Method for producing a peptide containing a non-natural amino acid

Abstract

To provide efficient methods for producing peptides comprising unnatural amino acids, and provide engineered L31 proteins for use in these methods and ribosomes comprising the same.SOLUTION: A method for producing a peptide comprises a step of translating an mRNA encoding a peptide comprising one or more types of unnatural amino acids in a translation system that comprises a ribosome having an engineered L31 protein, wherein the ribosome having the engineered L31 protein has greater activity for translation of the peptide comprising the unnatural amino acids, as compared to a ribosome comprising wild-type Escherichia coli L31 that comprises a specific amino acid sequence.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for producing a peptide containing a non-natural amino acid or a library containing the peptide. The present invention also relates to a modified L31 protein or the like for use in the method.

Background Art

[0002] In recent years, a drug discovery method has been considered in which a candidate substance for a pharmaceutical is selected from a library of various peptides containing a plurality of non-natural amino acids. Among them, an mRNA display library of peptides containing non-natural amino acids using a cell-free translation system has attracted expectations in terms of its diversity and simplicity of screening. Several methods for synthesizing peptides containing non-natural amino acids using a translation system have been reported (Non-Patent Documents 1 and 2). However, the methods described in these documents have low translation synthesis efficiency.

[0003] In vivo, a peptide is synthesized by polymerizing amino acids in accordance with the base sequence information of mRNA. This process of peptide synthesis is called translation. The ribosome plays a central role in the process of translation. Usually, purified ribosomes are added to the cell-free translation system used for preparing an mRNA display library.

[0004] As a protein constituting the ribosome, the L31 protein is known. The L31 protein forms inter subunit Bridge B1b and plays a role in stabilizing the association state (70S) of 30S and 50S (Non-Patent Document 3).

[0005] The L31 protein is known to be degraded by protease 7 during its purification. When comparing these activities for ribosomes prepared from Escherichia coli wild-type strains, ribosomes prepared from protease7 KO strains lacking protease 7, and ribosomes prepared from L31KO strains lacking the L31 protein, it has been reported that ribosomes prepared from protease 7-deficient strains have higher activity compared to ribosomes prepared from Escherichia coli wild-type strains or ribosomes prepared from L31-deficient strains (Non-Patent Document 3). It has also been reported that ribosomes prepared from L31-deficient strains have a 38% decrease in the initiation rate in vivo and a slower rate of 70S formation in vitro (Non-Patent Document 4). Furthermore, it has been reported that Fidelity decreases in L31-deficient strains (Non-Patent Document 4). Thus, in the translation of peptides composed of natural amino acids, ribosomes containing the L31 protein that have not been degraded by protease 7 are known to have higher activity than ribosomes containing the cleaved L31 protein or ribosomes without the L31 protein.

Prior Art Documents

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

[0007] In one aspect, the present invention aims to provide an efficient method for producing peptides containing unnatural amino acids, a modified L31 protein for use in the method, and a ribosome containing the same. [Means for Solving the Problems]

[0008] When the present inventors attempted to produce a peptide containing a non-natural amino acid using a translation system, they examined whether there was a difference in the translation efficiency of mRNA between ribosomes containing L31 protein that had not been degraded by protease 7 and ribosomes containing L31 protein cleaved by protease 7. As a result, the present inventors found that when translating an mRNA encoding a peptide containing a non-natural amino acid by the Initiation Suppression (iSP) method, using ribosomes containing L31 protein cleaved by protease 7 increased the amount of the target product translated compared to using ribosomes containing L31 protein that had not been degraded by protease 7. The present inventors also found that using ribosomes containing L31 protein cleaved by protease 7 made it possible to reduce the relative amount of by-products. These results were contrary to the facts known in the translation of peptides not containing non-natural amino acids. Furthermore, when the present inventors synthesized peptides by changing the magnesium ion concentration in the translation system, they clarified that when the magnesium ion concentration was within a certain concentration range, the amount of translation was large and the proportion of by-products was low.

[0009] The present invention is based on such findings, and specifically relates to the following [1] to

[37] . [1] A method for producing a peptide, comprising a step of translating an mRNA encoding a peptide containing one or more non-natural amino acids in a translation system containing ribosomes containing a modified L31 protein, wherein the ribosomes containing the modified L31 protein have a higher translation activity for the peptide containing the non-natural amino acid compared to ribosomes containing wild-type Escherichia coli L31 having the amino acid sequence of SEQ ID NO: 1. 〔2〕A method for producing a peptide, comprising a step of translating an mRNA encoding a peptide containing one or more non-natural amino acids in a translation system containing ribosomes containing a modified L31 protein, wherein the modified L31 protein is selected from the group consisting of the proteins described in the following (1) to (3): (1) A protein comprising an amino acid sequence in which 6 or more amino acid residues are deleted from the C-terminus in the amino acid sequence represented by SEQ ID NO: 1; (2) A protein comprising an amino acid sequence in which one or more amino acids are inserted, substituted, deleted and / or added in the amino acid sequence of the protein described in (1); (3) A protein comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence of the protein described in (1). 〔3〕A method for producing a peptide, comprising a step of translating an mRNA encoding a peptide containing one or more non-natural amino acids in a translation system containing ribosomes containing a modified L31 protein, wherein the modified L31 protein is selected from the group consisting of the proteins described in the following (1) to (3): (1) A protein consisting of an amino acid sequence in which 6 or more amino acid residues are deleted from the C-terminus in the amino acid sequence represented by SEQ ID NO: 1; (2) A protein consisting of an amino acid sequence in which one or more amino acids are inserted, substituted, deleted and / or added in the amino acid sequence of the protein described in (1); (3) A protein consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence of the protein described in (1). 〔4〕The method according to [2] or [3], wherein the amino acid sequence in which 6 or more amino acid residues are deleted from the C-terminus described in (1) is an amino acid sequence in which 8 or more amino acid residues are deleted from the C-terminus. 〔5〕The method according to [4], wherein the amino acid sequence in which 8 or more amino acid residues are deleted from the C-terminus in the amino acid sequence represented by SEQ ID NO: 1 described in (1) is an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 42 to 48. 〔6〕The method according to any one of 〔2〕 to 〔5〕, wherein the ribosome containing the protein described in 〔2〕 and 〔3〕 has a higher translation activity of a peptide containing a non-natural amino acid as compared with a ribosome containing wild-type E. coli L31 having the amino acid sequence of SEQ ID NO: 1. 〔7〕The method according to any one of 〔2〕 to 〔6〕, wherein the ribosome containing the protein described in 〔1〕 has a higher translation activity of a peptide containing a non-natural amino acid as compared with a ribosome containing wild-type E. coli L31 having the amino acid sequence of SEQ ID NO: 1. 〔8〕The method according to any one of 〔2〕 to 〔7〕, wherein the amino acid sequence having 6 or more amino acid residues deleted from the C-terminus is an amino acid sequence having 6 to 50 amino acid residues deleted from the C-terminus. 〔9〕The method according to any one of 〔2〕 to 〔7〕, wherein the amino acid sequence having 6 or more amino acid residues deleted from the C-terminus is an amino acid sequence having 8 to 50 amino acid residues deleted from the C-terminus. 〔10〕The method according to any one of 〔2〕 to 〔7〕, wherein the amino acid sequence having 6 or more amino acid residues deleted from the C-terminus is an amino acid sequence having 6 to 43 amino acid residues deleted from the C-terminus. 〔11〕The method according to any one of 〔2〕 to 〔7〕, wherein the amino acid sequence having 6 or more amino acid residues deleted from the C-terminus is an amino acid sequence having 8 to 43 amino acid residues deleted from the C-terminus. 〔12〕The method according to any one of 〔1〕 to 〔11〕, wherein the modified L31 protein is a protein consisting of an amino acid sequence having 8 or more amino acid residues deleted from the C-terminus in the amino acid sequence represented by SEQ ID NO: 1. 〔13〕The method according to any one of 〔1〕 to 〔12〕, wherein the modified L31 protein is a protein consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and 42 to 48. 〔14〕The method according to any one of 〔1〕 to 〔13〕, wherein the ratio of the ribosome containing the modified L31 protein to all ribosomes in the translation system is 50% or more. 〔15〕The method according to any one of 〔1〕~〔14〕, wherein the translation system further contains magnesium ions at 2~8 mM. 〔16〕The method according to any one of 〔1〕~〔15〕, further comprising the step of cyclizing the peptide. 〔17〕The method according to any one of 〔1〕~〔16〕, wherein the peptide contains a non-natural amino acid at the position of its starting amino acid. 〔18〕The method according to any one of 〔1〕~〔17〕, wherein the translation is performed by initiation suppression. 〔19〕The method according to any one of 〔1〕~〔18〕, wherein a non-natural amino acid is acylated to the starting tRNA contained in the translation system. 〔20〕The method according to any one of 〔1〕~〔19〕, wherein the translation activity is evaluated by the ratio (iRT ratio) of the translation products in which amino acid skipping occurs with respect to all translation products. 〔21〕The method according to any one of 〔1〕~〔20〕, wherein the translation activity is evaluated by translating the peptide of SEQ ID NO: 29 using the template mRNA of SEQ ID NO: 10. 〔22〕The iRT ratio of the ribosome containing the modified L31 protein is 15% or more lower than the iRT ratio of the ribosome containing wild-type E. coli L31 having the amino acid sequence of SEQ ID NO: 1. The method according to any one of 〔1〕~〔21〕. 〔23〕A library containing the peptide produced by the method according to any one of 〔1〕~〔22〕 or the peptide. 〔24〕A method for screening a peptide that binds to a target substance, comprising the steps described in the following (a) and (b); (a) A step of contacting the target substance with the peptide obtained by the method described in any one of 〔1〕~〔22〕 or the library containing the peptide, or the peptide or library described in 〔23〕, and (b) A step of selecting a peptide that binds to the target substance. 〔25〕The modified L31 protein according to any one of 〔2〕~〔24〕. The modified L31 protein as described in

[26]

[25] (however, excluding the L31 protein consisting of the amino acid sequence represented by SEQ ID NO: 2). An isolated nucleic acid encoding the modified L31 protein as described in

[27]

[25] or

[26] . A vector or cell containing the nucleic acid as described in

[28]

[27] . 〔29〕A method for producing ribosomes containing a modified L31 protein, comprising the steps described in (a) to (c) below; (a) A step of culturing the cell as described in

[28] , (b) A step of generating a lysate from the culture of the cell, and (c) A step of purifying ribosomes from the lysate. 〔30〕A method for producing a modified L31 protein, comprising the steps described in (a) and (b) below; (a) A step of culturing the cell as described in

[28] , and (b) A step of isolating the expression product from the culture of the cell. 〔31〕A method for producing ribosomes, comprising the steps described in (a) and (b) below; (a) A step of generating a lysate from a culture of wild-type Escherichia coli using a French press under conditions where the magnesium ion concentration is 5 mM or less, and (b) A step of purifying ribosomes from the lysate. 〔32〕Ribosomes containing the modified L31 protein as described in

[25] or

[26] . 〔33〕A composition containing the ribosomes as described in

[32] . 〔34〕The composition as described in

[33] , wherein the ratio of the ribosomes containing the modified L31 protein to all ribosomes is 50% or more. 〔35〕The composition as described in

[33] or

[34] , further comprising (a) and / or (b) below; (a) Aminoacyl-tRNA formed by binding a non-natural amino acid and tRNA, (b) mRNA encoding a peptide containing one or more non-natural amino acids. The composition according to any one of

[33] to

[35] , further comprising an initiation tRNA acylated with a non-natural amino acid. 〔37〕A method for producing a cell-free translation system, comprising the following steps; (a) A step of producing ribosomes by the method described in

[29] or

[31] , and (b) A step of mixing the ribosomes with an initiation tRNA acylated with a non-natural amino acid.

Advantages of the Invention

[0010] The present invention provides an efficient method for producing a peptide containing a non-natural amino acid and a library containing the peptide. The present invention also provides a modified L31 protein for use in the method, and ribosomes containing the same. By using the method of the present invention, a peptide containing a non-natural amino acid and its library can be efficiently produced. Conventionally, it has been suggested that ribosomes containing the L31 protein cleaved by protease 7 are less active than ribosomes containing the uncleaved L31 protein. Based on this fact, it was a surprising effect that a peptide containing a non-natural amino acid could be efficiently translated by using ribosomes containing the modified L31 protein in this specification.

Brief Description of the Drawings

[0011]

Figure 1

Figure 2

Figure 3

Mode for Carrying Out the Invention

[0012] The present invention relates to a method for producing a peptide or a library of peptides, which comprises a step of translating an mRNA encoding a peptide containing one or more non-natural amino acids in a translation system containing ribosomes containing a modified L31 protein. In the present invention, a peptide or a library of peptides can be produced by mixing a translation system containing ribosomes containing a modified L31 protein with an mRNA encoding a peptide containing one or more non-natural amino acids. Therefore, the present invention relates to a method for producing a peptide or a library of peptides, which comprises mixing a translation system containing ribosomes containing a modified L31 protein with an mRNA encoding a peptide containing one or more non-natural amino acids and translating the mRNA. The production method of the present invention is a method capable of efficiently translating a peptide containing a non-natural amino acid as compared with the case of using ribosomes containing a wild-type L31 protein having the amino acid sequence set forth in SEQ ID NO: 1 derived from wild-type Escherichia coli.

[0013] The L31 protein is one of the proteins constituting ribosomes and plays an important role in the association of the 50S subunit and the 30S subunit. The present inventors have found that when synthesizing a peptide containing one or more non-natural amino acids in a translation system containing ribosomes containing a modified L31 protein in which the C-terminal amino acid residue is deleted in the wild-type L31 protein having the amino acid sequence set forth in SEQ ID NO: 1 derived from wild-type Escherichia coli, the amount of the translation product increases as compared with the case of synthesizing the peptide in a translation system containing ribosomes containing the wild-type Escherichia coli L31 protein. Furthermore, the present inventors have found that when using a translation system containing ribosomes containing a modified L31 protein, it is possible to reduce the relative production amount of by-products as compared with the case of using a translation system containing ribosomes containing the wild-type Escherichia coli L31 protein.

[0014] In this specification, the "E. coli wild-type L31 protein" refers to L31 having the amino acid sequence set forth in SEQ ID NO: 1, and may be referred to as "E. coli wild-type L31", "intact L31", "L31intact", etc. Further, in this specification, the "modified L31 protein" refers to a modified L31 protein in which six or more amino acid residues are deleted from the C-terminus of the E. coli wild-type L31 protein. In this specification, among such proteins, the "modified L31 protein in which amino acid residues after the 63rd position are deleted from the E. coli wild-type L31 protein", specifically, L31 having the amino acid sequence set forth in SEQ ID NO: 2, may be referred to as "shortL31", "L31short", etc. Further, the "modified L31 protein in which amino acid residues after the (X + 1)th position are deleted from the E. coli wild-type L31 protein" may be referred to as "L31(1-X)". That is, the shortL31 is also referred to as "L31(1-62)". Further, in this specification, a ribosome containing a certain L31 protein may be denoted by attaching "ribosome" after the name of the L31 protein. For example, ribosomes containing "intact L31", "L31intact", "shortL31", "L31(1-62)" or "L31short" may be referred to as "intactL31 ribosome", "L31intact ribosome", "shortL31 ribosome", "L31(1-62) ribosome" or "L31short ribosome", respectively. Further, a ribosome purified from an E. coli wild-type strain may be referred to as "WT ribosome", etc.

[0015] Modified L31 protein In the production method according to the present disclosure, by using a ribosome containing a modified L31 protein, it is possible to efficiently translate a peptide containing a non-natural amino acid as compared with the case of using a ribosome containing E. coli wild-type L31. In one aspect of the present invention, the modified L31 protein is (1) A protein comprising an amino acid sequence in which 6 or more amino acid residues are deleted from the C-terminus in the amino acid sequence represented by SEQ ID NO:1, (2) A protein comprising an amino acid sequence in which one or more amino acids are inserted, substituted, deleted and / or added in the amino acid sequence of the protein according to (1) above, (3) A protein comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence of the protein according to (1) above, can be exemplified.

[0016] As the protein of (1) above, it may be a protein containing an amino acid sequence in which 8 or more amino acid residues, or 9 or more amino acid residues are deleted from the C-terminus in the amino acid sequence represented by SEQ ID NO: 1. For example, it may be a protein containing an amino acid sequence in which the number of deleted amino acid residues is within the range of the lower limit value selected from 6, 7, 8, 9, 10, 11, 12, 13 from the C-terminus and the upper limit value selected from 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38. In one aspect, the protein of (1) above may be a protein containing an amino acid sequence in which any number of amino acid residues within the range of 6 to 50, any number within the range of 6 to 43, any number within the range of 8 to 50, or any number within the range of 8 to 43 are deleted from the C-terminus in the amino acid sequence represented by SEQ ID NO: 1. In a further aspect, as the protein of (1) above, a protein containing an amino acid sequence in which 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 27, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acid residues are deleted from the C-terminus in the amino acid sequence represented by SEQ ID NO: 1 can be mentioned. More specifically, the modified L31 protein in the present disclosure includes proteins containing amino acid sequences in which 8, 43, 38, 33, 28, 23, 18, or 13 amino acids are deleted from the C-terminus in the amino acid sequence represented by SEQ ID NO: 1, and proteins in which amino acid residues are modified (deleted, substituted, and / or inserted) in these proteins. That is, the modified L31 protein in the present disclosure includes (1) a protein containing an amino acid sequence represented by any one selected from SEQ ID NO: 2 and 42 to 48, (2) A protein comprising an amino acid sequence in which one or more amino acids are deleted, inserted, substituted, and / or added in the amino acid sequence represented by any one selected from SEQ ID NO: 2 and SEQ ID NOs: 42 to 48, and (3) A protein comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by any one selected from SEQ ID NO: 2 and SEQ ID NOs: 42 to 48 are also included. The modified L31 protein of the present invention is preferably a protein that is functionally equivalent to any one of SEQ ID NO: 2 and SEQ ID NOs: 42 to 48, or a protein containing the amino acid sequence set forth in SEQ ID NO: 2. In the present specification, "comprising" means both "comprising" and "consisting of".

[0017] In some embodiments, the modified L31 protein of the present disclosure is (i) a protein comprising an amino acid sequence in which 6 or more or 8 or more amino acid residues are deleted from the C-terminus in the amino acid sequence represented by SEQ ID NO: 1, or (ii) in the amino acid sequence of a protein comprising an amino acid sequence represented by any one selected from SEQ ID NO: 2 and SEQ ID NOs: 42 to 48, one or more (for example, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, or 3 or less, or 2, 3, 4, 5, 6, 7, 8, 9, or 10, or more than 10) amino acids are inserted, substituted, deleted, and / or added. The addition, deletion, substitution, and / or insertion of amino acids can be carried out by methods known in the art. For example, site-directed mutagenesis (Kunkel et al., Proc. Natl. Acad. Sci. USA 82, 488-492 (1985)) or Overlap extension PCR can be performed on the nucleic acid encoding the amino acid sequence. These can be carried out alone or in combination as appropriate.

[0018] Generally, modification (e.g., conservative substitution, deletion, insertion, and / or addition) of one or more (e.g., 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, or 3 or less, or 2, 3, 4, 5, 6, 7, 8, 9, or 10, or more than 10) amino acids in a protein is known not to affect the function of the peptide or even to enhance the function of the original protein. Amino acids are classified into hydrophobic amino acids (A, I, L, M, F, P, W, Y, V) and hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T) according to the characteristics of their side chains. Also, the side chains of amino acids can be classified into aliphatic side chains (G, A, V, L, I, P), side chains containing a hydroxyl group (S, T, Y), side chains containing a sulfur atom (C, M), side chains containing a carboxylic acid and an amide (D, N, E, Q), side chains containing a base (R, K, H), and side chains containing an aromatic group (H, F, Y, W). Proteins in which the amino acids contained in a protein having an amino acid sequence represented by any of SEQ ID NOs: 1, 2, and 42 to 48 are modified with other amino acids classified into groups having the same characteristics are also included in the modified L31 protein of the present invention. However, the modified L31 protein of the present invention may include non-conservative modifications as long as it is functionally equivalent to a protein containing any of SEQ ID NOs: 2 and 42 to 48 or the amino acid sequence set forth in SEQ ID NO: 2.

[0019] Also, in one aspect of the present invention, a protein comprising an amino acid sequence in which 6 or more, or 8 or more amino acid residues are deleted from the C-terminus in the amino acid sequence represented by SEQ ID NO: 1, or an amino acid sequence having a high sequence identity with the amino acid sequence represented by any one of SEQ ID NO: 1, 2, and 42 to 48 is also included in the modified L31 protein in the present disclosure. In the present disclosure, high identity refers to at least 50% or more, more preferably 70% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90% or more (for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity in the entire amino acid sequence or the entire nucleotide sequence. The sequence identity can be determined using the algorithm BLAST by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990, Proc Natl Acad Sci USA 90: 5873, 1993). Programs called BLASTN and BLASTX based on the BLAST algorithm have been developed (Altschul SF, et al: J Mol Biol 215: 403, 1990). When analyzing a nucleotide sequence using BLASTN, the parameters are, for example, score = 100 and wordlength = 12. When analyzing an amino acid sequence using BLASTX, the parameters are, for example, score = 50 and wordlength = 3. When using BLAST and the Gapped BLAST program, the default parameters of each program are used. Specific methods of these analysis methods are known.

