Cyclization method for peptide compounds

Abstract

To provide a novel cyclization method for peptide compounds to construct a peptide display library, and to provide methods for the synthesis of branched peptides.SOLUTION: Provided is a method for producing a peptide compound having a cyclic portion, the method comprising: 1) translating and synthesizing an acyclic peptide compound from a nucleic acid encoding the peptide compound composed of an amino acid residue and / or amino acid analog residue, or an amino acid residue and / or amino acid analog residue and N-terminal carboxylic acid analog, the acyclic peptide compound containing an amino acid residue or amino acid analog residue having a reactive site in one of its side chains, and an amino acid, amino acid analog residue or N-terminal carboxylic acid analog with another reactive site at the N-terminus; and 2) forming a carbon-carbon bond between the reactive site of the N-terminal amino acid residue, N-terminal amino acid analog residue, or N-terminal carboxylic acid analog and the reactive site of the amino acid residue or amino acid analog having a reactive site in one of its side chains.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a novel method for cyclizing peptide compounds, and to novel peptide compounds and libraries containing them. [Background technology]

[0002] In recent years, the development of drug discovery technologies using medium-sized molecules (molecular weight 500-2000) to enable drug discovery for tough targets, such as protein-protein interaction inhibitors, agonists, and molecular chaperones, has attracted attention (Non-Patent Document 1). It has been suggested that compounds with molecular weights of 500-2000 can effectively inhibit tough targets, which have previously been considered difficult to discover using anything other than antibodies (Non-Patent Document 2). Furthermore, examples have been reported, primarily of natural products, that fall outside the scope of Lipinski's rule of 5 (many of which have molecular weights exceeding 500) and are capable of inhibiting oral agents or intracellular targets (Non-Patent Document 3). Medium-sized molecules are valuable molecular species because they can access tough targets, something that small molecules cannot, and because they can be transported into cells (enabling intracellular drug discovery and oral administration), potentially enabling things that antibodies cannot.

[0003] Since most small molecule drug discovery has traditionally been carried out in the molecular weight range of less than 500, most of the medium molecules that make it possible to develop drugs for humanity's tough targets (a general term for drug targets that are difficult to find hit compounds for using traditional small molecule compounds using HTS (high throughput screening). These targets are characterized by not having deep cavities that small molecules can bind to. For example, protein-protein interaction inhibitors, such as the binding inhibitor between IL-6 and IL-6R, are examples. Other examples include RNA-protein interaction inhibitors and nucleic acid-nucleic acid interaction inhibitors.) are limited to natural products. Drugs derived from natural products are currently estimated to account for 30% of first-in-class (FIC) compounds, making it an effective method. However, even if all known compounds are combined, only 10 6Since the diversity is limited to the level of compounds, the targets from which active compounds can be obtained are limited. In addition, there are many cases where the membrane permeability and metabolic stability of active compounds are difficult, and there are only 10 types of membrane-permeable molecules that enable drug discovery in areas where neither small molecules nor antibodies can be used. 6 Furthermore, even when it is desired to improve the membrane permeability or metabolic stability of natural product hits, it is often difficult to improve them through chemical modification because complex chemical synthesis is required. For this reason, many natural product medicines are marketed without chemical modification.

[0004] By utilizing in vitro display technology that has been put to practical use in biodrug discovery, it is possible to create a group of novel polymer compounds with high diversity in a short period of time, but there are currently various limitations to applying this technology to the development of tough target drug discovery using medium-sized molecules. These limitations are easy to understand when compared to antibody drug discovery, a form of biodrug discovery that has already been put to practical use. Antibodies, which can create molecules for any target from a large library, are large protein scaffolds with long variable regions, and can form secondary and tertiary structures, allowing them to form binding surfaces with diverse three-dimensional structures. For this reason,10 10 A library of about 100 types can be used to create compounds that strongly bind to and inhibit many extracellular proteins. On the other hand, cyclic peptides, which are medium-sized molecules obtained using biotechnology, are required to have membrane permeability that antibodies cannot, so there are restrictions on chain length (molecular weight). In addition, current biotechnology is limited to natural amino acids, so there are also limits on stereodiversity.

[0005] It would be highly valuable to develop a technology that can easily synthesize and chemically modify molecules, has membrane permeability and metabolic stability, and can rapidly generate a large number of structurally diverse medium-sized molecules, and can then evaluate their efficacy. One such technology is the Display Library technology mentioned above (10 12(It is possible to synthesize and evaluate multiple compounds at once.) Currently, compounds that can be acquired from a display library are limited to peptides, but peptide drugs are highly valuable chemical species, with over 40 types already on the market (Non-Patent Document 4). Cyclosporin A is a representative example; it is an 11-residue peptide produced by microorganisms that inhibits an intracellular target (cyclophilin) ​​and can be administered orally. Peptides have generally been considered to have low metabolic stability and membrane permeability, but it has become clear that these properties can be improved by non-natural modification such as peptide cyclization and N-methylation (Non-Patent Document 5).

[0006] However, because the structure of natural peptides is altered by backbone modifications such as N-methylation, it is extremely difficult to use natural peptides as lead compounds to confer both membrane permeability and metabolic stability to peptides while maintaining their efficacy. One successful example has been reported in which an integrin inhibitory peptide was cyclized and then denaturalized to enter clinical trials as an oral drug (Non-Patent Document 6). However, this type of drug development is rare and requires a long period of research. For example, the development of oral formulations of valuable pharmaceutical injectables such as insulin, GLP-1 (glucagon-like peptide-1), PTH (parathyroid hormone), and calcitonin has yet to be successfully achieved.

[0007] In recent years, it has been reported that peptides containing unnatural amino acids or hydroxycarboxylic acid derivatives (Non-Patent Document 22) can be synthesized on the ribosome, making the realization of a display library containing unnatural amino acids more realistic. In particular, it has been reported that peptides containing N-methyl amino acids can be synthesized on the ribosome using a cell-free translation system such as PureSystem® and tRNA bound to an unnatural amino acid (Non-Patent Documents 7, 8, 9, 10, 11). There have also been reports of attempts to create a display library containing one unnatural amino acid (Non-Patent Documents 12 and 13). There have also been reports of the creation of a display library containing an N-methyl amino acid (Non-Patent Document 23).

[0008] Research is also underway to identify conditions for achieving both membrane permeability and metabolic stability in medium-sized peptides. Lokey et al. performed proline or N-methylation of cyclic peptides consisting of six amino acids and identified factors affecting membrane permeability using PAMPA (Parallel Artificial Membrane Permeation Assay) measurement (Non-Patent Document 14). Furthermore, they created a peptide with a BA (bioavailability) of 28% in rats (Non-Patent Document 15). Kessler et al. reported a review in which they performed N-methylation of cyclic peptides consisting of five or six amino acids to identify conditions favorable for membrane permeability and metabolic stability (Non-Patent Documents 16 and 17). However, to our knowledge, there have been no reports examining what kind of peptides generally achieve both membrane permeability and metabolic stability, or examining the broad range of druglikeness conditions for larger molecular weight peptides (seven or more amino acids), which are expected to have higher hit generation rates due to their greater diversity.

[0009] To obtain mid-sized hit compounds from a display library, there is also room for improvement in cyclization methods. For example, cyclization using conventional PhageDisplay was limited to disulfide bonds between two Cys residues (Non-Patent Document 18). Cyclic peptides generated by disulfide bond-based cyclization methods not only have a short blood half-life due to metabolic instability, but also suffer from degradation due to reduction and cleavage in the weakly acidic environment within cells, difficulty in oral absorption, and the resulting sulfhydryl groups may randomly form covalent bonds with endogenous proteins, potentially resulting in toxicity. Therefore, various improvements are required to develop drug-like mid-sized peptides. Recently, a technique for cyclizing two Cys residues via mesitylene has been reported as an improvement over this technique (Non-Patent Document 19). While this method allows for more stable cyclization via a thioether, its effectiveness is limited and there is still room for improvement. For example, it is widely known that thioethers are susceptible to oxidative metabolism. It has been reported that it is degraded by cytochrome P450 to RSCH2R' → RSH+R'CHO and metabolized to sulfoxide by flavin-containing monooxygenase (Non-Patent Document 20). The former becomes a reactive metabolite when generated, which can lead to toxicity. On the other hand, groundbreaking reports have been made on the realization of amide cyclization, a drug-like cyclization method for peptide cyclization (Non-Patent Documents 21, 25, 26, and 27). However, all of these methods involve the chemical reaction of the main-chain amino group of an amino acid with the active species obtained by cleaving the main-chain amide bond, and therefore cannot be directly applied as a cyclization method for display libraries. These methods are useful for condensation cyclization of the main-chain carboxylic acid and main-chain amino group of many natural products, such as cyclosporine A. However, in display libraries, the main-chain carboxylic acid terminus must be bound to mRNA, so methods that generate active species by decomposition of the main-chain amide bond cannot be used.

[0010] Two new cyclization methods have been proposed for mRNA display, but no display method has yet been established that addresses these issues. One method involves crosslinking the amino group of the N-terminal methionine with the amino group of a downstream (C-terminal) lysine using disuccinimidyl glutarate (DSG) (Non-Patent Document 12). Another method involves introducing an amino acid derivative bearing a chloroacetyl group as the N-terminal translation initiation amino acid, placing Cys downstream, and forming a thioether via intramolecular cyclization (Non-Patent Document 11, Patent Document 1). Even these methods have not sufficiently improved these issues, and the development of a new alternative cyclization method is needed. For example, the SS cyclization method using two cysteines requires two specific amino acids (cysteines), whereas the DSG crosslinking cyclization method requires three specific amino acids, including the lysine, resulting in reduced structural diversity in peptide libraries with a fixed number of residues. It has also been reported that altering the structure of the cyclization site significantly reduces the activity (strength of drug efficacy) of a peptide having the cyclization site (Non-Patent Document 24). This example shows that after obtaining a peptide having a cyclization site, it is difficult to modify the cyclization site to obtain a peptide with excellent membrane permeability and metabolic stability. Peptide libraries with cyclic moieties that are membrane permeable and metabolically stable are needed for pharmaceutical development, but there is room for improvement in various aspects in establishing such peptide libraries.

[0011] [Prior art documents] [Patent documents]

[0012] [Patent Document 1] International Publication No. 2008 / 117833 [Non-patent literature]

[0013] [Non-Patent Document 1] Satyanarayanajois, SD, Hill, RA Medicinal chemistry for 2020, Future Med. Chem. 2011, 3, 1765 [Non-patent document 2] Wells, JA, McClendon, CL,Reaching for high-hanging fruit in drug discovery at protein-protein interfaces. Nature, 2007, 450, 1001 [Non-patent document 3] Ganesan, A. The impact of natural products upon modern drug discovery. Curr. Opin. Chem. Bio. 2008, 12, 306. [Non-patent document 4] Gracia, SR, Gaus, K., Sewald,N. Synthesis of chemically modified bioactive peptides: recent advances, challenges and developments for medicinal chemistry. Future Med. Chem. 2009, 1,1289 [Non-patent document 5] Chatterjee, J., Gilon, C., Hoffman, A., Kessler, H., N-Methylation of peptides: A new perspective inmedicinal chemistry. 2008, 41, 1331. [Non-patent document 6] Kessler, H. et. al., Cilengitide:The first anti-angiogenic small molecule drug candidate. Design, synthesis and clinical evaluation. Anti-cancer Agents in Medicinal Chemistry 2010, 10, 753 [Non-Patent Document 7] Roberts, RW, et al. Encodamers:Unnatural peptide oligomers encoded in RNA. Chem. Bio. 2003, 10, 1043. [Non-patent document 8] Forster, AC et. al., Specificity of translation for N-alkyl amino acids. J. Am. Chem. Soc. 2007, 129, 11316. [Non-Patent Document 9] Merryman, C., Green, R.Transformation of aminoacyl tRNAs for the in vitro selection of"Drug-like" molecules. Chem. Bio. 2004, 11, 575. [Non-Patent Document 10] Szostak, JW et al. Ribosomalsynthesis of N-methyl peptides. J. Am. Chem. Soc. 2008, 130, 6131. [Non-Patent Document 11] Suga, H. et. al., MessengerRNA-programmed incorporation of multiple N-methyl-amino acids into linear and cyclic peptides. Chem. Bio. 2008, 15, 32. [Non-Patent Document 12] Roberts, R. et. al., In vitro selection of mRNA display libraries containing an unnatural amino acid. J. Am.Chem. Soc. 2002, 124, 9972 [Non-Patent Document 13] Roberts, R. et. al. Design of cyclic peptides that bind protein surfaces with antibody-like affinity. ACSchem. Bio. 2007, 9, 625. [Non-Patent Document 14] Lokey, RS et. al., Testing theconformational hypothesis of passive membrane permeability using synthetic cyclic peptide diastereomers. J. Am. Chem. Soc. 2006, 128, 2510 [Non-Patent Document 15] Lokey, RS et. al., On-resinN-methylation of cyclic peptides for discovery of orally bioavailable scaffolds. Nature Chem. Bio. 2011, 7, 810 [Non-Patent Document 16] Kessler, H. et. al., Improvementof drug-like properties of peptides: the somatostatin paradigm. Expert Opin.Drug Discov. 2010, 5, 655. [Non-Patent Document 17] Kessler, H. et. al., The impact of amino acid side chain mutations in conformational design of peptides and proteins. Chem. Eur. J. 2010, 16, 5385. [Non-Patent Document 18] Comb Chem High Throughput Screen.2010;13:75-87Phage-displayed combinatorial peptide libraries in fusion tobeta-lactamase as reporter for an accelerated clone screening: Potential usesof selected enzyme-linked affinity reagents in downstream applications.ShuklaGS, Krag DN. [Non-Patent Document 19] Heinis, C., Rutherford, T.Freund, S. Winter, G., Phage-encoded combinatorial chemical libraries based onbicyclic peptides. Nature Chem. Bio. 2009, 5, 502. [Non-Patent Document 20] Drug Metabolism: A Fundamental Study of Medical Pharmacy and Toxicology, 2nd Edition, edited by Ryuichi Kato and Tetsuya Kamataki [Non-Patent Document 21] Kawakami T, Diversebackbone-cyclized peptides via codon reprogramming. Nat Chem Biol. 2009, 5,888-90. [Non-Patent Document 22] Ohta A, et al., Synthesis of polyester by means of genetic code reprogramming. Chem Biol., 2007, 14,1315-22. Goto Y, et al., Flexizymes for genetic code reprogramming. Nat Protoc. 2011, 6, 779-90. [Non-Patent Document 23] Yamagishi Y. et al., Naturalproduct-like macrocyclic N-methyl-peptide inhibitors against a ubiquitin ligaseuncovered from a ribosome-expressed de novo library. Chem Biol. 2011, 18,1562-70. [Non-Patent Document 24] Chen S, et al.Structurally diversecyclization linkers impose different backbone conformations in bicyclic peptides.Chembiochem. 2012, 13, 1032-8 [Non-Patent Document 25] Parthasarathy, R. Subramanian, S.,Boder, ET Bioconjugate Chem., 2007, 18, 469-476. [Non-Patent Document 26] Tsukiji S., Nagamune T.,ChemBioChem, 2009, 10, 787-798. [Non-Patent Document 27] Katoh, Takayuki; Goto, Yuki; Reza,Md. Shamim; Suga, Hiroaki. Chemical Communications (Cambridge, United Kingdom)(2011), 47(36), 9946-9958) Summary of the Invention [Problem to be solved by the invention]

[0014] To obtain peptide compounds for clinical development that possess all the properties of efficacy, membrane permeability, and metabolic stability, a display library technology would be effective. This would allow for the design of peptides with membrane permeability and metabolic stability, display a group of these compounds, and then select peptides with medicinal properties from the group. Unlike natural products, hit compounds with a certain degree of membrane permeability and metabolic stability can be easily synthesized and chemically modified. This would allow for structural optimization of hit compounds while minimizing structural changes, similar to the conventional rule of 5 for small molecule drug discovery. Establishing such drug discovery technology and methods requires two key elements: identifying the conditions for satisfying the druglikeness (herein, preferably, membrane permeability and metabolic stability) of medium-sized peptides, which are outside the scope of the rule of 5, and constructing a display library containing unnatural amino acids consisting of molecules that satisfy these conditions. On the other hand, to effectively utilize the limited scale of mid-sized peptide display (which refers to the restriction on peptide chain length due to drug-likeness considerations), it is essential to include many peptides with completely different structures (peptides containing unnatural amino acids) in the library. In addition to introducing a variety of amino acids with different side chain properties, reducing fixed sites within a limited molecular weight to expand the variable region and including peptides with different main chain structures are valuable because they increase the likelihood of obtaining peptides that bind to various targets. From this perspective, effective methods include displaying peptides with various cyclization sites and branched peptides. The present invention has been made in view of the above circumstances, and aims to provide a novel method for cyclizing peptide compounds and a method for synthesizing branched peptides for constructing a peptide display library, and also aims to provide a drug-like display library and a drug-like peptide compound using these methods. [Means for solving the problem]

[0015] As a result of investigations aimed at solving the above-mentioned problems, the present inventors have, for the first time, clarified the conditions required for drug-like cyclic peptides. They have also discovered a method for synthesizing a display library consisting of highly diverse peptide compounds with cyclized moieties that satisfy these conditions. Specifically, the present inventors have discovered a method for synthesizing a library of peptide compounds with cyclized moieties using a novel translation method and post-translational modification, and a method for synthesizing a library of peptide compounds with linear moieties that further increases the likelihood of obtaining drug-like peptides with the desired activity. These methods enable the preparation of compounds that bind to and inhibit target molecules, leading to the completion of the present invention.

[0016] That is, the present invention includes the following. [1] A method for producing a peptide compound having a cyclic portion, comprising the steps of: 1) synthesizing an acyclic peptide compound composed of amino acid residues and / or amino acid analog residues, or amino acid residues and / or amino acid analog residues and an N-terminal carboxylic acid analog, by translating a nucleic acid encoding the peptide compound, the non-cyclic peptide compound comprises an amino acid residue or amino acid analog residue having a reactive site at one side chain on the C-terminal side, and an amino acid residue, amino acid analog residue, or N-terminal carboxylic acid analog having another reactive site on the N-terminal side; 2) A step of bonding the reactive site of the N-terminal amino acid residue, amino acid analog residue, or N-terminal carboxylic acid analog with the reactive site of the amino acid residue or amino acid analog residue in the side chain on the C-terminal side to form an amide bond or a carbon-carbon bond. The method comprising: [2] A method for producing a peptide compound having a cyclic portion, comprising the steps of: 1) synthesizing an acyclic peptide compound composed of amino acid residues and / or amino acid analog residues, or amino acid residues and / or amino acid analog residues and an N-terminal carboxylic acid analog, by translating a nucleic acid encoding the peptide compound, the non-cyclic peptide compound comprises an amino acid residue or an amino acid analog residue having an active ester group in the side chain, and an amino acid residue, an amino acid analog residue, or an N-terminal carboxylic acid analog having a reaction promoting group near the amine; 2) A step of forming an amide bond between the amino acid residue, amino acid analog residue, or N-terminal carboxylic acid analog having the reaction promoting group and an amino acid residue or amino acid analog residue having an active ester group in the side chain to obtain a cyclic compound. The method according to [1], comprising: [3] The method according to [2], wherein the active ester is a thioester. [4] The method according to [2] or [3], wherein the reaction promoting group is an SH group. [5] The method according to [3] or [4], further comprising a step of removing the reaction promoting group after the step of obtaining the cyclic compound. [6] The method according to any one of [2] to [5], wherein the amino acid, amino acid analog, or N-terminal carboxylic acid analog having a reaction promoting group near the amine is Compound N-1 or Compound N-2 represented by the following general formula: [ka] (wherein R1 represents a hydrogen atom, S-R23 (R23 represents an alkyl group, an aryl group, or an aralkyl group which may have a substituent), or a protecting group for an HS group; R2 and R3 each independently represent a hydrogen atom, or an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an aralkyl group, or a cycloalkyl group, each of which may have a substituent; or R2 and R3 form a ring, or R2 or R3 form a ring with R4, each of which may have a substituent; R4 represents an alkylene group which may have a substituent, an arylene group which may have a substituent, or a divalent aralkyl group which may have a substituent; R11 and R12 each independently represent a single bond, an alkylene group which may have a substituent, an arylene group which may have a substituent, or a divalent aralkyl group which may have a substituent. [7] The method according to any one of [2] to [6], wherein the amino acid or amino acid analog having an activated ester group in the side chain is compound C-1 represented by the following general formula: [ka] (wherein R25 represents OH, a halogen atom, OR, or SR1 (R represents Bt, At, NSu, or Pfp, and R1 represents a hydrogen atom, an optionally substituted alkyl group, an optionally substituted aryl group, an optionally substituted aralkyl group, an optionally substituted cycloalkyl group, an optionally substituted heteroaryl group, an optionally substituted alkenyl group, or an optionally substituted alkylene group); R26 represents an alkylene group which may have a substituent, an arylene group which may have a substituent, or a divalent aralkyl group which may have a substituent; R2 and R3 each independently represent a hydrogen atom or an alkyl group which may have a substituent. [8] The method according to any one of [1] to [7], wherein the total number of amino acid residues and / or amino acid analog residues, or amino acid residues and / or amino acid analog residues and N-terminal carboxylic acid analogs, constituting the cyclic portion of the peptide compound having a cyclic portion is 5 to 12. [9] The method according to any one of [1] to [8], wherein the total number of amino acid residues and / or amino acid analog residues, or amino acid residues and / or amino acid analog residues and N-terminal carboxylic acid analogs constituting the peptide compound having a cyclic portion, is 9 to 13.

[10] A method for producing a peptide compound having a cyclic portion, comprising the steps of: 1) synthesizing an acyclic peptide compound composed of amino acid residues and / or amino acid analog residues, or amino acid residues and / or amino acid analog residues and an N-terminal carboxylic acid analog, by translating a nucleic acid encoding the peptide compound, the non-cyclic peptide compound comprises an amino acid residue or an amino acid analog residue having an active ester group in the side chain, and an amino acid residue having an N-terminal main chain amino group, or an amino acid analog residue having an amino group in the main chain or side chain, or an N-terminal carboxylic acid analog; 2) A step of forming an amide bond between an N-terminal amino acid residue, an N-terminal amino acid analog residue, or an N-terminal carboxylic acid analog and an amino acid residue or amino acid analog having an active ester group in the side chain to obtain a cyclic compound. The method according to [1], comprising:

[11] The method according to

[10] , wherein the active ester group is an alkylthioester group or an aralkylthioester group, and comprises a step of converting the active ester group into a more active ester group by adding an activator after the translational synthesis in step 1).

[12] The method according to

[11] , wherein the activator is an aryl thiol or N-hydroxysuccinimide.

[13] The method according to

[12] , wherein an activating agent highly reactive with the translated thioester and an activating agent highly reactive with the amine to be cyclized are added to convert the thioester into a more active ester group.

[14] The method according to

[13] , which comprises a step of converting the arylthioester into an active ester group, and then converting the arylthioester into a more active ester group with an oxyma or a derivative thereof.

[15] The method according to any one of [1] to

[14] , wherein the translational synthesis of the N-terminal site in step 1) is carried out by introducing a translation initiation tRNA acylated with a translatable amino acid other than formylmethionine, a translatable amino acid analog, or a translatable N-terminal carboxylic acid analog.

[16] The method according to any one of

[10] to

[14] , wherein the translational synthesis of the N-terminal site in step 1) is carried out by skipping the initiation codon and introducing a translatable amino acid other than Met, a translatable amino acid analog, or a translatable N-terminal carboxylic acid analog at the N-terminus.

[17] The method according to any one of

[10] to

[14] , wherein the translational synthesis of the N-terminal portion in step 1) is carried out by cleaving the N-terminal amino acid, amino acid analog, or carboxylic acid analog with aminopeptidase.

[18] The method according to

[17] , wherein the translational synthesis of the N-terminal site is carried out by treating with methionine aminopeptidase to remove the N-terminal formyl Met and then introducing another translatable amino acid, translatable amino acid analog, or translatable N-terminal carboxylic acid analog to the N-terminus.

[19] The method according to any one of

[10] to

[14] , wherein the translational synthesis of the N-terminal site is carried out by a method in which translation is carried out in a translation system containing norleucine instead of Met, and the N-terminal formylnorleucine is removed by treatment with methionine aminopeptidase, and another translatable amino acid, translatable amino acid analog, or translatable N-terminal carboxylic acid analog is introduced at the N-terminus.

[20] The method according to any one of

[17] to

[19] , wherein peptide deformylase is further used to remove the N-terminal amino acid, amino acid analog, or carboxylic acid analog. 〔twenty one〕 The method according to any one of [1] to [2], wherein the peptide compound having a cyclic portion further has a linear portion. 〔twenty two〕 The method according to any one of [1] to

[21] , wherein the non-cyclic peptide compound comprises an α-hydroxycarboxylic acid and an amino acid or amino acid analog having an optionally protected amino group at the side chain, and the method comprises, after the step 2) of forming a cyclic compound, a step 3) of chemically reacting the α-hydroxycarboxylic acid moiety with the amino acid or amino acid analog moiety having an optionally protected amino group at the side chain to form a branching moiety. 〔twenty three〕 A method for producing a peptide compound having a cyclic portion and a linear portion, comprising: 1) A step of translating a nucleic acid encoding an acyclic peptide compound composed of amino acid residues and / or amino acid analog residues, or amino acid residues and / or amino acid analog residues, an N-terminal carboxylic acid analog, and an α-hydroxycarboxylic acid, to synthesize the acyclic peptide compound, i) containing an amino acid residue (or amino acid analog residue) having a reactive site at one side chain on the C-terminal side, and an amino acid residue, amino acid analog residue, or N-terminal carboxylic acid analog having another reactive site on the N-terminal side, ii) an α-hydroxycarboxylic acid having Rf5 at the α-position between the two reaction sites described in i) (Rf5 is selected from a hydrogen atom, an optionally substituted alkyl group, an aralkyl group, a heteroaryl group, a cycloalkyl group, an alkenyl group, and an alkynyl group), and an amino acid residue or an amino acid analog residue having an optionally protected amino group in the side chain in a non-cyclic peptide compound, a non-cyclic peptide compound; 2) a cyclization reaction step in which the reactive site of the N-terminal amino acid residue, amino acid analog residue, or N-terminal carboxylic acid analog is bonded to the reactive site of the amino acid residue or amino acid analog residue in the side chain on the C-terminal side; 3) A step of cleaving the ester bond of the α-hydroxycarboxylic acid described in ii) of step 1) to generate a thioester group 4) a cyclization reaction step of bonding the thioester group generated in step 3) with the amino group described in ii) of step 1); A method comprising: 〔twenty four〕 The method according to

[23] , wherein the number of amino acid residues and / or amino acid analog residues contained between the α-hydroxycarboxylic acid described in step 1) ii) and the amino acid residue or amino acid analog residue having an amino group in the side chain is 7 or less. 〔twenty five〕 The method according to

[23] or

[24] , wherein the α-hydroxycarboxylic acid described in step 1) ii) is contained in the acyclic peptide compound as Cys-Pro-α-hydroxycarboxylic acid.

[26] The method according to any one of

[23] to

[25] , wherein the amino acid residue, amino acid analog residue, or N-terminal carboxylic acid analog having another reactive site on the N-terminal side described in i) of step 1) has a reaction promoting group.

[27] The method of any of

[23] to

[26] , wherein the amino acid residue, amino acid analog residue, or N-terminal carboxylic acid analog having another reactive site on the N-terminus described in i) of step 1) does not have a reaction promoting group, the amino group of the amino acid residue or amino acid analog residue having an amino group at its side chain described in ii) has a protecting group, the cyclization reaction of step 2) is carried out by adding an activating agent, and the method comprises a step of removing the protecting group of the amino group of the amino acid residue or amino acid analog residue having an amino group at its side chain described in ii) after the cyclization reaction of step 2) and before the cyclization reaction of step 3).

[28] A method for producing a peptide compound having a cyclic portion, comprising the steps of: 1) synthesizing an acyclic peptide compound composed of amino acid residues and / or amino acid analog residues, or amino acid residues and / or amino acid analog residues and an N-terminal carboxylic acid analog, by translating a nucleic acid encoding the peptide compound, the non-cyclic peptide compound comprises an amino acid residue or amino acid analog residue having a reactive site at one side chain and an amino acid, amino acid analog residue or N-terminal carboxylic acid analog having another reactive site at the N-terminus; 2) forming a carbon-carbon bond between the reactive site of the N-terminal amino acid residue, the N-terminal amino acid analog residue, or the N-terminal carboxylic acid analog and the reactive site of the amino acid residue or amino acid analog having one reactive site in the side chain; The method according to [1], comprising:

[29] The method according to

[28] , comprising the steps of selecting a carbon-carbon double bond as the reactive site of an N-terminal amino acid residue, an N-terminal amino acid analog residue, or an N-terminal carboxylic acid analog, selecting an aryl halide as the reactive site of an amino acid residue or an amino acid analog residue having one reactive site in the side chain, and carrying out a cyclization reaction by a carbon-carbon bond reaction using a transition metal as a catalyst.

[30] The method according to

[29] , wherein the carbon-carbon bond-forming reaction catalyzed by a transition metal is a Heck-type chemical reaction catalyzed by Pd.

[31] The method according to any one of [1] to

[30] , wherein the peptide compound having a cyclic portion has a reactive site for carrying out a cyclization reaction at a position where the total number of amino acids or amino acid analogs forming the cyclic portion is 5 to 12.

[32] The method according to

[31] , wherein the total number of amino acid and amino acid analog residues in the peptide compound having a cyclic portion is 9 to 13.

[33] The method according to any one of [1] to

[32] , characterized in that a peptide compound-nucleic acid complex is produced in which the C-terminus of the peptide compound and the template used in translational synthesis are linked via a spacer.

[34] The method according to

[33] , wherein the peptide compound-nucleic acid conjugate is synthesized using a nucleic acid encoding the acyclic peptide compound used in translational synthesis, wherein puromycin is bound via a linker to the 3' end of the nucleic acid.

[35] The method according to

[33] or

[34] , wherein the spacer is a peptide, RNA, DNA, or hexaethylene glycol polymer, or a combination thereof.

[36] The method according to any one of [1] to

[35] , wherein the peptide compound is produced by translating a nucleic acid library consisting of a plurality of nucleic acids having different sequences.

[37] A peptide compound or a peptide compound-nucleic acid complex produced by the production method according to any one of [1] to

[36] .

[38] A library consisting of a plurality of peptide compounds or peptide compound-nucleic acid complexes according to

[37] , each having a different structure.

[39] A peptide compound having a cyclic portion, characterized by having the following (i) to (iv): (i) The compound contains a cyclic portion consisting of 5 to 12 amino acid and amino acid analog residues in total, and the total number of amino acids and amino acid analogs is 9 to 13. (ii) contains at least two N-substituted amino acids and at least one non-N-substituted amino acid; (iii) ClogP value is 6 or more; (iv) The bonds of the amino acids or amino acid analogs forming the cyclic moiety have at least one bond consisting of an activated ester group in the side chain of an amino acid or amino acid analog and an amine group of another amino acid or amino acid analog.

[40] The peptide compound according to

[39] , wherein the amino acids and amino acid analogs contained in the peptide compound are amino acids or amino acid analogs selected from amino acids or amino acid analogs that can be translationally synthesized, or amino acids or amino acid derivatives obtained by chemically modifying the side chain or N-substitution site of a translatable amino acid or amino acid analog.

