): 【Chemical 1】 【Chemistry 2】 (In the above formula (I), R 1 represents a hydrogen atom or an arbitrary substituent, and n represents an integer of 0 to 11. In the above formula (II), R 2 are each independently an arbitrary substituent, p is an integer of 0 to 4, and n is an integer of 0 to 11. The method includes a step of contacting a compound having at least one structure represented by the following formula (III) or (IV) with a prenyltransferase to introduce a prenyl group into the structure: 【Chemistry 3】 【Chemistry 4】 (In the above formula (III) and formula (IV), R 1 , R 2 and p have the same meanings as defined above. The prenyltransferase is LimF or an enzyme homologous thereto. Manufacturing method.

2. The production method according to claim 1, wherein a peptide or protein having an amino acid sequence containing at least one His, Tyr, or derivative thereof is contacted with a prenyltransferase to introduce a prenyl group into at least one His residue, Tyr residue, or derivative residue thereof.

3. The method comprises the steps of contacting a compound library containing a peptide or protein having an amino acid sequence containing at least one His, Tyr, or a derivative thereof with a prenyltransferase to introduce a prenyl group into at least one His residue, Tyr residue, or a derivative residue thereof; The prenyltransferase is LimF or an enzyme homologous thereto. A method for producing a compound library containing prenylated peptides or proteins.

4. The production method according to claim 3, further comprising a step of translating the mRNA library using a cell-free translation system to prepare the compound library containing peptides or proteins having an amino acid sequence containing at least one His, Tyr, or derivative thereof.

5. The method according to claim 3 , wherein the peptides or proteins in the compound library are genotype-bound peptides or proteins.

6. The method further comprises the step of preparing the compound library containing genotype-bound peptides or proteins by an mRNA display method; The process comprises: providing an mRNA library, each mRNA encoding a peptide or protein having an amino acid sequence including at least one His, Tyr, or derivative thereof; a step of binding puromycin to the 3' end of each mRNA in the mRNA library to produce a puromycin-bound mRNA library; The method according to claim 5 , further comprising: translating the puromycin-binding mRNA library using a cell-free translation system.

7. The production method according to any one of claims 1 to 6, wherein the prenyltransferase comprises a prenyltransferase derived from Limnothrix sp. or Symploca sp., or an enzyme having homology thereto.

8. The method according to any one of claims 1 to 6, wherein the prenyltransferase comprises an enzyme comprising an amino acid sequence corresponding to the following (1), (2), or (3): (1) An amino acid sequence represented by any one of SEQ ID NOs: 1 to 12 (2) An amino acid sequence represented by any one of SEQ ID NOs: 1 to 12, in which one or several amino acids are deleted, substituted, or added. (3) An amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO:

1.

9. The production method according to any one of claims 1 to 6, wherein the prenyltransferase is at least one selected from the group consisting of enzymes having an amino acid sequence selected from the group consisting of the amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence represented by SEQ ID NO: 2, and these amino acid sequences in which one or several amino acids are deleted, substituted, or added.

10. The method according to any one of claims 1 to 6, wherein the prenylated compound is a peptide or protein having a cyclic structure formed by four or more amino acids.

11. the compound to be contacted with the prenyltransferase is a peptide or a protein; The peptide or protein has a partial structure represented by Xaa1-Xaa2 or an amino acid sequence having His, Tyr, or a derivative thereof at the N-terminus, The method according to any one of claims 1 to 6, wherein Xaa1 is a neutral amino acid, and Xaa2 is His, Tyr, or a derivative thereof.

12. the compound to be contacted with the prenyltransferase is a peptide or a protein; The peptide or protein has a partial structure represented by Xaa2-Xaa3 or an amino acid sequence having His, Tyr, or a derivative thereof at the C-terminus, The method according to any one of claims 1 to 6, wherein Xaa2 is His, Tyr, or a derivative thereof, and Xaa3 is any amino acid other than Pro and derivatives thereof.

13. A compound library produced by the production method according to any one of claims 1 to 6.

14. a step of contacting a compound library produced by the production method according to any one of claims 1 to 6 with a target substance; and selecting a compound that binds to the target substance. A screening method for identifying compounds that bind to a target substance.