[0020] As used herein, "functionally equivalent" means that a ribosome containing a modified L31 protein exhibits translation activity equivalent to that of a ribosome containing an L31 protein consisting of an amino acid sequence in which 6 or more amino acid residues are deleted from the C-terminus in the amino acid sequence represented by SEQ ID NO: 1 (for example, the amino acid sequence set forth in any of SEQ ID NO: 2 and SEQ ID NOs: 42 to 48) with respect to the translation of an mRNA encoding a peptide containing a non-natural amino acid. Thus, when a ribosome containing a certain protein has the same level of activity with respect to the translation of an mRNA encoding a peptide containing a non-natural amino acid as compared to a ribosome containing an L31 protein consisting of an amino acid sequence in which 6 or more amino acid residues are deleted from the C-terminus in the amino acid sequence represented by SEQ ID NO: 1 (for example, the amino acid sequence set forth in any of SEQ ID NO: 2 and SEQ ID NOs: 42 to 48), the said certain protein can be referred to as "a protein functionally equivalent to a protein consisting of an amino acid sequence in which 6 or more amino acid residues are deleted from the C-terminus in the amino acid sequence represented by SEQ ID NO: 1 (for example, the amino acid sequence set forth in any of SEQ ID NO: 2 and SEQ ID NOs: 42 to 48)". The inventors of the present invention have clarified that a ribosome containing an L31 protein consisting of an amino acid sequence in which 6 or more amino acid residues are deleted from the C-terminus in the amino acid sequence represented by SEQ ID NO: 1 (for example, the amino acid sequence set forth in any of SEQ ID NO: 2 and SEQ ID NOs: 42 to 48) has higher translation activity for an mRNA encoding a peptide containing a non-natural amino acid as compared to a ribosome containing an L31 protein consisting of the amino acid sequence set forth in SEQ ID NO: 1. Thus, when a ribosome containing a certain protein has higher translation activity for an mRNA encoding a peptide containing a non-natural amino acid as compared to a ribosome containing an L31 protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, the said certain protein can be referred to as "a protein functionally equivalent to a protein consisting of an amino acid sequence in which 6 or more amino acid residues are deleted from the C-terminus in the amino acid sequence represented by SEQ ID NO: 1 (for example, the amino acid sequence set forth in any of SEQ ID NO: 2 and SEQ ID NOs: 42 to 48)".Alternatively, when a certain protein provides the translation activity of mRNA encoding a peptide containing a large unnatural amino acid when forming a ribosome, as compared with the L31 protein containing the amino acid sequence set forth in SEQ ID NO:1, the certain protein can be referred to as "a protein functionally equivalent to a protein consisting of an amino acid sequence in which 6 or more amino acid residues are deleted from the C-terminus in the amino acid sequence represented by SEQ ID NO:1 (for example, the amino acid sequence set forth in any one of SEQ ID NO:2 and 42 to 48)".

[0021] In the present disclosure, "the translation activity of mRNA encoding a peptide containing an unnatural amino acid" can also be expressed as "the translation efficiency of mRNA encoding a peptide containing an unnatural amino acid", and specifically includes the ease of incorporation of tRNA having an unnatural amino acid, the degree of skipping of codons encoding an unnatural amino acid, the amount of peptide translated, and / or the amount of by-products generated. In the present disclosure, "the translation activity of mRNA encoding a peptide containing an unnatural amino acid is high" can also be expressed as "the translation efficiency of mRNA encoding a peptide containing an unnatural amino acid is high", and specifically means that tRNA having an unnatural amino acid is easily incorporated, the skipping of codons encoding an unnatural amino acid is less, the amount of the target peptide produced is large, and / or the amount of by-products generated is small.

[0022] In the present disclosure, the "translation activity of mRNA encoding a peptide containing a non-natural amino acid" can be evaluated using, as an index, the ratio of translation products in which amino acid skipping has occurred with respect to the total translation products. When translating mRNA encoding a peptide containing a non-natural amino acid, in addition to the target product (TM), peptides such as those in which the starting amino acid is skipped and translation starts from the second amino acid (referred to as "1iRT" in the present disclosure) or those in which the starting amino acid and the second amino acid are skipped and translation starts from the third amino acid (referred to as "2iRT" in the present disclosure), i.e., initiation read-through (iRT) peptides, may be included. In the present disclosure, the "iRT ratio" calculated from the concentrations of initiation read-through (iRT) and the target product (TM) by the following formula can be adopted as the "translation activity of mRNA encoding a peptide containing a non-natural amino acid". The "iRT total concentration" in the formula can be calculated as the total value of the 1iRT (peptide translated starting from the second amino acid) concentration and the 2iRT (peptide translated starting from the third amino acid) concentration.

[0023] (Equation 1) iRT ratio = (iRT total concentration [nM]) / (iRT total concentration [nM] + TM concentration [nM]) × 100

[0024] Specifically, the iRT ratio can be evaluated by the method described in the Examples using aminoacyl tRNA, translation systems, etc. described in the Examples. As an example, by translating a peptide of any one of SEQ ID NOs: 29 to 31, preferably SEQ ID NO: 29, using the template mRNA of SEQ ID NO: 10, the "translation activity of mRNA encoding a peptide containing a non-natural amino acid" can be evaluated.

[0025] In the present disclosure, "the translation activity of mRNA encoding a peptide containing a non-natural amino acid is high" can mean that the iRT ratio is low. "The translation activity of the peptide containing the non-natural amino acid is high as compared with a ribosome containing wild-type Escherichia coli L31 having the amino acid sequence of SEQ ID NO: 1" can mean, for example, that the iRT ratio of the ribosome containing the modified L31 protein of the present disclosure is 15%, 20%, 25%, or 30% or more lower than the iRT ratio of the ribosome containing wild-type Escherichia coli L31 having the amino acid sequence of SEQ ID NO: 1.

[0026] Note that the ribosome of a prokaryotic cell includes a 50S large subunit and a 30S small subunit. The 50S subunit is further composed of 23S rRNA, 5S rRNA, and a plurality of proteins. The 30S subunit is composed of 16S rRNA and a plurality of proteins. On the other hand, the ribosome of a eukaryotic cell includes a 60S large subunit and a 40S small subunit. The 60S subunit is further composed of 28S rRNA, 5.8S rRNA, 5S rRNA, and a plurality of proteins. The 40S subunit is composed of 18S rRNA and a plurality of proteins. The components of the ribosome are known to those skilled in the art.

[0027] Amino acid In the present disclosure, the "amino acids" constituting the peptide include "natural amino acids" such as α-amino acids and "non-natural amino acids" such as β-amino acids and γ-amino acids. The steric structure of the amino acid may be either an L-type amino acid or a D-type amino acid. "Amino acid", "natural amino acid", and "non-natural amino acid" may also be referred to as "amino acid residue", "natural amino acid residue", and "non-natural amino acid residue", respectively.

[0028] In certain embodiments, the natural amino acids consist of the following 20 α - amino acids: glycine (Gly), alanine (Ala), serine (Ser), threonine (Thr), valine (Val), leucine (Leu), isoleucine (Ile), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), histidine (His), glutamic acid (Glu), aspartic acid (Asp), glutamine (Gln), asparagine (Asn), cysteine (Cys), methionine (Met), lysine (Lys), arginine (Arg), and proline (Pro). Alternatively, those obtained by removing any one or more amino acids from the above - mentioned 20 amino acids may also be used as the natural amino acids in the present disclosure. In one embodiment, the natural amino acids consist of 19 amino acids excluding isoleucine. In one embodiment, the natural amino acids consist of 19 amino acids excluding methionine. In a further embodiment, the natural amino acids consist of 18 amino acids excluding isoleucine and methionine. Natural amino acids are usually L - type amino acids.

[0029] Non-natural amino acid In the present disclosure, non-natural amino acids refer to all amino acids excluding the natural amino acids composed of the above 20 types of α-amino acids. Examples of non-natural amino acids include β-amino acids, γ-amino acids, D-type amino acids, α-amino acids with side chains different from natural amino acids, α,α-disubstituted amino acids, amino acids in which the amino group of the main chain has a substituent (N-substituted amino acids), and the like. The side chains of non-natural amino acids are not particularly limited, but in addition to a hydrogen atom, they may have, for example, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, cycloalkyl, and the like. In the case of α,α-disubstituted amino acids, the two side chains may form a ring. Furthermore, these side chains may have one or more substituents. In a specific embodiment, the substituent can be selected from any functional group containing a halogen atom, O atom, S atom, N atom, B atom, Si atom, or P atom. For example, in the present disclosure, "C1-C6 alkyl having a halogen as a substituent" means "C1-C6 alkyl" in which at least one hydrogen atom in the alkyl is substituted with a halogen atom. Specifically, for example, it includes trifluoromethyl, difluoromethyl, fluoromethyl, pentafluoroethyl, tetrafluoroethyl, trifluoroethyl, difluoroethyl, fluoroethyl, trichloromethyl, dichloromethyl, chloromethyl, pentachloroethyl, tetrachloroethyl, trichloroethyl, dichloroethyl, chloroethyl, and the like. Also, for example, "C5-C10 aryl C1-C6 alkyl having a substituent" means "C5-C10 aryl C1-C6 alkyl" in which at least one hydrogen atom in the aryl and / or alkyl is substituted with a substituent. Furthermore, "having two or more substituents" also includes having a certain functional group (for example, a functional group containing an S atom) as a substituent, and further that the functional group has another substituent (for example, a substituent such as amino or halogen). For specific examples of non-natural amino acids, reference can also be made to WO2013 / 100132, WO2018 / 143145, and the like.

[0030] The amino group in the main chain of the unnatural amino acid may be an unsubstituted amino group (NH2 group) or a substituted amino group (NHR group). Here, R represents alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, or cycloalkyl which may have a substituent. Also, like proline, a carbon chain bonded to the N atom of the amino group in the main chain and the α-carbon atom may form a ring. Examples of alkyl substitution of the amino group include N-methylation, N-ethylation, N-propylation, N-butylation, etc., and examples of aralkyl substitution include N-benzylation, etc. Specific examples of N-methyl amino acids include N-methyl alanine, N-methyl glycine, N-methyl phenylalanine, N-methyl tyrosine, N-methyl-3-chlorophenylalanine, N-methyl-4-chlorophenylalanine, N-methyl-4-methoxyphenylalanine, N-methyl-4-thiazolealanine, N-methyl histidine, N-methyl serine, N-methyl aspartic acid, etc.

[0031] Examples of the substituent containing a halogen include an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an aralkyl group, etc. having a halogen as a substituent. More specifically, fluoroalkyl, difluoroalkyl, trifluoroalkyl, etc. are exemplified.

[0032] Examples of the substituent containing an O atom include hydroxyl (-OH), oxy (-OR), carbonyl (-C=O-R), carboxyl (-CO2H), oxycarbonyl (-C=O-OR), carbonyloxy (-O-C=O-R), thiocarbonyl (-C=O-SR), carbonylthio group (-S-C=O-R), aminocarbonyl (-C=O-NHR), carbonylamino (-NH-C=O-R), oxycarbonylamino (-NH-C=O-OR), sulfonylamino (-NH-SO2-R), aminosulfonyl (-SO2-NHR), sulfamoylamino (-NH-SO2-NHR), thiocarboxyl (-C(=O)-SH), carboxylcarbonyl (-C(=O)-CO2H).

[0033] Examples of oxy (-OR) include alkoxy, cycloalkoxy, alkenyloxy, alkynyloxy, aryloxy, heteroaryloxy, aralkyloxy, etc.

[0034] Examples of carbonyl (-C=O-R) include formyl (-C=O-H), alkylcarbonyl, cycloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, arylcarbonyl, heteroarylcarbonyl, aralkylcarbonyl, etc.

[0035] Examples of oxycarbonyl (-C=O-OR) include alkyl oxycarbonyl, cycloalkyl oxycarbonyl, alkenyl oxycarbonyl, alkynyl oxycarbonyl, aryl oxycarbonyl, heteroaryl oxycarbonyl, aralkyl oxycarbonyl, etc. (-C=O-OR)

[0036] Examples of carbonyl oxy (-O-C=O-R) include alkylcarbonyl oxy, cycloalkylcarbonyl oxy, alkenylcarbonyl oxy, alkynylcarbonyl oxy, arylcarbonyl oxy, heteroarylcarbonyl oxy, aralkylcarbonyl oxy, etc.

[0037] Examples of thiocarbonyl (-C=O-SR) include alkylthiocarbonyl, cycloalkylthiocarbonyl, alkenylthiocarbonyl, alkynylthiocarbonyl, arylthiocarbonyl, heteroarylthiocarbonyl, aralkylthiocarbonyl, etc.

[0038] Examples of carbonyl thio (-S-C=O-R) include alkylcarbonyl thio, cycloalkylcarbonyl thio, alkenylcarbonyl thio, alkynylcarbonyl thio, arylcarbonyl thio, heteroarylcarbonyl thio, aralkylcarbonyl thio, etc.

[0039] Examples of aminocarbonyl (-C=O-NHR) include alkylaminocarbonyl, cycloalkylaminocarbonyl, alkenylaminocarbonyl, alkynylaminocarbonyl, arylaminocarbonyl, heteroarylaminocarbonyl, aralkylaminocarbonyl, and the like. In addition to these, groups in which the H atom bonded to the N atom in -C=O-NHR is further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl are also included.

[0040] Examples of carbonylamino (-NH-C=O-R) include alkylcarbonylamino, cycloalkylcarbonylamino, alkenylcarbonylamino, alkynylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, aralkylcarbonylamino, and the like. In addition to these, groups in which the H atom bonded to the N atom in -NH-C=O-R is further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl are also included.

[0041] Examples of oxycarbonylamino (-NH-C=O-OR) include alkoxycarbonylamino, cycloalkoxycarbonylamino, alkenyloxycarbonylamino, alkynyloxycarbonylamino, aryloxycarbonylamino, heteroaryloxycarbonylamino, aralkyloxycarbonylamino, and the like. In addition to these, groups in which the H atom bonded to the N atom in -NH-C=O-OR is further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl are also included.

[0042] Examples of sulfonylamino (-NH-SO2-R) include alkylsulfonylamino, cycloalkylsulfonylamino, alkenylsulfonylamino, alkynylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino, aralkylsulfonylamino, etc. In addition to these, groups in which the H atom bonded to the N atom in -NH-SO2-R is further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl are also included.

[0043] Examples of aminosulfonyl (-SO2-NHR) include alkylaminosulfonyl, cycloalkylaminosulfonyl, alkenylaminosulfonyl, alkynylaminosulfonyl, arylaminosulfonyl, heteroarylaminosulfonyl, aralkylaminosulfonyl, etc. In addition to these, groups in which the H atom bonded to the N atom in -SO2-NHR is further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl are also included.

[0044] Examples of sulfamoylamino (-NH-SO2-NHR) include alkylsulfamoylamino, cycloalkylsulfamoylamino, alkenylsulfamoylamino, alkynylsulfamoylamino, arylsulfamoylamino, heteroarylsulfamoylamino, aralkylsulfamoylamino, etc. Further, the two H atoms bonded to the N atom in -NH-SO2-NHR may be substituted with substituents independently selected from the group consisting of alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl, and these two substituents may form a ring.

[0045] Examples of substituents containing an S atom include thiol (-SH), thio (-S-R), sulfinyl (-S=O-R), sulfonyl (-S(O)2-R), and sulfo (-SO3H).

[0046] Examples of thio (-S-R) include alkylthio, cycloalkylthio, alkenylthio, alkynylthio, arylthio, heteroarylthio, aralkylthio, and the like.

[0047] Examples of sulfinyl (-S=O-R) include alkylsulfinyl, cycloalkylsulfinyl, alkenylsulfinyl, alkynylsulfinyl, arylsulfinyl, heteroarylsulfinyl, aralkylsulfinyl, and the like.

[0048] Examples of sulfonyl (-S(O)2-R) include alkylsulfonyl, cycloalkylsulfonyl, alkenylsulfonyl, alkynylsulfonyl, arylsulfonyl, heteroarylsulfonyl, aralkylsulfonyl, and the like.

[0049] Examples of substituents containing an N atom include azide (-N3, also referred to as "azide group"), cyano (-CN), primary amino (-NH2), secondary amino (-NH-R), tertiary amino (-NR(R')), amidino (-C(=NH)-NH2), substituted amidino (-C(=NR)-NR'R''), guanidino (-NH-C(=NH)-NH2), substituted guanidino (-NR-C(=NR''')-NR'R''), aminocarbonylamino (-NR-CO-NR'R'').

[0050] Examples of secondary amino (-NH-R) include alkylamino, cycloalkylamino, alkenylamino, alkynylamino, arylamino, heteroarylamino, aralkylamino, and the like.

[0051] Examples of tertiary amino (-NR(R')) include, for example, alkyl(aralkyl)amino and the like, and amino groups having any two substituents independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, etc., and any two of these substituents may form a ring.

[0052] Examples of the substituted amidino (-C(=NR)-NR'R'') include groups in which the three substituents R, R', and R'' on the N atom are each independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, such as alkyl(aralkyl)(aryl)amidino and the like.

[0053] Examples of the substituted guanidino (-NR-C(=NR''')-NR'R'') include groups in which R, R', R'', and R''' are each independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, or groups formed by these groups forming a ring and the like.

[0054] Examples of the aminocarbonylamino (-NR-CO-NR'R'') include groups in which R, R', and R'' are each independently selected from a hydrogen atom, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, or groups formed by these groups forming a ring and the like.

[0055] Examples of the substituent containing a B atom include boryl (-BR(R')) and dioxaborolyl (-B(OR)(OR')). The two substituents R and R' are each independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, etc., or they may form a ring.

[0056] At least one atom constituting the "amino acid" constituting the peptide may be an atom (isotope) having the same atomic number (number of protons) and different mass numbers (sum of the number of protons and neutrons). Examples of the isotopes contained in the "amino acid" constituting the peptide include a hydrogen atom, a carbon atom, a nitrogen atom, an oxygen atom, a phosphorus atom, a sulfur atom, a fluorine atom, a chlorine atom, etc., respectively, 2 H, 3 H, 13 C, 14 C, 15 N, 17 O, 18O, 31 P, 32 P, 35 S, 18 F, 36 Cl, etc. are included.

[0057] In this specification, examples of the "halogen atom" include F, Cl, Br, or I.

[0058] In this specification, "alkyl" is a monovalent group derived by removing any one hydrogen atom from an aliphatic hydrocarbon, which does not contain a heteroatom (an atom other than a carbon and hydrogen atom) or an unsaturated carbon-carbon bond in the skeleton, and has a subset of a hydrocarbyl or hydrocarbon group structure containing hydrogen and carbon atoms. Alkyl includes not only linear ones but also branched-chain ones. Specifically, alkyl is an alkyl having 1 to 20 carbon atoms (C1-C 20 , hereinafter "C p -C q " means having p to q carbon atoms), preferably C1-C 10 alkyl, more preferably C1-C6 alkyl. Specifically, examples of alkyl include methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, isobutyl (2-methylpropyl), n-pentyl, s-pentyl (1-methylbutyl), t-pentyl (1,1-dimethylpropyl), neopentyl (2,2-dimethylpropyl), isopentyl (3-methylbutyl), 3-pentyl (1-ethylpropyl), 1,2-dimethylpropyl, 2-methylbutyl, n-hexyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1,1,2,2-tetramethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, etc.

[0059] In this specification, "alkenyl" means having at least one double bond (two adjacent SP 2A monovalent group having a carbon atom). Depending on the arrangement of the double bond and the substituent (if present), the geometric form of the double bond can take an entgegen (E) or zusammen (Z), cis or trans configuration. Alkenyl includes not only linear ones but also branched ones. Preferably, alkenyl is C2-C 10 Alkenyl, more preferably C2-C6 alkenyl, and specifically, for example, vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl (including cis and trans), 3-butenyl, pentenyl, 3-methyl-2-butenyl, hexenyl, etc.

[0060] As used herein, "alkynyl" is a monovalent group having at least one triple bond (two adjacent sp carbon atoms). Alkynyl includes not only linear ones but also branched ones. Preferably, alkynyl is C2-C 10 Alkynyl, more preferably C2-C6 alkynyl, and specifically, for example, 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, 3-methyl-(5-phenyl)-4-pentynyl, etc.

[0061] As used herein, "cycloalkyl" means a saturated or partially saturated cyclic monovalent aliphatic hydrocarbon group, including monocyclic, bicyclic, and spiro rings. Preferably, cycloalkyl is C3-C8 cycloalkyl, and specifically, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[2.2.1]heptyl, spiro[3.3]heptyl, etc.

[0062] As used herein, "aryl" means a monovalent aromatic hydrocarbon ring, preferably C6-C 10Aryl is exemplified. Specifically, aryl includes, for example, phenyl, naphthyl (e.g., 1-naphthyl, 2-naphthyl), and the like.