[41] The peptide compound according to

[39] or

[40] , further comprising at least one linear portion consisting of a total of 1 to 8 amino acid and amino acid analog residues.

[42] The peptide compound according to any one of

[39] to

[41] , wherein the bond between the amino acids or amino acid analogs forming the cyclic moiety is an amide bond or a carbon-carbon bond.

[43] The peptide compound according to any one of

[39] to

[42] , wherein the cyclic portion contains an intersection unit represented by the following general formula (I): [ka] (The symbols in the formula have the following meanings: R51 is a C1-C6 alkyl group, a C5-C10 aryl group, an aralkyl group, an ester group, or an amide represented by Formula 1, which may have a substituent; R52 is a C1-C6 alkyl group, an aryl group, or an aralkyl group which may have a substituent; R53 is a C1-C6 alkyl group which may have a substituent, or a hydrogen atom, or R53 and R51 may be bonded to each other to form a C3-C5 alkylene group and a 5- to 7-membered ring containing a nitrogen atom; R54 is a peptide consisting of 0-8 amino acid residues, R55 is a C1-C6 alkyl group which may have a substituent, a C5-C10 aryl group, an aralkyl group, an ester group, or an amide group which may have a substituent, * indicates the binding site in the ring.

[44] A pharmaceutical composition comprising the peptide compound according to any one of

[39] to

[43] .

[45] The pharmaceutical composition according to

[44] , wherein the pharmaceutical composition is an oral agent.

[46] A method for producing the peptide compound according to any one of

[39] to

[45] , comprising steps (i) to (v): (i) translationally synthesizing an acyclic peptide compound containing a total of 9 to 13 amino acids and amino acid analogs, and forming an acyclic peptide compound-nucleic acid complex in which the acyclic peptide compound and a complex having a nucleic acid sequence encoding the acyclic peptide compound are linked via a linker; (ii) cyclizing the non-cyclic peptide compound of the complex translationally synthesized in step (i) via an amide bond or a carbon-carbon bond to form a cyclic compound having a total of 5 to 12 amino acid and amino acid analog residues in the cyclic portion; and (iii) A step of contacting the library of peptide compound-nucleic acid complexes having a cyclic portion obtained in step (ii) with a biological molecule and selecting a complex having binding activity to the biological molecule.

[47] The method for producing the compound according to

[46] , further comprising the steps of: (iv) obtaining sequence information of the peptide compound from the nucleic acid sequence of the complex selected in step (iii); and (v) A step of chemically synthesizing a peptide compound based on the sequence information obtained in the step (iv).

[48] The method according to

[46] or

[47] , wherein the non-cyclic peptide compound comprises an α-hydroxycarboxylic acid and an amino acid or amino acid analog having an amino group in the side chain, which may be protected, and the method further comprises, after the step (ii) of forming the cyclic compound, a step of chemically reacting the α-hydroxycarboxylic acid moiety with the amino acid or amino acid analog moiety having an amino group in the side chain to form a branching moiety.

[49] The method of any one of

[46] to

[48] , wherein the biological molecule is a molecule that does not have a region to which a compound with a molecular weight of less than 500 can bind.

[50] A method for producing a complex according to any one of

[46] to

[49] , wherein the complex has binding activity to a biological molecule and further has activity to inhibit the binding of the biological molecule to other biological molecules.

[51] The production method according to any one of

[46] to

[50] , wherein the amino acid or amino acid analog on the N-terminal side to be cyclized is an amino acid or amino acid analog selected from the compounds represented by Compound N-1 or Compound N-2, and the amino acid or amino acid analog on the C-terminal side is an amino acid or amino acid analog selected from the compounds represented by Compound C-1, and the method comprises a step of removing the reaction promoting group after the step of obtaining the cyclic compound in step (ii).

[52] The method according to any one of

[46] to

[51] , wherein the nucleic acid sequence has a spacer at the 3' end, and the C-terminus of the peptide compound to be translationally synthesized forms a complex with the nucleic acid sequence via the spacer.

[53] The method according to

[52] , wherein the peptide compound-nucleic acid conjugate is synthesized using a nucleic acid encoding the acyclic peptide compound used in translational synthesis, wherein puromycin is bound via a linker to the 3' end of the nucleic acid.

[54] The method for production according to

[52] or

[53] , wherein the spacer is a peptide, RNA, DNA, or a polymer of hexaethylene glycol. [Effects of the Invention]

[0017] The present invention provides drug-like peptide compounds having a cyclic portion (excellent membrane permeability and metabolic stability) or peptide compounds having a cyclic portion and a linear portion that can be translationally synthesized, as well as a display library of such compounds. Furthermore, because the display library of the present invention is a highly diverse library, there is a high probability of obtaining hit compounds for desired target molecules. Furthermore, because the hit compounds obtained from the display library of the present invention already have excellent membrane permeability and metabolic stability, they can be efficiently optimized as pharmaceuticals in the same way as conventional small molecule compounds, without requiring major structural transformations. Thus, the present invention provides scaffolds for new pharmaceuticals that differ from the small molecule compounds and antibodies currently known as pharmaceuticals, and a new drug discovery system for efficiently creating pharmaceuticals. [Brief explanation of the drawings]

[0018] [Figure 1] FIG. 1 shows a general method for synthesizing a group of aminoacylated pdCpA compounds in which the side chain carboxylic acid is converted into an active ester. [Figure 2] FIG. 1 shows mass spectrometry analysis of the translation product of mRNA encoding the peptide sequence P-1 containing Glu(SBn). [Figure 3] This figure shows the mass spectrometry analysis of the translation product of mRNA encoding the peptide sequence P-2 containing Asp(SMe). During translation of Asp(SMe), a compound that was de-MeSHed from the translated full-length peptide was detected as the major product (translated peptide P-3). [Figure 4] FIG. 1 shows the production of peptide P-4 by hydrolysis of translated peptide P-6. [Figure 5] FIG. 1 shows the translation of a peptide sequence that does not contain Tyr but contains a thioester. [Figure 6] FIG. 1 shows the translational synthesis of a thioester-containing peptide that does not have an N-terminal amino group. [Figure 7]FIG. 1 shows the translational synthesis of a model peptide in which an N-alkylated amino acid has been introduced as the C-terminal amino acid following an amino acid with a thioesterified side chain. [Figure 8-1] FIG. 1 shows a comparison of the chemical reactivity of thioesters with glycine derivatives or cysteine ​​derivatives. [Figure 8-2] This is a figure showing a continuation of Figure 8-1. [Figure 9] FIG. 1 shows the synthesis of amide 5d-1 by the reaction of thioester 5b-1 with a glycine derivative under imidazole-added conditions. [Figure 10] FIG. 1 shows a general method for synthesizing aminoacylated pdCpA of a cysteine ​​derivative. [Figure 11] FIG. 1 shows an example of the synthesis of aminoacylated pdCpA, a cysteine ​​derivative. [Figure 12] FIG. 1 shows the translational synthesis of a peptide with Cys(StBu) at the N-terminus. [Figure 13] FIG. 1 shows the stability of Compound 2n-A and Compound 2e-A in a translation simulation solution. [Figure 14] This figure shows the efficient translational incorporation of N-terminal Cys by actively utilizing initiation read-through. [Figure 15-1] This figure shows the mass spectra of translation products in which various amino acids are encoded in the third codon immediately following Cys (P-15: Phe). [Figure 15-2] This figure shows the mass spectra of translation products in which various amino acids are encoded in the third codon immediately following Cys (P-16:Leu). [Figure 16-1] This figure shows the mass spectra of translation products in which various amino acids are encoded in the third codon immediately following Cys (P-17: Tyr). [Figure 16-2] This figure shows the mass spectra of translation products in which various amino acids are encoded in the third codon immediately following Cys (P-18: Cys). [Figure 17-1]This figure shows the mass spectra of translation products in which various amino acids are encoded in the third codon immediately following Cys (P-19:Trp). [Figure 17-2] This figure shows the mass spectra of translation products in which various amino acids are encoded in the third codon immediately following Cys (P-20:Leu). [Figure 18-1] This figure shows the mass spectra of translation products in which various amino acids are encoded in the third codon immediately following Cys (P-21:Leu). [Figure 18-2] This figure shows the mass spectra of translation products in which various amino acids are encoded in the third codon immediately following Cys (P-22: Pro). [Figure 19-1] This figure shows the mass spectra of translation products in which various amino acids are encoded in the third codon immediately following Cys (P-23: His). [Figure 19-2] This figure shows the mass spectra of translation products in which various amino acids are encoded in the third codon immediately following Cys (P-24: Gln). [Figure 20-1] This figure shows the mass spectra of translation products in which various amino acids are encoded in the third codon immediately following Cys (P-25: Arg). [Figure 20-2] This figure shows the mass spectra of translation products in which various amino acids are encoded in the third codon immediately following Cys (P-26: Arg). [Figure 21-1] This figure shows the mass spectra of translation products in which various amino acids are encoded in the third codon immediately following Cys (P-27: Ile). [Figure 21-2] This figure shows the mass spectra of translation products in which various amino acids are encoded in the third codon immediately following Cys (P-29: Asn). [Figure 22] This figure shows the mass spectra of translation products in which various amino acids are encoded in the third codon immediately following Cys (P-30: Ser). [Figure 23-1] This figure shows the mass spectra of translation products in which various amino acids are encoded in the third codon immediately following Cys (P-32: Val). [Figure 23-2] This figure shows the mass spectra of translation products in which various amino acids are encoded in the third codon immediately following Cys (P-33: Ala). [Figure 24] FIG. 1 shows a general method for synthesizing non-natural amino acids with SH groups other than cysteine ​​and their aminoacylated pdCpAs. [Figure 25] This figure shows the mass spectra of peptides translated using stabilized aminoacylated tRNA containing an SH group (P-34: tBuSSEtGly, P-35: tBuSSEtβAla, P-36: tBuSSEtGABA, [M+H] is the target compound, S-deprotection is the compound in which the protecting group attached to the SH group of the target compound has been removed, indicating that the target compound has been translated, and Read-through is the translation product initiated from the second Thr, a by-product). [Figure 26] Figure 1 shows mass spectra of peptides translated using stabilized aminoacylated tRNA containing an SH group (P-41: Cys(StBu), P-40: PenCys(StBu), P-38: NVOC-Cys(StBu)). [Figure 27] FIG. 1 shows mass spectra of translationally synthesized and amide-cyclized peptides using a method for introducing an amino acid, amino acid analog, or N-terminal carboxylic acid analog other than methionine into the N-terminus. [Figure 28] This figure shows the mass spectrum of the translated and amide-cyclized peptide using initiation read-through (NCL: target compound). [Figure 29] This figure shows the mass spectrum of the translated and amide-cyclized peptide using initiation read-through (NCL: target compound). [Figure 30-1] FIG. 1 shows MS and MS / MS analysis for structural determination of translated and amide-cyclized peptides using initiation read-through. [Figure 30-2] This is a figure showing a continuation of Figure 30-1. [Figure 31]FIG. 1 shows a radical desulfurization reaction using a model substrate. [Figure 32] FIG. 1 shows a diagram illustrating an investigation of conditions for radical desulfurization using a model substrate. [Figure 33] FIG. 1 shows the desulfurization reaction conditions for translated peptide P-50 and the mass spectrum of the resulting peptide P-51. [Figure 34] FIG. 1 shows an evaluation of the effect of desulfurization on proteins. [Figure 35-1] FIG. 1 shows mass chromatograms of metabolites. [Figure 35-2] FIG. 1 shows the MS / MS spectrum of Peak 1. [Figure 35-3] FIG. 1 shows the MS / MS spectrum of Peak 2. [Figure 35-4] FIG. 1 shows the MS / MS spectrum of Peak 3. [Figure 35-5] FIG. 1 shows the MS / MS spectrum of Peak 4. [Figure 36] FIG. 1 shows the results of LCMS analysis of the reaction mixture produced by cyclization reaction to produce compound P-136. [Figure 37] FIG. 1 shows the results of LCMS analysis of the reaction mixture produced by cyclization reaction to produce compound P-136. [Figure 38] FIG. 1 shows the results of LCMS analysis of the reaction solution in which compound P-137 was produced by cyclization reaction. [Figure 39] FIG. 1 shows the results of mass spectrometry analysis of translation reaction products obtained by translational synthesis of peptides containing benzylthioesterified aspartic acid derivatives. [Figure 40] FIG. 1 shows the results of mass spectrometry analysis of the product obtained in an amide cyclization experiment of a peptide using the thioester on the translated peptide P141 and the N-terminal α-amino group. [Figure 41] FIG. 1 shows a comparison of synthesis conditions for compound 10. [Figure 42] FIG. 1 shows the results of LCMS analysis of the production reaction of compound P-151. [Figure 43]FIG. 1 shows the results of a reverse transcription reaction using the mRNA-peptide fusion molecule after the cyclization reaction as a template. [Figure 44] FIG. 1 shows the results of MALDI-MS analysis of peptides containing N-terminal Phe, Ala and a benzylthioesterified aspartic acid derivative. [Figure 45] FIG. 1 shows the results of MALDI-MS analysis of peptides containing N-terminal Phe, Ala and a benzylthioesterified aspartic acid derivative. [Figure 46] FIG. 1 shows the results of electrophoretic evaluation of RNA stability under cyclization reaction conditions. [Figure 47] FIG. 1 shows the results of electrophoretic analysis of reaction products of peptide cyclization reactions. [Figure 48] FIG. 1 shows the results of MALDI-MS analysis of cyclized peptides obtained by post-translational removal of the initiating amino acid and peptide cyclization without using a reaction promoter, using a peptide whose translation is initiated with norleucine and contains a benzylthioesterified aspartic acid derivative in the side chain. [Figure 49] FIG. 1 shows the results of LCMS analysis of the reaction solution for producing compound SP-606. [Figure 50] FIG. 1 shows the results of MALDI-MS analysis of a cyclic peptide having a cysteinylprolyl ester sequence and a side chain amino group (compound P-H1). [Figure 51] FIG. 1 shows the results of MALDI-MS analysis of the product from the intramolecular branched peptide (linear portion 2) production reaction using translated peptide P-H1. [Figure 52] FIG. 1 shows the results of LCMS analysis of the reaction solution for producing compound SP-606. [Figure 53] FIG. 1 shows the results of LCMS analysis of the reaction solution for producing compound SP-606. [Figure 54] FIG. 1 shows the results of LCMS analysis of the reaction solution for producing compound SP-606. [Figure 55] FIG. 1 shows the results of LCMS analysis of the reaction solution for producing compound SP-607. [Figure 56] This figure shows the mass chromatogram (top) before the desulfurization reaction (compound SP606) and the mass chromatogram (bottom) after 3 hours of desulfurization reaction, averaged over the retention time range of 0.34 to 0.39 minutes (analysis condition SQDFA05). By integrating within this range, not only the target compound and reaction raw materials but also any by-products with similar structures should be observed, making it possible to accurately evaluate the overall reaction selectivity. This is true not only for Figure 56, but for all mass spectra where a certain time range is integrated. [Figure 57] FIG. 1 shows the results of LCMS analysis of the reaction solution for producing compound SP-607. [Figure 58] This shows the mass chromatogram (top) before the desulfurization reaction (compound SP606) and the mass chromatogram (bottom) obtained by integrating and averaging the data from retention times 0.34 to 0.39 minutes 3 hours after the desulfurization reaction (analysis condition SQDFA05). [Figure 59] FIG. 1 shows the results of LCMS analysis of the reaction solution for producing compound SP618. [Figure 60] FIG. 1 shows the results of LCMS analysis of the reaction solution for producing compound SP619. [Figure 61] Figure 61 shows the LCMS mass chromatogram from 0.3 to 0.6 minutes. Note that under these analytical conditions, all peptide components are eluted between 0.3 and 0.6 minutes. Therefore, to evaluate the selectivity of the reaction, the mass chromatogram from 0.3 to 0.6 minutes was integrated and averaged. This figure shows that the reaction proceeded selectively. [Figure 62] Figure 62 shows the results of MALDI-MS analysis of peptide P-E1 (Peak I), which contains N-terminal formylmethionine and is thioester-cyclized between the side chain thiol group and carboxylic acid, and compound P-E2 (Peak II), which has been amide-cyclized between the nitrogen atom of the exposed N-terminal amino group and the side chain carboxylic acid of Asp after the N-terminal formylmethionine has been removed. [Figure 63] FIG. 1 shows the results of LCMS analysis of the reaction solution for producing compound SP618. [Figure 64] FIG. 1 shows the results of LCMS analysis of the reaction solution for producing compound SP619. [Figure 65] Figure 65 shows the LCMS mass chromatogram from 0.3 to 0.6 minutes. Note that under these analytical conditions, all peptide components are eluted between 0.3 and 0.6 minutes. Therefore, in order to evaluate the selectivity of the reaction, the mass chromatogram from 0.3 to 0.6 minutes was integrated and averaged. [Figure 66] This is a graph showing the predicted average and distribution of CLOGP values, NMe amino acid numbers, and molecular weights based on a virtual library using computer simulation. [Figure 67] This is a graph showing the predicted average and distribution of CLOGP values, NMe amino acid numbers, and molecular weights based on a virtual library using computer simulation. [Figure 68] This is a mass chromatogram obtained by integrating and averaging the entire LCMS range. [Figure 69] This is a mass chromatogram obtained by integrating and averaging the entire LCMS range. [Figure 70] This is a mass chromatogram obtained by integrating and averaging the entire LCMS range. [Figure 71] FIG. 1 shows the results of LCMS analysis of the reaction solution for producing compound SP664. [Figure 72] FIG. 1 shows the results of LCMS analysis of the reaction solution for producing compound SP672. [Figure 73] FIG. 1 shows the inhibitory activity of the compounds obtained in Example 26 (SP854, SP855, SP857, and SP859) on hIL-6- and shIL-6R-mediated cell proliferation. [Figure 74] FIG. 1 shows the results of electrophoretic analysis of reaction products containing a peptide-RNA complex having an intramolecularly branched peptide (linear portion 2). [Figure 75] FIG. 1 shows the results of MALDI-MS analysis of translation reaction products. [Figure 76] FIG. 1 shows the results of analysis of translation reaction products by mass chromatogram and mass spectrum. [Figure 77]FIG. 1 shows the results of analysis of translation reaction products by mass chromatogram and mass spectrum. [Figure 78] FIG. 1 shows the results of analysis of translation reaction products by mass chromatogram and mass spectrum. [Figure 79] FIG. 1 shows the results of analysis of translation reaction products by mass chromatogram and mass spectrum. [Figure 80] FIG. 1 shows the results of electrophoretic evaluation of RNA stability under side chain amino group deprotection conditions. [Figure 81] FIG. 1 shows the results of LC / MS analysis of samples obtained by treating RNase-treated samples. [Figure 82-1] FIG. 1 shows the interaction between a protein and a peptide consisting of 13 residues, with a 10 amino acid ring forming a ring and a 3 amino acid branch. [Figure 82-2] The figure shows the interaction between a protein and a peptide consisting of 13 residues, with 10 amino acids forming a ring and three amino acids branching off (left). The figure shows the interaction between a protein and a peptide consisting of 13 amino acids forming a ring (right). [Figure 83] FIG. 1 shows the results of electrophoretic evaluation of RNA stability under side chain amino group deprotection conditions. DETAILED DESCRIPTION OF THE INVENTION

[0019] <Peptide compounds> Peptide compounds with cyclic moieties The peptide compounds of the present invention having a cyclic portion are compounds formed by amide or ester bonds between amino acids or amino acid analogs, and the cyclic portion is cyclized via a covalent bond such as an amide bond or a carbon-carbon bond-forming reaction. Compounds obtained by further chemically modifying such compounds are also included in the peptide compounds of the present invention. The peptide compounds of the present invention may have a linear portion, and can be depicted, for example, as shown in Scheme A (Schemes A-1 and A-2). The peptide compounds of the present invention may further have a linear portion. The number of amide or ester bonds (number and length of amino acids or amino acid analogs) is not particularly limited. However, when a linear portion is present, the total number of amino acids in the cyclic portion and the linear portion is preferably 30 or less. To achieve high membrane permeability, the total number of amino acids in the cyclized and linear portions is preferably 13 or less. To achieve high metabolic stability, the total number of amino acids is preferably 9 or more. Considering both membrane permeability and metabolic stability (drug-likeness), the number of amino acids and amino acid analogs constituting the cyclic portion is preferably 5 to 12. In addition to the above, the number of amino acids and amino acid analogs constituting the cyclic portion is more preferably 5 to 11, and even more preferably 7 to 11 residues. 9 to 11 residues are particularly preferred. The number of amino acids and amino acid analogs (number of units) in the linear portion is preferably 0 to 8, and even more preferably 0 to 3. Unless otherwise specified in the present application, amino acids may also include amino acid analogs. Here, "drug-likeness" or "drug-like" means that the peptide compound has membrane permeability and metabolic stability sufficient to be used as a pharmaceutical, at least when targeted to an oral agent, an intracellular protein, a nucleic acid, an intracellular region of a membrane protein, or a transmembrane domain of a membrane protein.

[0020] The cyclic moiety of the peptide compound of the present invention having a cyclic portion is not particularly limited as long as it is a peptide that forms a ring. However, the post-translation cyclization portion must be a cyclization unit that forms a functional group that can achieve both membrane permeability and metabolic stability (drug-likeness). There is no particular limitation on the cyclization method. Examples include an amide bond formed from a carboxylic acid and an amine, and carbon-carbon bond reactions catalyzed by transition metals, such as the Suzuki reaction, the Heck reaction, and the Sonogashira reaction. Therefore, the peptide compound of the present invention contains at least one pair of functional groups capable of these bonding reactions. From the perspective of metabolic stability in particular, it is preferable that the peptide compound contains a functional group that forms an amide bond by bonding reaction.

[0021] The cyclic moiety of the peptide compound of the present invention is preferably formed by cyclization through a chemical reaction after translational synthesis, such as that shown in Scheme A. Furthermore, a cyclic moiety that can be formed under reaction conditions that do not affect nucleic acids such as RNA and DNA after translation is preferred.

[0022] The formation of the cyclic moiety is preferably drug-like cyclization. Drug-like cyclization means that the resulting bond is drug-like. For example, it is preferable that the bond contains a heteroatom that can be easily oxidized and does not contain a bond that interferes with metabolic stability. Bonds formed by cyclization include, for example, an amide bond formed by bonding an activated ester with an amine, and a bond formed by a Heck reaction product formed by a carbon-carbon double bond with an aryl halide. These require reactive functional groups in the triangle unit (the N-terminal unit of the cyclization) or intersection unit as shown in Scheme A, so amino acids suitable for drug-like behavior are not necessarily selected for the triangle unit or intersection unit. However, after post-translational modification, they are converted into compounds having drug-like functional groups. In the present invention, such bonds are also included in the drug-like cyclization bonds.

[0023] The curved portion in Scheme A is the site that is cyclized after translation (post-translational cyclization portion), and this portion is bonded to form a cyclic portion by various post-translational modification chemical reactions, typified by amide bond or carbon-carbon bond forming reactions such as the Heck reaction. In this specification, "translational synthesis" refers to the synthesis of a peptide compound by translation from a nucleic acid (e.g., DNA, RNA) that encodes the peptide compound. Translation is a process in which a linear peptide is obtained by the action of ribosomes, using mRNA as a template, through repeated amide bond and ester bond reactions. Post-translational modification refers to a chemical reaction that occurs after translation automatically or by adding a different reagent other than through the action of ribosomes, and examples of such reactions include cyclization and deprotection. Post-translational cyclization is a post-translational modification that involves a ring-forming reaction. (Scheme A: A scheme illustrating the peptide compound of the present invention. The open circle units, filled circle units, triangle units, and square units each represent an amino acid or amino acid analog. Furthermore, each unit represents the same or different amino acid or amino acid analog. The triangle unit may be an N-terminal carboxylic acid analog. For example, the eight filled circle units may each represent a different type of amino acid or amino acid analog, or some or all of them may be the same. Furthermore, the amino acid or amino acid analog may be chemically converted or backbone-converted into a compound having a different skeleton by a chemical modification that can be performed post-translationally. Here, one unit corresponds to the amino acid or amino acid analog at the end of post-translational modification, but also includes an amino acid or amino acid analog translated by one tRNA that has been chemically converted or backbone-converted into a compound having a different skeleton by post-translational modification. The number of units is calculated in the same way. Unless otherwise specified, in the present application, amino acids include amino acid analogs. Furthermore, in the present specification, post-translational cyclization may be simply referred to as cyclization.)

[0024] For example, in Scheme A-1, the cyclic portion is a portion consisting of one triangle (▲) unit (residue) (cyclized N-terminal unit), eight black circle (●) units (cyclic main-chain units), and one white circle (○) unit (crossover unit), while the linear portion is a portion consisting of six square (■) units (linear main-chain units). In Scheme A-2, the cyclic portion is a portion consisting of one triangle unit, eight black circle units, and one crossover unit, while the linear portion is a portion consisting of four squares and three square units.

[0025] In the present invention, the intersection unit refers to an amino acid or amino acid analog that, in a peptide compound (non-cyclized peptide compound) formed after translation before cyclization, has a functional group in its amino acid side chain that is cyclized by chemical reaction with a functional group possessed by an amino acid or amino acid analog of a triangle unit in the peptide compound or a functional group possessed by an N-terminal carboxylic acid analog. The intersection unit is not particularly limited as long as it has the functional group necessary for cyclization with the triangle unit. The open circle unit in Scheme A corresponds to this. The intersection unit is preferably selected from the above amino acids or amino acid analogs and is translatable from nucleic acids. Furthermore, even if a compound itself is difficult to translate, it is not essential that it be translated if its derivative is translated. For example, when Asp(SBn) is translated, a compound in which the methylene chain of Asp's side chain is freely substituted is also acceptable as the intersection unit (e.g., R28 and R29 of Compound C-3 may not be translated). The intersection unit must have at least three functional groups, because the amino and carboxyl groups in the main chain are used for covalent bond formation during translational synthesis, and the third functional group is required for post-translational cyclization. The post-translational cyclization site is cyclized by a functional group in the side chain of the intersection unit.

[0026] Here, the amino acid or amino acid analog or N-terminal carboxylic acid analog having a functional group that can be cyclized with the intersection unit is not particularly limited as long as it has a functional group that can be cyclized with the intersection unit. This applies to the triangle unit in Scheme A. For example, in Scheme A, the triangle unit is positioned at the N-terminus. In such an example, when an amino acid is selected as the triangle unit, the main-chain amino group can be used as the cyclization functional group. For example, when an activated ester is used as the intersection unit, post-translational cyclization can be achieved through an amide bond with the main-chain amino group of the triangle unit. When the main-chain amino group is used as the reactive functional group, the side chain of the triangle unit does not need to have a reactive functional group. A reaction-promoting group such as an SH group (thiol group) can also be introduced into the side chain. Furthermore, when an amino acid analog is used as the triangle unit, the hydroxyl group of the main chain can be used as the reactive functional group, or a reactive functional group located in the side chain can be used. When an N-terminal carboxylic acid analog is used, an amino group or a hydroxyl group may be used as the reactive functional group as described above, but various functional groups can be introduced as free reactive units without an amino group or a hydroxyl group. The triangle unit is preferably selected from the above amino acids or amino acid analogs, or N-terminal carboxylic acid analogs, and is translated. As with the intersection unit, even if a unit itself is difficult to translate, it is not essential that the unit itself be translated as long as its derivative is translated.

[0027] The intersection unit and triangle unit can be incorporated at any desired position in the peptide compound before cyclization, as long as they are positions that allow cyclization, but they are preferably incorporated at positions such that the total number of amino acids, amino acid analogs, or N-terminal carboxylic acid analogs in the cyclic moiety after cyclization or post-translational modification after cyclization is 5 to 12. Furthermore, they are preferably incorporated at positions such that the total number of amino acids, amino acid analogs, or N-terminal carboxylic acid analogs in the cyclic moiety after cyclization or post-translational modification after cyclization is 5 to 11.

[0028] Although the triangle unit is placed at the N-terminus in Scheme A, it can also be placed at a position other than the N-terminus. In that case, it must be placed closer to the N-terminus than the intersection unit. When the triangle unit is placed at a position other than the N-terminus, the triangle unit is selected from amino acids and amino acid analogs and has a functional group in its side chain that undergoes a cyclization reaction with the intersection unit.

[0029] The black circle units and square units are selected from amino acids or amino acid analogs. Also included are chemical structures that can be generated by post-translational modification after translation of an amino acid or amino acid analog (e.g., the structure of linear portion 2). When an amino acid is selected, the black circle units are not particularly limited, but are preferably selected from drug-like amino acids or amino acids having reactive functional groups that can be converted into drug-like functional groups by chemical reaction in post-translational modification (e.g., lysine can be cited as an amino acid residue in linear portion 2). When an amino acid analog is selected, the black circle units are not particularly limited, but are preferably selected from drug-like amino acid analogs or amino acid analogs having various reactive functional groups in the side chain that are chemically modified by post-translational modification to convert the reactive functional groups into drug-like amino acid analogs.

[0030] The number of linear portions (number of branches) is not particularly limited and may be one as in Scheme A-1 or two or more as in Scheme A-2. Also, compounds in which the number of square units in Scheme A-1 is zero may be used, and compounds in which the number of linear portions 1 among the square units in Scheme A-2 is zero may be used. The presence of a linear portion can enhance the function of peptide compounds having a cyclic portion of the present invention. For example, when a peptide compound of the present invention is used to inhibit the binding of a certain receptor to a ligand, the presence of a linear portion in the peptide compound can enhance the binding activity of the peptide compound to the receptor or ligand compared to a peptide compound lacking a linear portion. This enhanced binding activity can enhance the inhibitory effect of receptor-ligand binding. In particular, the linear portion of the present invention can be added to a desired position in the cyclic portion, for example, according to the method described below. A peptide compound can be obtained in which the linear portion is added to an optimal position for enhanced functionality (hereinafter, referred to as linear portion 2).

[0031] These linear portions are also preferred for more efficiently obtaining peptide compounds with desired activity from a library of peptide compounds having a cyclic portion. Examples of peptide compounds with desired activity include those that have binding activity only to a target substance, those that inhibit the function of a target substance, those that activate the function of a target substance, and those that alter the function of a target substance. A function can be selected from these depending on the purpose. When the peptide compounds having a cyclic portion of the present invention have binding activity to a target substance, these peptide compounds have excellent membrane permeability and lipid stability. For example, labeling the peptide compounds enables real-time monitoring of the distribution of the target substance not only in vivo but also intracellularly. Furthermore, when the target substance is a causative factor of a disease, they may be useful for diagnosing the disease. Furthermore, when the peptide compounds having a cyclic portion of the present invention have the effect of inhibiting, activating, or altering the function of a target substance, for example, when the target substance is a causative factor of a disease, they can be used as a therapeutic agent for the disease. Furthermore, when the peptide compounds having a cyclic portion of the present invention have the effect of inhibiting, activating, or altering the function of a target substance, they can be used as a therapeutic agent for the disease. Furthermore, when the peptide compounds having a cyclic portion of the present invention have a linear portion 2, the yield of inhibitory compounds can be improved compared to when they do not have a linear portion. In the case of a peptide compound with a 13-residue cyclic region that binds to protein A and inhibits the protein-protein interaction between protein A and protein B, the contact site of the peptide in protein A to which the peptide compound binds is shown on the right side of Figure 82-2. Because it is a cyclic compound, the contact area between the peptide and protein A can be approximated by a circle with a diameter of approximately 3 to 5 residues. If protein B binds to this contact area, it is possible to effectively inhibit the protein-protein interaction between protein A and protein B. However, if protein B binds to protein A outside this contact area, it is possible that effective inhibition will not be obtained (examples of so-called allosteric inhibition have been reported for protein-protein interaction inhibition).