[0063] As used herein, "heteroaryl" means an aromatic cyclic monovalent group containing 1 to 5 heteroatoms in addition to carbon atoms. The ring may be a monocyclic ring, a fused ring with other rings, or may be partially saturated. The number of atoms constituting the ring is preferably 5 to 10 (5- to 10-membered heteroaryl), more preferably 5 to 7 (5- to 7-membered heteroaryl). Specifically, heteroaryl includes, for example, furyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, triazinyl, benzofuranyl, benzothienyl, benzothiadiazolyl, benzothiazolyl, benzoxazolyl, benzoxadiazolyl, benzimidazolyl, indolyl, isoindolyl, indazolyl, quinolyl, isoquinolyl, cinnolinyl, quinazolinyl, quinoxalinyl, benzodioxolyl, indolizinyl, imidazopyridyl, and the like.

[0064] As used herein, "alkoxy" means an oxy group to which the "alkyl" as defined above is bonded, and preferably C1-C6 alkoxy is exemplified. Specifically, alkoxy includes, for example, methoxy, ethoxy, 1-propoxy, 2-propoxy, n-butoxy, i-butoxy, s-butoxy, t-butoxy, pentyloxy, 3-methylbutoxy, and the like.

[0065] As used herein, "alkenyloxy" means an oxy group to which the "alkenyl" as defined above is bonded, and preferably C2-C6 alkenyloxy. Specific examples of alkenyloxy include, for example, vinyloxy, allyloxy, 1-propenyloxy, 2-propenyloxy, 1-butenyloxy, 2-butenyloxy (including cis and trans), 3-butenyloxy, pentyloxy, hexyloxy and the like.

[0066] As used herein, "cycloalkoxy" means an oxy group to which the "cycloalkyl" as defined above is bonded, and preferably C3-C8 cycloalkoxy. Specific examples of cycloalkoxy include, for example, cyclopropoxy, cyclobutoxy, cyclopentyloxy and the like.

[0067] As used herein, "aryloxy" means an oxy group to which the "aryl" as defined above is bonded, and preferably C6-C 10 aryloxy. Specific examples of aryloxy include, for example, phenoxy, 1-naphthyloxy, 2-naphthyloxy and the like.

[0068] As used herein, "amino" means -NH2 in a narrow sense and -NRR' in a broad sense, where R and R' are independently selected from hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, or R and R' together with the nitrogen atom to which they are bonded form a ring. Preferred examples of amino include -NH2, mono C1-C6 alkylamino, di C1-C6 alkylamino, 4- to 8-membered cyclic amino and the like.

[0069] As used herein, "monoalkylamino" means a group in which, among the "amino" groups as defined above, R is hydrogen and R' is "alkyl" as defined above, and preferably includes mono C1-C6 alkylamino. Specific examples of monoalkylamino include, for example, methylamino, ethylamino, n-propylamino, i-propylamino, n-butylamino, s-butylamino, t-butylamino, and the like.

[0070] As used herein, "dialkylamino" means a group in which, among the "amino" groups as defined above, R and R' are independently "alkyl" as defined above, and preferably includes di C1-C6 alkylamino. Specific examples of dialkylamino include, for example, dimethylamino, diethylamino, and the like.

[0071] As used herein, "aminoalkyl" means a group in which one or more hydrogens of the "alkyl" as defined above are replaced by the "amino" as defined above, and C1-C6 aminoalkyl is preferred. Specific examples of aminoalkyl include, for example, 1-pyridylmethyl, 2-(1-piperidyl)ethyl, 3-(1-piperidyl)propyl, 4-aminobutyl, and the like.

[0072] As used herein, "aralkyl (arylalkyl)" means a group in which at least one hydrogen atom of the "alkyl" as defined above is replaced by the "aryl" as defined above, and C7-C 14 aralkyl is preferred, and C7-C 10 aralkyl is more preferred. Specific examples of aralkyl include, for example, benzyl, phenethyl, 3-phenylpropyl, and the like.

[0073] Specific examples of non-natural amino acids can also be referred to, such as WO2013 / 100132 and WO2018 / 143145.

[0074] In one aspect, a peptide containing one or more non-natural amino acids obtained by the production method according to the invention of the present application can be used as a pharmaceutical. When using the peptide as a pharmaceutical, it is preferably excellent in metabolic stability and membrane permeability. Such properties are referred to herein as "drug-likeness" or "drug-like". As used herein, a "drug-like amino acid" is an α, β, or γ amino acid, and one of the two hydrogen atoms of the main-chain amino group (NH2 group), or one or two of the hydrogen atoms of the main-chain methylene group (-CH2- group) may be substituted with an alkyl group, cycloalkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, aralkyl group, etc. These substituents may further be substituted with "substituents contributing to drug-likeness". Amino acids having a "long side chain" disclosed in WO2018 / 225864 are preferably exemplified as drug-like amino acids. Also, the drug-like amino acids may be L-amino acids, D-amino acids, α,α-disubstituted amino acids, N-substituted amino acids, etc. Drug-like amino acids do not necessarily have to be translatable. Amino acids that can be chemically synthesized by optimizing the structure of the side-chain portion of a peptide obtained from a "translated amino acid" (for example, when a hit compound is obtained with D-tyrosine, the D-type amino acid chemically modified therefrom, or when a hit compound is obtained with β-alanine, the β-amino acid chemically modified therefrom), or the N-substituted portion by chemical conversion of N-methylamino acid are included. Since these amino acids function as components of drug-like peptides, they are selected from the range such that the peptide obtained by chemical modification performed after translation becomes drug-like. As described below, for example, lysine having an aminoalkyl group is not included in drug-like amino acids when the amino group is not involved in post-translational modification. However, when the amino group of lysine is utilized as a reactive functional group for post-translational modification (for example, a cross-linking unit), the lysine unit is included as a unit of drug-like amino acids. Thus, whether it corresponds to a "drug-like amino acid" is determined by the functional group after being converted by post-translational modification.Examples of such substituents include, among the substituents defined separately above, for example, an ester group (-CO-OR), a thioester group (-CO-SR), a thiol group (-SH), or a protected thiol group, an amino group (-NH2), a mono-substituted amino group (-NH-R) or a di-substituted amino group (-NRR'), or a protected amino group, a substituted sulfonylamino group (-NH-SO2-R), an alkyl borane group (-BRR'), an alkoxy borane group (-B(OR)(OR')), an azide group (-N3), a keto acid group (-CO-CO2H), a thiocarboxylic acid group (-CO-SH), a phosphoryl ester group (-CO-PO(R)(R')), an acyl hydroxyamino group (-NH-O-CO-R), and the like. One or two non-adjacent methylene groups contained in the side chain of the drug-like amino acid may be substituted with an oxygen atom, a carbonyl group (-CO-), or a sulfonyl group (-SO2-).

[0075] Examples of the "substituent contributing to drug-likeness" in the present specification include, for example, a halogen (F, Cl, Br, I, etc.), a hydroxy group (-OH), an alkoxy group (-OR), an oxy group (-OR), an amide (-NR-COR' or -CO-NRR'), a sulfonyl group (-SO2-R), a sulfinyl group (-SOR), an oxyamino group (-NR-OR'), an aminooxy group (-O-NRR'), an oxycarbonyl group (-CO-OR), a thiocarbonyl group (-CO-SR), a thiol group (-SH), a thio group (-SR), a primary amino group (-NH2), a secondary amino group (-NHR) or a tertiary amino group (-NRR'), a sulfonylamino group (-NH-SO2-R), a boryl group (-BRR'), a dioxiboryl group (-B(OR)(OR')), an azide (-N3), a carboxycarbonyl group (-CO-CO2H), a phosphorylcarbonyl group (-CO-PO(R)(R')), a carbonyloxyamino group (-NH-O-CO-R), a hydroxyamino group (-NR-OR'), an aminohydroxy group (-O-NRR'), and other substituents.

[0076] In addition, as particularly specific examples of the unnatural amino acids constituting the peptide containing one or more unnatural amino acids in the present disclosure, MeSer(tBuOH), BODIPYFL-4-AMF, MeCys(StBu), MeG, MeStBuOH, Nle, S3F5MePyr, SPh2Cl, MeF, MeHph, MeA3Pyr, SPh2Cl, Pic(2), MeHph, dA, etc. can be exemplified. These unnatural amino acids can be contained, for example, at the second or third position of the peptide.

[0077] Peptide In the present disclosure, a peptide refers to a compound in which two or more amino acids are linked by an amide bond and / or an ester bond. Although not intended to be limiting, the peptides in the present disclosure include linear peptides and cyclic peptides. In addition, the peptides in the present disclosure include peptides, peptide-nucleic acid complexes (peptide-nucleic acid complexes), peptide-ribosome-nucleic acid complexes, and the like. In addition, the "nucleic acids" in the present disclosure include DNA, mRNA, and tRNA. In addition, the "peptides" in the present disclosure may include pharmaceutically acceptable salts thereof.

[0078] In one aspect, the peptides in the present disclosure are composed of 2 to 100, 3 to 50, 4 to 30, or 5 to 30 amino acids linked by amide bonds and / or ester bonds. For example, in one aspect, when using a peptide containing one or more non-natural amino acids according to the present invention as a pharmaceutical, in order to obtain high membrane permeability, the number of amino acids constituting the peptide is preferably 20 or less, more preferably 18 or less, 16 or less, 15 or less, or 14 or less, particularly preferably 13 or less. Specifically, 9, 10, 11, 12, and 13 are exemplified. In addition, in order to obtain high metabolic stability, the number of amino acids constituting the peptide is preferably 8 or more, more preferably 9 or more, still more preferably 10 or more, and particularly preferably 11 or more. Considering the compatibility of membrane permeability and metabolic stability, the number of amino acids constituting the peptide is preferably 5 to 20, or 7 to 20, more preferably 7 to 17, 8 to 16, 9 to 16, or 10 to 16, still more preferably 8 to 13, 10 to 15, 11 to 15, 10 to 14, 10 to 13, or 11 to 14, and particularly preferably 11 to 13.

[0079] The number of amino acids constituting the cyclic part of the cyclic peptide in the present disclosure is not limited. For example, it may be 4 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 20 or less, 18 or less, 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16. Considering the compatibility of membrane permeability and metabolic stability, the number of amino acids constituting the cyclic part is preferably 5 to 15, more preferably 5 to 14, 7 to 14, or 8 to 14, still more preferably 8 to 13, 9 to 13, 8 to 12, 8 to 11, or 9 to 12, and particularly preferably 9 to 11. In the present specification, the "cyclic part" of a peptide means a cyclic part formed by linking two or more amino acid residues.

[0080] In a non-limiting aspect, the cyclic peptides in the present disclosure may have a linear portion. As used herein, the "linear portion" used to refer to a partial structure of a cyclic peptide is a portion not included in the main chain structure of the cyclic portion and having at least one amide bond and / or ester bond on the chain of the portion. The number of amino acids (number of units) in the linear portion is preferably from 0 to 8, more preferably from 0 to 5, and even more preferably from 0 to 3. In a non-limiting aspect, the linear portion in the present specification may include natural amino acids and non-natural amino acids (including amino acids with chemical modifications and backbone conversions).

[0081] In a non-limiting aspect, the number of non-natural amino acids contained in the peptides in the present disclosure is preferably 2 or more, more preferably 4 or more, 5 or more, or 6 or more, even more preferably 7 or more, and particularly preferably 8 or more. Also preferably exemplified are 20 or less, 15 or less, 14 or less, 13 or less, 12 or less, 10 or less, and 9 or less. Examples of the number of non-natural amino acids contained in the peptides in the present disclosure include 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, and 80% or more of the number of amino acids constituting the cyclic portion. Also, the number of types of non-natural amino acids contained in the peptides in the present disclosure is preferably 1 or more, more preferably 2 or more, 3 or more, or 4 or more, even more preferably 7 or more, and particularly preferably 8 or more. Also preferably exemplified are 20 or less, 15 or less, 14 or less, 13 or less, 12 or less, 10 or less, and 9 or less.

[0082] In the peptides containing one or more types of non-natural amino acids in the present disclosure, the sites containing non-natural amino acids are not limited. As one aspect, non-natural amino acids can be included at the position of the starting amino acid. Furthermore, non-natural amino acids can be included at the positions of the second and / or third amino acids.

[0083] When an amino acid other than fMet is introduced at the site of the start amino acid, a phenomenon defined as Initiation read through (iRT) is known to be observed, where translation starts from the second or third amino acid from the start (hereinafter, the peptide produced in this way is defined as an Initiation read through peptide (iRT peptide)). By using the modified L31 protein in the present disclosure, or a ribosome containing the same, it is possible to prevent the phenomenon of Initiation read through even when synthesizing a peptide in which a non-natural amino acid is introduced at the site of the start amino acid. Also, the iRT peptide is not cyclized. Therefore, when creating a library containing cyclic peptides, if most of the mRNA encoding them encodes iRT peptides, the number of displayable molecules will be limited, the number of displayed cyclic peptides will decrease, and the diversity of the library will decrease. By using the modified L31 protein in the present disclosure, or a ribosome containing the same, it is possible to prevent such a decrease in the quality of the library.

[0084] mRNA encoding peptide In the present disclosure, a peptide is produced by translating an mRNA that encodes a peptide containing one or more unnatural amino acids. An mRNA is an RNA that has genetic information that can be translated into a protein. Genetic information is encoded on an mRNA as codons, each of which corresponds to one of the 20 types of amino acids. Protein translation begins with an initiation codon and ends with a stop codon. In principle, the initiation codon in eukaryotes is AUG, but in prokaryotes (eubacteria and archaea), GUG, UUG, and other codons may be used as initiation codons in addition to AUG. AUG is a codon that encodes methionine (Met), and in eukaryotes and archaea, translation begins with methionine as it is. On the other hand, in eubacteria, only the initiation codon AUG corresponds to N-formylmethionine (fMet), so translation begins with formylmethionine. There are three types of stop codons: UAA (ochre), UAG (amber), and UGA (opal). When the stop codon is recognized by a protein called a translation release factor (RF), the peptide chain synthesized up to that point dissociates from the tRNA, and the translation process terminates. In one embodiment, an example of an mRNA encoding a peptide containing one or more unnatural amino acids in the present disclosure is one that contains a codon encoding a peptide containing an unnatural amino acid at least at the position of the start amino acid. In the present disclosure, the site containing an unnatural amino acid in an mRNA encoding a peptide containing one or more unnatural amino acids is not limited, but in one embodiment, the site can contain a codon encoding an unnatural amino acid at the position of the start amino acid.

[0085] Translation of peptide As described above, in the present disclosure, a peptide containing one or more non-natural amino acids can be produced by translation from mRNA encoding the peptide. As used herein, "translation" means synthesizing a peptide by translating it from a nucleic acid (e.g., DNA, RNA) encoding the peptide. Translation is a process of obtaining a linear peptide by repeating amide bond and / or ester bond reactions using mRNA as a template by the action of ribosomes.

[0086] In one aspect, the present disclosure provides a method for producing a peptide or a library containing a peptide, which contains at least one, two or more, three or more, four or more, or five or more non-natural amino acids. Without limitation, such a production method may include the following (i) and (ii): (i) preparing at least one, two or more, three or more, four or more, or five or more tRNAs to which non-natural amino acids are attached; (ii) translating a nucleic acid containing at least one codon corresponding to the anticodon of the tRNA in a translation system to obtain the peptide. Here, the nucleic acid may contain at least one codon corresponding to the anticodon of the tRNA. In one non-limiting embodiment, the mRNA encoding a peptide containing one or more non-natural amino acids in the present disclosure may contain a codon encoding a peptide containing a non-natural amino acid at least at the position of the starting amino acid.

[0087] In the translation of natural amino acids, each of the 64 codons is assigned 20 proteinogenic amino acids and translation termination. When a specific amino acid is removed from the translation system, the codon corresponding to the amino acid becomes an empty codon. Therefore, if a desired non-natural amino acid is linked to a tRNA having an anticodon complementary to the empty codon and added for translation, the amino acid will be encoded by the codon, and a peptide in which the desired non-natural amino acid is introduced instead of the removed amino acid will be translated.

[0088] In one aspect of the present disclosure, it is preferable to use the Initiation Suppression (iSP) method for peptide translation. In normal translation, methionine is generally translated as the N-terminal amino acid as the translation initiation amino acid. There is a dedicated "initiation tRNA" for the initiation of translation. When the initiation tRNA binds to methionine (formylmethionine in prokaryotes) and is transported to the ribosome, translation is initiated, and the N-terminal amino acid becomes methionine (formylmethionine in prokaryotes). In contrast, by removing (or not generating) the initiation tRNA aminoacylated with methionine from the translation system and adding instead the initiation tRNA aminoacylated with a desired amino acid prepared in advance to the translation system, the method of translating a peptide having the desired amino acid at the N-terminus is called the initiation suppression method. The tolerance of unnatural amino acids is higher in introduction to the N-terminus than during amino acid elongation, and it is known that unnatural amino acids with a significantly different structure from natural amino acids can be used as the N-terminal amino acid (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.). In one aspect of the present disclosure, the initiation tRNA contained in the translation system may be acylated with an unnatural amino acid.

[0089] Translation system In the present disclosure, the "translation system" is defined as a concept including both a method for translating a peptide and a composition for translating a peptide. In the present disclosure, the "translation system" is not limited as long as it includes ribosomes containing the modified L31 protein in the present disclosure. The "translation system" in the present disclosure preferably further includes a combination of protein factors involved in translation, tRNA, amino acids, an energy source such as ATP, and its regeneration system, and includes those capable of translating mRNA into protein. Also, a system during translation may be included in the "translation system" in this specification. The translation system in this specification may contain a nucleic acid serving as a template when translating a peptide, and in addition to these, may include initiation factors, elongation factors, dissociation factors, aminoacyl-tRNA synthetases, and the like. These factors can be obtained by purification from extracts of various cells. Examples of cells for purifying the factors include prokaryotic cells or eukaryotic cells. Examples of prokaryotic cells include Escherichia coli cells, hyperthermophilic bacterial cells, or Bacillus subtilis cells. Examples of eukaryotic cells include those prepared using yeast cells, wheat germ, rabbit reticulocytes, plant cells, insect cells, or animal cells. In addition to naturally occurring tRNA and aminoacyl-tRNA synthetase (ARS), artificial tRNA and artificial aminoacyl-tRNA synthetase that recognize unnatural amino acids can also be used. By using artificial tRNA and artificial aminoacyl-tRNA synthetase, a peptide into which an unnatural amino acid has been site-specifically introduced can be synthesized. If necessary, an RNA polymerase such as T7 RNA polymerase can be added to the translation system to perform transcription from template DNA.

[0090] In this specification, "a translation system contains a certain substance" includes a mode in which the substance is not contained at the start of translation but is synthesized and contained within the system during the translation process. For example, when aminoacylated tRNA is synthesized during the translation process, the translation system is understood to contain the aminoacyl tRNA.

[0091] The main types of translation systems include translation systems using living cells and translation systems using cell extracts (cell-free translation systems). Examples of translation systems using living cells include systems in which desired aminoacyl-tRNAs and mRNAs are introduced into living cells such as Xenopus laevis oocytes and mammalian cells by microinjection or lipofection methods to perform peptide translation (Nowak et al., Science (1995) 268: 439-442). Examples of cell-free translation systems include translation systems using extracts from Escherichia coli (Chen et al., Methods Enzymol (1983) 101: 674-690), yeast (Gasior et al., J Biol Chem (1979) 254: 3965-3969), wheat germ (Erickson et al., Methods Enzymol (1983) 96: 38-50), rabbit reticulocytes (Jackson et al., Methods Enzymol (1983) 96: 50-74), HeLa cells (Barton et al., Methods Enzymol (1996) 275: 35-57), or insect cells (Swerdel et al., Comp Biochem Physiol B (1989) 93: 803-806). Such translation systems can be appropriately prepared by methods known to those skilled in the art or methods equivalent thereto. Cell-free translation systems also include translation systems (reconstituted cell-free translation systems) constructed by isolating and purifying the factors necessary for peptide translation and then reconstituting them (Shimizu et al., Nat Biotech (2001) 19: 751-755). Reconstituted cell-free translation systems usually include ribosomes, amino acids, tRNAs, aminoacyl-tRNA synthetases (aaRSs), translation initiation factors (e.g., IF1, IF2, IF3), translation elongation factors (e.g., EF-Tu, EF-Ts, EF-G), translation termination factors (e.g., RF1, RF2, RF3), ribosome recycling factor (RRF), NTPs as an energy source, an energy regeneration system, and other factors necessary for translation.When performing a transcription reaction from DNA, RNA polymerase and the like may further be included. The various factors contained in the cell-free translation system can be isolated and purified by methods well known to those skilled in the art, and a reconstituted cell-free translation system can be appropriately constructed using them. Alternatively, commercially available reconstituted cell-free translation systems such as PUREfrex (registered trademark) of Gene Frontier and PURExpress (registered trademark) of New England BioLabs can also be used. In the case of a reconstituted cell-free translation system, only the necessary components among the components of the translation system can be reconstituted to construct a desired translation system.

[0092] PURESYSTEM (registered trademark) (BioComber, Japan) is a reconstituted cell-free translation system in which protein factors, energy regeneration system enzymes, and ribosomes necessary for translation in Escherichia coli are extracted and purified and then mixed with tRNA, amino acids, ATP, GTP, etc. Not only is the content of impurities low, but since it is a reconstituted system, a system that does not contain protein factors and amino acids that are desired to be excluded can be easily prepared ((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. PUREtechnology. Shimizu Y, Ueda T.).