[0032] Considering this, it is important to obtain peptide compounds that bind to proteins over as wide an area as possible. However, because the total number of amino acids that can permeate the membrane is limited, linear portions are effective. For example, Figure 82-1 shows the same 13-residue cyclic peptide as above, but with 10 residues forming the ring and the remaining three branched. In particular, linear portion 2 can be added to any position on the peptide compound using the method described below. Therefore, more branching points than the four shown in Figure 82-1 can be obtained. If four of them are overlapped, the contact area expands to the area shown on the left of Figure 82-2. Even though they act on the same location (hole), overlapping with the binding regions of Protein A and Protein B increases the chances of obtaining peptide compounds with inhibitory functions.

[0033] In this specification, the "amino acids" and "amino acid analogs" that constitute the peptide compounds may be referred to as "amino acid residues" and "amino acid analog residues," respectively. The amino acids are α-, β-, and γ-amino acids, and are not limited to naturally occurring amino acids (in this application, naturally occurring amino acids refer to the 20 types of amino acids contained in proteins, specifically Gly, Ala, Ser, Thr, Val, Leu, Ile, Phe, Tyr, Trp, His, Glu, Asp, Gln, Asn, Cys, Met, Lys, Arg, and Pro), but may also be unnatural amino acids. In the case of α-amino acids, they may be L-amino acids or D-amino acids, or α,α-dialkylamino acids. There are no particular restrictions on the selection of amino acid side chains, and in addition to hydrogen atoms, they may be freely selected from, for example, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, aralkyl groups, and cycloalkyl groups. Each of these may have a substituent, and the substituent is also freely selected from any functional group containing, for example, an N atom, an O atom, an S atom, a B atom, a Si atom, or a P atom (i.e., an optionally substituted alkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, aralkyl group, cycloalkyl group, etc.). The "amino acids" and "amino acid analogs" that constitute the peptide compounds include all corresponding isotopes. Isotopes of "amino acids" and "amino acid analogs" are those in which at least one atom has been replaced with an atom having the same atomic number (number of protons) but a different mass number (sum of the number of protons and neutrons). Examples of isotopes contained in the "amino acids" and "amino acid analogs" that constitute the peptide compounds of the present invention include hydrogen atoms, carbon atoms, nitrogen atoms, oxygen atoms, phosphorus atoms, sulfur atoms, fluorine atoms, and chlorine atoms, including 2H, 3H, 13C, 14C, 15N, 17O, 18O, 31P, 32P, 35S, 18F, and 36Cl, respectively.

[0034] Examples of the substituent include halogen-derived substituents such as fluoro (-F), chloro (-Cl), bromo (-Br), and iodine (-I). Examples of the substituent include alkyl groups, cycloalkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, and aralkyl groups, which are substituted with one or more of these and may have a halogen as a substituent.

[0035] Examples of substituents derived from O atoms that form ethers include alkoxy groups (-OR), which are selected from alkylalkoxy groups, cycloalkylalkoxy groups, alkenylalkoxy groups, alkynylalkoxy groups, arylalkoxy groups, heteroarylalkoxy groups, aralkylalkoxy groups, etc. Examples of substituents that form alcohol moieties include hydroxyl groups (-OH), and examples of substituents that form carbonyl groups include carbonyl groups (-C=OR), which are selected from hydrocarbonyl groups (-C=OH, which yield aldehydes as compounds), alkylcarbonyl groups (which yield ketones as compounds), cycloalkylcarbonyl groups, alkenylcarbonyl groups, alkynylcarbonyl groups, arylcarbonyl groups, heteroarylcarbonyl groups, aralkylcarbonyl groups, etc. An example of a substituent (-COH) that forms a carboxylic acid is a carboxyl group, and examples of substituents that form an ester group are an oxycarbonyl group (-OC=OR) and a carbonylalkoxy group (-C=O-OR). The carbonylalkoxy group is selected from an alkyloxycarbonyl group, a cycloalkyloxycarbonyl group, an alkenyloxycarbonyl group, an alkynyloxycarbonyl group, an aryloxycarbonyl group, a heteroaryloxycarbonyl group, an aralkyloxycarbonyl group, and the like. The oxycarbonyl group is selected from an alkylcarbonyloxy group, a cycloalkylcarbonyloxy group, an alkenylcarbonyloxy group, an alkynylcarbonyloxy group, an arylcarbonyloxy group, a heteroarylcarbonyloxy group, an aralkylcarbonyloxy group, and the like.

[0036] Substituents that form thioesters include a mercaptocarbonyl group (-SC=OR) and a carbonylalkylmercapto group (-C=O-SR), and are selected from a mercaptoalkylcarbonyl group, a mercaptocycloalkylcarbonyl group, a mercaptoalkenylcarbonyl group, a mercaptoalkynylcarbonyl group, a mercaptoarylcarbonyl group, a mercaptoheteroarylcarbonyl group, a mercaptoaralkylcarbonyl group, and the like, and alternatively include a carbonylalkylmercapto group, a carbonylcycloalkylmercapto group, a carbonylalkenylmercapto group, a carbonylalkynylmercapto group, a carbonylarylmercapto group, a carbonylheteroarylmercapto group, and a carbonylaralkylmercapto group.

[0037] Examples of the substituent forming an amide group include an aminoalkylcarbonyl group (-NH-CO-R), an aminocycloalkylcarbonyl group, an aminoalkenylcarbonyl group, an aminoalkynylcarbonyl group, an aminocycloalkylcarbonyl group, an aminoarylcarbonyl group, an aminoheteroarylcarbonyl group, an aminoaralkylcarbonyl group, etc., or a carbonylalkylamino group (-CO-NHR), a carbonylcycloalkylamino group, a carbonylalkenylamino group, a carbonylalkynylamino group, a carbonylarylamino group, a carbonylheteroarylamino group, a carbonylaralkylamino group, etc. Further examples include compounds in which the H atom bonded to the N atom is replaced with an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or an aralkyl group.

[0038] Examples of substituents that form a carbamate group include an aminoalkyl carbamate group (—NH—CO—OR), an aminocycloalkyl carbamate group, an aminoalkenyl carbamate group, an aminoalkynyl carbamate group, an aminocycloalkyl carbamate group, an aminoaryl carbamate group, an aminoheteroaryl carbamate group, an aminoaralkyl carbamate group, etc. Further examples include compounds in which the H atom bonded to the N atom is replaced with an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or an aralkyl group.

[0039] Examples of the substituent forming a sulfonamide group include an aminoalkylsulfonyl group (-NH-SO-R), an aminocycloalkylsulfonyl group, an aminoalkenylsulfonyl group, an aminoalkynylsulfonyl group, an aminocycloalkylsulfonyl group, an aminoarylsulfonyl group, an aminoheteroarylsulfonyl group, an aminoaralkylsulfonyl group, etc., or a sulfonylalkylamino group (-SO-NHR), a sulfonylcycloalkylamino group, a sulfonylalkenylamino group, a sulfonylalkynylamino group, a sulfonylarylamino group, a sulfonylheteroarylamino group, a sulfonylaralkylamino group, etc. Further examples include compounds in which the H atom bonded to the N atom is replaced with an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or an aralkyl group.

[0040] Examples of the substituent forming a sulfamide group include an aminoalkylsulfamoyl group (-NH-SO2-NHR), an aminocycloalkylsulfamoyl group, an aminoalkenylsulfamoyl group, an aminoalkynylsulfamoyl group, an aminocycloalkylsulfamoyl group, an aminoarylsulfamoyl group, an aminoheteroarylsulfamoyl group, an aminoaralkylsulfamoyl group, etc. Further examples include compounds in which the H atom bonded to the N atom is replaced by any two of the same or different alkyl groups, cycloalkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, and aralkyl groups, or substituents in which these may form a ring.

[0041] An example of a substituent that forms a thiocarboxylic acid is a thiocarboxylic acid group (-C(=O)-SH), and an example of a functional group that forms a keto acid is a keto acid group (-C(=O)-CO2H).

[0042] Furthermore, examples of substituents derived from S atoms that form thiol groups include thiol groups (-SH), which form alkylthiols, cycloalkylthiols, alkenylthiols, alkynylthiols, arylthiols, heteroarylthiols, and aralkylthiols. Substituents that form thioethers (-SR) are selected from alkyl mercapto groups, cycloalkyl mercapto groups, alkenyl mercapto groups, alkynyl mercapto groups, aryl mercapto groups, heteroaryl mercapto groups, aralkyl mercapto groups, etc.; substituents that form sulfoxide groups (-S=OR) are selected from alkyl sulfoxide groups, cycloalkyl sulfoxide groups, alkenyl sulfoxide groups, alkynyl sulfoxide groups, aryl sulfoxide groups, heteroaryl sulfoxide groups, aralkyl sulfoxide groups, etc.; substituents that form sulfone groups (-S(O)-R) are selected from alkyl sulfone groups, cycloalkyl sulfone groups, alkenyl sulfone groups, alkynyl sulfone groups, aryl sulfone groups, heteroaryl sulfone groups, aralkyl sulfone groups, etc.; and substituents that form sulfonic acids include sulfonic acid groups (-SOH).

[0043] Examples of substituents derived from N atoms include an azide group (-N3) and a nitrile group (-CN). Examples of substituents that form primary amines include an amino group (-NH2). Examples of substituents that form secondary amines (-NH-R) include alkylamino groups, cycloalkylamino groups, alkenylamino groups, alkynylamino groups, arylamino groups, heteroarylamino groups, and aralkylamino groups. Examples of substituents that form tertiary amines (-NR(R')) include alkyl(aralkyl)amino groups, and any two or two identical substituents selected from alkyl groups, cycloalkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, and aralkyl groups, or substituents that may form a ring. Examples of substituents that form a tertiary amine (-NR(R')) include alkyl(aralkyl)amino groups, alkyl groups, cycloalkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, and aralkyl groups, or substituents selected from substituents that may form a ring. Examples of substituents that form a tertiary amine (-NR(R')) include alkyl(aralkyl)amino groups, alkyl groups, cycloalkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, and aralkyl groups. Examples of substituents that form a ═NR)-NR'R") include an amidino group (-C(═NH)-NH2), or an alkyl(aralkyl)(aryl)amidino group in which three substituents on the N atom are substituted with any three of the following groups, which may be the same or different: an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, and an aralkyl group. Examples of substituents that form a guanidino group (-NR-C(═NR''')-NR'R") include a guanidine group (-NH-C(═NH)-NH2), or any four of R, R', R", and R''' that may be the same or different and are selected from alkyl groups, cycloalkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, and aralkyl groups, or substituents in which these groups may form a ring.

[0044] An example of a substituent that forms a urea group is an aminocarbamoyl group (—NR—CO—NR′R″). R, R′, and R″ each represent a hydrogen atom, or any three of the following groups that may be the same or different: an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, and an aralkyl group; or substituents selected from substituents that may form a ring.

[0045] Examples of functional groups derived from B atoms include alkylborane (-BR(R')) and alkoxyborane (-B(OR)(OR')). These two substituents may be any two or two identical substituents selected from alkyl groups, cycloalkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, aralkyl groups, etc., or substituents selected from substituents which may form a ring.

[0046] In this way, one or more of various functional groups containing an O atom, N atom, S atom, B atom, P atom, Si atom, or halogen atom, such as a halogen group, which are typically used in low molecular weight compounds, may be added. In other words, one or more additional substituents may be added to the alkyl group, cycloalkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, or aralkyl group shown as one of these substituents. A functional group that satisfies all of these conditions is defined as the ability to freely select substituents. In the case of β- and γ-amino acids, any stereoconfiguration is allowed, just as in the case of α-amino acids, and the selection of their side chains is also not particularly limited, just like in the case of α-amino acids. Furthermore, the amino group site on the main chain of the amino acid may be free (NH group) or may be N-alkylated, such as N-methylated (NHR group: R represents an alkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, aralkyl group, or cycloalkyl group that may be freely substituted, and the carbon atom coming from the N atom and the carbon atom from the α-position may form a ring, as in proline. The substituent can be freely selected, and examples thereof include a halogen group, an ether group, and a hydroxyl group.).

[0047] As used herein, a "translatable amino acid" or a "translatable amino acid" refers to an "amino acid" having a side chain that can be translated. As described hereinafter in the specification, an alkyl group, an alkenyl group, or a hydroxyl group (-OH), an alkoxy group (-OR), an ester group (-C(=O)-OR), a thioester group (-C(=O)-SR), a carboxyl group (-COH), an amide group (-CO-NRR" or -NR-CO-R'), a thiol group (-SH), an alkylthio group (-SR), a sulfoxide group (-S(=O)-R), a sulfone group (-SO2-R), an amino group (-NH2), a monosubstituted amino group (-NHR), a disubstituted amino group (-NRR'), an azide group (-N3), a nitrile group (-CN), an amidino group (-NC(=N)-NH2), ... These include L-type α-amino acids with alkynyl, aralkyl, aryl, heteroaryl, or cycloalkyl groups, N-methylated L-type α-amino acids, N-ethylated or N-propylated C1-C4 alkyl-substituted or N-benzylated N-aralkyl-substituted glycine derivatives, and L-type α-amino acids with various reactive functional groups that can be used in triangle or intersection units, such as substituents with reaction promoting groups such as thiol groups, or amino groups. Also included are some D-type α-amino acids such as D-tyrosine, β-amino acids such as β-alanine, and α,α-dialkylamino acids such as α-methyl-alanine (Aib).

[0048] As used herein, "drug-like amino acids" refer to amino acids with the same skeleton as "amino acids," i.e., α, β, and γ amino acids, in which one of the two hydrogen atoms in the main chain amino group (NH group) and one or two hydrogen atoms in the methylene group (-CH- group) may be substituted with an alkyl group, cycloalkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, aralkyl group, etc. The group in which the hydrogen atom in the CH group is substituted with one of the above groups forms a side chain. These substituents also include those substituted with substituents that function as building blocks of drug-like peptide compounds. These are preferably selected from the substituents separately defined above, and may be substituted with one or more substituents selected from, for example, a hydroxyl group (-OH), an alkoxy group (-OR), an amide group (-NR-CO-R' or -CO-NRR'), a sulfone group (-SO2-R), a sulfoxide group (-SO-R), a halogen group, a hydroxylamino group (-NR-OR'), and an aminohydroxy group (-O-NRR'). Furthermore, they may correspond to L-amino acids, D-amino acids, and α,α-dialkylamino acids. Drug-like amino acids do not necessarily need to be translatable. They include the side chain portion of a peptide obtained from a "translated amino acid" (for example, if a hit compound is obtained using D-tyrosine, a D-amino acid chemically modified from D-tyrosine; if a hit compound is obtained using β-alanine, a β-amino acid chemically modified from D-alanine), or all amino acids that can be chemically synthesized by structural optimization of the N-substituted portion through chemical conversion of N-methylamino acids. These amino acids function as building blocks of drug-like peptide compounds, and are selected from a range that allows the resulting peptide compound to be drug-like through post-translational chemical modification. As described below, for example, lysine having an aminoalkyl group is not included in drug-like amino acids if the amino group is not involved in post-translational modification. However, when the amino group of lysine is utilized as a reactive functional group in post-translational modification (e.g., a crossover unit), the lysine unit is included as a drug-like amino acid unit.Thus, whether an amino acid is a "drug-like amino acid" is determined by the functional group after post-translational modification. Examples of such substituents include, from the substituents defined separately above, an ester group (-CO-OR), a thioester group (-CO-SR), a thiol group (-SH), or a protected thiol group, an amino group (-NH), a mono-substituted amino group (-NH-R) or a di-substituted amino group (-NRR'), or a protected amino group, a substituted sulfonylamino group (-NH-SO-R), an alkylborane group (-BRR'), an alkoxyborane group (-B(OR)(OR')), an azide group (-N), a keto acid group (-CO-COH), a thiocarboxylic acid group (-CO-SH), a phosphoryl ester group (-CO-PO(R)(R')), and an acylhydroxylamino group (-NH-O-CO-R).

[0049] The amino acid analog of the present invention preferably refers to an α-hydroxycarboxylic acid. The side chain of the α-hydroxycarboxylic acid may have various substituents other than hydrogen atoms, as in amino acids (it may have a free substituent). The three-dimensional structure of the α-hydroxycarboxylic acid may correspond to either the L-type or D-type of an amino acid, and the side chain is not particularly limited and may be freely selected from, for example, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, aralkyl groups, cycloalkyl groups, and the like, which may be freely substituted. The number of substituents is not limited to one, and may be two or more. For example, the α-hydroxycarboxylic acid may have an S atom and may further have a functional group such as an amino group or a halogen group.

[0050] As used herein, "translatable amino acid analog" or "translatable amino acid analog" refers to an amino acid analog that can be translated among "amino acid analogs." Specific examples include compounds in which the main chain amino group of an L-amino acid is replaced with a hydroxyl group. Examples include L-lactic acid, α-hydroxyacetic acid, and L- or D-phenyllactic acid. Furthermore, the term "drug-like amino acid analog" is not particularly limited as long as it is an "amino acid analog" that functions as a component of a drug-like peptide compound. This scope is the same as the definition of the side chain or N-substituted portion of a drug-like amino acid described above. Specific examples include L- or D-lactic acid or compounds in which various drug-like substituents (e.g., halogen groups, hydroxyl groups, optionally substituted alkyl groups, alkenyl groups, alkynyl groups, cycloalkyl groups, aralkyl groups, aryl groups, heteroaryl groups, etc.) have been added to the side chain methyl group; α-hydroxyacetic acid, L- or D-phenyllactic acid, or compounds in which various drug-like substituents (e.g., halogen groups, hydroxyl groups, optionally substituted alkyl groups, alkenyl groups, alkynyl groups, cycloalkyl groups, aralkyl groups, aryl groups, heteroaryl groups, etc.) have been added to the side chain benzyl group; and the like. Drug-like amino acid analogs do not necessarily need to be translatable. When a peptide compound becomes drug-like through post-translational chemical modification of a "translated amino acid analog," the amino acid analog is also included in the "drug-like amino acid analog." Examples of such amino acid analogs include α-hydroxycarboxylic acids with an SH group attached to the side chain and α-hydroxycarboxylic acids with an amino group or a protected amine moiety attached to the side chain. For example, the SH group can be removed by desulfurization after post-translational modification, and the amino group can be converted to an amide or other form by post-translational modification. A specific example is R-2-hydroxy-3-sulfanylpropanoic acid.

[0051] The N-terminal carboxylic acid analogs of the present invention may be compounds having both an amino group and a carboxyl group and having three or more atoms between them, various carboxylic acid derivatives without an amino group, peptides consisting of two to four residues, or amino acids in which the main chain amino group is chemically modified by an amide bond with a carboxylic acid. They may also have a boric acid or borate ester moiety that can be used for cyclization at curved portions. They may also be carboxylic acids having a double bond or triple bond, or carboxylic acids having a ketone or halide. In addition, the moieties (free substituents) of these compounds other than the specified functional groups may be selected from a wide range of groups, including optionally substituted alkyl groups, aralkyl groups, aryl groups, cycloalkyl groups, heteroaryl groups, alkenyl groups, and alkynyl groups.

[0052] As used herein, "translatable N-terminal carboxylic acid analogs" or "translatable N-terminal carboxylic acid analogs" refer to N-terminal carboxylic acid analogs that can be translated among "N-terminal carboxylic acid analogs." Specific examples include compounds in which a double bond and a carboxylic acid are connected via an alkyl group (e.g., but-3-enoic acid, pent-4-enoic acid), L-amino acids whose N-termini are amidated by acetylation or other methods (e.g., Ac-Phe, Ac-Ala, Ac-Leu), and α-hydroxycarboxylic acid derivatives, dipeptides, and tripeptides whose OH groups are alkylated. Furthermore, "drug-like N-terminal carboxylic acid analogs" are not particularly limited as long as they function as components of drug-like peptide compounds among "N-terminal carboxylic acid analogs." These substituents include the same as those defined for the side chains of drug-like amino acids. Specifically, examples of such compounds include compounds in which a double bond and a carboxylic acid are connected by an alkyl group (e.g., but-3-enoic acid, pent-4-enoic acid), in which a substituent has been added to the carbon atom in a drug-like range; L-amino acids (e.g., Ac-Phe, Ac-Ala, Ac-Leu) in which the N-terminus is amidated by acetylation or the like, in which the acetyl group, the side chain, or the hydrogen atom at the α-position is substituted in a drug-like range; α-hydroxycarboxylic acid derivatives in which the OH group is alkylated, in which the alkyl group at the OH group, the side chain of the hydroxycarboxylic acid, or the hydrogen atom at the α-position is substituted in a drug-like range; and dipeptides and tripeptides substituted in a drug-like range. Furthermore, drug-like N-terminal carboxylic acid analogs do not necessarily need to be translatable. When a "translated N-terminal carboxylic acid analog" is chemically modified after translation to make the peptide compound drug-like, the N-terminal carboxylic acid analog is also included in the "drug-like N-terminal carboxylic acid analog." Examples of such N-terminal carboxylic acid analogs include dipeptides, γ-aminocarboxylic acids, and δ-aminocarboxylic acids, all of which retain an amino group at the N-terminus.

[0053] Membrane permeability of peptide compounds In order for the peptide compound of the present invention to have good membrane permeability, the total number of amino acids contained in the peptide compound is preferably 13 or less. There are no particular restrictions on the selection of each unit (amino acid residue), but it is preferable to select the units so that the CLogP (a computer-calculated partition coefficient calculated using Daylight Version 4.9 from Daylight Chemical Information Systems, Inc.) of the resulting molecule exceeds 6, taking into consideration the shape of the molecule (main chain structure) after all post-translational chemical modifications have been completed. In particular, to ensure good membrane permeability, it is preferable to select the CLogP to be greater than 8 but not greater than 15.

[0054] To ensure membrane permeability, the amino acid side chain is preferably selected from drug-like substituents. For example, optionally substituted alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and heteroaryl groups are preferred, including those with halogen, hydroxyl (-OH), amide (-CO-NR-R' or -NR-CO-R'), sulfone (-SO-R), and ether (-OR) groups as substituents (R and R' are similarly selected from optionally substituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl groups). The aryl moiety of the aryl and aralkyl groups may be, in addition to a phenyl group, a basic group such as a pyridine group, a group containing two or more heteroatoms such as a thiazole group, a hydrogen atom donor such as an imidazole group, or a fused aromatic ring such as an indole group.

[0055] On the other hand, polar functional groups that are undesirable for achieving membrane permeability include alkylamino groups and alkylguanidine groups, which are highly ionized in vivo (around pH 7), and it is preferable that these functional groups are not included.

[0056] To achieve membrane permeability, amino acids or amino acid analogs constituting a peptide compound may be N-methylated or N-alkylated, such as by cyclization with the α-carbon atom, as in proline. Preferably, two or more N-alkylated amino acids are present per peptide molecule. Furthermore, it is preferred that at least one non-N-alkylated amide bond is present per peptide molecule. Desirably, three or more N-alkylated and three or more non-N-alkylated amino acids are present per peptide molecule. This N-alkylation includes all chemical modifications other than NH, and therefore may be selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl groups, which may be substituted (the selection of substituents is similar to that for the amino acid side chain substituents for ensuring membrane permeability). Furthermore, N-alkylation also includes those that form a ring structure between the N atom and the α-carbon, as in proline.

[0057] It is more preferable that the C-terminal portion of the peptide compound is chemically modified rather than remaining as a carboxylic acid. For example, the carboxylic acid portion can be converted into an optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or aralkyl amide compound, such as piperidine amide, by reacting the carboxylic acid portion with piperidine or the like (-CO-NRR': one of R and R' may be a hydrogen atom and the other may be chemically modified, or both R and R' may be chemically modified, or R and R' may form a ring, such as piperidine amide, or both R and R' may be hydrogen atoms). Alternatively, the carboxylic acid group can be converted into various nonionic functional groups, such as optionally substituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl groups, such as methyl or trifluoromethyl groups. The substituents can be selected in the same manner as for the amino acid side chain substituents for ensuring membrane permeability. Furthermore, the amino acids constituting the peptide compound can also be optimized for translationally synthesized compounds. Furthermore, the amino acids constituting the resulting peptide compound are not limited to those synthesized by translational synthesis.

[0058] Here, optimization refers to chemical modification of the amino acids in a compound translationally synthesized from "translated amino acids" to produce a more drug-like peptide compound, a peptide compound with stronger activity against a pharmacological target, and / or a peptide compound with reduced toxicity. Examples of toxicity that should be avoided or are preferable include hERG inhibition (cardiac toxicity), AMES (carcinogenicity test), CYP inhibition (drug-drug interaction test), CYP induction, and GSH binding assay (a test for covalent bond formation between a peptide or peptide metabolite and glutathione). These assays may involve both the active peptide compound itself and its metabolites. To ensure negative results, particularly in AMES, CYP induction, and GSH binding assays, it is preferable to avoid metabolites that may form covalent bonds. Therefore, it is preferable that all peptide compounds in the display library be cyclized by a cyclization reaction that does not form such potential functional groups, particularly one that has a certain degree of stability against oxidation. For example, if phenylalanine is identified as the translated amino acid, various chemical modifications such as alkyl and halogen substitutions can be performed on the phenyl group, as is the case with many small molecule compounds. These modifications differ significantly from traditional chemical modifications of natural peptides, such as N-methylation and cyclization, in that they can be performed without significantly disrupting the three-dimensional structure of the translationally synthesized compound. Furthermore, in natural peptides, highly ionized arginine and lysine residues often contribute to activity, making it difficult to easily convert these residues to drug-like functional groups. By utilizing this technology, only drug-like functional groups are pre-defined, conferring activity against the target and cyclizing the resulting peptides using a drug-like cyclization method. Therefore, chemical modifications can be performed as is typically done in small molecule chemistry, enabling the development of clinical candidate compounds with the same accuracy and success rate as small molecule chemistry. Meanwhile, the CLogP value, an indicator of lipophilicity, can be easily adjusted during the optimization process.In addition, alkylation and halogenation, which are commonly performed in the optimization of small molecules, can enhance target binding activity. A 10- to 50-fold increase in activity can typically be expected. These common conversions can also simultaneously increase the CLogP value. For example, chlorination can increase it by approximately 0.7, and methylation can increase it by approximately 0.5. In the case of compounds with highly ionized functional groups, increasing the CLogP value during optimization is insufficient to achieve excellent membrane permeability. However, in the case of compounds without highly ionized functional groups, a CLogP value sufficient for membrane permeability can be achieved during optimization. Therefore, for example, if a hit compound with a CLogP value of 5 is obtained from a display library with an average CLogP value of around 6, it is likely feasible to further increase the CLogP value and optimize it to the optimal range of 8 to 15 for membrane permeability.

[0059] The membrane permeability of the peptide compounds of the present invention can be confirmed using known methods, such as the rat intestinal method, the cultured cell (Caco-2, MDCK, HT-29, LLC-PK1, etc.) monolayer membrane method, the immobilized artificial membrane chromatography method, the method using partition coefficients, the ribosomal membrane method, and the parallel artificial membrane permeation assay (PAMPA) method. Specifically, when using the PAMPA method, for example, confirmation can be performed according to the description of Holger Fischer et al. (Non-patent document: H. Fischer et al. Permeation of permanently positively charged molecules through artificial membranes—influence of physicochemical properties. Eur J. Pharm. Sci. 2007, 31, 32-42). More specifically, confirmation can be performed according to the method described in Example 19-2.

[0060] When the membrane permeability of oral pharmaceuticals, hydrochlorothiazide, furosemide, and metoprolol, was measured by the PAMPA method, the iPAMPA Pe values ​​were 0.6X10 -6 , 1.5X10 -6 and 2.9X10 -5 The membrane permeability of the peptide compound of the present invention is, for example, measured by the PAMPA method, typically expressed as an iPAMPA Pe of 1.0 x 10 -6 When the membrane permeability is 1.0x 10 or more, it can be considered that the drug can be used as a pharmaceutical. -6 It is preferable that it is 1.0X10 or more. -5 More preferably, it is 1.5X10 or more. -5 More preferably, it is 2.0X10 or more. -5 It is even more preferable that the above is true.

[0061] ·Metabolic stability of peptide compounds In order for the peptide compound of the present invention to have good metabolic stability, the total number of amino acids contained in the peptide compound is preferably 9 or more, and more preferably 11 or more. If the total number of amino acids in the peptide compound is 9 or more, the above-mentioned conditions for membrane permeability do not affect the metabolic stability of the peptide compound.

[0062] The metabolic stability of the peptide compounds of the present invention can be confirmed by known methods, for example, using hepatocytes, small intestinal cells, liver microsomes, small intestinal microsomes, liver S9, etc. Specifically, for example, the stability of the peptide compounds in liver microsomes can be confirmed by measuring the stability according to the description in the literature of LL von Moltke et al. (Midazolam hydroxylation by human liver microsomes in vitro: inhibition by fluoxetine, norfluoxetine, and by azole antifungal agents. J Clin Pharmacol, 1996, 36(9), 783-791). More specifically, the stability can be confirmed according to the method described in Example 18-2.

[0063] Regarding metabolic stability, for example, when stability in liver microsomes is measured according to the method described above, a liver intrinsic clearance (CLh int (μL / min / mg protein)) of 150 or less can be considered to be metabolically stable enough for use as an oral pharmaceutical, and preferably 100 or less. For drugs metabolized by CYP3A4, a CLh of 78 or less is preferred to avoid small intestinal metabolism in humans (Non-patent document: M. Kato et al., The intestinal first-pass metabolism of substances of CYP3A4 and P-glycoprotein - quantitative analysis based on information from the literature. Drug Metab. Pharmacokinet. 2003, 18(6), 365-372.), and a CLh of 35 or less (assuming FaFg = 1, 0% protein binding) is even more preferred to demonstrate bioavailability of approximately 30% or more in humans.

[0064] Scheme A [ka]

[0065] Method for producing peptide compounds having a cyclic portion The peptide compound having a cyclic portion of the present invention can be produced using the method described below. For example, the following manufacturing method can be mentioned. 1) synthesizing an acyclic peptide compound composed of amino acid residues and / or amino acid analog residues, or amino acid residues and / or amino acid analog residues and an N-terminal carboxylic acid analog, by translating a nucleic acid encoding the peptide compound, the non-cyclic peptide compound comprises an amino acid residue or amino acid analog residue having a reactive site at one side chain on the C-terminal side, and an amino acid residue, amino acid analog residue, or N-terminal carboxylic acid analog having another reactive site on the N-terminal side; 2) A step of bonding the reactive site of the N-terminal amino acid residue, amino acid analog residue, or N-terminal carboxylic acid analog with the reactive site of the amino acid residue or amino acid analog residue in the side chain on the C-terminal side to form an amide bond or a carbon-carbon bond. The method comprising: For the translational synthesis of the present invention, known methods can be used.