[0093] For example, although many methods have been reported for using a stop codon as a codon for introducing a non-natural amino acid, a synthetic system excluding natural amino acids and ARS can be constructed by using the aforementioned PURESYSTEM. By doing so, a non-natural amino acid can be associated with the codon encoding the natural amino acid to be excluded (J Am Chem Soc. 2005;127:11727-35. Ribosomal synthesis of unnatural peptides. Josephson K, Hartman MC, Szostak JW.). Furthermore, by resolving the degeneracy of the codon, a non-natural amino acid can be added without excluding natural amino acids (Kwon I, et al. Breaking the degeneracy of the genetic code. J Am Chem Soc. 2003, 125, 7512-3.). A peptide containing an N-methyl amino acid can be synthesized by ribosome using a cell-free translation system such as PURESYSTEM.

[0094] More specifically, for example, in the case of in vitro translation, the translation components required for translation in E. coli (protein factors such as methionyl-tRNA transformylase, EF-G, RF1, RF2, RF3, RRF, IF1, IF2, IF3, EF-Tu, EF-Ts, ARS (select as necessary 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, tRNA derived from E. coli, creatine phosphate, potassium glutamate, HEPES-KOH pH 7.6, magnesium acetate, spermidine, dithiothreitol, GTP, ATP, CTP, UTP, etc. are appropriately selected and mixed, and mRNA is added to a known cell-free translation system such as PURESYSTEM. In addition, if T7 RNA polymerase is added, transcription and translation from template DNA containing a T7 promoter can be coupled. Further, by adding a desired group of aminoacyl-tRNAs and a group of unnatural amino acids (e.g., F-Tyr) tolerated by aminoacyl-tRNA synthetase (ARS) to the system, a peptide compound containing an unnatural amino acid can be translationally synthesized (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.). Furthermore, by including a modified form of ARS in the system, either instead of or in addition to natural ARS, and by including a group of unnatural amino acids in the system, mRNA encoding a peptide containing an unnatural amino acid can also be translated.Alternatively, by using mutants such as ribosomes and EF-Tu, it is also possible to enhance the efficiency of translation of mRNA encoding peptides containing unnatural amino acids and the accompanying introduction of unnatural amino acids (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 with phosphoserine. Science. 2011, 333, 1151-4.).

[0095] In addition, the translation system in the present disclosure preferably contains ribosomes containing the modified L31 protein in the present disclosure at a ratio of at least 50% or more, preferably 60% or more, more preferably 70% or more, still more preferably 80% or more, particularly preferably 90% or more (for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more) in terms of the molecular number ratio with respect to all ribosomes contained in the translation system. The "all ribosomes" is not particularly limited as long as it is a ribosome contained in the translation system, and examples thereof include the total of ribosomes containing the modified L31 protein in the present disclosure and other ribosomes (for example, ribosomes containing wild-type E. coli L31 and ribosomes functionally equivalent thereto). The ratio of the modified L31 protein to all ribosomes contained in the translation system can be calculated, for example, based on the ratio of the intensity of the modified L31 protein to the intensity of all ribosomes by a mass spectrometer.

[0096] Also, the translation system in the present disclosure preferably contains magnesium ions. It is known that magnesium ions are required to maintain the association state of the small and large subunits of ribosomes. The L31 protein is one of the proteins that form the interaction between the large and small subunits of ribosomes. It has been clarified that ribosomes lacking the L31 protein or ribosomes containing shortL31 have a higher magnesium ion concentration required to maintain the associated state than ribosomes containing intact L31. Therefore, in the production method in the present disclosure in which ribosomes containing a modified L31 protein are used, it is preferable to set the magnesium ion concentration higher than the magnesium ion concentration in the translation system using ribosomes containing intact L31. To prepare such a translation system, the method of the present invention may further include a step of adding magnesium ions to the translation system in the present invention. Alternatively, in the present invention, a translation system with magnesium ions added in advance can also be used. The amount of magnesium added is not particularly limited, and for example, it can be exemplified by a range specifiable by any combination of a lower limit selected from values of 1 mM or more, 2 mM or more, 3 mM or more, 4 mM or more, 5 mM or more, 6 mM or more, or 7 mM or more, and an upper limit selected from 9 mM or less, 8 mM or less, 7 mM or less, 6 mM or less, 5 mM or less, 4 mM or less, 3 mM or less.

[0097] tRNA In order to introduce unnatural amino acids into peptides by translating mRNA encoding peptides containing unnatural amino acids, aminoacylation of tRNAs ( (i) Biochemistry. 2003;42:9598-608. Adaptation of an orthogonal archaeal 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.) that are orthogonal and efficiently incorporated into ribosomes is required. Five methods can be used as methods for aminoacylating tRNAs.

[0098] Inside cells, aminoacyl-tRNA synthetases (ARSs) are prepared for each amino acid as enzymes for aminoacylation of tRNA. Therefore, as the first method, there are methods that utilize the fact that certain ARSs accept unnatural amino acids such as N-Me His, or methods that prepare mutant aminoacyl-tRNA synthetases that accept unnatural amino acids and utilize them ((i) Proc Natl Acad Sci U S A. 2002;99:9715-20. An engineered Escherichia coli tyrosyl-tRNA synthetase for site-specific incorporation of an unnatural amino acid 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.). Second, a method of aminoacylating tRNA in vitro and then chemically modifying 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.).Thirdly, aminoacyl-tRNA can be obtained by ligating the CCA sequence at the 3'-end of tRNA with the CA removed and 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.). There is also aminoacylation by Flexizyme, a ribozyme that attaches the active esters of various unnatural amino acids to tRNA (J Am Chem Soc. 2002; 124: 6834-5. Aminoacyl-tRNA synthesis by a resin-immobilized ribozyme. Murakami H, Bonzagni NJ, Suga H.). Fourthly, the method of ultrasonic mixing of tRNA and amino acid active ester in cationic micelles can also be used (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.). Fifthly, aminoacylation is also possible by adding to tRNA a compound in which an amino acid active ester is bound to PNA complementary to the vicinity of the 3'-end of tRNA (J Am Chem Soc. 2004; 126: 15984-9. In situ chemical aminoacylation with amino acid thioesters linked to a peptide nucleic acid. Ninomiya K, Minohata T, Nishimura M, Sisido M.).

[0099] More specifically, aminoacyl-tRNA can be prepared using the following methods. Prepare template DNA encoding the desired tRNA sequence with a T7, T3, or SP6 promoter placed upstream, and use an RNA polymerase adapted to the promoter, such as T7 RNA polymerase, T3 RNA polymerase, or SP6 RNA polymerase, to synthesize RNA by transcription. tRNA can be extracted and purified from cells, and the target generated tRNA can also be extracted using a probe of the complementary sequence of the tRNA sequence. At this time, cells transformed with the expression vector of the target tRNA can also be used as a source. RNA of the target sequence can also be synthesized by chemical synthesis. For example, aminoacyl-tRNA can be obtained by ligating the tRNA with the CA removed from the CCA sequence at the 3' end obtained in this way to separately prepared aminoacylated pdCpA or pCpA with RNA ligase (pdCpA method, pCpA method). The said tRNA is useful in the production of peptides. Alternatively, it is also possible to perform aminoacylation with Flexizyme, a ribozyme that prepares full-length tRNA and attaches active esters of various unnatural amino acids to the tRNA. Also, although not intended to be limiting, aminoacyl-tRNA can also be prepared using a natural ARS or a modified variant thereof. When using a natural ARS or a modified variant thereof, aminoacyl-tRNA once consumed in the translation system can be regenerated by the natural ARS or its modified variant, so it is not necessary to have a large amount of pre-prepared aminoacyl-tRNA present in the translation system. Such ARS variants are described in WO2016 / 148044. These methods for preparing aminoacyl-tRNA can also be appropriately combined.

[0100] Cyclization of peptide In a non-limiting aspect, in the method for producing a peptide or a library containing a peptide in the present disclosure, a step of cyclizing the translated peptide can be further included. As modes of cyclization, for example, cyclization using an amide bond, a carbon-carbon bond, a thioether bond, a disulfide bond, an ester bond, a thioester bond, a lactam bond, a bond via a triazole structure, a bond via a fluorophore structure, etc. can be mentioned. Among them, an amide bond is preferable because of its high metabolic stability. The peptide translation step and the cyclization reaction step may proceed separately or continuously. Cyclization can be carried out by methods known to those skilled in the art described in, for example, WO2013 / 100132, WO2008 / 117833, WO2012 / 074129, etc.

[0101] The mode of the bond in ring formation is not limited, but it may be any of the bond between the N-terminus and C-terminus of the peptide, the bond between the N-terminus of the peptide and the side chain of another amino acid residue, the bond between the C-terminus of the peptide and the side chain of another amino acid residue, or the bond between the side chains of amino acid residues, and two or more of these may be used in combination.

[0102] In a non-limiting aspect, the present invention relates to a method for producing a peptide or a library containing a peptide, which includes, for example, the following steps: (1) A step of producing an acyclic peptide containing one or more non-natural amino acids by the method described herein, wherein the acyclic peptide contains an amino acid residue having a reaction point on one of the side chains on the C-terminal side and another amino acid residue having a reaction point on the N-terminal side; and (2) A step of bonding the reaction point of the amino acid residue on the N-terminal side to the reaction point of the amino acid residue having a reaction point on the side chain on the C-terminal side to form an amide bond, a carbon-carbon bond or a thioether bond. It should be noted that these steps (1) and (2) may proceed separately or continuously.

[0103] Specifically, as a non-limiting aspect of the method for cyclizing a peptide by an amide bond, there is a cyclization method in which the amino group of the N-terminal methionine and the amino group of lysine arranged downstream (C-terminal side) are cross-linked with disuccinimidyl glutarate (DSG), or an amino acid derivative having a chloroacetyl group is introduced as the translation initiation amino acid at the N-terminal, and Cys is arranged downstream to form a thioether by an intramolecular cyclization reaction, and there is a method of translating a peptide having cysteine or a cysteine analog at the N-terminal and an active ester in the side chain of the C-terminal amino acid and cyclizing it using native chemical ligation.

[0104] In a non-limiting aspect, the C-terminal site of the peptide in the present disclosure may not remain as a carboxylic acid but may be chemically modified. For example, the carboxylic acid site may be reacted with piperidine or the like to be converted into a piperidine amide or the like.

[0105] Library The present invention also relates to a peptide produced by the method for producing a peptide described in this specification and a library containing the peptide. The present invention also relates to a library containing the peptide, which includes a step of producing the peptide by the method for producing a peptide described in this specification. The library in the present disclosure includes a library containing the peptide in the present disclosure and a library containing a nucleic acid encoding the peptide in the present disclosure. The library in the present disclosure includes a library of the peptide in the present disclosure and a library of peptide-nucleic acid complexes. As the library, a display library is preferable. Examples of the display library include libraries using display, and among them, an mRNA display library, a DNA display library, and a ribosome display library are preferable, and an mRNA display library is more preferable.

[0106] Display library A display library is a library in which a peptide that is a phenotypic form is associated with an RNA or DNA encoding a peptide that is its genotypic form. Using this library, a peptide that can specifically bind to a target molecule can be identified. For example, 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 the peptide that binds to the target (panning method). By analyzing the genetic information associated with the peptide selected through such a process, the sequence of the peptide that binds to the target can be revealed. For example, there is a method that utilizes the fact that the antibiotic puromycin, which is an analog of aminoacyl tRNA, is non-specifically linked to a protein during mRNA translation elongation by ribosomes, which is 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.) or in vitro virus (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.).

[0107] A spacer such as puromycin is bound to the 3'-end of an mRNA library obtained by transcription from a DNA library containing a promoter such as the T7 promoter. When the mRNA is translated into protein in a cell-free translation system, puromycin is misincorporated into the protein by the ribosome as an amino acid, and the mRNA and the protein encoded thereby are ligated, resulting in a library in which the mRNA and its product are associated. Since this process does not involve transformation such as that of Escherichia coli, high efficiency is achieved, and a large-scale display library can be constructed. For the molecules enriched and selected by panning, cDNA can be synthesized from the mRNA, which is a tag containing the genetic information of the molecule, amplified by PCR, and the sequence of the bound peptide can be elucidated by analyzing the nucleotide sequence.

[0108] Cell-free translation system-based display libraries include, in addition to mRNA display, cDNA display, which is a library consisting of cDNA encoding peptides to which complexes of peptides and puromycin are bound (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 fact that ribosomes and translation products form a relatively stable complex 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.), covalent display, which utilizes the fact that bacteriophage endonuclease P2A forms a covalent bond with 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, which utilizes the fact that the replication initiation protein RepA of a microbial plasmid binds to the replication origin 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. Also, in vitro compartmentalization (Nat Biotechnol. 1998;16:652-6. Man-made cell-like compartments for molecular evolution. Tawfik DS, Griffiths AD.) in which a transcription-translation system is encapsulated in a water-in-oil emulsion or liposome for each molecule of DNA constituting a DNA library and a translation reaction is performed is also known. The above methods can be used as appropriate using known methods.

[0109] Nucleic acid library The "nucleic acid" in the present disclosure can also include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or nucleotide derivatives having artificial bases. It can also include peptide nucleic acid (PNA). In the present disclosure, the nucleic acid can be any of these nucleic acids or a hybrid as long as the target genetic information is retained. That is, chimeric nucleic acids in which different nucleic acids such as DNA-RNA hybrid nucleotides or DNA and RNA are linked in a single strand are also included in the nucleic acid in the present disclosure.

[0110] Examples of the nucleic acid library serving as a template for the peptides contained in the peptide library include an mRNA library, a DNA library, and the like. A nucleic acid library can be obtained by synthesizing a mixture of bases at positions where amino acid residues are not fixed on the peptide sequence. For example, as a DNA library, a repetition of a multiple of 3 of a mixture of 4 bases (N) of A, T, G, C, and as an RNA library, a repetition of a multiple of 3 of a mixture of 4 bases (N) of A, U, G, C, or the first and second letters of the codon are N, and the third letter is a mixture of 2 bases such as W, M, K, S. Further, if the number of types of amino acids to be introduced is suppressed to 16 or less, there is also a method of making the third letter a single type of base. In addition, a codon unit corresponding to 3 letters of the codon is prepared, and by mixing this at an arbitrary ratio and using it for synthesis, the appearance frequency of amino acid residues can be freely adjusted.

[0111] These nucleic acid libraries can be translated using a cell-free translation system. When using a cell-free translation system, it is preferable to include a sequence encoding a spacer downstream of the nucleic acid of interest. Examples of the spacer sequence include, but are not limited to, sequences containing glycine or serine. Further, between the nucleic acid library and a compound incorporated into the peptide during translation by ribosomes, such as puromycin and its derivatives, it is preferable to include a linker formed of RNA, DNA, a polymer of hexaethylene glycol (spc18) (for example, 5 polymers).

[0112] The production of the library in the present disclosure can be carried out according to the method for producing a peptide in the present disclosure and can be combined with publicly known methods as appropriate. In one aspect, a peptide library in the present disclosure can be produced using the cell-free translation system in the present disclosure. That is, the method for producing a library in the present disclosure may include a step of synthesizing a peptide using the cell-free translation system in the present disclosure. In one aspect, the examples, preferred ranges, and aspects described in the cell-free translation system in the present disclosure can be directly applied to the method for producing a library in the present disclosure.

[0113] Screening method In a non-limiting embodiment, peptides that can specifically bind to a target molecule can be selected by screening using the library in the present disclosure.

[0114] In a non-limiting embodiment, the screening method in the present disclosure involves contacting a library of peptides containing one or more non-natural amino acids in the present disclosure with a target molecule, and washing away peptides that do not bind to the target molecule to concentrate peptides that bind to the target molecule (panning). In one embodiment, cDNA is synthesized from mRNA, which is a tag containing the nucleotide sequence information included in the peptides thus selected, amplified by PCR, and the nucleotide sequence is analyzed to reveal the amino acid sequence of the bound peptides. Note that, in one embodiment, an mRNA library may be obtained by transcribing the cDNA amplified above, and a peptide library may be produced again using these as templates. Since this library is enriched with peptides that can bind to the target molecule, panning can be performed again using this library to further enrich peptides that can specifically bind to the target molecule. By repeating this process multiple times, the target peptide can be further enriched. In one embodiment, the amino acid sequence can be identified based on the nucleotide sequence information contained in the peptides thus enriched, and peptides that can specifically bind to the target molecule can be produced. In one embodiment, the screening method in the present disclosure can be performed in vitro.

[0115] In a non-limiting embodiment, the peptides obtained by the screening method in the present disclosure may be optimized by chemically modifying them by known methods, etc. As used herein, "optimization" means chemically modifying by converting the structure of each amino acid in the translated peptide to obtain a more drug-like peptide, chemically modifying to obtain a peptide with stronger activity against the drug efficacy target, and / or chemically modifying to obtain a peptide with more avoided toxicity.

[0116] In a non-limiting aspect, the screening method in the present disclosure includes the following steps: (a) contacting a peptide contained in the library in the present disclosure with a target molecule; (b) selecting a peptide that can bind to the target molecule. The screening method in the present disclosure may include, before the step (a), a step of obtaining a library by the method described herein. The library can be obtained following the method for producing a peptide in the present disclosure.

[0117] In one aspect, the screening method in the present disclosure may concentrate a peptide that can specifically bind to a target molecule by repeating the steps (a) and (b) above two or more times.

[0118] Target molecule The target molecule used in the screening method in the present disclosure is not particularly limited, and examples include proteins, peptides, nucleic acids, sugars, lipids, etc., and among them, it is preferable to target proteins. Also, the location of the target molecule in vivo is not particularly limited. In one aspect, it is also possible to target intracellular proteins.

[0119] In a non-limiting aspect, the target molecule used in the screening method in the present disclosure is used after being immobilized on a carrier. The carrier is not particularly limited as long as it can immobilize the target molecule, and examples include beads or resins. The target molecule can be immobilized on the carrier by a known method.

[0120] As described above, the target molecules of the library and screening method in the present disclosure are not particularly limited, and examples thereof include GTPase KRas (KRAS), Dual specificity mitogen-activated protein kinase kinase 1 (MEK1), Mitogen-sctivated protein kinase 3 (ERK1), and interleukin 6 receptor (IL-6R). As described in the examples, since the library in the present disclosure contains peptides that can specifically bind to various target molecules, in one aspect, it may enable drug discovery for tough targets for which drug discovery has been difficult heretofore.

[0121] In one non-limiting aspect, the method for producing a peptide in the present disclosure may include the following steps: (i) A step of contacting a peptide contained in the library in the present disclosure with a target molecule; (ii) A step of selecting a peptide that can bind to the target molecule; and (iii) A step of producing a peptide based on the amino acid sequence of the peptide selected in (ii). The above method may include a step of obtaining a library by the method described in this specification.

[0122] In one non-limiting aspect, the method for producing a library, the method for producing a peptide, and / or the screening method in the present disclosure may be performed in vitro.

[0123] In one aspect, the peptide in the present disclosure may be cyclized.

[0124] Nucleic acid encoding modified L31 protein, etc. The present invention also relates to a modified L31 protein of the present disclosure, a ribosome containing the protein, and an isolated nucleic acid encoding the protein. The present invention also relates to a vector or cell containing the nucleic acid. Examples of the modified L31 protein of the present disclosure may include the following (1) to (6). (1) A protein comprising an amino acid sequence in which 6 or more amino acid residues, 8 or more amino acid residues, or 9 or more amino acid residues are deleted from the amino acid sequence represented by SEQ ID NO: 1, counted from the C-terminus, (2) A protein comprising an amino acid sequence in which one or more amino acids are inserted, substituted, deleted, and / or added in the amino acid sequence of the protein described in (1) above, (3) A protein comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence of the protein described in (1) above, (4) A protein comprising an amino acid sequence selected from SEQ ID NO: 2 and SEQ ID NOs: 42 to 48, (5) A protein comprising an amino acid sequence in which one or more amino acids are deleted, inserted, substituted, and / or added in the amino acid sequence selected from SEQ ID NO: 2 and SEQ ID NOs: 42 to 48, and which is a protein functionally equivalent to the protein comprising the amino acid sequence represented by SEQ ID NO: 2, and (6) A protein comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence selected from SEQ ID NO: 2 and SEQ ID NOs: 42 to 48, and which is a protein functionally equivalent to the protein comprising the amino acid sequence represented by SEQ ID NO: 2. The modified L31 protein, ribosome containing the protein, isolated nucleic acid encoding the protein, etc. in the present disclosure can be used, for example, in the production of peptides containing unnatural amino acids in the present disclosure and libraries containing such peptides.

[0125] In the present disclosure, an "isolated" nucleic acid refers to a nucleic acid molecule separated from the elements of the natural environment. An isolated nucleic acid includes nucleic acid molecules contained in cells that normally contain the nucleic acid molecule, provided that the nucleic acid molecule is present extrachromosomally or at a position different from its natural chromosomal position. In the case of a nucleic acid that does not exist in the natural chromosome, the isolated nucleic acid may be present at any position within the cell. The "nucleic acids" in the present disclosure include DNA (genomic DNA, cDNA) and RNA.

[0126] As used interchangeably herein, "polynucleotide" or "nucleic acid" refers to a polymer of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substance that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. Polynucleotides can include modified nucleotides such as methylated nucleotides and their analogs. Non-nucleotide components may be interspersed within the nucleotide sequence. Polynucleotides can include modifications made after synthesis, such as conjugation to a label. Other types of modifications include, for example, "caps", substitution of one or more naturally occurring nucleotides with analogs, internucleotide modifications, such as those involving uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) and charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those including pendant moieties such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those involving intercalating agents (e.g., acridines, psoralens, etc.), those including chelating agents (e.g., metals, radioactive metals, boron, metal oxides, etc.), those including alkylating agents, those involving modified linkages (e.g., alpha anomeric nucleic acids, etc.) and those involving polynucleotides in unmodified form. Further, any hydroxyl group normally present on the sugar can be substituted, for example, with a phosphonate group, a phosphate group, protected by a standard protecting group, or activated to create further linkages to additional nucleotides, or conjugated to a solid or semi-solid support. The OH at the 5' and 3' termini can be phosphorylated or substituted with an amine or an organic cap group moiety of 1 to 20 carbon atoms. Other hydroxyls can also be derivatized with standard protecting groups.Polynucleotides can also include analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example: 2'-O-methyl-, 2'-O-allyl-, 2'-fluoro-, or 2'-azido-ribose, carbocyclic sugar analogs, α-anomer sugars, epimeric sugars such as arabinose or xylose or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acyclic analogs, and basic nucleoside analogs such as methyl riboside. One or more phosphodiester bonds can be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which the phosphate is replaced by: P(O)S (“thioate”), P(S)S (“dithioate”), (O)NR2 (“amidate”), P(O)R, P(O)OR', CO, or CH2 (“formacetal”), where each R or R' is independently H, or substituted or unsubstituted alkyl (1-20C) optionally including an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. Not all linkages in the polynucleotide need to be the same. The above description applies to all polynucleotides referred to herein, including RNA and DNA.

[0127] As a method for preparing a nucleic acid encoding a modified L31 protein in the present disclosure, for example, the site-directed mutagenesis method (Kramer, W. and Fritz, H.-J. (1987) Oligonucleotide-directed construction of mutagenesis via gapped duplex DNA. Methods in Enzymology, 154: 350-367) can be mentioned. Alternatively, by hybridization technology (Southern, E.M. (1975) Journal of Molecular Biology, 98, 503), it is also possible to obtain a nucleic acid encoding a protein functionally equivalent to the modified L31 protein described in any of SEQ ID NOs: 2 and 42-48. That is, the nucleic acid encoding the modified L31 protein in the present disclosure may hybridize under stringent conditions with a nucleic acid encoding the amino acid sequence described in any of SEQ ID NOs: 2 and 42-48. Stringent hybridization conditions can be appropriately selected by those skilled in the art. For example, prehybridization is carried out overnight at 42°C in a hybridization solution containing 25% formamide, and for more stringent conditions, 50% formamide, 4×SSC, 50 mM Hepes pH 7.0, 10×Denhardt's solution, and 20 μg / ml denatured salmon sperm DNA. Then, a labeled probe is added and hybridization is carried out by incubating overnight at 42°C. The washing solution and temperature conditions in the subsequent washing can be, for example, about "2×SSC, 0.1% SDS, 50°C", "2×SSC, 0.1% SDS, 42°C", "1×SSC, 0.1% SDS, 37°C", and for more stringent conditions, about "2×SSC, 0.1% SDS, 65°C", "0.5×SSC, 0.1% SDS, 42°C", and for even more stringent conditions, about "0.2×SSC, 0.1% SDS, 65°C". Thus, the more stringent the hybridization conditions, the more likely it is to isolate a nucleic acid having high homology with the nucleic acid sequence encoding the amino acid sequence described in any of SEQ ID NOs: 2 and 42-48.However, the combination of the above SSC, SDS, and temperature conditions is exemplary, and those skilled in the art can appropriately combine the above or other elements (e.g., probe concentration, probe length, hybridization reaction time, etc.) that determine the stringency of hybridization to achieve the same stringency as above.

[0128] The nucleic acid isolated thereby is considered to have high homology with the modified L31 protein described in any one selected from SEQ ID NOs: 2 and 42 to 48 at the amino acid level. Also, at the nucleotide sequence level, it is considered to have high homology with the nucleotide sequence of the nucleic acid encoding the amino acid sequence described in any one selected from SEQ ID NOs: 2 and 42 to 48. As described above, high homology refers to sequence identity of at least 50% or more, more preferably 70% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90% or more (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) for the entire amino acid sequence or the entire nucleotide sequence.

[0129] The nucleic acid of the present invention can be used, for example, in the preparation of a modified L31 protein or ribosomes containing the same. When preparing a recombinant protein of modified L31 or ribosomes containing the same, usually, a nucleic acid encoding the modified L31 protein is inserted into an appropriate expression vector, the vector is introduced into an appropriate cell, and the transformed cell is cultured to isolate, purify, and culture the expressed modified L31 protein or ribosomes containing the same. The modified L31 protein can also be expressed as a fusion protein with another protein for the purpose of facilitating purification. For example, a method of preparing it as a fusion protein with a maltose-binding protein using Escherichia coli as a host (vector pMAL series sold by New England BioLabs, USA), a method of preparing it as a fusion protein with glutathione-S-transferase (GST) (vector pGEX series sold by Amersham Pharmacia Biotech), a method of preparing it by adding a histidine tag (pET series of Novagen), etc. can be used.

[0130] In a non-limiting embodiment, the vector may be a knock-in vector into which the nucleic acid of the present invention has been knocked in by homologous recombination. That is, the modified L31 protein of the present invention, or the ribosome containing the same, can also be prepared by knocking in the nucleic acid of the present invention into the vector by homologous recombination. Such a vector may be constructed such that the nucleic acid of the present invention for preparing the modified L31 protein, or the ribosome containing the same, is inserted into the same reading frame of the target gene in a host such as Escherichia coli. In one embodiment, it is preferable that the nucleic acid of the present invention is inserted into an exon containing the translation start point such that the translation start point thereof coincides with the translation start point of the target gene in the knock-in vector. In this case, in the knock-in vector, it is preferable that a nucleotide sequence upstream of the translation start point of the target gene is arranged on the 5'-side of the translation start point of any foreign gene. In another embodiment, when an exon-intron structure sequence is added to the 5'-side of any foreign gene of the knock-in vector, it is preferable that the 5'-terminal end of the exon-intron structure is inserted into an exon containing the translation start point such that it coincides with the translation start point of the target gene. In this case, in the knock-in vector, it is preferable that a nucleotide sequence upstream of the translation start point of the target gene is arranged on the 5'-upstream side of the 5'-terminal end of the exon-intron structure.

[0131] Also, the knock-in vector preferably has the ability to replicate in the host cell. The knock-in vector is not particularly limited as long as it is a vector used in genetic engineering. Examples of known vectors include plasmid vectors, cosmid vectors, bacterial artificial chromosome (BAC) vectors, yeast artificial chromosome (YAC) vectors, retroviral vectors, lentiviral vectors, and other viral vectors.

[0132] The host cell is not particularly limited as long as it is suitable for the expression of recombinant proteins. In addition to the above-mentioned Escherichia coli, for example, yeast, various animal and plant cells, insect cells, etc. can be used. For introducing the vector into the host cell, various methods known to those skilled in the art can be used. For example, for introduction into Escherichia coli, an introduction method using calcium ions (Mandel, M., Higa, A. (1970) Journal of Molecular Biology, 53, 158-162, Hanahan, D. (1983) Journal of Molecular Biology, 166, 557-580) can be used. The modified L31 protein expressed in the host cell can be purified and recovered from the host cell, its cell culture, or culture supernatant by methods known to those skilled in the art. When the modified L31 protein is expressed as a fusion protein with the above-mentioned maltose-binding protein, etc., affinity purification can be easily performed.

[0133] In one aspect of the present disclosure, the nucleic acid of the present invention may be inserted into a vector. Examples of vectors include, for example, when Escherichia coli is used as a host, in order to amplify and prepare a large amount of the vector in Escherichia coli (e.g., JM109, DH5α, HB101, XL1Blue, etc.), the vector has an "ori" for amplification in Escherichia coli, and further, a selection gene for transformed Escherichia coli (e.g., a drug resistance gene that can be discriminated by a drug (such as ampicillin, tetracycline, kanamycin, chloramphenicol, etc.)). Examples of such vectors include, but are not limited to, M13-based vectors, pUC-based vectors, pBR322, pBluescript, pCR-Script, etc. Also, for the purpose of cDNA subcloning or excision, in addition to the above vectors, for example, pGEM-T, pDIRECT, pT7, etc. can be mentioned. When a vector is used for the purpose of producing a modified L31 protein, in particular, an expression vector is useful. Examples of expression vectors include, for example, when the purpose is expression in Escherichia coli, in addition to having the above characteristics such that the vector can be amplified in Escherichia coli, when the host is Escherichia coli such as JM109, DH5α, HB101, XL1-Blue, etc., a promoter that can be efficiently expressed in Escherichia coli, for example, the lacZ promoter (Ward et al., Nature (1989) 341, 544-546; FASEB J. (1992) 6, 2422-2427), the araB promoter (Better et al., Science (1988) 240, 1041-1043), or the T7 promoter, etc. is essential. Examples of such vectors include, in addition to the above vectors, pGEX-5X-1 (manufactured by Pharmacia), the "QIAexpress system" (manufactured by Qiagen), pEGFP, or pET, etc.

[0134] In addition, the vector may contain a signal sequence for polypeptide secretion. When producing in the periplasm of Escherichia coli, the pelB signal sequence (Lei, S. P. et al J. Bacteriol. (1987) 169, 4379) may be used as the signal sequence for polypeptide secretion. Introduction of the vector into the host cell can be carried out using, for example, the calcium chloride method or the electroporation method. Examples of vectors that are expressed in plants include vectors such as pMH1, pMH2, and pCAMBIA.

[0135] In addition to Escherichia coli, for example, as vectors for producing a modified L31 protein, expression vectors derived from mammals (for example, pcDNA3 (manufactured by Invitrogen), pEGF - BOS (Nucleic Acids. Res. 1990, 18(17), p5322), pEF, pCDM8), expression vectors derived from insect cells (for example, "Bac - to - BAC baculovairus expression system" (manufactured by Gibco BRL), pBacPAK8), expression vectors derived from plants (for example, pMH1, pMH2), expression vectors derived from animal viruses (for example, pHSV, pMV, pAdexLcw), expression vectors derived from retroviruses (for example, pZIPneo), expression vectors derived from yeast (for example, "Pichia ExpressionKit" (manufactured by Invitrogen), pNV11, SP - Q01), expression vectors derived from Bacillus subtilis (for example, pPL608, pKTH50), etc. can be mentioned.

[0136] When aiming for expression in animal cells such as CHO cells, COS cells, NIH3T3 cells, etc., it is essential to have promoters necessary for intracellular expression, for example, the SV40 promoter (Mulligan et al., Nature (1979) 277, 108), MMLV-LTR promoter, EF1α promoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322), CMV promoter, etc. It is more preferable to have a gene for selecting cell transformation (for example, a drug resistance gene that can be discriminated by a drug (neomycin, G418, etc.)). Examples of vectors having such characteristics include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, pOP13, etc.

[0137] The transformed cells of the present invention can be used, for example, as a production system for producing or expressing a modified L31 protein or ribosomes containing the same. Production systems for protein production include in vitro and in vivo production systems.

[0138] When using eukaryotic cells, for example, animal cells, plant cells, or fungal cells can be used as hosts. Examples of animal cells include mammalian cells (for example, in addition to the above-mentioned CHO cells, COS cells, NIH3T3 cells, 3T3, myeloma cells, BHK (baby hamster kidney), HeLa, Vero, etc.), amphibian cells (for example, Xenopus laevis oocytes (Valle, et al., Nature (1981) 291, 358 - 340)), or insect cells (for example, sf9, sf21, Tn5, etc.). As CHO cells, in particular, dhfr-CHO (Proc. Natl. Acad. Sci. USA (1980) 77, 4216 - 4220) and CHO K-1 (Proc. Natl. Acad. Sci. USA (1968) 60, 1275), which are CHO cells lacking the DHFR gene, can be preferably used. CHO cells are particularly preferred when aiming for large-scale expression.

[0139] As plant cells, in addition to the cells derived from plants described below, for example, cells derived from Nicotiana tabacum are known as a protein production system, and these can be subjected to callus culture. As fungal cells, yeast, for example, the genus Saccharomyces, for example, Saccharomyces cerevisiae, filamentous fungi, for example, the genus Aspergillus, for example, Aspergillus niger are known, but are not limited thereto.

[0140] In the present disclosure, the modified L31 protein, or the ribosome containing the same, can also be obtained by purifying from a culture of wild-type Escherichia coli. That is, by suspending the cultured wild-type Escherichia coli in a buffer containing a low concentration of magnesium ions and disrupting it, a lysate containing the modified L31 protein can be obtained. Specifically, by disrupting wild-type Escherichia coli in a buffer containing magnesium ions of, for example, 5 mM or less (more specifically, for example, 4 mM or less, 3 mM or less, 2 mM or less, 1 mM or less, or 0 mM), a lysate containing the wild-type L31 protein of Escherichia coli described in SEQ ID NO: 1 in the present specification and the modified L31 protein in the present disclosure can be obtained. Disruption of Escherichia coli cells can be performed using methods known to those skilled in the art, such as disruption by a French press, disruption by ultrasonic treatment, disruption by a homogenizer, disruption by glass beads, disruption by a mortar, etc., but in the present disclosure, disruption by a French press is preferred. When the lysate thus prepared is purified by a method known to those skilled in the art, the modified L31 protein, or the ribosome containing the same, can be isolated and obtained.

[0141] In another aspect of the present invention, there is provided a composition comprising ribosomes containing the modified L31 protein disclosed herein. The composition disclosed herein can be used, for example, as a translation system or a part thereof in the production of peptides containing non-natural amino acids. Thus, the composition disclosed herein preferably further comprises, in addition to ribosomes containing the modified L31 protein disclosed herein, for example, aminoacyl-tRNA formed by binding of a non-natural amino acid and tRNA, mRNA encoding a peptide containing one or more non-natural amino acids, and the like. The composition disclosed herein preferably contains ribosomes containing the modified L31 protein disclosed herein at a ratio of at least 50% or more, preferably 60% or more, more preferably 70% or more, still more preferably 80% or more, particularly preferably 90% or more (for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more) in terms of the molecular number ratio with respect to all ribosomes contained in the composition.

[0142] All prior art documents cited herein are incorporated herein by reference.

Examples

[0143] The present invention will be described in more detail with reference to the following examples, but the content of the present invention is not necessarily limited only to the following examples. The following abbreviations were used in the examples. AA Ammonium acetate CH2CN Cyanomethyl group CH3CN Acetonitrile CTACl N,N,N-Trimethylhexadecane-1-aminium chloride DBU 1,8-Diazabicyclo[5.4.0]-7-undecene DCM Dichloromethane DIPEA N,N-Diisopropylethylamine DMF Dimethylformamide DMSO Dimethyl sulfoxide FA Formic acid Fmoc 9-Fluorenylmethyloxycarbonyl group F-Pnaz 4-(2-(4-Fluorophenyl)acetamido)benzyloxycarbonyl group:

Chem.

[0144] MePhe may be abbreviated as MeF, Pic(2) as Pic2, Gly as G, Ile as I, Pro as P, Thr as T, MeSer(tBuOH) as MeStBuOH, D-Ala as dA, Leu as L, BODIPYFL-4-AMF as Bdp4AMF, Acbz-D-MeCys(StBu) as AcbzdMeCStBu, Acbz-MeCys(StBu) as AcbzMeCStBu, and Acbz-Cys(StBu) as AcbzCStBu.

[0145] Example 1. Construction of Escherichia coli strain A defective strain of ompT (defined as the L31intact strain) and an L31short strain expressing up to the N-terminal 62 amino acids of the L31 protein were constructed. During the purification process of ribosomes, Protease7 is known to cleave between the 62nd and 63rd amino acids of the L31 protein. Escherichia coli strains were constructed according to the kit of Quick and Easy Conditional Knockout Kit(loxP / Cre) (Gene Bridges).

[0146] Creation of functional cassette with added homology arm Using the functional cassette loxP-PGK-gb2-neo-loxP (Gene Bridges, A003) as a template, a PCR reaction was performed with PrimeSTAR HS DNA Polymerase (Takara Bio, R010A). The obtained PCR product was purified using the QIAquick PCR Purification Kit (QIAGEN, 28104). The concentration of the functional cassette was about 200 ng / μL. For the functional cassette Cassette-1 for creating the ompT (Protease7) - deficient strain, PCR was performed using the primers Oligo1 (SEQ ID NO: 3) and Oligo2 (SEQ ID NO: 4). For the functional cassette Cassette-2 for creating the L31short strain, PCR was performed using the primer set of Oligo3 (SEQ ID NO: 5) and Oligo4 (SEQ ID NO: 6).

[0147] Creation of Escherichia coli strain It was carried out according to the protocol described in the Quick and Easy Conditional Knockout Kit (loxP / Cre) (Gene Bridges). Using Escherichia coli K-12 W3110 strain, pRed / ET was transformed to prepare competent cells expressing Red / ET. The functional cassette Casette-1 or Casette-2 was introduced into the prepared competent cells by electroporation to induce homologous recombination. Colony PCR was performed on the obtained colonies, and the introduction of the kanamycin cassette and the deletion or partial deletion of the target gene were confirmed from the length of the amplified DNA. According to the protocol described in the kit, the kanamycin resistance gene cassette flanked by Loxp sequences was deleted from the obtained Escherichia coli strain of interest. The obtained Escherichia coli was single - colonized, and the completion of the target strain was confirmed by reading the sequence of the genomic DNA at the gene recombination site.

[0148] Example 2. Preparation method of ribosome Culture of Escherichia coli The W3110 strain (WT) was cultured. The cells of the W3110 strain were inoculated into the pre-culture medium (Glycerol 5 g / L, Yeast Extract 6 g / L, KH2PO 4, 4 g / L, K2HPO4 9.3 g / L) and pre-cultured. The pre-cultured bacteria were adjusted to OD 600 = 0.1 and added to 30 L of the main-culture medium (Glycerol 10 g / L, Yeast Extract 10 g / L, Polypeptone N 15 g / L, KH2PO4 4 g / L, MgSO4·7H2O 2.4 g / L, FeSO4·7H2O 0.04 g / L, CaCl2·2H2O 0.04 g / L, Adekanol LG-109 0.24 g / L). The culture was carried out in a 50 L culture tank. The cells were cultured at 37°C for 5.4 hours and harvested when OD 600 reached 33.5. The culture broth was dispensed into 500 ml aliquots and allowed to stand at room temperature for 1 hour and then at 4°C for 1 hour. After standing, the cells were centrifuged at 6000×g for 10 min, and the precipitate was suspended in D-PBS(-) (Takara Bio Inc., T9181) and centrifuged again at 6000×g for 10 min. The harvested cells were frozen in liquid nitrogen and stored at -80°C.

[0149] Culture of L31short strain or L31intact strain LB medium, Miller (Nacalai Tesque Inc., 20068-75) was used for the culture. The cells were pre-cultured. The pre-cultured cells were added to the main-culture medium so that OD600 = 0.05. The culture was carried out in a 3 L baffled flask (Corning Inc., 431253) with 1 L of LB medium added. Using a Climo-shaker ISF-1-X, the cells were cultured at 37°C and 100 rpm for about 2 hours and 40 minutes until OD600 reached about 1.0. Then the cells were taken out and allowed to stand at room temperature for 1 hour and then at 4°C for 1 hour. The cells were harvested by centrifugation at 5000×g for 10 min. The cells were suspended in PBS (Takara Bio Inc., T9181) equal to the volume of the medium and centrifuged again at 5000×g for 10 min. The cells were frozen in liquid nitrogen and stored at -80°C.

[0150] Disruption of Escherichia coli Disruption using FP in L31intact strain and L31short strain The recovered cells were suspended at 0.004 mL of Lysis Buffer (10 mM HEPES-KOH, pH 7.6, 5 - 10 mM MgCl2, 50 mM KCl, 1 mM DTT, 10 μg / mL DNaseI) per 1 OD of the cells. The cells were disrupted using a French Press (Emulsi Flex B15, AVESTIN). The pressure was set at 40 Bar. The disruption rate was controlled to be approximately 1 ml / min. Centrifugation was performed at 20,000×g for 30 minutes at 4°C, and the supernatant was collected. Disruption using FP in WT strain Three types of Lysis Buffer with the following compositions were prepared. Mg10 Lysis Buffer (10 mM HEPES-KOH, pH 7.6, 50 mM KCl, 10 mM MgCl2, 1 mM DTT, 10 μg / mL DNaseI) Mg5 Lysis Buffer (10 mM HEPES-KOH, pH 7.6, 50 mM KCl, 5 mM MgCl2, 1 mM DTT, 10 μg / mL DNaseI) Mg0 Lysis Buffer (10 mM HEPES-KOH, pH 7.6, 50 mM KCl, 1 mM DTT, 10 μg / mL DNaseI) Using each type of Lysis Buffer, the cells were suspended at 0.004 mL of Lysis Buffer per 1 OD of the recovered cells. The cells were disrupted using a French Press (Emulsi Flex B15, AVESTIN). The pressure was set at 40 Bar. The disruption rate was controlled to be approximately 1 ml / min. Centrifugation was performed at 20,000×g for 30 minutes at 4°C, and the supernatant was collected.