[0066] Introduction of a translatable amino acid, a translatable amino acid analog, or a translatable N-terminal carboxylic acid analog into the N-terminus Normally, methionine is generally used as the translation initiation amino acid for translation at the N-terminus. However, a method can also be used in which a translation initiation tRNA aminoacylated with another desired amino acid is used to translate the desired amino acid at the N-terminus. The tolerance for introducing an unnatural amino acid into the N-terminus is higher than that during elongation, and it is known that amino acids or amino acid analogs with structures significantly different from those of natural amino acids are used (Non-Patent Document: J Am Chem Soc. 2009 Apr 15;131(14):5040-1. Translation initiation with initiator tRNA charged with exotic peptides. Goto Y, Suga H.). For example, in the present invention, the following methods can be used to introduce a translatable amino acid other than methionine, a translatable amino acid analog, or a translatable N-terminal carboxylic acid derivative into the N-terminus. A tRNA acylated with a triangle unit as a translation initiation tRNA is added to a translation system lacking methionine, formyl donor, or methionyltransferase, and the triangle unit is encoded by a translation initiation codon (e.g., ATG) and translated to construct a pre-cyclization peptide compound or peptide compound library with a triangle unit at the end. Using various combinations of translation initiation tRNAs with anticodons other than CAU and the codons corresponding to the anticodons as combinations of translation initiation tRNAs and initiation codons can create diversity at the N-terminus. In other words, by aminoacylating desired amino acids, amino acid analogs, or N-terminal carboxylic acid analogs with multiple types of translation initiation tRNAs with different anticodons and translating mRNA or an mRNA library with the corresponding codon as the initiation codon, a library of pre-cyclization peptide compounds or peptide compounds with a non-unique N-terminal residue can be produced.Specifically, for example, a peptide compound or a peptide compound library before cyclization can be prepared using the method described in Mayer C, et al. Anticodon sequence mutants of Escherichia coli initiator tRNA: effects of overproduction of aminoacyl-tRNA synthetases, methionyl-tRNA formyltransferase, and initiation factor 2 on activity ininitiation. Biochemistry. 2003, 42, 4787-99. (translation initiation from an amino acid other than f-Met using mutant Escherichia coli initiator tRNA with an anticodon other than CAU and expression of a protein containing the same codon midway.)

[0067] Furthermore, examples of methods for bonding the reactive site of an N-terminal amino acid residue, amino acid analog residue, or N-terminal carboxylic acid analog with the reactive site of an amino acid residue or amino acid analog residue in a side chain on the C-terminal side include a method of amide-bonding an amino acid residue, amino acid analog residue, or N-terminal carboxylic acid analog having an amino group and a reaction promoting group in the N-terminal side chain with an amino acid residue or amino acid analog residue having an activated ester group in the side chain to obtain a cyclic compound; a method of amide-bonding an N-terminal amino acid residue, amino acid analog residue, or N-terminal carboxylic acid analog having an amino group with an amino acid residue or amino acid residue having an activated ester group in the side chain to obtain a cyclic compound; and a method of forming a carbon-carbon bond between the reactive site of an N-terminal amino acid residue, N-terminal amino acid analog residue, or N-terminal carboxylic acid analog with the reactive site of an amino acid residue or amino acid analog having one reactive site in the side chain. Furthermore, without being limited to the above examples, the functional group arrangement may be reversed from that described above, for example, by having an active ester group on the N-terminal amino acid residue, N-terminal amino acid analog, or N-terminal carboxylic acid side, and an amino group (which may have a reaction promoting group) on the C-terminal side side via an amide bond.

[0068] The following describes the reaction design using amide cyclization as an example. To achieve reaction selectivity between the desired amino group of the triangle unit and the undesired basic functional group, it may be necessary to improve the reactivity of the desired amine relative to other functional groups. A typical method for activating an amine is to introduce a reaction promoter near the amine. The reaction promoter is not particularly limited as long as it activates the amine to a degree that prevents RNA reaction. For example, a mercaptoethyl or mercaptopropyl group can be introduced from the terminal amine. It is also possible to position a thiol moiety at the α-position of the amine, as in cysteine ​​(see Scheme C). These thiol groups can be protected or unprotected during translational incorporation; however, deprotection reactions can be performed during or before the reaction to generate thiols, as needed. Reaction of the activated amine with a carboxylic acid activated ester yields a peptide amide-cyclized at the desired position. The SH groups of the resulting cyclic peptides can be desulfurized under mild reaction conditions that do not react with RNA, such as by adding reagents such as TCEP (tris(2-carboxyethyl)phosphine) and VA-044 (2,2'-azobis-2-(2-imidazolin-2-yl)propane).

[0069] When creating a display library by translation, a thioester is preferred as the active ester of the intersection unit to be translationally introduced. When an SH group is used as the reaction promoting group, the following combinations are used, for example: When translational synthesis is performed with the SH group unprotected, it is preferable to use an aspartic acid derivative as the thioester intersection unit. In this case, all of the black circle units and square units can be arbitrarily selected from translated amino acids or translated amino acid analogs. On the other hand, when translational synthesis is performed with the SH group protected, it is preferable to select the square unit adjacent to the C-terminus of the aspartic acid thioester from N-alkylated amino acids (e.g., proline, N-methylalanine, etc.).

[0070] Scheme C [ka]

[0071] [ka]

[0072] As a general formula, an amino acid having a reaction promoting group near the amine as a triangle unit, an N-terminal carboxylic acid analog, for example, an N-terminal amino acid, can be written as compound N-1 or N-2. The substituents of these compounds N-1 and N-2, among the substituents represented by R, preferably, the substituents used other than protecting groups (R1, R23, trityl group, etc.) are the same as those defined above for the side chain of a drug-like amino acid. Furthermore, it is more preferable that the compound obtained as a result of their introduction can be translationally synthesized. However, even if the derivative itself is not translationally synthesized, its analog can also be translationally synthesized (see the following paragraph (e.g., the paragraph three after)).

[0073] R1 is selected from a hydrogen atom, an S-R23 group, or a protecting group for an SH group represented by a C(Phe)3 group (trityl group), etc. R23 is selected from alkyl groups such as methyl, ethyl, isopropyl, and tert-butyl; aryl groups such as phenyl, p-trifluoromethylphenyl, and p-fluorophenyl; aralkyl groups such as benzyl and phenethyl; heteroaryl groups; alkenyl groups; and alkynyl groups. These groups are selected from substituents that allow the resulting compound N-1 or N-2 to be translationally synthesized. For example, an N,N-dimethylaminoethylmercapto group, in which the ethyl group in R23 is replaced with a dimethylamino group, may be used. When R1 has a protecting group (other than a H atom), the protecting group may be selected from protecting groups that allow translational synthesis and that are deprotected in the translational synthesis solution to become a hydrogen atom before cyclization, without any particular limitation. Protecting groups such as the S-R23 group are slowly deprotected in the translation synthesis solution, so deprotection can be performed without the need to actively determine separate deprotection conditions. If necessary, a deprotecting agent can be added under the various reaction conditions described herein (see the description of protecting groups for SH groups).

[0074] R2 and R3 are defined in the same way as the side chain of a drug-like amino acid. For example, R2 and R3 preferably represent a hydrogen atom, an optionally substituted alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an aralkyl group, or a cycloalkyl group, or a substituent in which R2 and R3 form a ring, or a substituent in which R2 or R3 form a ring with R4. More preferably, they are selected from a hydrogen atom, a C1-C4 alkyl group optionally substituted with a C1-C4 alkyl group, an alkoxy group, a halogen group, or the like. The configuration of the R3 group is acceptable for both L-amino acids and D-amino acids. Preferably, the configuration of the R3 group corresponds to an L-amino acid when the R3 group is assumed to be a hydrogen atom.

[0075] R4 is a unit that links the S (sulfur) atom and the amino acid moiety. Representative structures are shown below. Both units can be linked by C1-C6 units such as an optionally substituted methylene group (partial structure N-3, C1 unit), an optionally substituted ethylene group (partial structure N-4, C2 unit), or an optionally substituted propylene group (partial structure N-5, C3 unit). Examples of substituents in the optionally substituted methylene, ethylene, and propylene groups include compound N-1 in which R13 is methylated (R14=H) and compound N-1 in which R13 is dimethylated, such as R13=R14=Me. If any of these derivatives is translationally synthesized, all other derivatives are included in this definition, even if they are not translationally synthesized. For example, if R13=R14=H is translationally synthesized, a derivative such as R13=R14=Me is included as a triangle unit because these substituents are included in the definition of a drug-like amino acid side chain, even if they are not translationally synthesized. R13, R14, R15, R16, R17, R18, R19, R20, R21, and R22 are defined similarly to R4, but are selected from, for example, a hydrogen atom, a C1-C4 alkyl group, an alkoxy group, a C1-C4 alkyl group optionally substituted with a halogen atom, or the like. A cyclized structure may be formed between these groups. Particularly preferred are hydrogen atoms and methyl groups. They may also be directly linked to the aryl carbon of an aromatic compound (partial structure N-6). They may also be linked via an aralkyl structure (partial structures N-7 and N-8). In partial structure N-7, either the nitrogen atom or the sulfur atom of the divalent ring may be linked. In the scheme below, the linking position is limited to ortho, but is not limited to ortho and can also be meta or para. While a phenyl group is shown as the aryl group, the phenyl group may be substituted with a halogen atom, an alkoxy group, a trifluoromethyl group, or other substituent. Furthermore, aryl groups other than phenyl (i.e., various aromatic rings, including heteroaryl groups) may also be used.

[0076] [ka]

[0077] R11 and R12 are also selected from the same partial structures as R4. For example, they can be selected from partial structures N-3, N-4, N-5, N-6, N-7, and N-8. R12 can also include a CO unit (when the linking site is directly bonded).

[0078] Preferred structural formulas of compound structure N-1 and compound structure N-2 are shown in compound structures N-9, N-10, N-11, and N-12. In compound N-9, the R12 moiety of compound N-1 is directly linked to the C0 unit. In compound N-10, the R11 moiety of compound N-2 is linked to the C1 unit (corresponding to partial structure N-3). In compound N-11, the R11 moiety of compound N-2 is linked to the C2 unit (corresponding to partial structure N-4). In compound N-12, the R11 moiety of compound N-2 is linked to the C3 unit (corresponding to partial bond N-5). [ka] R5 to R10 have the same definitions as R13 to R18.

[0079] Furthermore, the preferred structural formulas of these compounds are shown below as compounds N-13, N-14, N-15, N-16, N-17, N-18, N-19, and N-20. In compound N-13, the R4 moieties of compound N-9 are linked by a C1 unit (corresponding to partial structure N-3). In compound N-14, the R4 moieties of compound N-10 are linked by a C2 unit (corresponding to partial structure N-4). In compound N-15, the R4 moieties of compound N-11 are linked by a C2 unit (corresponding to partial structure N-4). In compound N-16, the R4 moieties of compound N-12 are linked by a C2 unit (corresponding to partial structure N-4). In compound N-17, the R4 moieties of compound N-9 are linked by a C2 unit (partial structure N-4). In compound N-18, the R4 moieties of compound N-10 are linked by a C3 unit (partial structure N-5). In compound N-19, the R4 moieties of compound N-11 are linked by a C3 unit (partial structure N-5), and in compound N-20, the R4 moieties of compound N-12 are linked by a C3 unit (partial structure N-5). [ka]

[0080] Although the chemical structures represented by general formula compounds N-1 and N-2 have been described so far, the definition of the triangle unit containing the reaction promoter group SH is not limited to these. That is, the triangle unit has an amino group and a reaction promoter group, which are units that react with the intersection unit, and can be selected from any structure that can be translated into synthesis. The amino group may be derived from either the main chain or the side chain. The triangle unit does not necessarily have to be located at the N-terminus; a square unit (linear portion) may be located N-terminally of the triangle unit. A chemical structure in which the positional relationship between the SH group and the amino group is β (two linking atoms between the two functional groups) or γ (three linking atoms), with 2 to 6 linking atoms, is preferred. More preferably, the positional relationship between the SH group and the amino group is β or γ. Furthermore, even if a unit with an active ester functional group is located in the triangle unit, an amine-containing unit may be located on the intersection unit side.

[0081] The general formula of an amino acid residue having an activated ester group in the side chain can be represented as Compound C-1. Preferably, the substituents excluding the activated ester moiety are the same as those defined for the side chain of a drug-like amino acid, as described above. Even if the derivative itself is not translationally synthesized, its analogue may be translationally synthesized. In this application, the term "activated ester" refers to a carboxylic acid derivative that can react with an amino group moiety directly or via a reaction promoting group. It is not particularly limited as long as it has such properties as an activated ester or activated thioester. R25 is selected from a hydrogen atom or an activated ester group. Examples of activated esters include the widely used N-hydroxysuccinimide (ONSu) group, OAt group, OBt group, methyl thioester, aryl thioester, aralkyl thioester, etc. All derivatives exhibiting similar reactivity are included, even if they have been given a commonly used compound substituent at these active ester sites (for example, examples of the substituent include electron-withdrawing groups such as halogen groups, nitro groups, trifluoromethyl groups, and nitrile groups that are often used to increase reactivity, electron-donating groups such as alkoxy groups such as methoxy groups and alkyl groups such as methyl groups that are often used to reduce reactivity and increase reaction selectivity, bulky substituents typified by t-butyl groups and isopropyl groups, sulfonic acid groups and di-substituted amino groups such as dimethylamino groups that take into account affinity with water for implementation in water, and conversely, highly lipophilic groups such as long-chain alkyl groups that take into account affinity with lipophilicity).

[0082] R2 and R3 are as defined above for the amine moiety.

[0083] R26 has the same definition as R4, and its representative structure is shown below. Both units can be linked by a C1-C6 unit such as a methylene group (partial structure N-3), an ethylene group (partial structure N-4), or a propylene group (partial structure N-5). For R13, R14, R15, R16, R17, R18, R19, R20, R21, and R22, these substituents are preferably the same as those defined for the side chains of drug-like amino acids as above. These include derivatives that are not translationally synthesized, but whose analogs are translationally synthesized. For example, they are selected from hydrogen atoms, C1-C4 alkyl groups, and C1-C4 alkyl groups optionally substituted with halogen atoms. A cyclized structure may be formed between these. More preferably, they are selected from a methylene group (C1 unit, partial structure N-3), a C4 unit, a C5 unit, and a C-6 unit. Even more preferably, a C1 unit (partial structure N-3) is selected. It can also be linked directly from the aryl carbon of an aromatic compound (partial structure N-6). It can also be linked via an aralkyl structure (partial structures N-7 and N-8). In the scheme below, the linking position is limited to ortho, but it is not limited to ortho and meta, para, etc. are also possible. Although a phenyl group is shown as the aryl group, the phenyl group may be substituted with a substituent such as a halogen group or an alkoxy group, and aryl groups other than phenyl may also be used.

[0084] Among Compound C-1, a preferred structure is shown in Compound C-2. R27 is selected from a hydrogen atom, an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted alkynyl group, an optionally substituted aryl group, an optionally substituted heteroaryl group, an optionally substituted cycloalkyl group, and an aralkyl group to which an optionally substituted alkyl group may be attached. These substituents are not particularly limited, as long as Compound C-2, which is obtained as a result of the selection of the substituent, can be translationally synthesized. For example, the substituent may be selected from electron-withdrawing groups such as halogen groups, nitro groups, trifluoromethyl groups, and nitrile groups, which are often used to enhance reactivity; electron-donating groups such as alkoxy groups such as methoxy groups and alkyl groups such as methyl groups, which are often used to reduce reactivity and enhance reaction selectivity; bulky substituents such as t-butyl groups and isopropyl groups; sulfonic acid groups and disubstituted amino groups such as dimethylamino groups, which are used in consideration of affinity with water for use in water; and conversely, highly lipophilic groups such as long-chain alkyl groups, which are used in consideration of lipophilicity. Preferably, the substituent is selected from an optionally substituted alkyl group, an optionally substituted cycloalkyl group, or an optionally substituted aralkyl group. More preferably, it is selected from alkyl groups and aralkyl groups in which the aryl moiety may be substituted. R3 is defined in the same way as the side chain of a drug-like amino acid, and is selected from, for example, a C1-C4 alkyl group, a C1-C4 alkyl group optionally substituted with a halogen, etc., and is particularly preferably a hydrogen atom. The configuration of the R3 group is acceptable for both L- and D-amino acids when the R3 group is assumed to be a hydrogen atom, but is preferably one corresponding to an L-amino acid.

[0085] A more preferred structure is shown in Compound C-3. R28 and R29 are each defined in the same way as for the side chain of a drug-like amino acid, but are selected from, for example, a hydrogen atom, an optionally substituted C1-C6 alkyl group, an optionally substituted C2-C6 alkenyl group, an optionally substituted C2-C6 alkynyl group, an optionally substituted aryl group, an optionally substituted heteroaryl group, an aralkyl group to which an optionally substituted C1-C6 alkyl group may be attached, and an optionally substituted cycloalkyl group. Examples of these substituents include monomethylation (R28 = Me, R29 = H), dimethylation (R28 = R29 = Me), and monotrifluoromethylation (R28 = CF3, R29 = H).

[0086] R3 is selected from a hydrogen atom, a C1-C4 alkyl group, a C1-C4 alkyl group optionally substituted with halogen, etc., and the like, with a hydrogen atom being particularly preferred. Assuming that the R3 group is a hydrogen atom, the conformation of the R3 group may be either an L- or D-amino acid, but is preferably an L-amino acid. As with Compound C-1, Compound C-2, and Compound C-3, they can also be selected from Compound COH-1, Compound COH-2, and Compound COH-3.

[0087] [ka]

[0088] When Compound C-2 or Compound C-3 is used, the reaction with Compound N-1 or Compound N-2 can proceed mildly and selectively. The reaction can proceed smoothly even in a translation solution (e.g., 37°C, pH around 7.3). Removal of the reaction promoter can also proceed easily under reaction conditions in which RNA is stable.

[0089] When an activated ester is placed on the N-terminal side (triangle unit), an amine unit having a reaction promoting group may be placed on the C-terminal side (intersection unit). In this case, an amino group and a thiol group are placed on the side chain of the intersection unit. These may be protected during the translation stage, but are deprotected immediately before the reaction. There are no particular restrictions on the amino group and the thiol group as long as they are located nearby, but it is preferable that the relationship between them be the β- or γ-position.

[0090] That is, the present application also encompasses any method in which a triangle unit has an activated ester such as a thioester in the side chain and another (intersection unit) undergoes drug-like cyclization by an amide condensation reaction with an amino group (having a reaction promoting group such as a thiol nearby) in the side chain, and either functional group may be located in the triangle unit or the intersection unit.

[0091] Below, specific examples of structures different from those of Compounds N-1 and N-2 are shown as amine moieties having a reaction promoting group. In both cases, the amino group and thiol group may be protected as necessary. The methods described herein can be used to select the protecting group and deprotection reaction conditions. For Compound Na-10, as shown in the figure, a structure with two carbon atoms between the amino group and the thiol group can be selected. For Compound Na-11, as shown in the figure, a structure with three carbon atoms between the amino group and the thiol group can be selected. An Na-7 group, Na-8 group, or Na-9 group is placed as a substituent in one of Ra20 to Ra25. The definition of Ra7 is as described above, but only in the case of Compound Na-10 or Compound Na-11, an Na-7 group, Na-8 group, or Na-9 group can be selected for Ra7. The Na-7 group, Na-8 group, or Na-9 group can be selected to be limited to only one of Ra-7 or Ra20 to Ra25. Ra20 to Ra25 other than the substituent selected from Na-7, Na-8, and Na-9 groups are selected from hydrogen atoms, alkyl groups which may be substituted with drug-like functional groups, aryl groups, heteroaryl groups, aralkyl groups, etc. Preferably, they are selected from hydrogen atoms and alkyl groups.

[0092] When a unit having an active ester group such as a thioester group is selected for the triangle unit (N-terminal side), it can be selected from, for example, Compounds C-1, C-2, C-3, as well as Compounds Ca-1, COH-1, COH-2, and COH-3. Compounds C-1 and the like retain the main chain amino group after cyclization, whereas Compounds Ca-1, COH-1, COH-2, and COH-3 lack the amino group, making them more drug-like and more preferred. In this case, the intersection unit is selected from Compounds Na-10 (Na-7 group or Na-8 group) and Na-11 (Na-7 group or Na-8 group). These compounds may be translated in a protected state. The methods described herein can be used for the translatable protecting group and deprotection conditions under RNA-stable reaction conditions. More preferably, the Na-7 group is used, rather than the Na-8 group, due to its higher metabolic stability.

[0093] Furthermore, when Compounds C-1, C-2, and C-3, or Compounds COH-1, COH-2, and COH-3, each having an activated ester group at the intersection unit (C-terminal side), are selected, Compounds Na-10 and Na-11 can be selected for the triangle unit (N-terminal side), in addition to Compounds N-1 and N-2. When the triangle unit is at the N-terminus, Compounds Na-10 (Na-8 and Na-9 groups) and Na-11 (Na-8 and Na-9 groups) are preferably selected in addition to Compounds N-1 and N-2. This is because the use of the Na-7 group retains the main chain amine, reducing drug-likeness. On the other hand, when the triangle unit is not at the N-terminus, Compounds Na-10 (Na-7 and Na-8 groups) and Compounds Na-11 (Na-7 and Na-8 groups) can be used. In this case, it is more preferable to use an amino acid derivative or an N-terminal carboxylic acid derivative at the N-terminus. This is to prevent the main chain amino group from being retained at the N-terminus.

[0094] Furthermore, the groups imparted to Compounds Na-10 and Na-11 are not limited to Na-7, Na-8, and Na-9. For example, the Na-7 group is derived from an α-amino acid skeleton, but may be a β-amino acid skeleton.

[0095] [ka]

[0096] The method of activating a normal amine is not limited to the aforementioned method of containing an SH group as a reaction promoter. Alternatively, a heteroatom can be directly introduced into the amine to improve its reactivity. Examples of such methods include hydroxyamines (Compounds F-1, F-4, F-5, and F-7), alkoxyamines (Compounds F-2, F-14, F-15, and F-16), and azides (Compounds F-3, F-9, F-10, and F-11). Thus, all methods of activating an amino group by introducing a heteroatom that can function as a reaction promoter into the vicinity of the amino group, either directly or via a linker, are encompassed within the scope of the present application. For the reaction with hydroxyamine, compounds F-7 and F-8 are selected as the activated ester moiety. For the reaction with alkoxyamine, compounds F-17 and F-18 are selected. For the reaction with azide, compounds F-12 and F-13 are selected.

[0097] R101, R102, R103, R104, R105, R106, and R107 are substituents that are commonly used in amino acid side chains and are not limited to naturally occurring amino acids, i.e., selected from a hydrogen atom, an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted alkynyl group, an optionally substituted aryl group, an optionally substituted heteroaryl group, and an optionally substituted aralkyl group. It is more preferable that one of R101 and R102 is a hydrogen atom, one of R103 and R104 is a hydrogen atom, and one of R106 and R107 is a hydrogen atom. Furthermore, it is preferable that the hydrogen atoms are arranged so that they have the same configuration as an L-amino acid.

[0098] R105 is selected from an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted alkynyl group, an optionally substituted aryl group, and an optionally substituted aralkyl group.

[0099] [ka]

[0100] [ka]

[0101] [ka]

[0102] [ka]

[0103] When an activated amine such as the above is selected, possible combinations with an activated ester that can serve as a corresponding intersection unit include, for example, a combination of a thioester and an amine having a thiol nearby, or an α-ketoester and an azide. [ka]

[0104] By utilizing initiation read-through (skipping the initiation codon), there is a method for synthesizing peptide compounds or peptide compound libraries with diverse termini by introducing amino acids, amino acid analogs, or N-terminal carboxylic acid analogs other than methionine into the N-terminus, eliminating the need to prepare multiple types of aminoacyl translation initiation tRNAs. Generally, proteins and peptides are translated from the translation initiation amino acid methionine encoded by the AUC codon. However, in a cell-free translation system, when a translation initiation methionyl tRNA is not present or when an unnatural amino acid with low translation efficiency is added to the translation initiation tRNA, translation products are produced from amino acids encoded by the second or subsequent codons.

[0105] Initiation read-through can be used to generate peptides or peptide libraries with a triangle unit at the N-terminus by encoding a triangle unit in the second codon following the start codon of the peptide-encoding mRNA and translating the peptide in a translation system that does not contain methionine or a translation initiation methionine tRNA. Another method is known in which the N-terminal methionine of a peptide is removed by the action of enzymes such as peptide deformylase and methionine aminopeptidase (see, e.g., Meinnel, T., et al., Biochimie (1993) 75, 1061-1075, "Methionine astranslation start signal: A review of the enzymes of the pathway in Escherichia coli"). Alternatively, a library of peptides starting with a translation initiation methionine can be prepared, and then treated with methionine aminopeptidase to remove the N-terminal methionine, thereby preparing a library with random N-termini. We also demonstrated that N-terminal amino acids such as methionine can be removed by cyclization using the second amino acid following the translation initiation methionine, followed by aminopeptidase treatment. As a result, the methionine residue is not included in the unit of Scheme A (it has already been defined that the number of units is determined by the chemical structure after all post-translational modifications have been completed), and the amino acid residue corresponding to the triangle unit becomes the second encoded amino acid. Consequently, the triangle unit can be coded by multiple codons, expanding the degree of freedom to two or more types and increasing the variable position by one. Because it is possible to use two or more types of amino acids or amino acid analogs as triangle units, it is possible to create peptide compounds or peptide compound libraries of the present invention with greater diversity.It is now possible to use up to the same number of amino acids or amino acid analogs as in the random region, and it is even possible to expand the number of degrees of freedom to the same number as in the random region. Here, the term "random region" refers to a region in the peptide compound of the present invention from which amino acids or amino acid analogs can be freely selected. In other methods, this refers to the region other than the intersection unit and triangle unit in Scheme A (i.e., the black circle unit and square unit). However, in this method, the triangle unit also becomes a random region. The structural diversity of the black circle unit and square unit can be ensured while maintaining the reactivity of the triangle unit with the intersection unit. In other words, using this method, while two amino acids (corresponding to the triangle unit and intersection unit) previously required to be fixed in a display library for post-translational cyclization, this method can reduce the number of fixed amino acids to one (the intersection unit). For example, since most of the amino acids and amino acid analogs selected for the random region have a main-chain amino group, applying this method to the triangle unit also results in a random amino acid sequence with an amino group at the N-terminus. Amide cyclization can be achieved by selective amide bond formation with the common main-chain amino group in this region. For example, this is particularly useful when constructing a display library that does not incorporate basic amino acids such as lysine or arginine. Our results indicate that the number of residues with drug-likeness is 13 or less. Therefore, reducing the number of units to be fixed from 2 to 1 means increasing the number of units in the random region from 11 to 12. The value of increasing the number of residues that can be randomized by one is extremely high in terms of maximizing the degree of freedom under limited conditions for maintaining drug-likeness.

[0106] Specifically, a carboxylic acid or carboxylic acid activated ester can be translationally introduced into the side chain of the intersection unit (the amino acid at the intersection of the linear, cyclic, and triangle units), allowing for amide bond formation between the fixed intersection unit and a triangle unit selected from random amino acids (Scheme B). When constructing a library, the intersection unit does not need to be of one type; two or more types can be selected. Specifically, a library can be constructed using mRNA as a template, with the intersection unit codons at the desired positions, along with the (amino)acylated tRNAs encoding each intersection unit. Scheme B [ka]

[0107] Amide cyclization is an example of a library construction method that cyclizes without immobilizing the N-terminus (triangle unit). (This method can also be used when the N-terminus is immobilized.) While an aspartic acid derivative is used as the intersection unit, other examples are not limited and can be selected from the group of compounds represented by Compounds C-1, C-2, and C-3. Any amino acid or amino acid analog with a carboxylic acid in the side chain, such as N-methylaspartic acid or a glutamic acid derivative, can be used. (i) An amino acid with a carboxylic acid in the side chain can be introduced and post-translationally converted into an active ester. For example, as shown in Compound E-1, aspartic acid itself can be translationally introduced. The resulting translated peptide can be amide cyclized by condensation of a carboxylic acid with the N-terminal amine. For example, it can be converted to an N-hydroxysuccinimide activated ester, as shown in Compound E-2, or an activated ester such as HOBt or HOAt. The resulting activated ester can be easily reacted with amines, resulting in an amide cyclization reaction with a randomized N-terminus. The key to achieving this method is to select a method that converts only the carboxylic acid moiety into an activated ester and leaves RNA (and other nucleic acid moieties) unreacted. (ii) An amino acid bearing a carboxylic acid activated ester in its side chain can be translationally introduced, followed by reaction of the activated ester with an amine. This method involves translationally synthesizing an activated ester, such as compound E-2, in advance. In addition to compound E-2, it is also possible to translationally introduce benzyl thioesters, such as compound E-3, aryl thioesters, and alkyl thioesters, such as compound E-4. While phenyl groups are used in compounds E-4 and E-3, any aryl or heteroaryl group is acceptable. Furthermore, the aryl or heteroaryl group may contain, for example, electron-withdrawing groups such as halogen, nitro, trifluoromethyl, and nitrile groups, or electron-donating groups such as alkoxy groups, such as methoxy, and alkyl groups, such as methyl. Considering the rate of the thioester exchange reaction, the reactivity of the thioester with amines after the exchange reaction, and the selectivity of side reactions with water, a well-balanced substituent is preferred.The substituents are selected from bulky groups such as t-butyl and isopropyl groups, sulfonic acid groups and disubstituted amino groups such as dimethylamino groups, which are suitable for aqueous synthesis, and lipophilic groups such as long-chain alkyl groups, which are suitable for hydrophilicity. Various substituents, such as nitro, trifluoromethyl, and halogens, may also be simultaneously introduced. A thioaryl activated ester, more active than compound E-3, may be pretranslationally introduced, as in compound E-4. Thioesters can be selected from aralkyl and aryl thioesters, as well as alkyl thioesters. The key to achieving this method is the combination of two properties: stability during translational synthesis and sufficient reactivity with amines lacking a reaction promoter. (iii) It is also possible to translationally introduce an activated ester, such as a thioester, that is sufficiently stable during translational synthesis. After translation, an additive can be added to generate a more active activated ester in situ, which can then be cyclized with amines lacking a reaction promoter. For example, a more electron-deficient thiol can be added externally to a translationally incorporated thioester to generate a more active thioester in situ, which can then undergo cyclization with an amine. For example, in the case of a thioester such as compound E-3, it can be reacted directly with an amino group after translation. Alternatively, a more reactive thiol, such as trifluoromethylphenylthiol, can be added to the translation system to convert it to a more activated activated ester, E-4, which can then be reacted with an amino group. Alternatively, various raw materials known to form activated esters, such as HOBt, HOAt, and HONSu, can also be added. One or more of these additives may be selected. The advantage of adding two or more additives is improved reactivity. Converting a sufficiently stable and translatable activated ester in the translation solution to an activated ester sufficiently reactive with all amino groups is energetically unfavorable, and such a reaction may be difficult in a single step. In such cases, it is possible to first convert the translatable, stable activated ester to a more active, exchangeable activated ester, and then further convert this to an activated ester sufficiently reactive with all amino groups.Such multi-step activation of activated esters may enable amidation reactions with less reactive amino groups. Additives such as these activated esters may contain substituents, such as electron-withdrawing groups (e.g., halogen, nitro, trifluoromethyl, and nitrile), electron-donating groups (e.g., alkoxy groups (e.g., methoxy) and alkyl groups (e.g., methyl). The substituents may be bulky, such as t-butyl and isopropyl; disubstituted amino groups (e.g., sulfonic acid, carboxyl, hydroxy, and dimethylamino) that are suitable for aqueous synthesis; or lipophilic groups (e.g., long-chain alkyl groups) that are suitable for lipophilicity. (iv) A stable activated ester can be introduced translationally as in (iii), and then post-translationally subjected to a chemical reaction (e.g., deprotection) to activate it via an intramolecular reaction, followed by cyclization with an amine lacking a reaction-promoting group. For example, as in compound E-5, a more stable thioester can be introduced translationally, followed by post-translational deprotection of the SS bond and intramolecular conversion to a more reactive arylthiophenol, etc., before reacting with an amine. This objective can also be achieved by combining two or more of the concepts (i) to (iv). Thus, one effective way to utilize the initiation read-through method is to react the common amino group at the N-terminus with a crossover unit to construct a more diverse display library with a drug-like cyclization site.