[0151] Purification of Escherichia coli Ammonium sulfate precipitation of L31short strain or L31intact strain To the supernatant of the E. coli lysate, an equal volume of 2× Buffer for ammonium sulfate precipitation (10 mM HEPES-KOH, pH 7.6, 5 - 10 mM MgCl2, 50 mM KCl, 3.24 M ammonium sulfate, 1 mM DTT) was added, and the mixture was stirred at 4°C for 30 minutes. Centrifugation was performed at 4°C, 20000×g for 40 minutes, and the supernatant was collected. It was filtered through a 0.22 μm (Millipore) filter.

[0152] Ammonium sulfate precipitation of WT strain To 1 ml of the supernatant of the E. coli lysate, 0.222 g of ammonium sulfate finely ground in a mortar was added while stirring using a stirrer. The mixture was stirred at 4°C for 30 minutes. Centrifugation was performed at 4°C, 20380×g for 40 minutes, and the supernatant was collected. It was filtered through a 0.22 μm (Millipore) filter.

[0153] Butyl sepharose purification A column filled with Butyl Sepharose 4 FastFlow (GE Healthcare, 17098002) in an XK50 column (GE Healthcare, 28988952) was used. The column was equilibrated with Buffer A (20 mM HEPES-KOH, pH 7.6, 10 mM Mg(OAc)2, 1.5 M (NH4)2SO4, 1 mM DTT). The sample after ammonium sulfate precipitation was added to the column at 2 ml / min. After washing the column with Buffer A, it was further washed successively with 30% Buffer A and 70% Buffer B (20 mM HEPES-KOH, pH 7.6, 10 mM Mg(OAc)2, 1 mM DTT). Then, it was eluted with 50% Buffer A and 50% Buffer B.

[0154] Ultracentrifugation purification Add half of the volume of the ultracentrifugation tube with 30% sucrose Buffer (20 mM HEPES-KOH, pH 7.6, 10 mM Mg(OAc)2, 30 mM NH4Cl, 30% Sucrose, 1 mM DTT), and then add Butyl SepharoseThe purified eluate was added. Centrifugation was performed at 99700×g for 20 hours at 4°C. The pellet was suspended in storage Buffer (20 mM HEPES-KOH, pH 7.6, 6 mM Mg(OAc)2, 30 mM KCl, 1 mM DTT). It was prepared to a final concentration of 20 - 30 μM. Finally, five types of ribosomes, namely L31short ribosome, L31intact ribosome, WT-10MG ribosome (using Mg10 Lysis Buffer), WT-5MG ribosome (using Mg5 Lysis Buffer), and WT-0MG ribosome (using Mg0 Lysis Buffer) were prepared.

[0155] Example 3. Analysis of degradation rate of L31 The degradation rates of L31 in WT-10MG ribosome, WT-5MG ribosome, and WT-0MG ribosome were analyzed.

[0156] Sample preparation method 40 pmol of ribosome was diluted with 38 μL of water. Trifluoroacetic acid was added to a concentration of 1% to precipitate ribosomal RNA. Centrifugation was carried out, and the supernatant was mixed with matrix (50% acetonitrile, 5 mg / ml sinapic acid) at a ratio of 1:1, and 1 μL was spotted onto a plate for MALDI / MS and crystallized.

[0157] Analysis by MALDI / MS Using a mass spectrometer (ABS CIEX·TOF / TOF 5800), measurements were performed in the Liner Positive mode. Calibration was carried out using ribosomal protein as an internal standard. The measurement results with correct calibration were used. The ratio of intact L31 was determined by dividing the MS intensity of Intact L31 by the sum of the MS intensities of Intact L31 and Short L31. Measurements were carried out with N = 3, and the average value was taken as the ratio of intact L31.

[0158] Results of analysis of L31 degradation rate In the WT-10MG ribosome, 71% was intact L31. Similarly, in the WT-5MG ribosome, 20% was intact L31, and in the WT-0MG ribosome, 7% was intact L31.

[0159] Example 4. Synthesis of pCpA-amino acid (also called aminoacyl pCpA) used in cell-free translation system The analysis conditions of LCMS are as follows.

Table 1

[0160] Synthesis of Pnaz-MeSer(tBuOH)-pCpA (TS01) The synthesis of compound TS01 was carried out according to the following scheme.

Chemical formula

[0161] Synthesis of N-(((4-(2-(4-fluorophenyl)acetamido)benzyl)oxy)carbonyl)-O-(2-hydroxy-2-methylpropyl)-N-methyl-L-serine (TS01-3)

Chemical formula

[0162] (2S)-2-[[(9H-Fluoren-9-ylmethoxy)carbonyl](methyl)amino]-3-(2-hydroxy-2-methylpropoxy)propanoic acid (TS01-1, 41.0 mg, 0.10 mmol) synthesized based on WO2018225864 was dissolved in DCM (0.10 mL), 4-(3-phenylpropyl)piperidine (32.0 uL, 0.15 mmol) was added, and the mixture was stirred at room temperature for 15 hours. DCM was removed by concentration under reduced pressure to obtain (2S)-3-(2-hydroxy-2-methyl-propoxy)-2-(methylamino)propanoic acid (TS01-2) as a crude product. The obtained (2S)-3-(2-hydroxy-2-methyl-propoxy)-2-(methylamino)propanoic acid (TS01-2, crude product) and (4-nitrophenyl) 4-(2-(4-fluorophenyl)acetamido)benzyl carbonate synthesized based on WO2018225864 (110.0 mg, 0.26 mmol) were dissolved in DMSO (0.50 mL), triethylamine (41.8 uL, 0.30 mmol) was added, and the mixture was stirred at 50 °C for 1 hour. The reaction solution was purified by reverse-phase column chromatography (0.1% FA in H2O / 0.1% FA in CH3CN) to obtain the title compound (TS01-3, 40 mg, 84%). LCMS(ESI) m / z = 475.4 (M-H)- Retention time: 0.64 minutes (analysis condition SQDFA05)

[0163] Synthesis of cyanomethyl N-(((4-(2-(4-fluorophenyl)acetamido)benzyl)oxy)carbonyl)-O-(2-hydroxy-2-methylpropyl)-N-methyl-L-serinate (TS01-4) [Chemical formula]

[0164] N-(((4-(2-(4-Fluorophenyl)acetamido)benzyl)oxy)carbonyl)-O-(2-hydroxy-2-methylpropyl)-N-methyl-L-serine (TS01-3, 19.0 mg, 0.04 mmol) was dissolved in CH3CN (0.20 mL), and DIPEA (10.48 uL, 0.06 mmol) and 2-bromoacetonitrile (3.22 uL, 0.048 mmol) were added, followed by stirring at room temperature for 12 h. The reaction mixture was concentrated under reduced pressure to obtain the title compound (TS01-4) as a crude product. LCMS(ESI) 514(M-H)- Retention time: 0.73 min (analysis condition SQDFA05)

[0165] (2R,3S,4R,5R)-2-((((((2R,3S,4R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4-hydroxy-2-((phosphonooxy)methyl)tetrahydrofuran-3-yl)oxy)(hydroxy)phosphoryl)oxy)methyl)-5-(6-amino-9H-purin-9-yl)-4-hydroxy-tetrahydrofuran-3-yl N-(((4-(2-(4-fluorophenyl)acetamido)benzyl)oxy)carbonyl)-O-(2-hydroxy-2-methylpropyl)-N-methyl-L-serinate (TS01) synthesis

Chemical formula

[0166] To Buffer A (10 mL), dihydrogen phosphate ((2R,3R,4R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-3-(((((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-4-((tetrahydrofuran-2-yl)oxy)tetrahydrofuran-2-yl)methyl (28.9 mg, 0.04 mmol) synthesized by the method described in the literature (Helv. Chim. Acta, 90, 297 - 310) was dissolved, and an acetonitrile solution (0.021 mg, 0.04 mmol, 0.50 mL) of cyanomethyl N-(((4-(2-(4-fluorophenyl)acetamido)benzyl)oxy)carbonyl)-O-(2-hydroxy-2-methylpropyl)-N-methyl-L-serinate (TS01-4, crude product) was added dropwise, followed by stirring at room temperature for 60 minutes. After cooling the reaction solution to 0 °C, trifluoroacetic acid (0.50 mL) was added. After stirring the reaction solution at 0 °C for 60 minutes, the reaction solution was purified by reverse-phase silica gel column chromatography (0.05% aqueous trifluoroacetic acid / 0.05% trifluoroacetic acid acetonitrile) to obtain the title compound (TS01, 8.0 mg, 18.0%). LCMS(ESI) m / z = 1109.7 (M-H)- Retention time: 0.50 minutes (analysis condition SQDFA05)

[0167] Buffer A was adjusted as follows. Acetic acid was added to an aqueous solution of N,N,N-trimethylhexadecane-1-aminium chloride (6.40 g, 20 mmol) and imidazole (6.81 g, 100 mmol) to obtain Buffer A (1 L) with pH 7.9, 20 mM N,N,N-trimethylhexadecane-1-aminium, and 100 mM imidazole.

[0168] Synthesis of BdpFL-(4-AMF)-pCpA(MT01) (2S)-3-[4-(Aminomethyl)phenyl]-2-[[4-[[2-(4-Fluorophenyl)acetyl]amino]phenyl]methoxycarbonylamino]propanoic acid (Compound MT02) Synthesis

Chemical Structure

[0169] Under nitrogen atmosphere, a solution of 4N HCl in 1,4-dioxane was added to a suspension of (2S)-3-[4-(aminomethyl)phenyl]-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonylamino]propanoic acid (500 mg, 0.968 mmol) in dichloromethane (4.5 ml) at room temperature. After stirring at room temperature for 1 hour, the solvent was distilled off under reduced pressure. To the resulting residue and a suspension of [4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methyl (4-nitrophenyl) carbonate (411 mg, 0.968 mmol) in DMSO (5 mL), DIPEA (413 mg, 3.19 mmol) was added at room temperature. After stirring at room temperature for 2 hours, piperidine (400 mg, 4.7 mmol) was added at room temperature and stirred for 15 minutes. The reaction solution was purified by reverse-phase column chromatography (0.1% FA CH3CN / H2O) to obtain (2S)-3-[4-(aminomethyl)phenyl]-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonylamino]propanoic acid (Compound MT02) (83.3 mg, 18% yield, 3 steps). LCMS(ESI) m / z = 480.3 (M+H)+ Retention time: 0.46 minutes (analysis condition SQDFA05)

[0170] (S)-3-(4-((3-(5,5-Difluoro-7,9-dimethyl-5H-4λ 4 ,5λ 4 -Dipyrrolo[1,2-c:2’,1’-f][1,3,2]diazaborinin-3-yl)propanamide)methyl)phenyl)-2-((((4-(2-(4-Fluorophenyl)acetamido)benzyl)oxy)carbonyl)amino)propanoic acid (Compound MT03) Synthesis

Chemical Structure

[0171] Nitrogen atmosphere, a solution of (2S)-3-[4-(aminomethyl)phenyl]-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonylamino]propanoic acid (Compound MT02) (11.8 mg, 0.030 mmol) in NMP (500 ml) and a solution of 3-(3-((2,5-dioxopyrrolidin-1-yl)oxy)-3-oxopropyl)-5,5-difluoro-7,9-dimethyl-5H-5λ 4 -dipyrrolo[1,2-c:2’,1’-f][1,3,2]diazaborinin-4-ium in NMP (500 ml) were added at room temperature. After stirring at 40 °C for 10 minutes, the reaction solution was purified by reverse-phase column chromatography (0.1% FA CH3CN / H2O) to obtain (S)-3-(4-((3-(5,5-difluoro-7,9-dimethyl-5H-4λ 4 ,5λ 4 -dipyrrolo[1,2-c:2’,1’-f][1,3,2]diazaborinin-3-yl)propanamide)methyl)phenyl)-2-((((4-(2-(4-fluorophenyl)acetamido)benzyl)oxy)carbonyl)amino)propanoic acid (Compound MT03) (14.6 mg, 64% yield). LCMS(ESI) m / z = 752.2(M-H)- Retention time: 0.79 minutes (analysis conditions SQDFA05)

[0172] (S)-3-(4-((3-(5,5-Difluoro-7,9-dimethyl-5H-4λ 4 ,5λ 4 -Dipyrrolo[1,2-c:2’,1’-f][1,3,2]diazaborinin-3-yl)propanamide)methyl)phenyl)-2-((((4-(2-(4-Fluorophenyl)acetamido)benzyl)oxy)carbonyl)amino)propanoic acid cyanomethyl ester (Compound MT04) Synthesis [Chemical formula]

[0173] Under a nitrogen atmosphere, (S)-3-(4-((3-(5,5-difluoro-7,9-dimethyl-5H-4λ 4 ,5λ 4(S)-3-(4-((3-(5,5-difluoro-7,9-dimethyl-5H-[1,4,5]triazolo[1,5-a]pyrimidin-2-yl)propanamido)methyl)phenyl)-2-(((4-(2-(4-fluorophenyl)acetamido)benzyl)oxy)carbonyl)amino)propanoic acid (50 mg, 0.066 mmol) and N-ethyl-isopropylpropan-2-amine (DIPEA) (23.2 μL, 0.133 mmol) were dissolved in acetonitrile (200 μL). 2-Bromoacetonitrile (9.0 μL, 0.133 mmol) was added at 0 °C, and the mixture was stirred at 40 °C for 3.5 h. The reaction mixture was concentrated to give (S)-3-(4-((3-(5,5-difluoro-7,9-dimethyl-5H-4λ 4 ,5λ 4 -dipyrrolo[1,2-c:2’,1’-f][1,3,2]diazaborinin-3-yl)propanamido)methyl)phenyl)-2-(((4-(2-(4-fluorophenyl)acetamido)benzyl)oxy)carbonyl)amino)propanoic acid cyanomethyl ester (Compound MT04) as a crude product. The obtained crude product was used in the next step as it was. LCMS(ESI) 791.4(M-H)- Retention time: 0.90 min (analysis condition SQDFA05)

[0174] [(2R,3S,4R,5R)-2-((((((2R,3S,4R,5R)-5-(4-Amino-2-oxopyrimidin-1(2H)-yl)-4-hydroxy-2-((phosphonooxy)methyl)tetrahydrofuran-3-yl)oxy)(hydroxy)phosphoryl)oxy)methyl)-5-(6-Amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-3-yl (2S)-3-(4-((3-(5,5-Difluoro-7,9-dimethyl-5H-4λ 4 ,5λ 4 -Dipyrrolo[1,2-c:2’,1’-f][1,3,2]diazaborinin-3-yl)propanamide)methyl)phenyl)-2-((((4-(2-(4-Fluorophenyl)acetamido)benzyl)oxy)carbonyl)amino)propanoate (Compound MT01, BdpFL-(4-AMF)-pCpA) Synthesis

Chemical Structure

[0175] Dissolve ((2R,3R,4R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-3-(((((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-4-((tetrahydrofuran-2-yl)oxy)tetrahydrofuran-2-yl)methyl (47.7 mg, 0.066 mmol) in buffer A (55 mL), and add an acetonitrile solution (5 mL) of (S)-3-(4-((3-(5,5-difluoro-7,9-dimethyl-5H-4λ 4 ,5λ 4 -dipyrrolo[1,2-c:2’,1’-f][1,3,2]diazaborinin-3-yl)propanamide)methyl)phenyl)-2-(((4-(2-(4-fluorophenyl)acetamido)benzyl)oxy)carbonyl)amino)propanoic acid cyanomethyl ester (compound MT03) (52.3 mg, 0.066 mmol) in three portions. After addition, stir at room temperature for 90 minutes. Add TFA (3 mL) to the reaction solution at 0 °C, stir for 5 minutes, and then stir at room temperature for 40 minutes. Purify the reaction solution by reverse-phase silica gel column chromatography (0.05% TFA CH3CN / H2O) to obtain the title compound (compound MT01, BdpFL-(4-AMF)-pCpA) (8.3 mg, 9.1% yield). LCMS (ESI) m / z = 1386.7 (M-H)- Retention time: 0.65 minutes (analysis condition SQDFA05)

[0176] Example 5. Aminoacyl-tRNA Synthesis Sequence of tRNAGlu(-CA) The following tRNAGluCUU(-CA) was prepared by a conventional method. Sequence TR-1 (SEQ ID NO: 7) tRNAGluCUU(-CA) RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCCUCUUACGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGC The following tRNAGluCUG(-CA) was prepared by a conventional method. Sequence TR-2 (SEQ ID NO: 8) tRNAGluCUG(-CA) RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCCUCUGACGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGC The following tRNAfMet(CAU)(-CA) was prepared by a conventional method. Sequence TR-3 (SEQ ID NO: 9) GGCGGGGUGGAGCAGCCUGGUAGCUCGUCGGGCUCAUAACCCGAAGAUCGUCGGUUCAAAUCCGGCCCCCGCAAC

[0177] Aminoacyl-tRNA Synthesis Using Aminoacyl pCpA: Part 1 To 50 μM transcribed tRNAGluCUG(-CA) (SEQ ID NO: 8) (20 μl), 10X ligation buffer (500 mM HEPES-KOH pH 7.5, 200 mM MgCl2) (4 μl), 10 mM ATP (4 μl), and nuclease-free water (5.6 μl) were added. After heating at 95°C for 2 minutes, it was left at room temperature for 5 minutes to perform refolding of the tRNA. 10 unit / μl T4 RNA ligase (New england bio lab.) (2.4 μL) and a DMSO solution of 2.5 mM aminoacyl pCpA (TSO1) (4 μL) were added, and a ligation reaction was carried out at 16°C for 45 minutes. Sodium acetate was added to the ligation reaction solution to a final concentration of 0.3 M, followed by phenol-chloroform extraction, and aminoacyl tRNA (compound AAtR-1) was recovered by ethanol precipitation. The recovered aminoacyl tRNA (compound AAtR-1) was dissolved in 1 mM sodium acetate immediately before adding it to the translation mixture. Compound AAtR-1 MeSer(tBuOH)- tRNAGluCUG

[0178] Aminoacyl-tRNA Synthesis Using Aminoacyl pCpA: Part 2 To 50 μM transcribed tRNAGluCUU(-CA) (SEQ ID NO: 7) (20 μl), 10X ligation buffer (500 mM HEPES-KOH pH 7.5, 200 mM MgCl2) (4 μl), 10 mM ATP (4 μl), and nuclease-free water (5.6 μl) were added. After heating at 95°C for 2 minutes, it was left at room temperature for 5 minutes to perform refolding of tRNA. 10 unit / μl T4 RNA ligase (New england bio lab.) (2.4 μL) and a DMSO solution of 2.5 mM aminoacyl pCpA (MT01) (4 μL) were added, and a ligation reaction was carried out at 16°C for 45 minutes. Sodium acetate was added to the ligation reaction solution to a final concentration of 0.3 M, followed by phenol-chloroform extraction, and aminoacyl tRNA (Compound AAtR-2) was recovered by ethanol precipitation. The recovered aminoacyl tRNA (Compound AAtR-2) was dissolved in 1 mM sodium acetate immediately before adding to the translation mixture. Compound AAtR-2 BODIPYFL-4-AMF-tRNAGluCUU

[0179] Aminoacyl-tRNA Synthesis Using Aminoacyl pCpA: Part 3 To 50 μM of transcribed tRNAfMetCAU(-CA) (SEQ ID NO: 9) (20 μl), 10X ligation buffer (500 mM HEPES-KOH pH 7.5, 200 mM MgCl2) (4 μl), 10 mM ATP (4 μl), and nuclease-free water (5.6 μl) were added. After heating at 95°C for 2 minutes, it was left at room temperature for 5 minutes to perform refolding of tRNA. 10 unit / μl of T4 RNA ligase (New england bio lab.) (2.4 μL) and a DMSO solution of 2.5 mM of (Acbz-MeCys(StBu)-pCpA (compound nk14 described in Patent Document WO2017150732) (4 μL) were added, and a ligation reaction was carried out at 16°C for 45 minutes. 0.3 M sodium acetate was added to the ligation reaction solution, followed by phenol-chloroform extraction, and aminoacyl tRNA (compound AAtR-3) was recovered by ethanol precipitation. The recovered aminoacyl tRNA (compound AAtR-3) was dissolved in 1 mM sodium acetate immediately before adding to the translation mixture.) Compound AAtR-3 Acbz-MeCys(StBu)- tRNAfMetCAU

[0180] Example 6. Synthesis of LCT12 Synthesis of (2S,3R)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-((tetrahydro-2H-pyran-2-yl)oxy)butanoic acid (Fmoc-Thr(THP)-OH) for peptide synthesis using the LCT-12 peptide synthesizer [Chemical formula]

[0181] (2S,3R)-2-(((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-hydroxybutanoic acid monohydrate (monohydrate of Fmoc-Thr-OH, purchased from Tokyo Chemical Industry, 5.0 g, 13.9 mmol) and pyridinium p-toluenesulfonate (PPTS, 0.175 g, 0.70 mmol) were added to toluene (50 mL), and the toluene was distilled off under reduced pressure to remove the water contained by azeotropy. Ultra-dehydrated tetrahydrofuran (THF, 28 mL) and 3,4-dihydro-2H-pyran (8.8 mL, 97 mmol) were added to the obtained residue, and the mixture was stirred at 50 °C for 4 hours under a nitrogen atmosphere. After confirming the disappearance of the raw material by LCMS (SQDFA05), the mixture was cooled to 25 °C, and ethyl acetate (30 mL) was added. Subsequently, a saturated aqueous sodium chloride solution (30 mL) was added to wash the organic layer, and the aqueous layer was extracted with ethyl acetate (30 mL). All the obtained organic layers were mixed and further washed twice with a saturated aqueous sodium chloride solution (30 mL). The organic layer was dried over sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude product (9.3 g).