[0108] [ka]

[0109] Among these, when an activated ester such as (iii) alkyl thioester or benzyl thioester that can be translationally introduced and can be sufficiently stable during translational synthesis is used, and an additive is added after translation to generate a more active activated ester in the system, which is then subjected to a cyclization reaction with an amine lacking a reaction promoting group, for example, an alkyl thioester such as methyl thioester (Asp(SMe)) or an aralkyl thioester such as benzyl thioester (Asp(SBn)) can be used as the activated ester for the side chain carboxylic acid of aspartic acid.

[0110] After translational synthesis of these as intersection units, additives added for the purpose of chemically reacting with triangle units lacking a reaction promoting group include, for example, aryl thiols and heteroaryl thiols such as 4-(trifluoromethyl)benzenethiol, which may be substituted with electron-withdrawing groups, electron-donating groups, lipophilic groups, water-soluble groups, etc., but are preferably electron-withdrawing groups. Examples of electron-withdrawing groups include trifluoromethyl, nitro, and fluoro groups, but are preferably electron-withdrawing groups similar to trifluoromethyl.

[0111] The amount of these thiols added is not particularly limited, but is preferably more than 10 mM for the purpose of sufficiently increasing reactivity, and is preferably less than 10 M for the purpose of dissolving the additive. A range of 50 mM to 5 M is more preferable, and 200 mM to 2 M is even more preferable. The additive can be added as a thiol (acidic) as is, but it is preferable to neutralize the acidic portion by adding an equivalent amount of a base such as triethylamine and add it under neutral conditions.

[0112] Highly reactive activated esters may react with various reagents present in the translation reaction system. For example, the amine component (trishydroxymethylaminoethane) in commonly used Tris buffer may be one of these. Therefore, it is preferable to use a buffer that does not contain reactive amine components for translation synthesis and chemical reactions. Examples of such buffers include HEPES buffer and phosphate buffer.

[0113] In addition, a buffer can be added to the reaction mixture to avoid a decrease in the amount of thiol required for the reaction due to the side reaction of oxidation in air (SS formation) as the reaction proceeds, and to avoid conditions in which hydrolysis becomes dominant due to increased basicity. It is also possible to add a reducing agent such as tris(2-carboxyethyl)phosphine. It is also effective to avoid exposing the cyclization reaction to oxygen in the air as much as possible.

[0114] The pH of the solvent during the chemical reaction is preferably 2 to 10 to ensure stable RNA. A pH of 7.8 or higher is preferred to ensure smooth progress of the chemical reaction, and it is preferably maintained at 9.2 or lower to prevent hydrolysis. The reaction may be carried out in a reaction translation solution such as PureSystem alone, to which an organic solvent such as DMF or NMP may be added. Alternatively, the translation solution may be purified by column purification or the like, and then the solvent may be changed. The reaction temperature is not particularly limited as long as it is within a range in which the chemical reaction can be carried out, but is preferably 15°C to 80°C, and more preferably 25°C to 50°C.

[0115] There are no particular limitations on the triangle unit that facilitates the cyclization reaction. The amino group can be either a primary amine or a secondary amine (e.g., an N-alkyl group such as N-methyl), and the substituent on the amino acid side chain is also not limited. In particular, when the carbon atom next to the amino group is unsubstituted (CH2), either a primary amine or a secondary amine is acceptable. Among secondary amines, a methyl group is more preferred. When a substituent is present on the carbon atom next to the amino group, a primary amine is more preferred. As for the substituent moiety, CH2 at the β position, such as Ala or Phe, is more preferred than Val or Thr. In addition, amino acids in which the nitrogen atom and the α-position carbon atom form a five-membered ring, such as proline, and similar cyclic secondary amines, such as four- or six-membered rings, are also preferred.

[0116] The above describes an example of a method for constructing a library in which cyclization is performed without immobilizing the N-terminus (triangle unit), but the use of this amide cyclization reaction by condensation of an amino group with an activated ester group without using a reaction promoter is not limited to reactions with the N-terminal main chain amino group. It can also be performed with any combination of an amino group in the side chain of an amino acid or amino acid analog, or an amino group in an N-terminal carboxylic acid derivative, and an activated ester in the side chain of an amino acid or amino acid analog, or an activated ester in an N-terminal carboxylic acid derivative.

[0117] In this method, as in Scheme C, a unit having an active ester may be placed in the triangle unit and an amine side chain may be placed in the intersection unit, or the active ester and amino group may be placed in either the triangle unit or the intersection unit.

[0118] Examples of such combinations are given below. When a unit having an activated ester group is selected as the intersection unit, the intersection unit can be selected from Compound C-1, Compound C-2, Compound C-3, or Compound COH-1, Compound COH-2, or Compound COH-3. When selecting these six compounds, it is preferable to select the amino acid immediately following the C-terminus from among units in which the N-alkylated unit is selected. This restriction is intended to avoid the side reaction of aspartimide formation and is a common preferred choice when these six compounds are selected. From the viewpoint of metabolic stability, Compound C-1, Compound C-2, or Compound C-3 is more preferable. In such cases, the triangle unit can also be selected from Compound Na-1, Compound Na-2, or Compound Na-3. When the N-terminus (triangle unit) is not fixed, multiple compounds can be selected simultaneously from these compounds. The side chain amino group in any of Compound Na-1, Compound Na-2, and Compound Na-3 may be protected. If protected, the amino group is deprotected before or simultaneously with the cyclization reaction, and then the cyclization reaction is carried out. The protecting group and deprotection conditions can be determined using the methods described herein.

[0119] Ra13 of compound Na-1 can be selected from a hydrogen atom, a C1-C6 alkyl group, or an aralkyl group. These may be substituted with a functional group defined as drug-like, such as a hydroxyl group, a fluoro group, or an ether group. A hydrogen atom, a methyl group, an ethyl group, an n-propyl group, or a benzyl group is preferred.

[0120] Ra1 of compound Na-2 can be selected in the same manner as Ra13. Particularly preferably, it is selected from a hydrogen atom or a methyl group. Ra2 can be selected in the same manner as Ra13, but is preferably a hydrogen atom or a methyl group. Particularly preferably, it is a hydrogen atom. When a hydrogen atom is selected, the configuration is acceptable as an amino acid, either L- or D-form. More preferably, it is an L-form configuration. Ra3 can be selected in the same manner as R13. Ra3 and Ra4 may form a ring. Particularly preferably, it is a hydrogen atom. Ra4 can be selected from a hydrogen atom, an alkyl group, a cycloalkyl group, an aralkyl group, a heteroaryl group, or an aryl group. These may be substituted with a functional group defined as drug-like. Furthermore, Ra4 may form a ring together with Ra1. For example, proline corresponds to this ring.

[0121] Ra11 of compound Na-3 can be selected in the same manner as Ra13, but is particularly preferably a hydrogen atom, or a methyl group, ethyl group, n-propyl group, or benzyl group. A hydrogen atom is particularly preferred. Ra9 can be selected in the same manner as Ra4. It is preferably selected from a hydrogen atom, a C1-C6 alkyl group, or an aralkyl group. These may be substituted with a functional group defined as drug-like. Ra9 and Ra11 may also form a ring. The ring size is preferably a 3- to 8-membered ring. Five- and six-membered rings are particularly preferred for ring formation. A hydrogen atom is particularly preferred as a substituent for Ra9. Ra10 and Ra12 can be selected in the same manner as Ra4. Preferably, they can be selected in the same manner as Ra13. More preferably, either Ra10 or Ra12 is a hydrogen atom. Particularly preferably, both Ra10 and Ra12 are hydrogen atoms. In this case, the N-terminus is preferably selected from amino acid derivatives or N-terminal carboxylic acid derivatives. The presence of an amino group in the N-terminal main chain is disadvantageous in terms of reaction selectivity, and the amino group is retained even after translational modification is completed, resulting in reduced drug-likeness.

[0122] When the intersection unit is selected from Compound C-1, Compound C-2, or Compound C-3, the triangle unit can be positioned other than at the N-terminus and fixed by selecting Compound Na-4 or Compound Na-5.

[0123] Ra5 of compound Na-4 can be selected in the same way as Ra1. Ra6 can be selected in the same way as Ra2. Ra7 can be selected in the same way as Ra4. A hydrogen atom or a methyl group is more preferable. A hydrogen atom is particularly preferable. Like R4, Ra8 can be selected from alkylene groups of C1 to C6 units such as partial structures N-3, N-4, and N-5, and N-6, N-7, and N-8 (including not only ortho-substitution but also meta- and para-substitution). Here, the substituents of R13 to R22 can be selected from functional groups that do not react with active esters or amino groups, among functional groups selected by drug-like reaction. Preferably, they are selected from partial structures N-6, N-7, and N-8 having C4 to C6 alkylene units or aryl groups, which may have a substituent.

[0124] Ra5 of compound Na-5 can be selected in the same way as Ra1. Ra6 can be selected in the same way as Ra2. Ra7 can be selected in the same way as Ra4. A hydrogen atom or a methyl group is more preferable. A hydrogen atom is particularly preferable. Like R4, Ra8 can be selected from alkylene groups of C1 to C6 units such as partial structures N-3, N-4, and N-5, and N-6, N-7, and N-8 (including not only ortho-substitution but also meta- and para-substitution). Here, the substituents of R13 to R22 can be selected from functional groups that do not react with active esters or amino groups, among functional groups selected by drug-like reaction. Preferably, they are selected from partial structures N-6, N-7, and N-8 having C4 to C6 alkylene units or aryl groups, which may have a substituent.

[0125] The side chain amino group in both Compound Na-4 and Compound Na-5 may be protected. If protected, the amino group is deprotected before or simultaneously with the cyclization reaction, and then the cyclization reaction is carried out. The protecting group and deprotection conditions can be determined using the methods described herein.

[0126] When the intersection unit is selected from Compound C-1, Compound C-2, or Compound C-3, the triangle unit can be placed at the N-terminus and cyclized with the amino group in the side chain. In addition to Compound Na-4 and Compound Na-5, the triangle unit can be selected from a wide range of compounds having an amino group and a carboxyl group. In translational synthesis, various units can be translationally synthesized as N-terminal carboxylic acid analogs. There are no particular limitations as long as they have an amino group that can be amide-cyclized and a carboxylic acid that can be translated into a peptide. Preferably, the functional group possessed by the divalent unit that connects the amino group and the carboxylic acid group is selected from drug-like functional groups. Compound Na-6 is an example of such a compound. Ra9 in Compound Na-6 may be selected from alkyl groups, cycloalkyl groups, aryl groups, heteroaryl groups, and aralkyl groups, each of which may be substituted with a drug-like functional group. Other acceptable groups include -NRCOR' (R and R' are drug-like substituents), -OR (R is a drug-like substituent), and NR (R includes amino acids, dipeptides, tripeptides, etc.).

[0127] On the other hand, it is also possible for the intersection unit to have a unit on the amino group side. For example, if Compound Na-4 or Compound Na-5 is selected, Compound C-1, Compound C-2, Compound C-3, Compound COH-1, Compound COH-2, Compound COH-3, etc. can also be selected as the triangle unit. In this case, it is preferable from the viewpoint of drug-likeness that a linear portion also exists on the N-terminal side of the triangle unit and that the N-terminus is selected from N-terminal carboxylic acid derivatives. For example, when Compound Na-4 or Compound Na-5 is selected, Compound COH-1, Compound COH-2, Compound COH-3, etc. can also be selected as the triangle unit. In this case, the triangle unit may be present at the N-terminus, or, alternatively, a linear portion may be present on the N-terminal side of the triangle unit, and the N-terminus is preferably selected from N-terminal carboxylic acid derivatives, from the viewpoint of drug-likeness.

[0128] Furthermore, when compounds Na-4, Na-5, etc. are selected as the intersection unit, compound Ca-1 can also be selected as the triangle unit.

[0129] [ka]

[0130] Addition of straight chain part 2 (branch part) As a method for generating the linear portion 2, it is possible to use a method in which both an amino acid analogue that does not have an amino group in the main chain, such as α-hydroxycarboxylic acid, but can form an ester bond by translational synthesis, and an amino acid that has an amino group side chain that may be protected (the protecting group is not particularly limited as long as it acts as a protecting group for the amino group and gives an amino acid that is translationally synthesized), are introduced into the translation and then post-translationally modified (Scheme E). When applied to a display library, it is selected from the units to be translated. However, peptide compounds obtained by subsequent optimization may include those that are obtained by post-translational modification after translational synthesis, even if the peptide compound itself is not translationally synthesized. There are no particular limitations on the side chain of the α-hydroxycarboxylic acid moiety, but in the method described in Scheme E, it is particularly advantageous from the perspective of translational synthesis when Re1 is a hydrogen atom (glycolic acid ( HOGly) itself) or one having a thiol group (SH group) or a protected thiol group in the side chain is preferred. The steric configuration of the side chain is not particularly limited, but if a hydrogen atom is present at the α-position, it is more preferred that it has a steric configuration similar to that of an L-amino acid. The configuration of the thiol group or protected thiol group is also not particularly limited, but it is particularly preferred that it is located at the β- or γ-position of the OH group (i.e., Re1 is an optionally protected mercaptomethyl or mercaptoethyl group). These thiol groups or protected thiol groups are located (added) from an optionally substituted alkyl group, alkenyl group, alkynyl group, cycloalkyl group, aralkyl group, aryl group, or heteroaryl group in the α-hydroxycarboxylic acid side chain. It is more preferred that the substituents on these α-hydroxycarboxylic acid side chains are the same as those defined for the side chains of drug-like amino acids, excluding the added thiol group or protected thiol group.

[0131] The type of amino acid having an amino side chain is not particularly limited, and may be any of optionally substituted alkylamino, alkenylamino, alkynylamino, aralkylamino, arylamino, heteroarylamino, and cycloalkylamino groups, as long as it has an amino group. However, the side chain may also contain a thiol group (SH group) or a protected thiol group. These substituents, excluding reaction-promoting groups (e.g., SH groups), are preferably those defined for the side chains of drug-like amino acids. Furthermore, it is more preferable that they be selected from substituents that allow translational synthesis. Furthermore, while one of the hydrogen atoms at the site represented by NH in the scheme is specified as a hydrogen atom, the other hydrogen atom may be substituted with an optionally substituted alkyl, alkenyl, alkynyl, aralkyl, aryl, heteroaryl, or cycloalkyl group, or these may be provided with a reaction-promoting group, preferably an NH group. These substituents, excluding reaction-promoting groups (e.g., SH groups), are preferably those defined for the side chains of drug-like amino acids. Furthermore, it is more preferable that the substituent be selected from those that can be translationally synthesized. A codon (A) encoding an amino acid analogue capable of forming an ester bond without an amino group, such as α-hydroxycarboxylic acid, and a tRNA acylated with the codon (A), a codon (B) encoding an amino acid with an amino side chain, and a tRNA aminoacylated with the codon (B), are prepared. A desired number of random codons (preferably 0 to 7, more preferably 0 to 2) are arranged between codons (A) and (B), and the resulting peptide is translated using mRNA as a template. The resulting peptide is then cyclized by the above-described method or an alternative method. Codon A is positioned N-terminally of codon B. The resulting cyclized peptide (e.g., the method of Scheme B or Scheme C can be used) is optionally deprotected if a protecting group is present on the side chain amino group. The ester moiety can be activated by hydrolysis or the addition of an external additive, resulting in hydrolysis or activation (active esterification or active thioesterification) of the ester bond without hydrolysis of the amide bond. By subjecting the obtained main chain carboxylic acid or activated (thio)ester to an intramolecular cyclization reaction with a side chain amine, a peptide having a linear portion 2, which is the desired branched peptide, can be obtained.For example, external additives include thiol compounds, compounds that form various active esters such as HONSu, HOBt, and HOAt, or mixtures of two or more of these. Scheme E shows an example in which Re1-substituted hydroxycarboxylic acid is used as codon A and lysine is used as codon B.

[0132] When Re1 is a hydrogen atom, the first cyclization reaction (cyclization of the triangle unit and the intersection unit) can be performed by selecting a triangle unit with or without a reaction promoter at the amine moiety. When a unit with a reaction promoter is selected as the triangle unit (e.g., compound N-1 or compound N-2), the first cyclization reaction can proceed easily. The reaction with the intersection unit having a thioester in its translationally synthesizable side chain can be performed at a pH of around 7 (translation conditions), with or without the addition of a reaction additive. On the other hand, when a unit without a reaction promoter is selected as the triangle unit (e.g., Ala or Phe as in compound Na-2), the addition of an additive such as trifluoromethylthiophenol is preferred to facilitate the first reaction. It is more preferable to raise the pH to around 7.8 and perform the reaction at a temperature of around 37°C to 50°C for approximately 6 to 10 hours. Furthermore, for the second branching reaction, the amine moiety may not have a reaction promoting group, but it is more preferable that the amine moiety have a reaction promoting group, in which case the NH group is preferably protected. In this case, a thiol is preferably added as an ester activator. As the thiol, alkyl thiols are preferred, and in particular, alkyl thiols with water-soluble substituents are preferred from the viewpoint of additive solubility, with 2-dimethylaminoethanethiol and 2-mercaptoethanesulfonic acid being preferred. The amount of these thiols added is not particularly limited, but is preferably more than 10 mM for the purpose of sufficiently increasing reactivity, and less than 10 M for the purpose of dissolving the additive. A range of 100 mM to 5 M is more preferred, and 500 mM to 4 M is even more preferred. The choice of amine moiety is not particularly limited, but the reaction conditions for deprotection are the same as those described in Scheme F2.

[0133] [ka]

[0134] Various modifications of the method of Scheme E are possible. For example, a hydroxycarboxylic acid bearing an SH group or a protected SH group at the Re1 site can be used to translationally introduce an amino acid with a reaction promoter, such as an SH group or a protected SH group, at the amine side chain site or near the amine at the protected amine side chain site. In this case, the protecting group attached to the SH group of the peptide obtained by translation, and the amine site (if protected) can be deprotected to easily undergo the ester-to-thioester exchange shown in Scheme F. The resulting thioester readily reacts with an amine bearing a reaction promoter, yielding a peptide with the desired branched peptide (linear portion 2). The SH group of the resulting branched peptide containing an SH group can be easily desulfurized under mild reaction conditions that do not involve RNA in the chemical reaction. In this case, an external additive can be added to further activate the (thio)ester. In the method of Scheme F, the post-translational cyclization reaction of the cyclized portion can be performed either before or after the linear portion 2 formation reaction. As described above, both the hydroxycarboxylic acid and the amine side chain moiety may have a reaction promoting group such as an SH group or a protected SH group, or only one of them may have a reaction promoting group, or neither may have a reaction promoting group. Also, the amine side chain moiety may or may not have a protecting group. In either case, an additive that accelerates the reaction may be added. Examples of the additive include an optionally substituted alkyl thiol, an optionally substituted alkenyl thiol group, an optionally substituted alkynyl thiol group, an optionally substituted aralkyl thiol, an optionally substituted aryl thiol, an optionally substituted heteroaryl thiol group, and an optionally substituted cycloalkyl thiol group. Also, a reagent that typically induces active esterification, such as HOBt, HONSu, HOAt, or paranitrophenol, or a derivative thereof, may be used. When an additive is added, it is preferable to carry out the linear portion 2 formation reaction after carrying out the cyclization reaction of the post-translational cyclization portion. The substituents of the additive can be freely selected, as defined for the side chain of the "amino acid" above.

[0135] In addition, as a method for generating the linear portion 2, a thioester is generated using an ester functional group of an α-hydroxycarboxylic acid as a stepping stone. However, a Cys and a Pro are placed in the two sequences immediately before the N-terminus of the α-hydroxycarboxylic acid, for example, Cys-Pro- HO A method of generating a thioester from a Gly sequence can also be used (see Scheme F2). In Scheme F2, an example is shown in which an active thioester-generating sequence for branching has Cys-Pro followed by an α-hydroxycarboxylic acid having Rf5 in the side chain, and lysine having an amino group in the side chain is used as the amino acid to react with this. A codon (A) encoding an amino acid analogue that can form an ester bond without an amino group, such as an α-hydroxycarboxylic acid, an acylated tRNA, a codon (B) encoding an amino acid with an amino side chain, and an aminoacylated tRNA are prepared. A peptide obtained by translation using mRNA as a template, in which a desired number of random codons (preferably 0 to 7, more preferably 0 to 2) are placed between codons (A) and (B), is first cyclized by the above method or another method (e.g., Scheme B or Scheme C). Codon A is positioned N-terminally of codon B. Cys-Pro- HOBranching sites such as Gly exist stably under the conditions of the first cyclization reaction without undergoing chemical reactions. Subsequently, if necessary, a thiol such as 4-trifluoromethylphenylthiol is added as an additive, and the pH is adjusted to the basic side (e.g., 9) to generate a new active thioester. This is then passed through the more active trifluoromethylphenylthioester to form an amide bond with the side chain amino group of lysine, etc., to generate a branched peptide (if a protecting group exists on the side chain amino group, a deprotection reaction is performed first). Subsequently, if necessary, a reaction to remove the reaction promoter, such as a SH deprotection reaction, may be performed. In this reaction, the Cys-Pro- that was present before the branched peptide was generated is removed. HO Of the Gly structures, Cys and Pro are eliminated and are not included in the branched peptide (after all post-translational modifications are completed). Therefore, they are not included in either the black circle unit or the square unit. Therefore, the number of drug-like units is calculated based on the number of units in the branched peptide.

[0136] The α-position (Rf5) of the α-hydroxycarboxylic acid moiety is selected from a hydrogen atom, an optionally substituted alkyl group, an aralkyl group, a heteroaryl group, a cycloalkyl group, an aryl group, etc. The substituent is preferably a drug-like functional group. In particular, this thioester generating site is stable under the reaction conditions of the first cyclization and is suitable for use as a precursor (e.g., Cys-Pro- HO It is preferable to maintain the (Gly) structure, and a hydrogen atom, an alkyl group such as a methyl group, or an aralkyl group such as a benzyl group is preferred.

[0137] Furthermore, the substituent of Rf5 in this portion can be either an L- or D-amino acid when the main-chain OH group is replaced with an amino group; however, an L-configuration is preferred in terms of translation efficiency. A unit that can be translated by ARS is particularly preferred, as it can improve translation efficiency. Examples of such a unit include Lac. The side chain of the amino acid bearing the amino side chain to be reacted with this thioester to form a branch is not particularly limited as long as it contains an amino group or an optionally protected amino group. However, if an unprotected amino group is used, it must be sufficiently less reactive than the amino group used in the first cyclization. The amino group may be a secondary or primary amine, but primary amines are preferred for efficient branching. A reaction-promoting group such as an SH group may be present in the vicinity of the amino group or the optionally protected amino group, and these may also be protected. The type of amino acid having an amino side chain is not particularly limited, and may be any of an optionally substituted alkylamino group, aralkylamino group, arylamino group, heteroarylamino group, or cycloalkylamino group, as long as it has an amino group. However, the side chain may also contain a thiol group (SH group) or a protected thiol group. These substituents, excluding reaction-promoting groups (e.g., SH group), are preferably those defined for the side chains of drug-like amino acids. Furthermore, it is more preferable that they be selected from substituents that allow translational synthesis. Furthermore, while one of the hydrogen atoms at the site represented by NH2 in the scheme is specified as a hydrogen atom, the other hydrogen atom may be substituted with an optionally substituted alkyl group, alkenyl group, alkynyl group, aralkyl group, aryl group, heteroaryl group, or cycloalkyl group, or these may also have a reaction-promoting group attached to them, preferably an NH2 group. These substituents, excluding reaction-promoting groups (e.g., SH group), are preferably those defined for the side chains of drug-like amino acids. Furthermore, it is more preferable that they be selected from substituents that allow translational synthesis.

[0138] Scheme F3 shows an example in which both the first cyclization and branching are amide bond reactions. To obtain this branched peptide, the triangle unit is selected from amino acids, amino acid derivatives, or N-terminal carboxylic acid derivatives with a reaction promoter, such as Compounds N-1 and N-2, or compounds Na-1, Na-2, Na-3, or Na-4, Na-5, and Na-6, which do not have a reaction promoter. The intersection unit is selected from activated esters, such as Compound C-1. The site converted to an activated ester in the second branching reaction is selected from α-hydroxycarboxylic acid derivatives, such as Compound e1, or a site consisting of the three components Cys-Pro-α-hydroxycarboxylic acid, as shown in Compound F5, or an activated ester, such as Compound C-1. (Cys, Pro, and α-hydroxycarboxylic acid are each translated by tRNA as separate units, but Cys and Pro are eliminated after translational modification, so these two do not belong to either the black circle unit or the square unit. α-Hydroxycarboxylic acid is a square unit.) The amino group-bearing site in this branching reaction is selected from compounds such as Compound Na-4, Compound Na-10, and Compound Na-11 (the H atom represented by NH may be protected during translational synthesis). Examples of such compounds include unprotected amines such as lysine, as well as Compounds tk100, tk101, tk102, tk103, tk104, tk34, tk7, tk14, tk105, tk106, tk107, and tk108.

[0139] This concept allows for branching, enabling the display of a wider variety of structures than previously possible. This is expected to increase the possibility of obtaining compounds with diverse functions, such as molecules that bind to or inhibit drug targets, which was previously difficult to achieve. Because this branching is possible, the value of this method is not limited to cases where the first cyclization reaction is a drug-like cyclization; any cyclization reaction can be used as long as a drug-like cyclization reaction is formed in the second cyclization reaction. This method is useful in that it allows for the formation of a wide variety of structures, even when the first cyclization reaction is not limited to a drug-like cyclization reaction. In such cases, all methods for generating active esters from translation products are included in the scope of this method. Examples include compound F5, compound e1, compound C-1, compound C-2, and compound C-3. Examples of the second amine group site include optionally protected compounds Na-4, Na-5, Na-10 (in the case of Na-7 and Na-8 groups), and Na-11 (in the case of Na-7 and Na-8 groups).

[0140] When a unit with a reaction promoter is selected as the triangle unit (e.g., compound N-1 or compound N-2), the first cyclization reaction can proceed easily. The reaction with the translationally synthesizable intersection unit bearing a thioester in the side chain can be carried out at a pH of around 7 (translation conditions), with or without the addition of a reaction additive. In this case, the site for the second branching reaction has a wider range of options. That is, compound e1 can be made stable, and in compound F5, Rf5 is preferably a non-hydrogen atom, but a hydrogen atom is also acceptable and stable. The amino group of the unit on the amino side can also be an unprotected unit, such as Lys, without a reaction promoter. The amino group can also be protected, or it can be protected with an amine bearing a reaction promoter. All of these options are possible. After removing the protecting group as needed, the desired branched peptide can be obtained by a branching reaction followed by a step of removing the reaction promoter.

[0141] The Rf1 group of compound e1 is selected as described above, but is more preferably selected from a hydrogen atom or an optionally protected mercaptoalkyl group (the protected SH group), and particularly preferably selected from a hydrogen atom, an optionally protected mercaptomethyl group, or a 2-mercaptoethyl group. The Rf2 group in compound F5 is selected from optionally protected mercaptoalkyl groups. The configuration of the amino acid containing Rf2 is preferably L-type. More preferably, it is selected from optionally protected mercaptomethyl groups or 2-mercaptoethyl groups. The Rf3 group can be selected in the same manner as Ra13. Preferably, it can be selected in the same manner as Ra1. Particularly preferably, it is a methyl group or preferably forms a ring together with Rf4. The ring size is preferably a 3- to 8-membered ring, more preferably a 4- to 6-membered ring. The carbon atoms forming these rings may be substituted with substituents defined for the side chains of drug-like amino acids. A representative example of a 5-membered ring is proline. Rf4 can be selected in the same manner as Ra4.

[0142] On the other hand, when a unit without a reaction promoter is selected as the triangle unit (e.g., Ala or Phe as contained in compound Na-2), it is preferable to add an additive such as trifluoromethylthiophenol to promote the first reaction. It is more preferable to raise the pH to around 7.8 and conduct the reaction for approximately 6 to 10 hours at a reaction temperature of around 37 to 50°C. Therefore, it is preferable that the branching site is stable and does not undergo chemical reactions under these reaction conditions. Therefore, it is preferable not to select a unit with an unprotected amino group such as Lys as the amino group site involved in amide bond formation in the branching reaction. In other words, it is preferable to protect the NH group (and the reaction promoter) of compounds Na-4, Na-10, and Na-11. As long as they have a protected amino group, they may or may not have an optionally protected reaction promoter (e.g., compound tk100) (e.g., compound tk104). These protecting groups must be stable during translation and the first cyclization reaction. Examples of such protecting groups include trifluoroacetyl, 4-azidobenzyloxycarbonyl, 3-nitro-2-pyridinesulfenyl, and thiazolidine ring protecting groups. The trifluoroacetyl group is selectively deprotected only when the pH is increased above 8. The 4-azidobenzyloxycarbonyl group is selectively deprotected only when the reducing agent tris(2-carboxyethyl)phosphine is added. The 3-nitro-2-pyridinesulfenyl group is selectively deprotected only when the additive 2-mercaptopyridine is added at pH 4. The thiazolidine ring protecting group is selectively deprotected only when the additive dithiodipyridine is added at pH 4 to open the thiazolidine ring, followed by the addition of tris(2-carboxyethyl)phosphine. Thus, a protecting group for an amino group that can be translationally synthesized can be selected from protecting groups that are stable during translational synthesis and the first cyclization reaction, and that can be selectively deprotected after the first cyclization under reaction conditions that are stable for RNA.Furthermore, when compound e1 is selected as the precursor site for the thioester generated during the second branching, it is preferable that the Rf1 site be a hydrogen atom or that a reaction promoting group (e.g., an SH group) be present at the Rf1 site, and it is preferable that the reaction promoting group be protected. When compound F5 is selected, Rf5 is preferably a group other than a hydrogen atom. Examples of such groups include a methyl group or a benzyl group. When the α-position (Rf5) of the hydroxyl site is a hydrogen atom, thioester formation is inevitable, making it impossible to selectively carry out the first cyclization reaction. However, when one or more carbon atoms are placed at this position, thioester formation can be suppressed under the conditions of the first cyclization reaction. When a branching site is selected in this way, a deprotection reaction of the amine site or thioester site is performed as necessary, and then the pH is raised to 8.2 or higher to carry out branching. In this way, the branched peptide is stably present during the first cyclization reaction, and the branching reaction is carried out via a deprotection step, and finally, the reaction promoting group is removed as necessary to obtain a branched peptide.

[0143] The chemical structure having an amino group that forms an amide bond in a branching reaction and the protecting group therefor are described below.

[0144] Amino-protecting groups used in chemical modification of peptide compound-nucleic acid complexes obtained by translational synthesis and methods for deprotecting them The amino-protecting group described herein is defined as not only a functional group that inactivates or reduces the reactivity of a single primary amine or secondary amine, but also a structure that simultaneously inactivates multiple amino groups or other heteroatoms with a single functional group. For example, thiazolidine ring, thiazinane ring, oxazolidine ring, and imidazolidine ring are also included as amino-protecting groups. The carbon atoms forming these protecting groups may be substituted. [ka]

[0145] The above-mentioned protecting group refers to a protecting group that can be removed by any one of 1) to 6) or a combination of multiple 1) to 6) of the following protecting groups: 1) a protecting group that is removable under acidic conditions, 2) a protecting group that is removable under basic conditions, 3) a protecting group that is removable under oxidative conditions, 4) a protecting group that is removable under reductive conditions, 5) a protecting group that is removable by irradiation with light, and 6) a protecting group that is removable by the addition of a nucleophile.

[0146] Protecting groups that can be removed under acidic conditions are those that can be removed in the pH range of 1 to 7, preferably in the pH range of 2 to 6, and include, for example, the following trityl group (Tr), N-(4-methoxyphenyl)diphenylmethyl group (MMTr), 3,5-dimethoxyphenylisopropoxycarbonyl group (Ddz), and 2-(4-biphenyl)isopropoxycarbonyl group (Bpoc) (Non-Patent Documents: i) Greene's Protective Groups in Organic Synthesis, Fourth Edition; ii) Chemical Reviews, 2009, 109(6), 2455-2504). The carbon atoms forming these protecting groups may be substituted.

[0147] [ka]

[0148] Protecting groups that can be removed under basic conditions are those that can be removed in the pH range of 7 to 14, preferably in the pH range of 7 to 10. Examples include the 2-[phenyl(methyl)sulfonio]ethoxycarbonyl group, which has an extremely electron-withdrawing group attached. Examples include trichloroacetyl groups, which have one or more halogen groups, nitro groups, or trifluoro groups introduced. A specific example is the trifluoroacetyl group (Tfa) shown below. (Non-patent documents: i) Greene's Protective Groups in Organic Synthesis, Fourth Edition; ii) Chemical Reviews, 2009, 109(6), 2455-2504) [ka]

[0149] An example of a protecting group that can be removed under oxidative conditions is the following pentenoyl group, which can be removed in the presence of iodine. (Non-Patent Documents: i) Greene's Protective Groups in Organic Synthesis, Fourth Edition; ii) The Journal of Organic Chemistry, 1997, 62, 778-779; iii) Method, 2005, 36, 245-251) [ka]

[0150] Protecting groups that can be removed under reductive conditions are those that can be removed in the presence of, for example, tris(2-carboxyethyl)phosphine (TCEP) or dithiothreitol (DTT), and examples thereof include an azide group (Non-Patent Document ChemBioChem, 2009, 10, 1186-1192), a 4-azidobenzyloxycarbonyl group (p-Acbz) (Non-Patent Document Journal of the Chemical Society, Perkin Transactions 1, 1996, 1205-1211), a 2-azidobenzyloxycarbonyl group (o-Acbz), an azidomethoxycarbonyl group (Azoc) (Non-Patent Document Organic Letters, 2007, 9(11), 2223-2225), a phenyldisulfanylethyloxycarbonyl group (Phdec), and a 2-pyridyldisulfanylethyloxycarbonyl group (Pydec) (Non-Patent Document Chemical Reviews, 2009, 109(6), 2455-2504). The carbon atoms forming these protecting groups may be substituted. [ka]

[0151] Protecting groups that can be removed by irradiation with light include, for example, o-nitrobenzyloxycarbonyl (oNz), 4,5-dimethoxy-2-nitrobenzoxycarbonyl (Nvoc), and 2-(2-nitrophenyl)propyloxycarbonyl (Nppoc) groups (Non-Patent Documents: i) Chemical Reviews, 2009, 109(6), 2455-2504; ii) The Journal of Organic Chemistry, 1997, 62, 778-779; iii) Bioorganic & Medicinal Chemistry, 2012, 20, 2679-2689). The carbon atoms forming these protecting groups may be substituted. [ka]

[0152] Protecting groups that can be removed by the addition of a nucleophile are, for example, protecting groups that can be deprotected in the presence of a thiol, such as o-nitrobenzenesulfonyl group (o-NBS), 2,4-dinitrobenzenesulfonyl group (dNBS), and dithiosuccinoyl group (Dts) (Non-Patent Document Chemical Reviews, 2009, 109(6), 2455-2504). The carbon atoms forming these protecting groups may be substituted. [ka]

[0153] Protecting groups that can be removed by combining multiple of the above-mentioned methods 1) to 6) include, for example, protecting groups that can be removed by adding thiol at a pH range of 2 to 6, as a deprotection method that combines 1) and 6), such as the 3-nitro-2-pyridinesulfenyl group (Npys) (Non-Patent Documents i) International Journal of Peptide and Protein Research, 1990, 35, 545-549; ii) International Journal of Peptide and Protein Research, 1980, 16, 392-401) and the 2-nitrophenylsulfenyl group (Nps) (Non-Patent Documents Chemical Reviews, 2009, 109(6), 2455-2504). The carbon atoms forming these protecting groups may be substituted. [ka]

[0154] The protecting group applicable to the deprotection method combining 1) and 3) is, for example, a protecting group that can be deprotected by adding a disulfide compound in the pH range of 2 to 6, such as a thiazolidine ring or a thiazinane ring. The carbon atoms forming these protecting groups may be substituted. [ka]

[0155] Deprotection method to amino group by ring opening of thiazolidine ring and thiazinane ring Native Chemical Ligation (NCL), which utilizes the nucleophilicity of thiols, is a useful technique for the chemical synthesis of peptides and proteins. When performing sequential amidation reactions or NCL at any desired timing, pre-protection of amino and thiol groups is a useful technique. For example, in the protein synthesis by multi-step amidation of peptide chains described in Non-Patent Document v1 and the chemical modification of proteins described in Non-Patent Document v2, a thiazolidine ring has been reported as a structure that simultaneously protects the amino and thiol groups of cysteine.

[0156] A known method for deprotecting a thiazolidine ring in a peptide structure to an amino group or a thiol group is to add methoxyamine to a buffer solution of pH 4 to pH 7 (for example, Non-Patent Document v1 (Angewandte Chemie, International Edition, 2006, 45(24), 3985-3988) and Non-Patent Document v2 (Journal of the American Chemical Society, 2011, 133, 11418-11421)). However, for example, in the structure in which an ester or thioester is contained in the peptide compound described in this patent, there is a risk of the formation of an alkoxyamide as a side reaction in which methoxyamine reacts with the ester moiety during the deprotection reaction or when the next reaction is carried out in one pot.

[0157] The present inventors have developed a method for deprotecting the thiazolidine ring and the thiazinane ring without using methoxyamine, in which a disulfide compound is added. This method is characterized by the ring-opening of the thiazolidine and thiazinane rings under acidic conditions, leading to the formation of aminodisulfide structures, which can then be converted to aminothiols by the addition of a reducing agent. This method is carried out in water, a buffer solution, or a water-miscible organic solvent. The organic solvent used must be miscible with water and not react with or precipitate the substrate. Examples include N,N-dimethylacetamide, N,N-dimethylformamide, and acetonitrile. The ratio of water to organic solvent is determined by the solubility of the substrate and disulfide compound. For example, when performing the reaction with 10 mM dithiodipyridine, it is preferable to use 5% or more N,N-dimethylacetamide due to the solubility of dithiodipyridine.

[0158] The pH range for opening the thiazolidine ring is preferably 1 to 5 when the reaction is to be completed within 12 hours, and more preferably 4 to 5 when a compound having an unstable thiazolidine ring is used. The reaction temperature for opening the thiazolidine ring is preferably 15° C. or higher when the reaction is to be completed within 12 hours, and more preferably in the range of 15 to 50° C. when a compound having a thermally unstable thiazolidine ring is used.

[0159] The disulfide compound to be added may be, for example, a dialkyl disulfide or a diaryl disulfide, with diaryl disulfide being preferred, and dithiodipyridine being more preferred.

[0160] The amount of dithiodipyridine used depends on the amount of thiazolidine derivative used. For example, when 30 mM dithiodipyridine is used for 1.0 mM thiazolidine derivative and the reaction is carried out at pH 4.2 and 36° C., the opening of the thiazolidine ring is completed within 12 hours.

[0161] As a reducing agent for converting an aminodisulfide structure to an aminothiol, alkylphosphines, arylphosphines, and thiol compounds with reducing power, such as TCEP and DTT, can be used. TCEP is preferred for rapid conversion to an aminothiol.

[0162] The amount of TCEP used depends on the amount of thiazolidine derivative and dithiodipyridine used. For example, if 1 mM of thiazolidine derivative is converted to 2-(2-pyridyldithio)ethylamine with 30 mM of dithiodipyridine, conversion to aminoethanethiol is completed within 1 hour if 40 mM of TCEP is used and the reaction is carried out at pH 4.5 and 24°C.

[0163] This technology for forming branched structures from linear peptide sequences translated from the primary sequence information of mRNA makes it possible to construct an mRNA display library of peptides with high structural diversity and branched structures that cannot be achieved by conventional techniques.

[0164] Although we have described a chemical space-expanding peptide synthesis method using hydroxycarboxylic acids as a stepping stone, this concept is not limited to the methods shown in Schemes E, F, F2, and F3. The first cyclization reaction is not limited to the above-mentioned cyclization reactions; it can be applied to any cyclization reaction that allows for branching (i.e., it must be stable during the first cyclization reaction and be activated for branching under RNA-stable reaction conditions). Compounds F5 or E1 can be used as the second activated ester generating site, and compounds Na-4, Na-5, Na-10 (Na-7 or Na-8 group), or Na-11 (Na-7 or Na-8 group) can be used as the second amino group site. The desired branched peptide (linear portion 2) can be obtained by placing an amino acid containing a hydroxycarboxylic acid and an amine moiety at the desired position and performing the appropriate chemical reaction. The hydroxycarboxylic acid and amine, and the respective amino acids or amino acid analogs can be freely selected as long as they have the necessary functional groups, but it is preferable for the hydroxycarboxylic acid moiety to be located on the N-terminus. The hydroxycarboxylic acid is not limited to α-hydroxycarboxylic acid, but can be a variety of hydroxycarboxylic acids, including β- and γ-hydroxycarboxylic acids. Furthermore, by translationally incorporating thiocarboxylic acids instead of hydroxycarboxylic acids, branched peptide formation reactions can be performed using thioesters rather than esters. This method realizes the expansion of chemical space, which is essential for creating hits with medium-sized molecules with a limited number of amino acids.

[0165] [ka] [ka] [ka]

[0166] Next, we will describe the reaction design using CC bond cyclization as an example. For example, a carboxylic acid with a double bond, an N-terminal carboxylic acid analog, can be translationally introduced into the triangle unit, and an amino acid with an iodophenyl group in the side chain can be translationally introduced into the intersection unit (○ unit) (Scheme C-2). In this way, if functional groups capable of condensation via Pd-catalyzed Heck reaction are introduced into both the triangle unit and the intersection unit, the CC bond cyclization product can be obtained by Pd-catalyzed reaction. Various Pd ligands are available. Phosphine ligands, phosphine oxide ligands, nitrogen atom ligands, arsenic atom ligands, and carbene-type ligands are commonly used in Pd-catalyzed reactions. These can be monodentate ligands with one functional group capable of coordinating within the molecule, or bidentate ligands combining two of these functional groups capable of coordinating to Pd. Since the reaction must be performed in water, ligands with water-soluble functional groups can also be used. Here, because Pd is inactivated by complex formation with nucleic acid components such as RNA components, GTP, and ATP contained in the translation solution, ligands that form strong coordinate bonds with Pd are preferred. Examples include bidentate phosphine ligands such as 1,1'-bis(diphenylphosphino)ferrocene, 2,2'-bis(diphenylphosphino)-1,1'-biphenyl, 2,2'-bis(diphenylphosphino)-1,1'-binaphthyl, 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene, and 1,3-bis(diphenylphosphino)propane, with 2,2'-bis(diphenylphosphino)-1,1'-biphenyl being more preferred in terms of solubility in aqueous solvents. In ordinary organic synthesis, a catalytic amount of Pd (often less than 1 mol%) is sufficient. However, when applied to display libraries after translational synthesis, Pd forms a complex with the RNA portion necessary for translational synthesis. Therefore, unless an excess of Pd is used, the desired chemical reaction will not proceed at a sufficient rate. On the other hand, if too much Pd is used, the solubility of the complex with RNA decreases, and the desired chemical reaction will not proceed at a sufficient rate. The amount of Pd used is preferably 1 nmol or more per translational synthesis product containing 1 pmol of m-RNA, and more preferably 60 nmol.As will be described in the Examples below, the use of micelles is preferred because it accelerates the reaction.

[0167] Micelles that can be used include anionic, cationic, zwitterionic, and nonionic micelles, but the use of polyoxyethanyl-α-tocopheryl sebacate (PTS), a nonionic micelle, is particularly preferred. Polyoxyethanyl-α-tocopheryl sebacate can be used at any concentration, but a final concentration of 1% or higher is preferred, and a final concentration of 7.5% or higher is more preferred. A base is required for the reaction to proceed, and a buffer containing a component that does not coordinate with Pd is preferred, such as a phosphate buffer or carbonate buffer. The pH of the reaction solvent is preferably adjusted to a range in which the mRNA can exist stably and the cyclization reaction can proceed; a pH of 7 or higher and 10 or lower is more preferred, and a pH of 7.5 or higher and 8.5 or lower is even more preferred. The reaction temperature is not particularly limited as long as it is within a range in which the mRNA can exist stably and a typical chemical reaction can be carried out, but is preferably 15°C to 80°C, and more preferably 40°C to 60°C.

[0168] [ka]

[0169] Although the Heck reaction using Pd has been described as an example of a CC bond bonding reaction, the CC bond cyclization reaction in this application is not limited to the Heck reaction using Pd. Various reactions using Pd, such as the Suzuki reaction and the Sonogashira reaction, can also be carried out in the same manner. In this case, the selection of the ligand and base may be important. Furthermore, the transition metal is not limited to Pd, and C-C bond reactions can be carried out using various metals such as Ni, Ru, and Co (see non-patent literature: Matthew S. Sigman et al., Advances in Transition Metal (Pd, Ni, Fe)-Catalyzed Cross-Coupling Reactions Using Alkyl-Organometallics as Reaction Partners, Chem. Rev., 2011, 111, 1417-1492; Lutz Ackermann et al., Ruthenium-Catalyzed Direct Arylations Through C-H Bond Cleavages, Top Curr Chem. 2010, 292, 21; Paul Knochel et al., Pd-, Ni-, Fe-, and Co-Catalyzed Cross-Couplings Using Functionalized Zn-, Mg-, Fe-, and In-Organometallics, Isr. J. Chem. 2010, 50, 547; Gwilherm Evano et al. Copper-MediatedCoupling Reactions and Their Applications in Natural Products and Designed Biomolecules Synthesis. Chem. Rev. 2008, 108, 3054).

[0170] The amino acids, amino acid analogs, and N-terminal carboxylic acid analogs that make up the triangle units in Scheme C-2 are not particularly limited as long as they have a reactive functional group. However, if an amino acid is selected, an amine residue will remain at the N-terminus, which is disadvantageous for membrane permeation compared to when this residue is not present. For this reason, triangle units with fewer heteroatoms are preferred. Examples of such N-terminal carboxylic acid analogs include compounds CC-1 to CC-4. In compound CC-1, the carbon-carbon double bond serves as the reactive site. In compound CC-2, the carbon-carbon triple bond serves as the reactive site. In compound CC-3, X serves as the reactive site. X is preferably a halogen, selected from Cl, Br, I, and F. From the standpoint of reactivity, Br and I are preferred, with I being most preferred. In compound CC-4, the borate ester moiety serves as the reactive site. R301, R302, and R303 are selected from hydrogen atoms, as well as optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and aralkyl groups. These substituents are not particularly limited as long as the resulting compounds CC-1 to CC-4 can be translationally synthesized. Examples of such substituents include halogen groups and alkoxy groups.

[0171] R304 is a unit that connects the reactive site and the translation site (carboxylic acid site). Representative structures are shown below. Both units can be connected by a C1-C6 unit such as a methylene group (partial structure N-3), an ethylene group (partial structure N-4), or a propylene group (partial structure N-5). They can also be connected directly from the aryl carbon of an aromatic compound (partial structure N-6). They can also be connected by an aralkyl structure (partial structures N-7 and N-8). The linking position is not limited to ortho, but can also be meta or para, and can also be a substituted aryl group or substituted aralkyl group other than a phenyl group. Substituents include, for example, a halogen group and an alkoxy group.

[0172] Furthermore, examples of amino acids in the triangle unit constituting Scheme C-2 are not particularly limited as long as they have a double bond, triple bond, halogen, or borate ester in the side chain. L-amino acids, D-amino acids, and α-α-dialkylamino acids may be used, but L-amino acids are particularly preferred. To obtain drug-like peptides, it is preferable that the N-terminal amino group be substituted. While alkylation such as N-methylation is also possible, the introduction of a substituent that eliminates the basicity of the nitrogen atom, such as amidation (e.g., acylation), is preferred.

[0173] The amino acid analog or N-terminal carboxylic acid analog of the triangle unit constituting Scheme C-2 may be a hydroxycarboxylic acid having a double bond, a triple bond, a halogen, or a borate ester in the side chain. As in the case of amino acids, the type of side chain is not particularly limited. In addition to α-hydroxycarboxylic acids, β- or γ-hydroxycarboxylic acids may also be used. Furthermore, dipeptides or tripeptides having a double bond, a triple bond, a halogen, or a borate ester in the side chain may also be used. [ka]

[0174] Examples of the intersection unit (○ unit) constituting Scheme C-2 include amino acids or α-hydroxycarboxylic acids having a double bond, triple bond, halogen, or borate ester in the side chain. The side chain of the amino acid or α-hydroxycarboxylic acid is not particularly limited as long as it has these reactive functional groups. The reactive functional groups are selected from optionally substituted alkyl groups, alkenyl groups, alkynyl groups, cycloalkyl groups, aryl groups, heteroaryl groups, aralkyl groups, and the like, each substituted with a reactive functional group. Examples of these substituents include halogen groups and alkoxy groups. Any of L-, D- and α-α-dialkyl forms is acceptable, but when a hydrogen atom is present at the α-position, an L-amino acid or amino acid analog is preferred.

[0175] The combination of the triangle unit and the intersection unit (◯) unit is not particularly limited as long as it is a combination that can undergo a condensation reaction with a transition metal such as Pd.

[0176] Chemical synthesis of peptide compounds The peptide compounds of the present invention can also be produced by chemical synthesis. Examples include Fmoc synthesis and Boc synthesis. In Fmoc synthesis, the base unit is an amino acid in which the main chain amino group is protected with an Fmoc group, the side chain functional groups are optionally protected with a protecting group that is not cleaved by a base, such as piperidine, and the main chain carboxylic acid is not protected. Other combinations are not particularly limited as long as they contain an Fmoc-protected amino group and a carboxylic acid group. For example, a dipeptide may be used as the base unit. The base unit placed at the N-terminus may be other than an Fmoc amino acid. For example, it may be a Boc amino acid or a carboxylic acid analog without an amino group. The main chain carboxylic acid group is supported on the solid phase by chemical reaction with a functional group on the solid phase support. Subsequently, the Fmoc group is deprotected with a base such as piperidine or DBU, and the newly generated amino group is condensed with the subsequently added base unit, a protected amino acid with a carboxylic acid, to form a peptide bond. Various combinations are possible for the condensation reaction, such as DIC and HOBt, DIC and HOAt, and HATU and DIPEA. The desired peptide sequence can be generated by repeated Fmoc group removal and subsequent peptide bond formation reactions. After the desired sequence is obtained, the peptide is cleaved from the solid phase and, if necessary, the protective groups on the side chain functional groups are removed. It is also possible to perform structural transformation or cyclization of the peptide before cleavage from the solid phase. Cleavage from the solid phase and deprotection can be performed under the same conditions, such as 90:10 TFA / HO, or, if necessary, under separate conditions. Cleavage from the solid phase can be performed using a weak acid such as 1% TFA, or by using a protecting group such as Pd to take advantage of the orthogonal nature of the chemical reactions. Cyclization or other steps can be performed between or after these steps. For example, a side-chain carboxylic acid can be condensed with the N-terminal amino group of the main chain, or a side-chain amino group can be condensed with the C-terminal carboxylic acid of the main chain. In this case, orthogonality of the reaction is required between the carboxylic acid on the C-terminus and the side-chain carboxylic acid to be cyclized, or between the main-chain amino group or hydroxyl group on the N-terminus and the side-chain amino group to be cyclized. As mentioned above, the protecting group is selected taking into consideration the orthogonality of the protecting group.The reaction product thus obtained can be purified using a reverse phase column, a molecular sieve column, etc. Details of these methods are described, for example, in the Solid Phase Synthesis Handbook published by Merck Ltd. on May 1, 2002.

[0177] Preparation of drug-like peptide compounds or peptide compound-nucleic acid complexes Furthermore, the present invention provides a method for producing a drug-like peptide compound or a peptide compound-nucleic acid complex having a desired activity.

[0178] A method for producing a drug-like peptide compound-nucleic acid complex having a desired activity can include, for example, a production method comprising the following steps: (i) a step of translating and synthesizing an acyclic peptide compound having a total of 9 to 13 amino acid and amino acid analog residues to form an acyclic peptide compound-nucleic acid complex comprising a complex in which the acyclic peptide compound and a nucleic acid sequence encoding the acyclic peptide compound are linked via a linker; (ii) a step of cyclizing the non-cyclic peptide compound of the complex translationally synthesized in step (i) via an amide bond or a carbon-carbon bond to form a cyclic compound having a total of 5 to 12 amino acid and amino acid analog residues in the cyclic portion. (iii) A step of contacting the library of peptide compound-nucleic acid complexes having a cyclic portion obtained in step (ii) with a biological molecule and selecting a complex having binding activity to the biological molecule.

[0179] Furthermore, a drug-like peptide compound having a desired activity can be produced from the complex selected in the above steps. For example, the production method may include the following steps: (iv) obtaining sequence information of the peptide compound from the nucleic acid sequence of the complex selected in step (iii); and (v) chemically synthesizing a peptide compound based on the sequence information obtained in step (iv); Furthermore, the above production process may include a step in which the non-cyclic peptide compound contains an α-hydroxycarboxylic acid and an amino acid or amino acid analog having an amino group in the side chain, which may be protected, and a step in which a branched portion is formed by chemically reacting the α-hydroxycarboxylic acid portion with the amino acid or amino acid analog portion having an amino group in the side chain, either before or after the step of forming the cyclic compound in step (ii). This step makes it possible to produce peptide compounds having the above-mentioned linear portion 2 at various positions in the cyclic portion.

[0180] Here, the total number of amino acids and amino acid analogs described in step (i) and the total number of amino acid and amino acid analog residues in the cyclic portion described in step (ii) do not include amino acids or amino acid analogs removed by post-translational modification. For example, in the above-mentioned scheme F3, the Cys-Pro sequence that is removed from the peptide during post-translational modification in the production of a peptide compound having linear portion 2 is not included in the number of amino acids and amino acid analogs in steps (i) and (ii). In addition, for example, when a Gly-Ser repeat structure is used as a linker site between a peptide compound and RNA, the Gly-Ser repeat structure, a fixed amino acid region, and a site intended to bind a peptide compound having a cyclic portion of the present invention, particularly a drug-like peptide compound portion, to a nucleic acid are included in the linker and are not included in the number of amino acids and amino acid analogs in steps (i) and (ii). In this case, the number of residues in the cyclic portion after cyclization in step (ii) is not 5 to 11 but is a longer number, as long as the number of residues in the cyclic portion after the reaction to form linear portion 2 is 5 to 11.

[0181] In this production method, chemical modification may be performed in steps (ii) and (v) to obtain the above-mentioned drug-like peptide compound or for optimization. Furthermore, in the present invention, the target substance is preferably a biological molecule. The "biological molecule" in the present invention is not particularly limited as long as it is a molecule present in the body, but is preferably a molecule that serves as a target for disease treatment, and particularly preferably a molecule that does not have a cavity to which conventional low-molecular-weight compounds with a molecular weight of less than 500 can bind, or an intracellular protein, nucleic acid, intracellular region of a membrane protein, or transmembrane domain of a membrane protein that cannot be accessed by high-molecular-weight compounds such as antibodies. Specific examples include transcription factors such as STAT, AP1, CREB, and SREBP; G protein-coupled receptors (GPCRs) such as muscarinic acetylcholine receptors, cannabinoid receptors, GLP-1 receptors, and PTH receptors; cytokines and their receptors such as TNF, TNFR, IL-6, and IL-6R; ionotropic receptors such as P2X receptors and nicotinic acetylcholine receptors; ion channels; transporters; and microRNAs such as miR-21 and miR206. The cyclic portion can be formed, for example, by utilizing the above-mentioned cyclization reaction, and the cyclization reaction can form an amide bond or a carbon-carbon bond.

[0182] Furthermore, in the synthesis process of the peptide compound-nucleic acid conjugate, known techniques such as a cell-free translation system can be used. Specifically, the conjugate can be prepared using the following method.

[0183] Transfer RNA (tRNA) is an RNA with a molecular weight of 25,000 to 30,000, consisting of 73 to 93 bases in length and including a CCA sequence at the 3' end. It is ester-linked to the carboxyl terminus of an amino acid via its 3' end as an aminoacylated tRNA, which is transported to the ribosome in a ternary complex with polypeptide elongation factor (EF-Tu) and GTP. During the translation of mRNA base sequence information into amino acid sequences by the ribosome, tRNA is involved in codon discrimination by base pairing between the anticodon in the tRNA sequence and the codon in the mRNA. tRNA synthesized in cells contains covalently modified bases, which may affect tRNA conformation and anticodon base pairing, thereby aiding tRNA codon discrimination. tRNA synthesized by typical in vitro transcription consists of the so-called nucleic acid bases adenine, uracil, guanine, and cytosine, but those prepared from within cells or chemically synthesized may contain other modified bases such as methylated forms, sulfur-containing derivatives, deaminated derivatives, and adenosine derivatives containing isopentenyl groups or threonine, and may also contain deoxy bases when using methods such as pdCpA.

[0184] RNA can be synthesized by transcription using a template DNA encoding the desired tRNA sequence and a T7, T3, or SP6 promoter upstream, using an RNA polymerase compatible with the promoter, such as T7 RNA polymerase, T3, or SP6 RNA polymerase. Alternatively, tRNA can be extracted and purified from cells, and the desired tRNA can be isolated using a probe complementary to the tRNA sequence. Cells transformed with an expression vector for the desired tRNA can also be used as the source. RNA of the desired sequence can also be synthesized chemically. For example, aminoacyl-tRNA can be obtained by ligating the thus-obtained tRNA, in which the CA has been removed from the 3'-terminal CCA sequence, with separately prepared aminoacylated pdCpA using RNA ligase (the pdCpA method). Aminoacylation can also be performed using full-length tRNA with flexizyme, a ribozyme that can attach activated esters of various unnatural amino acids to tRNA. Alternatively, acylated tRNA can be obtained using the methods described below.

[0185] Protein factors required for translation in E. coli (methionyl-tRNA transformylase, EF-G, RF1, RF2, RF3, RRF, IF1, IF2, IF3, EF-Tu, EF-Ts, ARS (select from AlaRS, ArgRS, AsnRS, AspRS, CysRS, GlnRS, GluRS, GlyRS, HisRS, IleRS, LeuRS, LysRS, MetRS, PheRS, ProRS, SerRS, ThrRS, TrpRS, TyrRS, ValRS)), ribosomes, amino acids, creatine kinase, myokinase, inorganic pyrophosphatase, nucleoside diphosphate kinase, E. coli-derived tRNA, creatine phosphate, potassium glutamate, HEPES-KOH Peptide translation can be achieved by adding mRNA to the PURE system, which contains a pH 7.6 mixture of magnesium acetate, spermidine, dithiothreitol, GTP, ATP, CTP, and UTP. Furthermore, by adding T7 RNA polymerase, coupled transcription and translation from a template DNA containing a T7 promoter can be performed. Adding the desired acylated tRNAs or unnatural amino acids (e.g., F-Tyr) that are tolerated by the ARS to the system allows translational synthesis of peptides containing unnatural amino acids (Kawakami T, et al., Ribosomal synthesis of polypeptoids and peptoid-peptide hybrids. J Am Chem Soc. 2008, 130, 16861-3., Kawakami T, et al., Diverse backbone-cyclized peptides via codon reprogramming. Nat Chem Biol. 2009, 5, 888-90.).Alternatively, the efficiency of translational incorporation of unnatural amino acids can be increased by using mutants such as ribosomes or EF-Tu (Dedkova LM, et al. Construction of modified ribosomes for incorporation of D-amino acids into proteins. Biochemistry. 2006, 45, 15541-51; Doi Y, et al. Elongation factor Tu mutants expand amino acid tolerance of protein biosynthesis system. J Am Chem Soc. 2007, 129, 14458-62; Park HS, et al. Expanding the genetic code of Escherichia coli withphosphoserine. Science. 2011, 333, 1151-4).

[0186] To create an mRNA display library, first chemically synthesize a DNA library with the desired sequence downstream of a promoter such as the T7 promoter. This is then used as a template to create double-stranded DNA through a primer extension reaction. This is then transcribed into mRNA using an RNA polymerase such as T7 RNA polymerase. A linker (spacer) containing the antibiotic puromycin, an aminoacyl-tRNA analog, is attached to the 3' end of this RNA. This is then added to a known cell-free translation system such as the PURE system mentioned above and incubated, whereupon the mRNA is translated and the mRNA and the peptide encoded thereby are linked via puromycin. In this way, a display library can be constructed consisting of complexes of mRNA and its products, where the mRNA and its product are associated with each other.

[0187] Furthermore, molecules that bind to the target can be enriched by contacting the library with a desired immobilized target and washing away molecules that do not bind to the target (panning). cDNA is synthesized from mRNA, which is a tag containing genetic information attached to the selected molecules, and then PCR-amplified and sequenced to reveal the sequence of the bound peptide.

[0188] In the present invention, the construction of a cyclized peptide compound-nucleic acid complex display library, and further the acquisition of a target-binding cyclized peptide compound (a peptide compound having a cyclic portion) or a cyclized branched peptide (a peptide compound having a cyclic portion and a linear portion 2) from the constructed display library, can be specifically carried out, for example, by the methods [I] to [XVII] shown in the following embodiments.