[0182] Of the obtained crude product, 4.65 g was dissolved in tetrahydrofuran (THF, 30 mL), and then a 1.0 M phosphate buffer solution adjusted to pH 8.0 (30 mL) was added. This mixture was stirred at 50 °C for 4 hours. After cooling to 25 °C, ethyl acetate (30 mL) was added, and the organic layer and the aqueous layer were separated. After adding ethyl acetate (30 mL) to the aqueous layer for extraction, all the obtained organic layers were mixed and washed twice with a saturated aqueous sodium chloride solution (30 mL). The organic layer was dried over sodium sulfate, the solvent was distilled off under reduced pressure, and further dried under reduced pressure with a pump at 25 °C for 30 minutes.

[0183] The obtained residue was dissolved in diethyl ether (50 mL), and then heptane (50 mL) was added. Under controlled reduced pressure (~100 hPa), only diethyl ether was distilled off, and the obtained mixture was filtered to obtain a solid. This washing operation with heptane was repeated twice. The obtained solid was dried under reduced pressure with a pump at 25 °C for 2 hours to obtain the sodium salt of Fmoc-Thr(THP)-OH (2.80 g, 6.26 mmol).

[0184] To the sodium salt of the total amount of Fmoc-Thr(THP)-OH obtained, ethyl acetate (50 mL) and 0.05 M aqueous phosphoric acid solution at pH 2.1 (140 mL) were added, and after stirring at 25 °C for 5 minutes, the organic layer and the aqueous layer were separated. After adding ethyl acetate (50 mL) to the aqueous layer for extraction, all the obtained organic layers were mixed and washed twice with a saturated aqueous sodium chloride solution (50 mL). The organic layer was dried over sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was dried under reduced pressure with a pump at 25 °C for 2 hours, and then the obtained solid was dissolved in t-butyl methyl ether (TBME, 50 mL), and the solvent was distilled off under reduced pressure. Further drying under reduced pressure with a pump at 25 °C for 1 hour gave (2S,3R)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-((tetrahydro-2H-pyran-2-yl)oxy)butanoic acid (Fmoc-Thr(THP)-OH, 2.70 g, with 30 mol% of t-butyl methyl ether (TBME) remaining) as a diastereomer derived from the asymmetric carbon on the THP protection. The obtained Fmoc-Thr(THP)-OH was stored in a freezer at -25 °C. LCMS(ESI) m / z = 424.2 (M-H)- Retention time: 0.84 min, 0.85 min (analysis condition SQDFA05_01)

[0185] Synthesis of peptide (LCT-12) with BdpFL at the N-terminus for use as an LC / MS standard

Chemical Structure

[0186] Using 2-chlorotrityl resin (100 mg) loaded with Fmoc-Ala-OH, peptide elongation was carried out on a peptide synthesizer using Fmoc-Gly-OH, Fmoc-Thr(THP)-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, and Fmoc-Pro-OH as Fmoc amino acids. Peptide elongation was carried out according to the peptide synthesis method by the Fmoc method (WO2013100132B2). After peptide elongation, removal of the Fmoc group at the N-terminus was carried out on the peptide synthesizer, and then the resin was washed with DCM.

[0187] TFE / DCM (1:1, v / v, 2 mL) was added to the resin and shaken for 1 hour to cleave the peptide from the resin. After completion of the reaction, the solution in the tube was filtered through a synthesis column to remove the resin, and the resin was washed twice with TFE / DCM (1:1, v / v, 1 mL). All the extracts were mixed, DMF (2 mL) was added, and then concentrated under reduced pressure. The resulting residue was dissolved in NMP (0.5 mL), and 1 / 4 (125 μL) of it was used for the next reaction. To the NMP solution of the peptide, BdpFL succinimide ester prepared to 76.5 mM (140 μL) was added at room temperature, stirred at 40 °C overnight, and then concentrated under reduced pressure. The resulting residue was dissolved in 0.05 M tetrabutylammonium hydrogensulfate in HFIP (1.2 ml, 0.060 mmol) and stirred at room temperature for 2 hours. The reaction solution was purified by reverse-phase silica gel column chromatography (0.1% FA MeCN / H2O) to obtain the title compound (LCT-12) (0.3 mg). The amino acid sequence of LCT-12 is shown in SEQ ID NO: 17. LCMS (ESI) m / z = 1972.9 (M-H)- Retention time: 0.74 minutes (analysis condition SQDFA05_01)

[0188] Example 7. In vitro translation of peptides Overview of Experiment 1 An experiment was conducted to compare the translation characteristics of two types of ribosomes, ribosomes prepared from the L31intact strain (L31intact ribosomes) and ribosomes prepared from the L31short strain (L31short ribosomes). Specifically, the template mRNA sequences mR-1 (SEQ ID NO: 10) or mR-2 (SEQ ID NO: 11) were translated using the compounds AAtR-1, AAtR-2, and Initiator-tRNA (AAtR-3) to translationally synthesize peptide compounds. As translation products, for mR-1, Acbz-MeCys(StBu):MeSer(tBuOH):MePhe:Ile:Ile:Gly:MePhe:BODIPYFL-4-AMF:Ile:Ile:Pro:Ile:Gly (SEQ ID NO: 14) and for mR-2, Acbz-MeCys(StBu):T:MePhe:Ile:Ile:Gly:MePhe:BODIPYFL-4-AMF:Ile:Ile:Pro:Ile:Gly (SEQ ID NO: 16) were designed to be translated. Hereinafter, amino acids are represented separated by colons. Quantification of the target product (TM) and by-products was performed using LC-MS. The main by-product observed was the Initiation Read through (iRT) peptide in which translation started from the third amino acid from the starting amino acid.

[0189] Translation conditions The translation system used the PURE system, a reconstituted cell-free protein synthesis system derived from prokaryotes. Specifically, the translation solution contained the following: 1 mM GTP, 1 mM ATP, 20 mM creatine phosphate, 50 mM HEPES-KOH pH 7.6, 100 mM potassium acetate, 2 mM spermidine, 1 mM dithiothreitol, 1.0 mg / ml tRNA from E. coli MRE600 (RNase-negative) (Roche) (a portion of the tRNA was removed by the method described in Non-Patent Document: Yokogawa T, Kitamura Y, Nakamura D, Ohno S, Nishikawa K. 2010. Nucleic acids research 38:e89), 3 μM in vitro transcribed E. coli tRNA Ala1B, 0.26 μM EF-G, 4 μg / ml creatine kinase, 3 μg / ml myokinase, 2 unit / ml inorganic pyrophosphatase, 1.1 μg / ml nucleoside diphosphate kinase, 2.7 μM IF1, 0.4 μM IF2, 1.5 μM IF3, 40 μM EF-Tu, 35 μM EF-Ts, 1 μM EF-P-Lys, 0.4 unit / μl RNasein Ribonuclease inhibitor (Promega, N2111), 0.4 - 0.5 μM Penicillin G Amidase (PGA), 2.7 μM AlaRS, 1 μM GlyRS, 0.4 μM IleRS, 0.5 μM mutant PheRS (WO2016 / 148044), 0.16 μM ProRS, 0.09 μM ThrRS, 1 μM mutant ValRS (WO2016 / 148044), 1 μM mutant SerRS (WO2016 / 148044), 250 μM Gly, 250 μM Lys, 100 μM Ile, 250 μM Pro, 250 μM Thr, 5 mM N-methylalanine, 5 mM N-methylphenylalanine, 5 mM N-methylserine, 5 mM N-methylvaline. The magnesium acetate concentration was adjusted at four points: 2 mM, 4 mM, 6 mM, and 8 mM. The initiator aminoacyl tRNA (compound AAtR-3) was added to the translation reaction mixture at 25 μM, and the aminoacyl tRNAs containing compounds AAtR-1 and AAtR-2 were each added at 10 μM.Also, mRNA (mR-1 or mR-2) was added to the translation reaction mixture to a concentration of 1 μM. The translation reaction mixture contained 185 μM of aminoacyl-tRNA that was not used for translation. L31short ribosome or L31incact ribosome was added to a concentration of 1.2 μM, and the mixture was allowed to stand at 37 °C for 1 hour. Then, the mixture was heated at 95 °C for 3 minutes and allowed to stand until it reached room temperature. Peptidyl-tRNA hydrolase (Pth) was added to a concentration of 8.58 μM, and the mixture was allowed to stand at 37 °C for 1 hour.

[0190] Preparation of mRNA Template mRNA, sequence mR-1 or sequence mR-2 was synthesized from template DNA (SEQ ID NO: 12 or SEQ ID NO: 13) by in vitro transcription reaction using RiboMAX LargeScale RNA production System T7 (Promega, P1280), and purified using RNeasy Mini kit (Qiagen).

[0191] Analysis overview A solution containing a peptide translation product containing unnatural amino acids was diluted 10-fold and analyzed using an LC-FLR-MS apparatus. The analysis data identified the retention time of the target translated peptide from the MS data, and the fluorescence peak at the corresponding retention time was quantified to evaluate the amount of peptide translation. For the quantitative evaluation, a calibration curve was prepared using LCT12 synthesized in Example 6 as a standard, and the content was calculated by relative quantification. LC-MS was performed under the following analysis conditions.

[0192] Analysis conditions

Table 2

[0193] Results In the translation experiments of the mRNA sequences of mR-1 and mR-2 at the optimal Mg concentration for each ribosome, the translation amount of the target product when translated by L31short ribosome was higher than that when translated using L31intact ribosome. For the translation of the mRNA sequences of mR-1 and mR-2 at the optimal Mg concentration for each ribosome, the ratio of the translation amount of the by-product iRT peptide to the target product was lower when using L31short ribosomes than when using L31intact ribosomes. The sequence of mR-1 with the second letter from the start amino acid being MeSer(tBuOH) had a greater improvement effect on the translation amount of the target product by L31short ribosomes at the optimal Mg 2+ concentration than the sequence of mR-2 with Thr. The usefulness of using L31short ribosomes prepared from the L31short strain during translation using the Initiation Suppression (iSup) method was shown.

[0194] The translation amounts of the target product and the iRT peptide when translating the mR-1 sequence and the mR-2 sequence using L31short ribosomes or L31intact ribosomes are shown below.

[0195]

Table 3-1

Table 3-2

Table 3-3

Table 3-4

[0196] Overview of Experiment 2 In Example 2, using wild-type Escherichia coli derived from the W3110 strain, the Mg in the disruption buffer 2+Ribosomes were prepared at three concentrations: 10 mM, 5 mM, and 0 mM. The ribosomes prepared by each preparation method were named WT-10MG ribosomes, WT-5MG ribosomes, and WT-0MG ribosomes. In Example 3, the ratio of degradation of the L31 protein in each ribosome during purification was measured using MALDI-MS. As a result, it was shown that the lower the Mg 2+ in the lysate, the more L31 was degraded. Using these ribosomes, a translation experiment was conducted using the sequence of mR-2 in the same manner as in Experiment 1. As a result, it was found that when using the WT-0MG ribosomes, in which L31 was most degraded, the amount of the target product was the largest.

[0197] Translation conditions The translation system used the PURE system, a reconstituted cell-free protein synthesis system derived from prokaryotes. Specifically, the translation solution contained the following: 1 mM GTP, 1 mM ATP, 20 mM creatine phosphate, 50 mM HEPES-KOH pH 7.6, 100 mM potassium acetate, 4 mM magnesium acetate, 2 mM spermidine, 1 mM dithiothreitol, 1.0 mg / ml tRNA from E. coli MRE600 (RNase negative) (Roche) (a portion of the tRNA was removed by the method described in the non-patent literature: Yokogawa T, Kitamura Y, Nakamura D, Ohno S, Nishikawa K. 2010. Nucleic acids research 38:e89), 3 μM in vitro transcribed E. coli tRNA Ala1B, 0.26 μM EF-G, 4 μg / ml creatine kinase, 3 μg / ml myokinase, 2 unit / ml inorganic pyrophosphatase, 1.1 μg / ml nucleoside diphosphate kinase, 2.7 μM IF1, 0.4 μM IF2, 1.5 μM IF3, 40 μM EF-Tu, 35 μM EF-Ts, 1 μM EF-P-Lys, 0.4 unit / μl RNasein Ribonuclease inhibitor (Promega, N2111), 0.5 μM Penicillin G Amidase (PGA), 2.7 μM AlaRS, 1 μM GlyRS, 0.4 μM IleRS, 0.5 μM mutant PheRS (WO2016 / 148044), 0.16 μM ProRS, 0.09 μM ThrRS, 1 μM mutant ValRS (WO2016 / 148044), 1 μM mutant SerRS (WO2016 / 148044). 250 μM Gly, 250 μM Lys, 100 μM Ile, 250 μM Pro, 250 μM Thr, 5 mM N-methylalanine, 5 mM N-methylphenylalanine, 5 mM N-methylserine, 5 mM N-methylvaline. 25 μM of the initiator aminoacyl tRNA (compound AAtR-3) and 10 μM each of the aminoacyl tRNAs containing compounds AAtR-1 and AAtR-2 were added to the translation reaction mixture. The translation reaction mixture contained 185 μM of the aminoacyl tRNAs not used for translation.WT-0MG ribosome, WT-5MG ribosome, or WT-10MG ribosome was added at 1.2 μM and allowed to stand at 37°C for 1 hour. Then, it was heated at 95°C for 3 minutes and allowed to stand until it reached room temperature. Peptidyl-tRNA hydrolase (Pth) was added to a concentration of 8.58 μM and allowed to stand at 37°C for 1 hour.

[0198] Analysis overview The solution containing the peptide translation product containing the unnatural amino acid was diluted 10-fold and analyzed using an LC-FLR-MS apparatus. The analysis data identified the retention time of the target translated peptide from the MS data and quantified the fluorescence peak at the corresponding retention time to evaluate the peptide translation amount. For the quantitative evaluation, a calibration curve was created using LCT12 synthesized in Example 6 as a standard, and the content was calculated by relative quantification. LC-MS was performed under the following analysis conditions. Analysis conditions

Table 4

[0199] Results In terms of the translation amount of the target product from high to low, they were WT-0MG ribosome, WT-5MG ribosome, and WT-10MG ribosome. Also, in ascending order of the ratio of the iRT peptide to the target product, they were WT-0MG ribosome, WT-5MG ribosome, and WT-10MG ribosome. From the results of Experiment 1 and Example 3, it was shown that when the Mg 2+ concentration was low, the ratio of ribosomes in which L31 was degraded increased, and the translation amount in the iSup method could be increased.

[0200]

Table 5

[0201] Example 8. Panning using L31short ribosomes Synthesis of acylated tRNA The acylated tRNA used for panning was prepared by the method described in Patent Document (WO2013 / 100132). Using 15 amino acids including Pic(2), MeAla(3-pyr), Ser(Ph-2-Cl), and MeGly described in Patent Document (WO2018 / 225864), an Elongator aminoacyl tRNA mixture was prepared. The final concentration of each acylated tRNA in the translation solution was 10 μM to 20 μM. For the Pnaz-protected pCpA amino acid, the operations after phenol extraction were carried out without deprotection. The Initiator aminoacyl tRNA was the same compound as compound AAtR-3 in Example 5 and was added to the translation solution at a final concentration of 25 μM for use.

[0202] Randomized double-stranded DNA library encoding a peptide compound library A DNA library was constructed by the method described in Patent Document (WO2013 / 100132). Twenty-four triplets including TTT, TTG, CTT, ATT, ATG, GTT, CCG, ACT, GCT, CAT, CAG, AAC, GAA, TGG, CGG, AGT, AGG, and GGT were prepared such that they randomly appeared 8 to 9 times in repetition.

[0203] Preparation of biotinylated target protein Glutathione S-transferase (GST) was used as the target protein for panning. The GST used was prepared by expression in Escherichia coli. Biotinylation was carried out according to Non-Patent Documents BMC biotechnology, 2008, 8, 41 and ProteinScience, 1990; 108(4): 673-6.

[0204] Translation solution used for panning The translation solution contains the following substances: 1 mM GTP, 1 mM ATP, 20 mM creatine phosphate, 50 mM HEPES-KOH pH 7.6, 100 mM potassium acetate, 6 mM magnesium acetate, 2 mM spermidine, 1 mM dithiothreitol, 1 mg / ml tRNA from E. coli MRE600 (RNase negative) (Roche) (a portion of tRNA has been removed by the method described in Non-Patent Document: Yokogawa T, Kitamura Y, Nakamura D, Ohno S, Nishikawa K. 2010. Nucleic acids research 38:e89), 4 μg / ml creatine kinase, 3 μg / ml myokinase, 2 unit / ml inorganic pyrophosphatase, 1.1 μg / ml nucleoside diphosphate kinase, 0.26 μM EF-G, 2.7 μM IF1, 0.4 μM IF2, 1.5 μM IF3, 40 μM EF-Tu, 49 μM EF-Ts, 1 μM EF-P-Lys, 1.2 μM ribosome (L31short or L31 intact), 2.73 μM AlaRS, 1 μM GlyRS, 0.4 μM IleRS, 0.5 μM mutant PheRS (WO2016 / 148044), 0.16 μM ProRS, 1 μM mutant SerRS (WO2016 / 148044), 0.09 μM ThrRS, 1 μM mutant ValRS (WO2016 / 148044), 0.11 μM LysRS, 3 μM in vitro transcribed E. coli tRNA Ala1B, 250 μM glycine, 100 μM isoleucine, 250 μM proline, 250 μM threonine, 250 μM lysine, 5 mM N-methylvaline, 5 mM N-methylserine, 5 mM N-methylalanine, 5 mM N-methylphenylalanine, Elongator aminoacyl tRNA mixture, 25 μM Initiator aminoacyl tRNA, 10 μM Penicillin G Amidase (PGA).

[0205] Performance of panning Using the translation solution containing the aforementioned double-stranded DNA library and L31short, or the translation solution containing L31intact, panning was carried out following the patent document (WO2013 / 100132). A TEV protease recognition sequence was introduced between GST and biotin, and elution was performed with TEV protease. After the peptide library was allowed to interact with the biotinylated protein, it was recovered with streptavidin-immobilized magnetic beads, washed, and then a TEV elution solution (50 mM Tris-HCl pH 8.0, 0.5 mM EDTA, 1 mM DTT, 0.1 U / μL AcTEV protease (Thermo Fisher Scientific, product number 12575015)) was added to the beads and reacted. After the reaction, the supernatant was recovered and PCR was performed.

[0206] Analysis of enriched sequences Sequences with an appearance frequency of rank A: 0.5% or more, rank B: 0.05% or more, or rank C: 50 or more NGS reads, and an increase in appearance frequency of 10 times or more when the target was added compared to when the pool of the previous round was divided and panned without adding the target were extracted after at least 1 round. As a result, in the case of using the translation system with L31short ribosome, there were 33 sequences in rank A, 253 sequences in rank B, and 396 sequences in rank C. On the other hand, in the case of using the translation system with L31intact ribosome, there were 32 sequences in rank A, 175 sequences in rank B, and 247 sequences in rank C. When panning was carried out using L31short ribosome, more types of sequences could be enriched compared to L31intact.

[0207] Example 9. Translation experiments using L31 mutant ribosomes Overview of Example 9 As described in Example 14 below, 8 types of L31 mutant strains with different lengths of L31 were constructed. As described in Example 15, ribosomes were prepared from these strains, the L31short strain, the L31intact strain, and the W3110 strain. Peptide translation synthesis was performed using the obtained 11 types of ribosomes, and the translation characteristics of ribosomes containing L31 mutants were examined. Specifically, using 11 types of ribosomes R1 to R11 shown in Table 6, the following three types of peptides with different second amino acids were translationally synthesized. In this specification, amino acids may be separated by colons to represent amino acid sequences as follows. AcbzMeCStBu:MeG:MeF:I:I:G:MeF:Bdp4AMF:I:I:P:I:G (SEQ ID NO: 29) AcbzMeCStBu:MeStBuOH:MeF:I:I:G:MeF:Bdp4AMF:I:I:P:I:G (SEQ ID NO: 30) AcbzMeCStBu:L:MeF:I:I:G:MeF:Bdp4AMF:I:I:P:I:G (SEQ ID NO: 31) Using the sequence mR-1 (SEQ ID NO: 10) of the template mRNA and MeG-tRNAGlu(CUG), MeSer(tBuOH)-tRNAGlu(CUG), and Leu-tRNAGlu(CUG) as aminoacyl-tRNAs that spin the second letter, respectively. The aminoacyl-tRNAs used were those prepared in Example 13.