[0189] initiation read through [I] The method for producing a peptide compound having a cyclic portion of the present invention can include one or more of the following steps: A) Providing pdCpA by aminoacylation of compound C-1 B) Providing a tRNA lacking a CA at the 3' end C) A step of linking the pdCpA of step A) with the tRNA of step B) to provide an initiator tRNA aminoacylated with compound C-1. D) providing a cell-free translation system containing the tRNA of step C) but not containing methionine, methionyl-tRNA synthetase (MetRS), translation initiation tRNA for methionine, formyl donor, or methionyl-tRNA transferase E) providing a template DNA library encoding peptide sequences containing, downstream of a promoter, a translation initiation ATG followed by a cysteine ​​codon UGU or UGC, and downstream of that a codon corresponding to the anticodon of the tRNA in step C); F) A step of providing an mRNA library from the template DNA library of step E). G) A step of attaching a spacer to the 3' end of the mRNA library of step F). H) A step of adding the spacer-bound mRNA library of step G) to the cell-free translation system of step D) and translating the mRNA to provide a pre-cyclized peptide compound-mRNA complex display library. I) Step of forming a cyclic structure In forming the circular structure, a desulfurization reaction can be carried out as necessary. Furthermore, in the present invention, after step G), a step of synthesizing cDNA using a primer that anneals to the 3' region of the mRNA library can be included. The method of the present invention may further comprise the following steps: J) A step of enriching the mRNA library that binds to the target substance by panning K) A process of synthesizing cDNA using reverse transcriptase L) A step of analyzing the base sequence

[0190] [II] Furthermore, the method for producing a peptide compound having a cyclic portion of the present invention can include one or more of the following steps: A) Providing pdCpA by aminoacylation of compound C-1 B) Providing a tRNA lacking a CA at the 3' end C) A step of linking the pdCpA of step A) with the tRNA of step B) to provide an initiator tRNA aminoacylated with compound C-1. D) Providing a cell-free translation system containing the tRNA of step C) E) providing a template DNA library encoding peptide sequences containing, downstream of a promoter, a translation initiation ATG followed by a cysteine ​​codon UGU or UGC, and downstream of that a codon corresponding to the anticodon of the tRNA in step C); F) A step of providing an mRNA library from the template DNA library of step E). G) A step of attaching a spacer to the 3' end of the mRNA library of step F). H) A step of adding the spacer-bound mRNA library of step G) to the cell-free translation system of step D) and translating the mRNA to provide a pre-cyclized peptide compound-mRNA complex display library. I) A step of treating the library of step H) with peptide deformylase and methionine aminopeptidase Steps H and I can also be carried out simultaneously by adding peptide deformylase and methionine aminopeptidase to the system during translation. J) Step of forming a cyclic structure In forming the circular structure, a desulfurization reaction can be carried out as necessary. Furthermore, in the present invention, after step H) to step I), a step of synthesizing cDNA using a primer that anneals to the 3' region of the mRNA library can be included. The method of the present invention may further comprise the following steps: J) A step of enriching the mRNA library that binds to the target substance by panning K) A process of synthesizing cDNA using reverse transcriptase L) A step of analyzing the base sequence

[0191] [III] Alternatively, the method for producing a peptide compound having a cyclic portion of the present invention can include one or more of the following steps: A) Providing pdCpA by aminoacylation of compound C-3 (R2 = R3 = R28 = R29 = H, L-aspartic acid derivative) B) Providing a tRNA lacking a CA at the 3' end C) A step of linking the pdCpA of step A) with the tRNA of step B) to provide an initiator tRNA aminoacylated with compound C-3. D) providing a cell-free translation system containing the tRNA of step C) but not containing methionine, methionyl-tRNA synthetase (MetRS), translation initiation tRNA for methionine, formyl donor, or methionyl-tRNA transferase E) providing a template DNA library that encodes peptide sequences, the template DNA library having, downstream of a promoter, a cysteine ​​codon following the translation initiation codon ATG and, further downstream of that, a codon corresponding to the anticodon of the tRNA in step C); F) A step of providing an mRNA library from the template DNA library of step E). G) A step of attaching a spacer to the 3' end of the mRNA library of step F). H) A step of adding the spacer-bound mRNA library of step G) to the cell-free translation system of step D) and translating the mRNA to provide a pre-cyclized peptide compound-mRNA complex display library. I) A step of forming a cyclic structure and then carrying out a desulfurization reaction In the present invention, after step G), a step of synthesizing cDNA using a primer that anneals to the 3' region of the mRNA library can be included. The method of the present invention can further include the following step: J) A step of enriching the mRNA library that binds to the target substance by panning. K) A process of synthesizing cDNA using reverse transcriptase L) A step of analyzing the base sequence

[0192] [IV] Alternatively, the method for producing a peptide compound having a cyclic portion of the present invention can include one or more of the following steps: A) Providing pdCpA by aminoacylation of compound C-3 (R2 = R3 = R28 = R29 = H, L-aspartic acid derivative) B) Providing a tRNA lacking a CA at the 3' end C) A step of linking the pdCpA of step A) with the tRNA of step B) to provide an initiator tRNA aminoacylated with compound C-3. D) Providing a cell-free translation system containing the tRNA of step C) E) providing a template DNA library that encodes peptide sequences, the template DNA library having, downstream of a promoter, a cysteine ​​codon following the translation initiation codon ATG and, further downstream of that, a codon corresponding to the anticodon of the tRNA in step C); F) A step of providing an mRNA library from the template DNA library of step E). G) A step of attaching a spacer to the 3' end of the mRNA library of step F). H) A step of adding the spacer-bound mRNA library of step G) to the cell-free translation system of step D) and translating the mRNA to provide a pre-cyclized peptide compound-mRNA complex display library. I) A step of treating the library of step H) with peptide deformylase and methionine aminopeptidase Steps H and I can also be carried out simultaneously by adding peptide deformylase and methionine aminopeptidase to the system during translation. I) A step of forming a cyclic structure and then carrying out a desulfurization reaction In the present invention, after step G), a step of synthesizing cDNA using a primer that anneals to the 3' region of the mRNA library can be included. The method of the present invention can further include the following step: J) A step of enriching the mRNA library that binds to the target substance by panning. K) A process of synthesizing cDNA using reverse transcriptase L) A step of analyzing the base sequence

[0193] [V-1] Introduction of amino acids other than methionine, amino acid analogs, or N-terminal carboxylic acid analogs to the N-terminus Alternatively, the method for producing a peptide compound having a cyclic portion of the present invention is a method for constructing an amide-cyclized peptide compound library, which can be carried out by using tBSSEtGABA or tBSSEtβAla, which are not easily tolerated in translation elongation reactions, at the N-terminus, and the method comprises one or more of the following steps: A) Providing pdCpA by aminoacylation of tBSSEtGABA or tBSSEtβAla B) Providing an initiator tRNA lacking a CA at its 3' end C) linking the pdCpA of step A) with the initiator tRNA of step B) to provide an initiator tRNA aminoacylated with tBSSEtGABA or tBSSEtβAla; D) A step of providing pdCpA by aminoacylation of compound C-1 E) Providing a tRNA lacking a CA at the 3' end F) a step of linking the pdCpA of step D) with the 3'-end tRNA of step E) to provide a tRNA aminoacylated with compound C-1; G) providing a cell-free translation system containing the initiation tRNA of step C) and the tRNA of step F) but not containing methionine, methionyl-tRNA synthetase (MetRS), translation initiation tRNA for methionine, formyl donor, or methionyl-tRNA transferase H) providing a template DNA library encoding peptide sequences having ATG as the first codon downstream of a promoter, and further downstream thereof a codon corresponding to the anticodon of the tRNA of step F), and further downstream thereof a codon for proline or an N-methyl amino acid that serves as a substrate for other ARSs on the 3' side of the codon; I) A step of providing an mRNA library from the template DNA library of step H). J) A step of attaching a spacer to the 3' end of the mRNA library of step I). K) adding the spacer-bound mRNA library of step J) to the cell-free translation system of step G) and translating it to provide a pre-cyclized peptide compound-mRNA complex display library; L) forming a cyclic portion and a linear portion 2. In the present invention, after step J), a step of synthesizing cDNA using a primer that anneals to the 3' region of the mRNA library can be included. M) A step of enriching the mRNA library that binds to the target substance by panning N) A process of synthesizing cDNA using reverse transcriptase O) A step of analyzing the base sequence

[0194] [V-2] Introduction of amino acids other than methionine, amino acid analogs, or N-terminal carboxylic acid analogs to the N-terminus Alternatively, the method for producing a peptide compound having a cyclic portion of the present invention is a method for constructing an amide-cyclized peptide compound library, which can be carried out by using tBSSEtGABA or tBSSEtβAla, which are not easily tolerated in translation elongation reactions, at the N-terminus, and the method comprises one or more of the following steps: A) Providing pdCpA by aminoacylation of tBSSEtGABA or tBSSEtβAla B) Providing an initiator tRNA lacking a CA at its 3' end C) linking the pdCpA of step A) with the initiator tRNA of step B) to provide an initiator tRNA aminoacylated with tBSSEtGABA or tBSSEtβAla; D) A step of providing pdCpA by aminoacylation of compound C-1 E) Providing a tRNA lacking a CA at the 3' end F) linking the pdCpA of step D) with the 3'-terminal tRNA of step E) to provide a tRNA aminoacylated with compound C-1, providing a pdCpA aminoacylated with any N-methyl amino acid, and then providing a tRNA lacking CA at the 3' terminal, linking the pdCpA and tRNA to provide a tRNA aminoacylated with an N-methyl amino acid; G) providing a cell-free translation system containing the initiation tRNA of step C) and the tRNA of step F) but not containing methionine, methionyl-tRNA synthetase (MetRS), translation initiation tRNA for methionine, formyl donor, or methionyl-tRNA transferase H) providing a template DNA library encoding peptide sequences having ATG as the first codon downstream of a promoter, further downstream thereof a codon corresponding to the anticodon of the tRNA of step F), and 3' to the codon for the N-methylaminoacylated tRNA of step F); I) A step of providing an mRNA library from the template DNA library of step H). J) A step of attaching a spacer to the 3' end of the mRNA library of step I). K) adding the spacer-bound mRNA library of step J) to the cell-free translation system of step G) and translating it to provide a pre-cyclized peptide compound-mRNA complex display library; L) forming a cyclic portion and a linear portion 2. In the present invention, after step J), a step of synthesizing cDNA using a primer that anneals to the 3' region of the mRNA library can be included. M) A step of enriching the mRNA library that binds to the target substance by panning N) A process of synthesizing cDNA using reverse transcriptase O) A step of analyzing the base sequence

[0195] [VI-1] Introduction of amino acids other than methionine, amino acid analogs, or N-terminal carboxylic acid analogs to the N-terminus Alternatively, the method for producing a peptide compound having a cyclic portion of the present invention is a method for constructing an amide-cyclized peptide compound library, which can be carried out by using tBSSEtGABA or tBSSEtβAla, which are not easily tolerated in translation elongation reactions, at the N-terminus, and the method comprises one or more of the following steps: A) Providing pdCpA by aminoacylation of tBSSEtGABA or tBSSEtβAla B) Providing an initiator tRNA lacking a CA at its 3' end C) linking the pdCpA of step A) with the initiator tRNA of step B) to provide an initiator tRNA aminoacylated with tBSSEtGABA or tBSSEtβAla; D) Providing Asp(SBn)-aminoacylated pdCpA E) Providing a tRNA lacking a CA at the 3' end F) a step of linking the pdCpA of step D) with the 3'-end tRNA of step E) to provide a tRNA aminoacylated with Asp(SBn); G) providing a cell-free translation system containing the initiation tRNA of step C) and the tRNA of step F) but not containing methionine, methionyl-tRNA synthetase (MetRS), translation initiation tRNA for methionine, formyl donor, or methionyl-tRNA transferase H) providing a template DNA library encoding peptide sequences having ATG as the first codon downstream of a promoter, and further downstream thereof a codon corresponding to the anticodon of the tRNA of step F), and further downstream thereof a codon for proline or an N-methyl amino acid that serves as a substrate for other ARSs on the 3' side of the codon; I) A step of providing an mRNA library from the template DNA library of step H). J) A step of attaching a spacer to the 3' end of the mRNA library of step I). K) adding the spacer-bound mRNA library of step J) to the cell-free translation system of step G) and translating it to provide a pre-cyclized peptide compound-mRNA complex display library; L) forming a cyclic portion and a linear portion 2. In the present invention, after step J), a step of synthesizing cDNA using a primer that anneals to the 3' region of the mRNA library can be included. M) A step of enriching the mRNA library that binds to the target substance by panning N) A process of synthesizing cDNA using reverse transcriptase O) A step of analyzing the base sequence

[0196] [VI-2] Introduction of amino acids other than methionine, amino acid analogs, or N-terminal carboxylic acid analogs to the N-terminus Alternatively, the method for producing a peptide compound having a cyclic portion of the present invention is a method for constructing an amide-cyclized peptide compound library, which can be carried out by using tBSSEtGABA or tBSSEtβAla, which are not easily tolerated in translation elongation reactions, at the N-terminus, and the method comprises one or more of the following steps: A) Providing pdCpA by aminoacylation of tBSSEtGABA or tBSSEtβAla B) Providing an initiator tRNA lacking a CA at its 3' end C) linking the pdCpA of step A) with the initiator tRNA of step B) to provide an initiator tRNA aminoacylated with tBSSEtGABA or tBSSEtβAla; D) Providing Asp(SBn)-aminoacylated pdCpA E) Providing a tRNA lacking a CA at the 3' end F) linking the pdCpA of step D) with the 3'-terminal tRNA of step E) to provide a tRNA aminoacylated with Asp(SBn), providing a pdCpA aminoacylated with any N-methyl amino acid, then providing a tRNA lacking CA at the 3' terminal, linking the pdCpA and tRNA to provide a tRNA aminoacylated with an N-methyl amino acid; G) providing a cell-free translation system containing the initiation tRNA of step C) and the tRNA of step F) but not containing methionine, methionyl-tRNA synthetase (MetRS), translation initiation tRNA for methionine, formyl donor, or methionyl-tRNA transferase H) providing a template DNA library encoding peptide sequences having ATG as the first codon downstream of a promoter, further downstream thereof a codon corresponding to the anticodon of the tRNA of step F), and 3' to the codon for the N-methylaminoacylated tRNA of step F); I) A step of providing an mRNA library from the template DNA library of step H). J) A step of attaching a spacer to the 3' end of the mRNA library of step I). K) adding the spacer-bound mRNA library of step J) to the cell-free translation system of step G) and translating it to provide a pre-cyclized peptide compound-mRNA complex display library; L) forming a cyclic portion and a linear portion 2. In the present invention, after step J), a step of synthesizing cDNA using a primer that anneals to the 3' region of the mRNA library can be included. M) A step of enriching the mRNA library that binds to the target substance by panning N) A process of synthesizing cDNA using reverse transcriptase O) A step of analyzing the base sequence

[0197] [VII-1] Introduction of amino acids other than methionine, amino acid analogs, or N-terminal carboxylic acid analogs to the N-terminus Alternatively, the method for producing a peptide compound having a cyclic portion of the present invention relates to a method for constructing an amide-cyclized peptide library, which can be carried out by using an amino acid that is poorly tolerated in a translation elongation reaction as the N-terminus, and includes one or more of the following steps: A) Providing pdCpA by aminoacylation of compound N-1 B) Providing an initiator tRNA lacking a CA at its 3' end C) linking the pdCpA of step A) with the initiator tRNA of step B) to provide an aminoacylated initiator tRNA in compound N-1 or compound N-2; D) A step of providing pdCpA by aminoacylation of compound C-1 E) Providing a tRNA lacking a CA at the 3' end F) linking the pdCpA of step D) with the tRNA of step E) to provide a tRNA aminoacylated with compound C-1; G) providing a cell-free translation system containing the initiation tRNA of step C) and the tRNA of step F) but not containing methionine, methionyl-tRNA synthetase (MetRS), translation initiation tRNA for methionine, formyl donor, or methionyl-tRNA transferase H) providing a template DNA library encoding peptide sequences downstream of a promoter, the template DNA library having as its first codon a codon corresponding to the anticodon of the translation initiation tRNA of step C) and further downstream thereof a codon corresponding to the anticodon of the tRNA of step F); I) A step of providing an mRNA library from the template DNA library of step H). J) A step of attaching a spacer to the 3' end of the mRNA library of step I). K) A step of adding the spacer-bound mRNA library of step J) to the cell-free translation system of step G) and translating the mRNA to provide a pre-cyclized peptide compound-mRNA complex display library. L) Step of forming a cyclic portion In the present invention, after step J), a step of synthesizing cDNA using a primer that anneals to the 3' region of the mRNA library can be included. Further, the method of the present invention can include the following steps. M) A step of enriching the mRNA library that binds to the target substance by panning N) A process of synthesizing cDNA using reverse transcriptase O) A step of analyzing the base sequence

[0198] [VII-2] Introduction of amino acids other than methionine, amino acid analogs, or N-terminal carboxylic acid analogs to the N-terminus Alternatively, the method for producing a peptide compound having a cyclic portion of the present invention relates to a method for constructing an amide-cyclized peptide library, which can be carried out by using an amino acid that is poorly tolerated in a translation elongation reaction as the N-terminus, and includes one or more of the following steps: A) Providing pdCpA by aminoacylation of compound N-2 B) Providing an initiator tRNA lacking a CA at its 3' end C) linking the pdCpA of step A) with the initiator tRNA of step B) to provide an aminoacylated initiator tRNA in compound N-1 or compound N-2; D) A step of providing pdCpA by aminoacylation of compound C-1 E) Providing a tRNA lacking a CA at the 3' end F) linking the pdCpA of step D) with the tRNA of step E) to provide a tRNA aminoacylated with compound C-1; G) providing a cell-free translation system containing the initiation tRNA of step C) and the tRNA of step F) but not containing methionine, methionyl-tRNA synthetase (MetRS), translation initiation tRNA for methionine, formyl donor, or methionyl-tRNA transferase H) providing a template DNA library encoding peptide sequences downstream of a promoter, the template DNA library having as its first codon a codon corresponding to the anticodon of the translation initiation tRNA of step C), further downstream a codon corresponding to the anticodon of the tRNA of step F), and further downstream a codon for proline or an N-methyl amino acid that serves as a substrate for other ARSs on the 3' side of the codon; I) A step of providing an mRNA library from the template DNA library of step H). J) A step of attaching a spacer to the 3' end of the mRNA library of step I). K) A step of adding the spacer-bound mRNA library of step J) to the cell-free translation system of step G) and translating the mRNA to provide a pre-cyclized peptide compound-mRNA complex display library. L) Step of forming a cyclic portion In the present invention, after step J), a step of synthesizing cDNA using a primer that anneals to the 3' region of the mRNA library can be included. Further, the method of the present invention can include the following steps. M) A step of enriching the mRNA library that binds to the target substance by panning N) A process of synthesizing cDNA using reverse transcriptase O) A step of analyzing the base sequence

[0199] [VII-3] Introduction of amino acids other than methionine, amino acid analogs, or N-terminal carboxylic acid analogs to the N-terminus Alternatively, the method for producing a peptide compound having a cyclic portion of the present invention relates to a method for constructing an amide-cyclized peptide library, which can be carried out by using an amino acid that is poorly tolerated in a translation elongation reaction as the N-terminus, and includes one or more of the following steps: A) Providing pdCpA by aminoacylation of compound N-2 B) Providing an initiator tRNA lacking a CA at its 3' end C) linking the pdCpA of step A) with the initiator tRNA of step B) to provide an aminoacylated initiator tRNA in compound N-1 or compound N-2; D) A step of providing pdCpA by aminoacylation of compound C-1 E) Providing a tRNA lacking a CA at the 3' end F) linking the pdCpA of step D) with the tRNA of step E) to provide a tRNA aminoacylated with compound C-1, further providing a pdCpA aminoacylated with any N-methyl amino acid, further providing a tRNA lacking CA at the 3' end, and further linking these pdCpAs with tRNA to provide a tRNA aminoacylated with an N-methyl amino acid; G) providing a cell-free translation system containing the initiation tRNA of step C) and the tRNA of step F) but not containing methionine, methionyl-tRNA synthetase (MetRS), translation initiation tRNA for methionine, formyl donor, or methionyl-tRNA transferase H) providing a template DNA library encoding peptide sequences downstream of a promoter, the template DNA library having as its first codon a codon corresponding to the anticodon of the translation initiation tRNA of step C), further downstream a codon corresponding to the anticodon of the tRNA of step F), and further downstream a codon for the N-methylaminoacylated tRNA of step F) on the 3' side of the codon; I) A step of providing an mRNA library from the template DNA library of step H). J) A step of attaching a spacer to the 3' end of the mRNA library of step I). K) A step of adding the spacer-bound mRNA library of step J) to the cell-free translation system of step G) and translating the mRNA to provide a pre-cyclized peptide compound-mRNA complex display library. L) Step of forming a cyclic portion In the present invention, after step J), a step of synthesizing cDNA using a primer that anneals to the 3' region of the mRNA library can be included. Further, the method of the present invention can include the following steps. M) A step of enriching the mRNA library that binds to the target substance by panning N) A process of synthesizing cDNA using reverse transcriptase O) A step of analyzing the base sequence

[0200] [VIII-1] Cyclization of peptides having an amino acid other than methionine at the N-terminus, or the method for producing a peptide compound having a cyclic portion of the present invention, is a method for constructing an amide-cyclized peptide library with different cyclization site structures, which can be carried out by simultaneously translating multiple types of peptides with different N-terminal amino acids, and relates to a method comprising one or more of the following steps: A) A step of providing pdCpA obtained by aminoacylation of Compound CX ("Compound CX" in this specification refers to any compound selected from Compound C-1, Compound C-2, and Compound C-3; the same applies hereinafter in this specification). B) Providing a tRNA lacking a CA at the 3' end C) Linking the pdCpA of step A) with the tRNA of step B) to provide an initiator tRNA aminoacylated with compound CX. D) Providing a cell-free translation system containing the tRNA of step C) E) providing a template DNA library downstream of a promoter, the template DNA library encoding peptide sequences containing a codon corresponding to the anticodon of the tRNA of step C) and a codon for proline or other N-methyl amino acid that serves as a substrate for ARS on the 3' side of the codon; F) A step of providing an mRNA library from the template DNA library of step E). G) A step of attaching a spacer to the 3' end of the mRNA library of step F). H) A step of adding the spacer-bound mRNA library of step G) to the cell-free translation system of step D) and translating the mRNA to provide a pre-cyclized peptide compound-mRNA complex display library. I) A step of treating the library of step H) with peptide deformylase and methionine aminopeptidase Steps H and I can also be carried out simultaneously by adding peptide deformylase and methionine aminopeptidase to the system during translation. J) Step of forming a cyclic structure In forming the circular structure, a desulfurization reaction can be carried out as necessary. Furthermore, in the present invention, after step H) to step I), a step of synthesizing cDNA using a primer that anneals to the 3' region of the mRNA library can be included. The method of the present invention may further comprise the following steps: J) A step of enriching the mRNA library that binds to the target substance by panning K) A process of synthesizing cDNA using reverse transcriptase L) A step of analyzing the base sequence

[0201] [IX] Cyclization of peptides having glycine, alanine, or phenylalanine at the N-terminus. Alternatively, the method for producing a peptide compound having a cyclic moiety of the present invention is a method for constructing an amide-cyclized peptide library with different cyclization moiety structures, which can be carried out by simultaneously translating multiple types of peptides with different N-terminal amino acids, and the method comprises one or more of the following steps: A) Providing pdCpA by aminoacylation of compound CX B) Providing a tRNA lacking a CA at the 3' end C) Linking the pdCpA of step A) with the tRNA of step B) to provide an initiator tRNA aminoacylated with compound CX. D) Providing a cell-free translation system containing the tRNA of step C) E) providing a template DNA library encoding peptide sequences containing, downstream of a promoter, a glycine, alanine, or phenylalanine codon immediately after the translation initiation ATG, a codon corresponding to the anticodon of the tRNA in step C), and a codon for proline or an N-methyl amino acid that serves as a substrate for other ARS on the 3' side of the codon; F) A step of providing an mRNA library from the template DNA library of step E). G) A step of attaching a spacer to the 3' end of the mRNA library of step F). H) A step of adding the spacer-bound mRNA library of step G) to the cell-free translation system of step D) and translating the mRNA to provide a pre-cyclized peptide compound-mRNA complex display library. I) A step of treating the library of step H) with peptide deformylase and methionine aminopeptidase Steps H and I can also be carried out simultaneously by adding peptide deformylase and methionine aminopeptidase to the system during translation. J) Step of forming a cyclic structure In forming the circular structure, a desulfurization reaction can be carried out as necessary. Furthermore, in the present invention, after step H) to step I), a step of synthesizing cDNA using a primer that anneals to the 3' region of the mRNA library can be included. The method of the present invention may further comprise the following steps: J) A step of enriching the mRNA library that binds to the target substance by panning K) A process of synthesizing cDNA using reverse transcriptase L) A step of analyzing the base sequence

[0202] [X] Cyclization of peptides having an amino acid other than methionine at the N-terminus, or a method for producing a peptide compound having a cyclic portion of the present invention, is a method for constructing an amide-cyclized peptide library with different cyclization site structures, which can be carried out by simultaneously translating multiple types of peptides with different N-terminal amino acids, and relates to a method comprising one or more of the following steps: A) Providing pdCpA aminoacylated with Asp(SBn) B) Providing a tRNA lacking a CA at the 3' end C) A step of linking the pdCpA of step A) with the tRNA of step B) to provide an initiator tRNA having Asp(SBn) aminoacylated. D) Providing a cell-free translation system containing the tRNA of step C) E) providing a template DNA library downstream of a promoter, encoding peptide sequences containing a codon corresponding to the anticodon of the tRNA in step C) and, on the 3' side thereof, a codon for proline or an N-methyl amino acid that serves as a substrate for other ARSs. F) A step of providing an mRNA library from the template DNA library of step E). G) A step of attaching a spacer to the 3' end of the mRNA library of step F). H) A step of adding the spacer-bound mRNA library of step G) to the cell-free translation system of step D) and translating the mRNA to provide a pre-cyclized peptide compound-mRNA complex display library. I) A step of treating the library of step H) with peptide deformylase and methionine aminopeptidase Steps H and I can also be carried out simultaneously by adding peptide deformylase and methionine aminopeptidase to the system during translation. J) Step of forming a cyclic structure In forming the circular structure, a desulfurization reaction can be carried out as necessary. Furthermore, in the present invention, after step H) to step I), a step of synthesizing cDNA using a primer that anneals to the 3' region of the mRNA library can be included. The method of the present invention may further comprise the following steps: J) A step of enriching the mRNA library that binds to the target substance by panning K) A process of synthesizing cDNA using reverse transcriptase L) A step of analyzing the base sequence

[0203] [XI] Alternatively, the method for producing a peptide compound having a cyclic portion of the present invention relates to a method for constructing a cyclic branched peptide library, which method comprises one or more of the following steps: A) Providing pdCpA by aminoacylation of Compound N-1 or Compound N-2 B) Providing an initiator tRNA lacking a CA at its 3' end C) linking the pdCpA of step A) with the initiator tRNA of step B) to provide an initiator tRNA aminoacylated with compound N-1 or compound N-2; D) A step of providing pdCpA by aminoacylation of compound C-1 E) Providing a tRNA lacking a CA at the 3' end F) linking the pdCpA of step D) with the tRNA of step E) to provide a tRNA aminoacylated with compound C-1; G) providing a cell-free translation system containing the initiation tRNA of step C) and the tRNA of step F) but not containing methionine, methionyl-tRNA synthetase (MetRS), translation initiation tRNA for methionine, formyl donor, or methionyl-tRNA transferase H) A step of providing a template DNA library encoding peptide sequences downstream of a promoter, which has as its first codon a codon corresponding to the anticodon of the translation initiation tRNA of step C), downstream of which are 0 to 2 arbitrary codons sandwiched between a HOGly codon and a lysine codon (alanine is closer to the N-terminus), and further downstream of which is a codon corresponding to the anticodon of the tRNA of step F), and when the SH group of compound N-1 or compound N-2 is protected, a codon for proline or another N-methyl amino acid that serves as a substrate for ARS is provided on the 3' side of the codon. I) A step of providing an mRNA library from the template DNA library of step H). J) A step of attaching a spacer to the 3' end of the mRNA library of step I). K) A step of adding the spacer-bound mRNA library of step J) to the cell-free translation system of step J) and translating the mRNA library to provide a peptide display library of pre-cyclized peptide compound-mRNA complexes. L) Ring formation step M) A step of activating the ester formed between HOGly and the immediately preceding N-terminal amino acid to generate a thioester. N) A step of forming an amide bond with the amino group of the lysine side chain to generate a linear portion 2 In the present invention, after step J), a step of synthesizing cDNA using a primer that anneals to the 3' region of the mRNA library can be included. Further, the method of the present invention can include the following steps. O) A step of enriching the mRNA library that binds to the target substance by panning P) A process of synthesizing cDNA using reverse transcriptase Q) The process of analyzing the base sequence

[0204] [XII] Alternatively, the method for producing a peptide compound having a cyclic portion of the present invention relates to a method for constructing a cyclic branched peptide library, which method comprises one or more of the following steps: A) Providing pdCpA by aminoacylation of Compound N-1 or Compound N-2 B) Providing a tRNA lacking a CA at the 3' end C) linking the pdCpA of step A) with the tRNA of step B) to provide tRNA aminoacylated with compound N-1 or compound N-2; D) A step of providing pdCpA by aminoacylation of compound C-1 E) Providing a tRNA lacking a CA at the 3' end F) linking the pdCpA of step D) with the tRNA of step E) to provide a tRNA aminoacylated with compound C-1; G) providing a cell-free translation system containing the tRNA of step C) and the tRNA and lactate of step F) H) providing a template DNA library encoding peptide sequences downstream of a promoter, which has as its second codon a codon corresponding to the anticodon of the tRNA in step C), followed downstream by a codon for cysteine, proline, and lactic acid, followed further downstream by lysine, followed further downstream by a codon corresponding to the anticodon of the tRNA in step F), and, when the SH group of compound N-1 or compound N-2 is protected, a codon for proline or an N-methyl amino acid serving as a substrate for other ARS on the 3' side of the codon I) A step of providing an mRNA library from the template DNA library of step H). J) A step of attaching a spacer to the 3' end of the mRNA library of step I). K) A step of adding the spacer-bound mRNA library of step J) to the cell-free translation system of step J) and translating the mRNA library to provide a peptide display library of pre-cyclized peptide compound-mRNA complexes. L) Ring formation step M) A process for generating active thioesters from cysteine, proline, and lactic acid moieties N) A step of forming an amide bond between the generated activated thioester and the amino group of the lysine side chain to generate the linear portion 2, followed by a step of carrying out a desulfurization reaction. In the present invention, after step J), a step of synthesizing cDNA using a primer that anneals to the 3' region of the mRNA library can be included. Further, the method of the present invention can include the following steps. O) A step of enriching the mRNA library that binds to the target substance by panning P) A process of synthesizing cDNA using reverse transcriptase Q) The process of analyzing the base sequence

[0205] [XIII] Alternatively, the method for producing a peptide compound having a cyclic portion of the present invention relates to a method for constructing a cyclic branched peptide library, which method comprises one or more of the following steps: A) Providing pdCpA by aminoacylation of Compound N-1 or Compound N-2 B) Providing a tRNA lacking a CA at the 3' end C) linking the pdCpA of step A) with the tRNA of step B) to provide tRNA aminoacylated with compound N-1 or compound N-2; D) A step of providing pdCpA by aminoacylation of compound C-1 E) Providing a tRNA lacking a CA at the 3' end F) linking the pdCpA of step D) with the tRNA of step E) to provide a tRNA aminoacylated with compound C-1; G) providing a cell-free translation system containing the tRNA of step C) and the tRNA and lactate of step F) H) providing a template DNA library encoding peptide sequences having, downstream of a promoter, a codon corresponding to the anticodon of the tRNA in step C) as the second codon, followed downstream by a codon for cysteine, proline, and lactic acid, followed downstream by a codon for lysine, followed further downstream by a codon corresponding to the anticodon of the tRNA in step F), and further downstream by a codon for proline or an N-methyl amino acid that serves as a substrate for other ARS on the 3' side of the codon; I) A step of providing an mRNA library from the template DNA library of step H). J) A step of attaching a spacer to the 3' end of the mRNA library of step I). K) A step of adding the spacer-bound mRNA library of step J) to the cell-free translation system of step J) and translating the mRNA library to provide a peptide display library of pre-cyclized peptide compound-mRNA complexes. L) A step of treating the library of step K) with peptide deformylase and methionine aminopeptidase Steps K and L can also be carried out simultaneously by adding peptide deformylase and methionine aminopeptidase to the system during translation. M) Step of forming a cyclic structure In forming the cyclic structure, a desulfurization reaction can be carried out as necessary. N) A process for generating an active thioester from cysteine, proline, and lactic acid units O) A step of converting the generated carboxylic acid into an active ester and forming an amide bond with the amino group of the lysine side chain to generate the linear portion 2, followed by a desulfurization reaction as needed. In the present invention, after step J), a step of synthesizing cDNA using a primer that anneals to the 3' region of the mRNA library can be included. Further, the method of the present invention can include the following steps. P) A step of enriching the mRNA library that binds to the target substance by panning P) A process of synthesizing cDNA using reverse transcriptase Q) The process of analyzing the base sequence

[0206] [XIV] Alternatively, the method for producing a peptide compound having a cyclic portion of the present invention relates to a method for constructing a cyclic branched peptide library, which method comprises one or more of the following steps: A) Providing pdCpA by aminoacylation of compound C-1 E) Providing a tRNA lacking a CA at the 3' end F) linking the pdCpA of step D) with the tRNA of step E) to provide a tRNA aminoacylated with compound C-1; G) providing a cell-free translation system containing the tRNA of step C) and the tRNA and lactate of step F) H) providing a template DNA library encoding peptide sequences having a cysteine ​​codon as the second codon downstream of a promoter, followed downstream by a codon for cysteine, proline, and lactic acid, followed by a codon for the protected amine compound Na-4, followed further downstream by a codon corresponding to the anticodon of the tRNA of step F), and further downstream by a codon for proline or another N-methyl amino acid that serves as a substrate for ARS on the 3' side of the codon; I) A step of providing an mRNA library from the template DNA library of step H). J) A step of attaching a spacer to the 3' end of the mRNA library of step I). K) A step of adding the spacer-bound mRNA library of step J) to the cell-free translation system of step J) and translating the mRNA library to provide a peptide display library of pre-cyclized peptide compound-mRNA complexes. L) Ring formation step M) A step of deprotecting the side chain amino group of compound Na-4 and generating an active thioester from the cysteine, proline, and lactic acid units. N) A step of forming an amide bond between the generated activated thioester and the side chain amino group of compound Na-4 to generate linear portion 2 In the present invention, after step J), a step of synthesizing cDNA using a primer that anneals to the 3' region of the mRNA library can be included. Further, the method of the present invention can include the following steps. O) A step of enriching the mRNA library that binds to the target substance by panning P) A process of synthesizing cDNA using reverse transcriptase Q) The process of analyzing the base sequence

[0207] [XV] Alternatively, the method for producing a peptide compound having a cyclic portion of the present invention relates to a method for constructing a cyclic branched peptide library, which method comprises one or more of the following steps: A) Providing pdCpA by aminoacylation of Compound N-1 or Compound N-2 B) Providing a tRNA lacking a CA at the 3' end C) linking the pdCpA of step A) with the tRNA of step B) to provide tRNA aminoacylated with compound N-1 or compound N-2; D) A step of providing pdCpA by aminoacylation of compound C-1 E) Providing a tRNA lacking a CA at the 3' end F) linking the pdCpA of step D) with the tRNA of step E) to provide a tRNA aminoacylated with compound C-1; G) A step of providing pdCpA by aminoacylation of Compound Na-4, Compound Na-10 (Na-7 group), or Compound Na-11 (Na-7 group), in which the amino group and, if necessary, the thiol group are also protected. H) Providing a tRNA lacking a CA at the 3' end I) linking the pdCpA of step G) with the tRNA of step H) to provide an aminoacylated tRNA with compound Na-4, compound Na-10 (Na-7 group), or compound Na-11 (Na-7 group), in which the amino group and, if necessary, the thiol group are also protected; J) providing a cell-free translation system containing the tRNA of step C), the tRNA of step F), and the tRNA of step I); K) providing a template DNA library encoding peptide sequences downstream of a promoter, which has as its second codon a codon corresponding to the anticodon of the tRNA in step C), downstream of which are codons corresponding to the anticodons of tRNAs for Cys, Pro, and lactate, downstream of which are codons corresponding to the anticodons of the tRNA in step G), and further downstream of which are codons corresponding to the anticodons of the tRNA in step D), and on the 3' side of which are codons for proline or an N-methyl amino acid that serves as a substrate for other ARSs; L) A step of providing an mRNA library from the template DNA library of step K). M) A step of attaching a spacer to the 3' end of the mRNA library of step L). N) A step of adding the spacer-bound mRNA library of step J) to the cell-free translation system of step M) and translating the mRNA to provide a peptide display library of pre-cyclized peptide compound-mRNA complexes. O) A step of treating the library of step K) with peptide deformylase and methionine aminopeptidase Steps K and L can also be carried out simultaneously by adding peptide deformylase and methionine aminopeptidase to the system during translation. P) Step of forming a cyclic structure Q) A step of deprotecting Compound Na-4, Compound Na-10 (Na-7 group), or Compound Na-11 (Na-7 group) in which the amino group and, if necessary, the thiol group are also protected. R) Step of generating linear portion 2 In forming the cyclic structure, a desulfurization reaction can be carried out as necessary. In the present invention, after step J), a step of synthesizing cDNA using a primer that anneals to the 3' region of the mRNA library can be included. Further, the method of the present invention can include the following steps. S) A process of enriching the mRNA library that binds to the target substance by panning. T) A process of synthesizing cDNA using reverse transcriptase U) A step of analyzing the base sequence

[0208] In the above [XIII] and [XV], the peptides having N-1, 2 as the N-terminus can also be prepared by the same method as in the above [XII].

[0209] [XVI] A method for constructing a cyclic branched peptide library by cyclization of peptides having an amino acid other than methionine at the N-terminus, the method comprising one or more of the following steps: The method for producing a peptide compound having a cyclic portion of the present invention relates to a method for constructing an amide-cyclized peptide library with different cyclization site structures, which can be carried out by simultaneously translating multiple types of peptides with different N-terminal amino acids, the method comprising one or more of the following steps: A) Providing pdCpA by aminoacylation of compound C-1 B) Providing a tRNA lacking a CA at the 3' end C) linking the pdCpA of step A) with the tRNA of step B) to provide a tRNA aminoacylated with compound C-1; D) A step of providing pdCpA by aminoacylation of Compound Na-4, Compound Na-10 (Na-7 group), or Compound Na-11 (Na-7 group), in which the amino group and, if necessary, the thiol group of the compound are also protected. E) Providing a tRNA lacking a CA at the 3' end F) linking the pdCpA of step D) with the tRNA of step E) to provide an aminoacylated tRNA with Compound Na-4, Compound Na-10 (Na-7 group), or Compound Na-11 (Na-7 group), in which the amino group and, if necessary, the thiol group are also protected; G) providing a cell-free translation system containing the tRNA of step C), the tRNA of step F), and the tRNA of step I); H) providing a template DNA library encoding peptide sequences containing, downstream of a promoter, a codon corresponding to the anticodon of the tRNA in step F), downstream of which are codons corresponding to the anticodons of the tRNAs of Cys, Pro, and lactate, and further downstream of which are codons corresponding to the anticodons of the tRNA in step C), and on the 3' side of which are codons for proline or other N-methyl amino acids that serve as substrates for ARSs; I) A step of providing an mRNA library from the template DNA library of step H). J) A step of attaching a spacer to the 3' end of the mRNA library of step I). K) A step of adding the spacer-bound mRNA library of step J) to the cell-free translation system of step J) and translating the mRNA library to provide a peptide display library of pre-cyclized peptide compound-mRNA complexes. L) A step of treating the library of step K) with peptide deformylase and methionine aminopeptidase Steps K and L can also be carried out simultaneously by adding peptide deformylase and methionine aminopeptidase to the system during translation. L) Step of forming a cyclic structure M) A step of deprotecting Compound Na-4, Compound Na-10 (Na-7 group), or Compound Na-11 (Na-7 group) in which the amino group and, if necessary, the thiol group have been protected. N) Step of generating linear portion 2 In forming the cyclic structure, a desulfurization reaction can be carried out as necessary. In the present invention, after step J), a step of synthesizing cDNA using a primer that anneals to the 3' region of the mRNA library can be included. Further, the method of the present invention can include the following steps. O) A step of enriching the mRNA library that binds to the target substance by panning P) A process of synthesizing cDNA using reverse transcriptase Q) The process of analyzing the base sequence In step G), normal leucine can also be added instead of methionine.

[0210] [XVII] A method for constructing a cyclic branched peptide library by cyclizing peptides having an amino acid other than methionine at the N-terminus, the method comprising one or more of the following steps: The method for producing a peptide compound having a cyclic portion of the present invention relates to a method for constructing an amide-cyclized peptide library with different cyclization site structures, which can be carried out by simultaneously translating multiple types of peptides with different N-terminal amino acids, the method comprising one or more of the following steps: A) Providing pdCpA by aminoacylation of compound C-1 B) Providing a tRNA lacking a CA at the 3' end C) linking the pdCpA of step A) with the tRNA of step B) to provide a tRNA aminoacylated with compound C-1; D) A step of providing pdCpA by aminoacylation of Compound Na-4, Compound Na-10 (Na-7 group), or Compound Na-11 (Na-7 group), in which the amino group and, if necessary, the thiol group of the compound are also protected. E) Providing a tRNA lacking a CA at the 3' end F) linking the pdCpA of step D) with the tRNA of step E) to provide tRNA aminoacylated with Compound Na-4, Compound Na-10 (Na-7 group), or Compound Na-11 (Na-7 group), in which the amino group and, if necessary, the thiol group of the compound are also protected; G) providing a cell-free translation system containing the tRNA of step C) and the tRNA and lactate of step F) H) providing a template DNA library encoding peptide sequences downstream of a promoter, which has as its second codon a codon corresponding to the anticodon of the tRNA in step C), downstream of which are codons for cysteine, proline, and lactic acid, and further downstream of which are codons corresponding to the anticodon of the tRNA in step F), and on the 3' side of which are codons for proline or an N-methyl amino acid that serves as a substrate for other ARSs; I) A step of providing an mRNA library from the template DNA library of step H). J) A step of attaching a spacer to the 3' end of the mRNA library of step I). K) A step of adding the spacer-bound mRNA library of step J) to the cell-free translation system of step J) and translating the mRNA library to provide a peptide display library of pre-cyclized peptide compound-mRNA complexes. L) A step of treating the library of step K) with peptide deformylase and methionine aminopeptidase Steps K and L can also be carried out simultaneously by adding peptide deformylase and methionine aminopeptidase to the system during translation. M) Step of forming a cyclic structure In forming the cyclic structure, a desulfurization reaction can be carried out as necessary. N) A step of deprotecting the side chain protecting group of Compound Na-4, Compound Na-10 (Na-7 group), or Compound Na-11 (Na-7 group), and a step of generating an active thioester from Cys, Pro, and lactic acid units. O) A step of forming an amide bond between the generated active thioester and the side chain amino group to generate a linear portion 2 In the present invention, after step J), a step of synthesizing cDNA using a primer that anneals to the 3' region of the mRNA library can be included. Further, the method of the present invention can include the following steps. P) A step of enriching the mRNA library that binds to the target substance by panning Q) The process of synthesizing cDNA using reverse transcriptase R) The process of analyzing the base sequence In step G), normal leucine can also be added instead of methionine. Preparation of aminoacyl-tRNA is not limited to the use of pdCpA, but also includes the use of aminoacyl-tRNA synthetase, Flexizyme, ultrasonic mixing in cationic micelles, PNA amino acid activated ester method, etc.

[0211] Aspartimide formation inhibition method When an aspartic acid-type thioester is introduced by translation, it reacts with a hydrogen atom at the amide bond immediately after the C-terminus to form aspartimide. Therefore, when an aspartic acid-type thioester is introduced by translation, the amino acid residue immediately after it is changed to an amino acid with an N-alkyl group (e.g., proline), thereby synthesizing a full-length peptide containing the desired thioester.

[0212] As used herein, the term "alkyl group" refers to a monovalent group derived from an aliphatic hydrocarbon by removing any one hydrogen atom, and does not contain heteroatoms or unsaturated carbon-carbon bonds in the backbone, but has a subset of hydrocarbyl or hydrocarbon group structures containing hydrogen and carbon atoms. The carbon chain length n ranges from 1 to 20. Examples of the alkyl group include a "C1 to C6 alkyl group", and specific examples thereof include a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, an isopropyl group, a t-butyl group, a sec-butyl group, a 1-methylpropyl group, a 1,1-dimethylpropyl group, a 2,2-dimethylpropyl group, a 1,2-dimethylpropyl group, a 1,1,2-trimethylpropyl group, a 1,2,2-trimethylpropyl group, a 1,1,2,2-tetramethylpropyl group, a 1-methylbutyl group, a 2-methylbutyl group, a 3-methylbutyl group, a 1,1-dimethylbutyl group, a 1,2-dimethylbutyl group, a 1,1-dimethylbutyl group, a 1,2-dimethylbutyl group, a 1,3-dimethylbutyl group, a 2,2-dimethylbutyl group, a 2,3-dimethylbutyl group, a 3,3-dimethylbutyl group, a 1-ethylbutyl group, a 2-ethylbutyl group, an isopentyl group, and a neopentyl group. In this specification, the above-mentioned "alkyl group" may include the below-mentioned "alkenyl group" and "alkynyl group".

[0213] As used herein, the term "alkenyl group" refers to a monovalent group having at least one double bond (two adjacent SP carbon atoms). Depending on the configuration of the double bond and any substituents (if present), the geometry of the double bond can be Entgegen (E) or Zusammen (Z), cis or trans. Alkenyl groups include linear or branched groups, including linear groups containing internal olefins. C2-C10 alkenyl groups are preferred, and C2-C6 alkenyl groups are more preferred. Specific examples of such alkenyl groups include vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl (including cis and trans), 3-butenyl, pentenyl, and hexenyl.

[0214] As used herein, "alkynyl" refers to a monovalent group having at least one triple bond (two adjacent SP carbon atoms). Examples include linear or branched alkynyl groups, including internal alkylene groups. C2-C10 alkynyl groups are preferred, and C2-C6 alkynyl groups are more preferred. Specific examples of alkynyl include ethynyl, 1-propynyl, propargyl, 3-butynyl, pentynyl, hexynyl, 3-phenyl-2-propynyl, 3-(2'-fluorophenyl)-2-propynyl, 2-hydroxy-2-propynyl, 3-(3-fluorophenyl)-2-propynyl, and 3-methyl-(5-phenyl)-4-pentynyl.

[0215] The term "cycloalkyl group" refers to a saturated or partially saturated cyclic monovalent aliphatic hydrocarbon group, including monocyclic, bicyclic, and spirocyclic rings. Preferred examples include C3-C10 cycloalkyl groups. Specific examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and bicyclo[2.2.1]heptyl groups.

[0216] The term "C1-C6 alkyl group optionally having a halogen atom(s)" refers to a "C1-C6 alkyl group" substituted with one or more halogen atoms. Examples include a trifluoromethyl group, a difluoromethyl group, a fluoromethyl group, a pentafluoroethyl group, a tetrafluoroethyl group, a trifluoroethyl group, a difluoroethyl group, a fluoroethyl group, a trichloromethyl group, a dichloromethyl group, a chloromethyl group, a pentachloroethyl group, a tetrachloroethyl group, a trichloroethyl group, a dichloroethyl group, and a chloroethyl group.

[0217] "Halogen" means fluorine (F), chlorine (Cl), bromine (Br) or iodine (I).

[0218] As used herein, the term "aryl group" refers to a monovalent aromatic hydrocarbon ring, preferably a C5-C10 aryl. Specific examples of aryl include phenyl and naphthyl (e.g., 1-naphthyl and 2-naphthyl). In this specification, the above-mentioned "aryl group" may include the below-mentioned "heteroaryl".

[0219] As used herein, the term "heteroaryl" refers to a monovalent aromatic ring containing preferably 1 to 5 heteroatoms among the ring-constituting atoms, and may be partially saturated. The ring may be a monocyclic ring or two condensed rings (e.g., a bicyclic heteroaryl condensed with a benzene ring or a monocyclic heteroaryl ring). The number of atoms constituting the ring is preferably 5 to 10 (C5-C10 heteroaryl). Specific examples of heteroaryl include a furyl group, a thienyl group, a pyrrolyl group, an imidazolyl group, a pyrazolyl group, a thiazolyl group, an isothiazolyl group, an oxazolyl group, an isoxazolyl group, an oxadiazolyl group, a thiadiazolyl group, a triazolyl group, a tetrazolyl group, a pyridyl group, a pyrimidyl group, a pyridazinyl group, a pyrazinyl group, a triazinyl group, a benzofuranyl group, a benzothienyl group, a benzothiadiazolyl group, a benzothiazolyl group, a benzoxazolyl group, a benzoxadiazolyl group, a benzimidazolyl group, an indolyl group, an isoindolyl group, an indazolyl group, a quinolyl group, an isoquinolyl group, a cinnolinyl group, a quinazolinyl group, a quinoxalinyl group, a benzodioxolyl group, an indolizinyl group, and an imidazopyridyl group.

[0220] The term "C5 to C10 aryl C1-C6 alkyl group (aralkyl group)" refers to the above-mentioned C1-C6 alkyl group in which one of the hydrogen atoms has been substituted with the above-mentioned C5 to C10 aryl group.

[0221] An arylalkyl group (aralkyl group) refers to a group containing both an aryl group and an alkyl group, and refers to, for example, a group in which at least one hydrogen atom of the alkyl group is substituted with an aryl group. A preferred example is a "C5-C10 aryl C1-C6 alkyl group." For example, it may be a benzyl group. An "optionally substituted C5-C10 aryl C1-C6 alkyl group" refers to a group in which at least one hydrogen atom of the aryl group and / or alkyl group of the "C5-C10 aryl C1-C6 alkyl group" is substituted with a substituent. Examples of these substituents include the various substituents defined for the side chain substituents of "amino acids."

[0222] The term "alkoxy group" refers to a group in which the hydrogen atom of a hydroxyl group is substituted with the above-mentioned alkyl group, and preferably includes, for example, a "C1-C6 alkoxy group."

[0223] As used herein, the term "active ester group" refers to a group containing a carbonyl group that reacts with an amino group to form an amide bond, and is a group in which, for example, OBt, OAt, OSu, OPfp, SR1, or the like is bound to the carbonyl group, and is a group that can promote the reaction with an amino group.

[0224] A "reaction promoting group" is a group that is introduced near a functional group to be bonded to activate the functional group for the bonding reaction in order to selectively cause a reaction at a desired position. For example, to react a carbonyl group with an amino group, a reaction promoting group can be introduced to either or both of the carbonyl group and the amino group. Examples of such reaction promoting groups include SH. Such reaction promoting groups can be removed during or after the bonding reaction. By "normal amine" is meant an amine that is not activated by a reaction promoting group.

[0225] mRNA display The "nucleic acid" of the present invention can include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or nucleotide derivatives containing artificial bases. It can also include peptide nucleic acid (PNA). The nucleic acid of the present invention can be any one of these nucleic acids or a mixture thereof, as long as the desired genetic information is retained. In other words, the nucleic acid of the present invention also includes DNA-RNA hybrid nucleotides and chimeric nucleic acids in which different nucleic acids such as DNA and RNA are linked in a single strand. In the present invention, nucleic acid libraries such as display libraries containing these nucleic acids as templates can be suitably used.

[0226] A display library is a library in which a peptide (phenotype) is associated with the RNA or DNA that encodes the peptide (genotype). By contacting the library with a desired immobilized target and washing away molecules that do not bind to the target, it is possible to enrich for peptides that bind (panning method). By analyzing the genetic information associated with the peptides selected through this process, the sequences of proteins that bind to the target can be determined. For example, methods utilizing the nonspecific binding of the antibiotic puromycin, an aminoacyl-tRNA analog, to proteins during mRNA translation elongation by ribosomes have been reported as mRNA display (Proc Natl Acad Sci USA. 1997;94:12297-302. RNA-peptide fusions for the in vitro selection of peptides and proteins. Roberts RW, Szostak JW.) and in vitro virus display (FEBS Lett. 1997;414:405-8. In vitro virus: bonding of mRNA bearing puromycin at the 3'-terminal end to the C-terminal end of its encoded protein on the ribosome in vitro. Nemoto N, Miyamoto-Sato E, Husimi Y, Yanagawa H.).

[0227] A spacer such as puromycin is attached to the 3' end of an mRNA library obtained by transcription from a DNA library containing a promoter such as the T7 promoter, and when the mRNA is translated into protein in a cell-free translation system, puromycin is mistaken for an amino acid and incorporated into the protein by the ribosome, linking the mRNA and the protein it encodes, resulting in a library in which the mRNA and its product are associated. This process does not involve transformation of E. coli or other organisms, making it highly efficient and allowing the creation of large-scale display libraries (10 12 -10 14It is possible to construct a variety of amino acids (types). After panning, cDNA is synthesized from mRNA, which is a tag containing genetic information attached to the selected molecules. The cDNA is then amplified by PCR and sequenced to reveal the sequence of the bound protein. The amino acid residues in the DNA library template can be freely mixed and synthesized. Alternatively, a mixture of four bases (A, T, G, and C) (N) can be synthesized in multiples of three, or the first and second letters of the codon can be N, and the third letter can be a mixture of two bases (W, M, K, S). Furthermore, if the number of amino acid types introduced is limited to 16 or less, the third letter can be a single base. Furthermore, as shown in the examples, the frequency of amino acid residues can be freely adjusted by preparing codon units corresponding to the three letters of a codon and mixing them in any ratio for synthesis.

[0228] In addition to mRNA display, other display libraries using cell-free translation systems include cDNA display, which is a library consisting of cDNAs encoding peptides bound to a peptide-puromycin complex (Nucleic Acids Res. 2009;37(16):e108. cDNA display: a novel screening method for functional disulfide-rich peptides by solid-phase synthesis and stabilization of mRNA-protein fusions. Yamaguchi J, Naimuddin M, Biyani M, Sasaki T, Machida M, Kubo T, Funatsu T, Husimi Y, Nemoto N.), ribosome display, which utilizes the relatively stable complex between ribosomes and translation products during mRNA translation (Proc Natl Acad Sci U S A. 1994;91:9022-6. An in vitro polysome display system for identifying ligands from very large peptide libraries. Mattheakis LC, Bhatt RR, Dower WJ), and bacteriophage endonuclease Covalent display utilizes the covalent bond formed between P2A and DNA (Nucleic Acids Res. 2005;33:e10. Covalent antibody display--an in vitro antibody-DNA library selection system. Reiersen H, Lobersli I, Loset GA, Hvattum E, Simonsen B, Stacy JE, McGregor D, Fitzgerald K, Welschof M, Brekke OH, Marvik OJ.), and CIS display utilizes the binding of the replication initiator protein RepA of microbial plasmids to the origin of replication, ori (Proc Natl Acad Sci U S A.2004;101:2806-10. CIS display: In vitro selection of peptides from libraries of protein-DNA complexes. Odegrip R, Coomber D, Eldridge B, Hederer R, Kuhlman PA, Ullman C, FitzGerald K, McGregor D.) is known. Another method known is in vitro compartmentalization, in which a transcription-translation system is encapsulated in a water-in-oil emulsion or liposome for each DNA molecule constituting a DNA library, and the translation reaction is carried out (Nat Biotechnol. 1998;16:652-6. Man-made cell-like compartments for molecular evolution. Tawfik DS, Griffiths AD.). The above methods can be used appropriately using known methods.

[0229] In the present invention, these nucleic acid libraries can be translated using the cell-free translation system described below. When using a cell-free translation system, it is preferable to include a sequence encoding a spacer downstream of the target nucleic acid. Examples of spacer sequences include, but are not limited to, sequences containing glycine or serine. It is also preferable to include a linker formed from RNA, DNA, or a polymer of hexaethylene glycol (SPC18) (e.g., a five-member polymer) between the nucleic acid library and a compound that is incorporated into peptides during ribosomal translation, such as puromycin or its derivatives.

[0230] Cell-free translation system The method for producing a peptide compound of the present invention preferably uses a protein production system such as a cell-free translation system. A cell-free translation system can translate mRNA into protein by combining ribosomes extracted from cells, protein factors involved in translation, tRNA, amino acids, an energy source such as ATP, and a regeneration system. The cell-free translation system of the present invention can also include initiation factors, elongation factors, release factors, aminoacyl-tRNA synthetases, methionyl-tRNA transformylase, and other factors. These factors can be obtained by purification from extracts of various cells. Cells for purifying factors include, for example, prokaryotic or eukaryotic cells. Examples of prokaryotic cells include Escherichia coli cells, extreme thermophilic bacteria cells, and Bacillus subtilis cells. Known eukaryotic cells include yeast cells, wheat germ, rabbit reticulocytes, plant cells, insect cells, and animal cells.

[0231] The cell-free translation system is prepared by disrupting the source cells, and then adding tRNA, amino acids, ATP, etc. to the extract prepared by centrifugation, dialysis, etc.For example, E. coli (Methods Enzymol. 1983;101:674-90. Prokaryotic coupled transcription-translation. Chen HZ, Zubay G.), yeast (J. Biol. Chem. 1979 254:3965-3969. The preparation and characterization of a cell-free system from Saccharomyces cerevisiae that translates natural messenger ribonucleic acid. E Gasior, F Herrera, I Sadnik, CS McLaughlin, and K Moldave), wheat germ (Methods Enzymol. 1983;96:38-50. Cell-free translation of messenger RNA in a wheat germ system. Erickson AH, Blobel G.), and rabbit reticulocytes (Methods Enzymol. 1983;96:50-74. Preparation and use of nuclease-treated Lysates made from rabbit reticulocytes for the translation of eukaryotic messenger RNA. Jackson RJ, Hunt T.), HeLa cells (Methods Enzymol. 1996;275:35-57. Assays for poliovirus polymerase, 3D(Pol), and authentic RNA replication in HeLa S10 extracts. Barton DJ, Morasco BJ, Flanegan JB.), and insect cells (Comp Biochem Physiol B. 1989;93:803-6. Cell-free translation in lysates from Spodoptera frugiperda (Lepidoptera: Noctuidae) cells. Swerdel MR, Fallon AM.) can be used.Addition of an RNA polymerase such as T7 RNA polymerase enables coupled transcription and translation from DNA. The PURE system is a reconstituted cell-free translation system in which protein factors, energy regeneration enzymes, and ribosomes required for translation in Escherichia coli are extracted and purified, and then mixed with tRNA, amino acids, ATP, GTP, and other components. Not only does it contain low impurities, but because it is a reconstituted system, it is easy to create a system that does not contain protein factors or amino acids that you want to eliminate. ((i) Nat Biotechnol. 2001;19:751-5. Cell-free translation reconstituted with purified components. Shimizu Y, Inoue A, Tomari Y, Suzuki T, Yokogawa T, Nishikawa K, Ueda T. (ii) Methods Mol Biol. 2010;607:11-21. PURE technology. Shimizu Y, Ueda T.) The above method can be implemented using known methods as appropriate.

[0232] Various factors contained in cell-free translation systems, such as ribosomes and tRNAs, can be purified from E. coli cells or yeast cells by methods well known to those skilled in the art. In addition to naturally occurring tRNAs and aminoacyl-tRNA synthetases, artificial tRNAs and artificial aminoacyl-tRNA synthetases that recognize unnatural amino acids can also be used. By using artificial tRNAs and artificial aminoacyl-tRNA synthetases, peptides containing unnatural amino acids can be synthesized in a site-specific manner.

[0233] Translational incorporation of unnatural amino acids into peptides requires aminoacylation of orthogonal tRNAs that are efficiently incorporated into ribosomes ((i) Biochemistry. 2003;42:9598-608. Adaptation of an orthogonalarchaeal leucyl-tRNA and synthetase pair for four-base, amber, and opal suppression. Anderson JC, Schultz PG., (ii) Chem Biol. 2003;10:1077-84. Using a solid-phase ribozyme aminoacylation system to reprogram the genetic code. Murakami H, Kourouklis D, Suga H.). Five methods for aminoacylation of tRNA are available:

[0234] Within cells, aminoacyl-tRNA synthetases are prepared to aminoacylate tRNA for each amino acid. One method utilizes the fact that certain aminoacyl-tRNA synthetases tolerate unnatural amino acids such as N-Me His, and the other method utilizes mutant aminoacyl-tRNA synthetases that tolerate unnatural amino acids. ((i) Proc Natl Acad Sci U S A. 2002;99:9715-20. An engineered Escherichia coli tyrosyl-tRNA synthetase for site-specific incorporation of unnatural amino acids into proteins in eukaryotic translation and its application in a wheat germ cell-free system. Kiga D, Sakamoto K, Kodama K, Kigawa T, Matsuda T, Yabuki T, Shirouzu M, Harada Y, Nakayama H, Takio K, Hasegawa Y, Endo Y, Hirao I, Yokoyama S. (ii) Science. 2003;301:964-7. An expanded eukaryotic genetic code. Chin JW, Cropp TA, Anderson JC, Mukherji M, Zhang Z, Schultz PG. Chin, JW. (iii) Proc Natl Acad Sci U S A. 2006;103:4356-61. Enzymatic aminoacylation of tRNA with unnatural amino acids. Hartman MC, Josephson K, Szostak JW.) A method of aminoacylation of tRNA in vitro followed by chemical modification of the amino acid can also be used (J Am Chem Soc. 2008;130:6131-6. Ribosomal synthesis of N-methylpeptides. Subtelny AO, Hartman MC, Szostak JW.).Aminoacyl-tRNA can be obtained by removing the CA from the CCA sequence at the 3' end of tRNA and ligating it with separately prepared aminoacylated pdCpA using RNA ligase (Biochemistry. 1984;23:1468-73. T4 RNA ligase-mediated preparation of novel "chemically misacylated" tRNAPheS. Heckler TG, Chang LH, Zama Y, Naka T, Chorghade MS, Hecht SM.). Aminoacylation can also be achieved using flexizyme, a ribozyme that can attach activated esters of various unnatural amino acids to tRNA (J Am Chem Soc. 2002;124:6834-5. Aminoacyl-tRNA synthesis by aresin-immobilized ribozyme. Murakami H, Bonzagni NJ, Suga H.). Another method is to ultrasonically mix tRNA and an amino acid activated ester in cationic micelles (Chem Commun (Camb). 2005;(34):4321-3. Simple and quick chemical aminoacylation of tRNA in cationic micellar solution under ultrasonic agitation. Hashimoto N, Ninomiya K, Endo T, Sisido M.). Aminoacylation can also be achieved by adding an amino acid activated ester bou...

Claims

[Claim 1] The invention described in the specification.

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