[0208] [Table 6]

[0209] Quantification of the target product (TM) and by-products was performed using LC-MS. The main by-products observed were Initiation Read through (iRT) peptides translated from the third amino acid from the start amino acid. In this specification, peptides starting translation from the second letter may be referred to as 1iRT, and peptides starting translation from the third letter may be referred to as 2iRT. Table 7 to Table 9 show TM (SEQ ID NO: 29 to 31) and 1iRT and 2iRT for each of them.

[0210] [Table 7]

[0211] [Table 8]

[0212]

Table 9

[0213] Translation conditions From the translation solution composition of Experiment 1 in Example 7, except that 0.5 uM HisRS was added in addition to in vitro transcribed E. coli tRNA Ala1B, Lys, and aminoacyl tRNA, 6.72 unit / ml myokinase, 59 μM EF-Ts, 4 mM magnesium acetate, 20 μM initiator aminoacyl tRNA (compound AAtR-3), 10 μM aminoacyl tRNA encoding various second letters, 1.2 μM, various ribosomes, and 1 μM mRNA, the translation conditions of Experiment 1 in Example 7 were followed.

[0214] Analysis overview Analysis was performed according to the method of Experiment 2 in Example 7, except that a calibration curve was created and quantitative evaluation was performed using LCT67 synthesized in Example 11 as a standard.

[0215] Results As shown in Table 10, in any of the three sequences, translation using ribosomes of R1 to R7 and R11 showed the translation characteristics of a high translation amount of TM and a low ratio of Initiation Read through (iRT) peptides. Ribosomes containing L31 mutants with 62 or fewer amino acid residues from the N-terminus were shown to have the characteristics of high translation activity and a low ratio of Initiation Read through (iRT) peptides. The iRT ratio was calculated by the following formula.

[0216] (Equation 1) iRT ratio = (iRT total concentration [nM]) / (iRT total concentration [nM] + TM concentration [nM]) × 100

[0217]

Table 10

[0218] Example 10. Effect of L31short ribosomes in in vitro translation of various peptides Experiment 1: The starting amino acid is AcbzdMeCStBu, and various amino acids are placed at the second position Overview of Experiment 1 Using the L31short ribosome and L31intact ribosome purified in Example 2, the translational synthesis of six types of peptides with different second amino acids was carried out, and it was confirmed that the superior translational characteristics of the L31short ribosome were observed in the translational synthesis of multiple peptides. Specifically, six types of peptides with different second amino acids (X) in the following amino acid sequence (SEQ ID NO: 36) were translationally synthesized. Here, X was Nle, S3F5MePyr, SPh2Cl, I, T, or L. AcbzdMeCStBu:X:MeF:I:I:G:MeF:Bdp4AMF:I:I:P:I:G (SEQ ID NO: 36)

[0219] Using the template mRNA sequence mR-1 (SEQ ID NO: 10) and Nle-tRNAGlu(CUG), S3F5MePyr-tRNAGlu(CUG), SPh2Cl-tRNAGlu(CUG), Ile-tRNAGlu(CUG), Thr-tRNAGlu(CUG), Leu-tRNAGlu(CUG) as aminoacyl-tRNAs that base-pair with the second letter, respectively. The aminoacyl-tRNAs used were those prepared in Example 13.

[0220] Quantification of the target product (TM) and by-products was performed using LC-MS. The main by-products observed were 1iRT and 2iRT. The iRT ratio was calculated by the following formula.

[0221] (Equation 1) iRT ratio = (total iRT concentration [nM]) / (total iRT concentration [nM] + TM concentration [nM]) × 100

[0222] Translation conditions Under the translation conditions of Example 9, except for HisRS, 8 mM magnesium acetate, 3 ug / ml myokinase, Translation was carried out according to the translation conditions of Example 9, except that the composition was 49 μM EF-Ts and 20 μM initiator aminoacyl tRNA (Acbz-D-MeCys(StBu)-tRNAfMetCAU) (Patent Document WO2017150732).

[0223] Analysis overview Analysis was performed according to the method of Example 9.

[0224] Results As shown in Table 11, in 4 sequences out of 6 types, the translation amount of TM increased by 2.8 to 3.9 times when using L31short ribosome compared to when using L31intact ribosome. In all sequences out of 6 types, the ratio of iRT decreased when using L31short ribosome compared to when using L31intact ribosome.

[0225]

Table 11

[0226] Experiment 2: The starting amino acid is AcbzMeCStBu, and various amino acids are placed at the second position Overview of Experiment 2 Using the L31short ribosome and L31intact ribosome purified in Example 2, the translational synthesis of 12 peptides with different second amino acids was carried out, and it was confirmed that the superior translational characteristics of the L31short ribosome were observed in the translational synthesis of multiple peptides. Specifically, 12 peptides with different second amino acids (X) of the following amino acid sequence (SEQ ID NO: 37) were translationally synthesized. Here, X was Nle, MeG, MeF, S3F5MePyr, MeHph, MeA3Pyr, SPh2Cl, G, I, T, MeStBuOH, or L. AcbzMeCStBu:X:MeF:I:I:G:MeF:Bdp4AMF:I:I:P:I:G (SEQ ID NO: 37)

[0227] The experiment was conducted in the same manner as in Experiment 1 of Example 10, except that MeG-tRNAGlu(CUG), MePhe-tRNAGlu(CUG), MeHph-tRNAGlu(CUG), MeA3Pyr-tRNAGlu(CUG), Gly-tRNAGlu(CUG), and MeSer(tBuOH)-tRNAGlu(CUG) were additionally used as the aminoacyl-tRNA that spins the second letter. The main by-products observed were 1iRT and 2iRT.

[0228] Translation conditions The translation conditions of Experiment 1 of Example 10 were followed, except for 4 mM magnesium acetate and 20 μM initiator aminoacyl-tRNA (compound AAtR-3).

[0229] Analysis overview Analysis was performed according to the method of Example 9.

[0230] Results As shown in Table 12, in 11 sequences out of 12, the translation amount of TM increased by 1.6 to 3.7 times when using the L31short ribosome compared to when using the L31intact ribosome. In all sequences out of 12, the ratio of iRT decreased when using the L31short ribosome compared to when using the L31intact ribosome.

[0231]

Table 12

[0232] Experiment 3: The starting amino acid is AcbzCStBu, and various amino acids are placed at the second position Overview of Experiment 3 Using the L31short ribosomes and L31intact ribosomes purified in Example 2, the translational synthesis of 12 peptides with different second amino acids was performed, and it was confirmed that the superior translational characteristics of L31short ribosomes were observed in the translational synthesis of multiple peptides. Specifically, 12 peptides with different second amino acids (X) in the following amino acid sequence (SEQ ID NO: 38) were translationally synthesized. Here, X was Nle, MeG, MeF, S3F5MePyr, MeHph, MeA3Pyr, SPh2Cl, G, I, T, MeStBuOH, or L. The experiment was conducted by a method according to Experiment 2 of Example 10. AcbzCStBu:X:MeF:I:I:G:MeF:Bdp4AMF:I:I:P:I:G (SEQ ID NO: 38)

[0233] Quantification of the target product (TM) and by-products was performed using LC-MS. The main by-products observed were 1iRT and 2iRT.

[0234] Translation conditions The translation conditions of Example 9 were followed, except that it was 0.23 μM EF-G, 5.92 unit / ml myokinase 2, 1.0 μg / ml nucleoside diphosphate kinase, 2.4 μM IF1, 1.3 μM IF3, 35 μM EF-Tu, 52 μM EF-Ts, and 20 μM initiator aminoacyl tRNA Acbz-Cys(StBu)-tRNAfMetCAU (WO2017 / 150732).

[0235] Analysis overview Analysis was performed according to the method of Example 9.

[0236] Results As shown in Table 13, in all sequences out of 12 types, the translation amount of TM increased by 1.7 to 3.8 times when using L31short ribosome compared to the case of using L31intact ribosome. Among all sequences out of 12 types, the ratio of iRT decreased when using L31short ribosome compared to the case of using L31intact ribosome.

[0237]

Table 13

[0238] Experiment 4: The starting amino acid is fMet, and various amino acids are placed at the second position Overview of Experiment 4 Using the L31short ribosome and L31intact ribosome purified in Example 2, the translation synthesis of 13 types of peptides with different second amino acids was carried out, and it was confirmed that the superior translation characteristics of the L31short ribosome were observed in the translation synthesis of multiple peptides. Specifically, 13 types of peptides with different second amino acids (X) in the following amino acid sequence (SEQ ID NO: 39) were translationally synthesized. Here, X was Nle, MeG, MeF, S3F5MePyr, Pic(2), MeHph, MeA3Pyr, SPh2Cl, G, I, T, MeStBuOH, or L. fMet:X:MeF:I:I:G:MeF:Bdp4AMF:I:I:P:I:G (SEQ ID NO: 39)

[0239] The experiment was carried out in a method according to Experiment 2 of Example 10, except that Pic(2)-tRNAGlu(CUG) was additionally used as the aminoacyl tRNA spinning the second letter. The target product (TM) and by-products were quantified using LC-MS. The main by-products observed were 1iRT and 2iRT.

[0240] Translation conditions Except for the initiator aminoacyl tRNA (compound AAtR-3), the translation conditions of Experiment 2 in Example 10 were followed, except that 0.03 μM MetRS, 0.6 μM methionyl-tRNA formyltransferase, 0.25 mM methionine, and 0.1 mM folate were added.

[0241] Analysis overview Analysis was performed according to the method of Example 9.

[0242] Results As shown in Table 14, in 12 out of 13 sequences, the translation amount of TM increased by 1.2 to 2 times when using L31short ribosomes compared to when using L31intact ribosomes. In all sequences out of 13, the ratio of iRT decreased when using L31short ribosomes compared to when using L31intact ribosomes.

[0243]

Table 14

[0244] Experiment 5: The starting amino acid is AcbzMeCStBu, the second amino acid is Thr, and various amino acids are placed at the third position Overview of Experiment 5 Using the L31short ribosome and L31intact ribosome purified in Example 15, the translational synthesis of 14 peptides with different third amino acids was carried out, and it was confirmed that the superior translational characteristics of the L31short ribosome were observed in the translational synthesis of multiple peptides. Specifically, 14 peptides with different third amino acids (X) of the following amino acid sequence (SEQ ID NO: 40) were translationally synthesized. Here, X was Nle, MeF, S3F5MePyr, Pic(2), MeHph, MeA3Pyr, SPh2Cl, G, I, P, T, MeStBuOH, dA, or L. AcbzMeCStBu:T:X:I:I:G:MeF:Bdp4AMF:I:I:P:I:G (SEQ ID NO: 40)

[0245] Using the template mRNA sequence mR-3 (SEQ ID NO: 28) and aminoacyl-tRNAs that encode the third letter as Nle-tRNAGlu(CUG), MePhe-tRNAGlu(CUG), S3F5MePyr-tRNAGlu(CUG), Pic(2)-tRNAGlu(CUG), MeHph-tRNAGlu(CUG), MeA3Pyr-tRNAGlu(CUG), SPh2Cl-tRNAGlu(CUG), Gly-tRNAGlu(CUG), Ile-tRNAGlu(CUG), Pro-tRNAGlu(CUG), Thr-tRNAGlu(CUG), MeSer(tBuOH)-tRNAGlu(CUG), D-Ala-tRNAGlu(CUG), Leu-tRNAGlu(CUG), respectively. The aminoacyl-tRNAs used were those prepared in Example 13.

[0246] Quantification of the target product (TM) and by-products was performed using LC-MS. The main by-products observed were 1iRT and 2iRT.

[0247] Preparation of mRNA The template mRNA, sequence mR-3 (SEQ ID NO: 28), was prepared by in vitro transcription reaction using RiboMAX Large Scale RNA production System T7 (Promega, P1280) from the template DNA (SEQ ID NO: 27) and purified using the RNeasy Mini kit (Qiagen).

[0248] Translation conditions The translation conditions of Example 9 were followed, except that 10 μM of various aminoacyl-tRNAs encoding the third letter were added.

[0249] Analysis overview Analysis was performed according to the method of Example 9.

[0250] Results As shown in Table 15, in all sequences out of 14 types, the translation amount of TM increased by 1.1 to 20.9 times when using L31short ribosome compared with the case of using L31intact ribosome. In all sequences out of 14 types, the ratio of iRT decreased when using L31short ribosome compared with the case of using L31intact ribosome. "1iRT + 2iRT concentration" in the table represents the combined concentration of 1iRT and 2iRT quantified from the sum of each peak when the peaks corresponding to 1iRT and 2iRT respectively did not separate in the chromatogram.

[0251]

Table 15

[0252] Experiment 6: The starting amino acid is fMet, the second amino acid is Thr, and various amino acids are placed at the third position Overview of Experiment 6 Using the L31short ribosome and L31intact ribosome purified in Example 15, the translation synthesis of 15 types of peptides with different third amino acids was carried out, and it was confirmed that the superior translation characteristics of L31short ribosome were recognized in the translation synthesis of multiple peptides. Specifically, 15 types of peptides with different third amino acids (X) of the following amino acid sequence (SEQ ID NO: 41) were translationally synthesized. Here, X was Nle, MeG, MeF, S3F5MePyr, Pic(2), MeHph, MeA3Pyr, SPh2Cl, G, I, P, T, MeStBuOH, dA, or L. fMet:T:X:I:I:G:MeF:Bdp4AMF:I:I:P:I:G (SEQ ID NO: 41)

[0253] The experiment was conducted in a method according to Experiment 5 of Example 10, except that MeG-tRNAGlu(CUG) was additionally used as the aminoacyl tRNA spinning the third letter. The target product (TM) and by-products were quantified using LC-MS. The main by-products observed were 1iRT and 2iRT.

[0254] Translation conditions It was carried out according to Experiment 4 of Example 10, except that it was 6.72 unit / ml myokinase, 59 μM EF-Ts, and 0.09 μM GlyRS.

[0255] Analysis overview Analysis was performed according to the method of Example 9.

[0256] Results As shown in Table 16, in all sequences out of 15 types, the translation amount of TM increased when using L31short ribosome compared to the case of using L31intact ribosome, and the translation amount of TM when using L31intact was below the detection limit. In all sequences out of 15 types, the ratio of iRT decreased when using L31short ribosome compared to the case of using L31intact ribosome. "1iRT + 2iRT concentration" in the table represents the combined concentration of 1iRT and 2iRT quantified from the combined value of each peak when the peaks corresponding to 1iRT and 2iRT respectively did not separate in the chromatogram.

[0257]

Table 16

[0258] Example 11. Synthesis of LCT-67 A peptide (LCT-67) having BdpFL at the N-terminus used as a standard for LC / MS was synthesized by the following procedure. Using 2-chlorotrityl resin (100 mg) loaded with Fmoc-Gly-OH, peptide elongation was carried out on a peptide synthesizer using Fmoc-Gly-OH as the Fmoc amino acid, Fmoc-Thr(THP)-OH (aa01) synthesized by the method described in Patent Document (WO2018225864), Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-MePhe-OH, and Fmoc-Pro-OH. (The abbreviations of amino acids are described separately in this specification.) Peptide elongation was carried out according to the peptide synthesis method by the Fmoc method (see, for example, WO2013100132). After peptide elongation, removal of the N-terminal Fmoc group was carried out on the peptide synthesizer, and then the resin was washed with DCM.

[0259] TFE / DCM (1:1, v / v, 2 mL) was added to the resin and shaken for 1 hour to cleave the peptide from the resin. After completion of the reaction, the resin was removed by filtering the solution in the tube through a synthesis column, and the resin was washed twice with TFE / DCM (1:1, v / v, 1 mL). All the extracts were mixed, DMF (2 mL) was added, and then concentrated under reduced pressure. The obtained residue was dissolved in NMP (1 mL), and N-succinimidyl 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionate (5 mg, 0.013 mmol) was added at room temperature. After stirring for 19 hours, the reaction solution was passed through a reverse-phase silica gel column chromatography (0.1% FA MeCN / H2O), and the fraction containing the intermediate was concentrated under reduced pressure. The obtained residue was dissolved in 5% TFA in DCM (2 mL) and stirred at room temperature for 2 hours. After concentrating the reaction solution under reduced pressure, the obtained residue was purified by reverse-phase silica gel column chromatography (0.1% FA MeCN / H2O) to obtain the title compound (LCT-67) (13 mg). The amino acid sequence of LCT-67 is shown in SEQ ID NO: 223. LCMS (ESI) m / z = 1751.2 (M-H)- Retention time: 0.97 minutes (analysis condition SQDFA05_02)

[0260] Example 12. Synthesis of pCpA amino acids In this example, the following abbreviations were used: Gly or G (glycine), Ile or I (isoleucine), Leu or L (leucine), Phe or F (phenylalanine), Pro or P (proline), Thr or T (threonine). The analysis conditions of LCMS are shown in Table 17 below.

[0261]

Table 17

[0262] Aminoacyl pCpA (ST04, ST08, ST11, ST14, ST17, ST20, ST23, ST28, ST31, ST32, ST33, ST34) was synthesized according to the following scheme.

Chemical formula

[0263] Synthesis of (2S)-2-aminohexanoic acid (Compound ST01, Nle-OH)

Chemical formula

[0264] Under a nitrogen atmosphere, DCM (0.2 ml) and H2O (0.8 ml) were added to (2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoic acid (35.3 mg, 0.10 mmol) synthesized by the method described in Patent Document (WO2018225851A1) at room temperature. Then, 4-(3-phenylpropyl)piperidine (0.212 ml, 1.00 mmol) was added at room temperature and stirred for 30 minutes. The reaction mixture was allowed to stand, and the aqueous layer was purified by reverse-phase silica gel column chromatography (0.1% aqueous formic acid solution / 0.1% formic acid acetonitrile solution) to obtain (2S)-2-aminohexanoic acid (Compound ST01, Nle-OH) (10 mg, 76%). LCMS (ESI) m / z = 130.0 (M-H)- Retention time: 0.14 minutes (analysis conditions SQDFA05_02)

[0265] Synthesis of (2S)-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonylamino]hexanoic acid (Compound ST02, F-Pnaz-Nle-OH)

Chem.

[0266] Under a nitrogen atmosphere, DMSO (150 μL) and triethylamine (9.62 μL, 0.07 mmol) were added to a mixture of (2S)-2-aminohexanoic acid (Compound ST01, Nle-OH) (3.94 mg, 0.03 mmol) and 4-(nitrophenyl) 4-[[2-(4-fluorophenyl)acetyl]amino]benzyl carbonate (12.7 mg, 0.03 mmol) synthesized by the method described in Patent Document (WO2018143145A1) at room temperature. After the reaction mixture was stirred at room temperature for 1 hour, it was purified by reverse-phase silica gel column chromatography (0.1% aqueous formic acid solution / 0.1% formic acid acetonitrile solution) to obtain (2S)-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonylamino]hexanoic acid (Compound ST02, F-Pnaz-Nle-OH) (9 mg, 72%). LCMS (ESI) m / z = 415.3 (M-H)- Retention time: 0.74 minutes (analysis conditions SQDFA05_02)

[0267] Cyanomethyl (2S)-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonylamino]hexanoate (Compound ST03, F-Pnaz-Nle-OCH 2 CN) Synthesis

Chem.

[0268] Under a nitrogen atmosphere, to an acetonitrile solution (0.1 ml) of (2S)-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonylamino]hexanoic acid (Compound ST02, F-Pnaz-Nle-OH) (8.3 mg, 0.02 mmol), 2-bromoacetonitrile (2.0 μL, 0.03 mmol) and N-ethyl-isopropylpropan-2-amine (DIPEA) (7.0 μL, 0.04 mmol) were added in sequence at room temperature. After the reaction mixture was stirred at room temperature for 16 hours, the reaction solution was concentrated to obtain the crude product cyanomethyl (2S)-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonylamino]hexanoate (Compound ST03, F-Pnaz-Nle-OCH2CN). The obtained crude product was dissolved in acetonitrile (0.45 mL) and used as it was in the next step. LCMS(ESI) m / z = 454.4 (M-H)- Retention time: 0.83 minutes (analysis condition SQDFA05_02)

[0269] [(2R,3S,4R,5R)-2-[[[(2R,3S,4R,5R)-5-(4-amino-2-oxopyrimidin-1-yl)-4-hydroxy-2-(phosphonooxymethyl)oxolan-3-yl]oxy-hydroxyphosphoryl]oxymethyl]-5-(6-aminopurin-9-yl)-4-hydroxyoxolan-3-yl] (2S)-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonylamino]hexanoate (Compound ST04, F-Pnaz-Nle-pCpA) Synthesis

Chemical Structure

[0270] Dissolve dihydrogen phosphate ((2R,3R,4R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-3-(((((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-4-((tetrahydrofuran-2-yl)oxy)tetrahydrofuran-2-yl)methyl (22 mg, 0.03 mmol), synthesized by the method described in the literature (Helv. Chim. Acta, 90, 297-310), in buffer A (9 mL), and add an acetonitrile solution (0.45 mL, 0.02 mmol) of cyanomethyl (2S)-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonylamino]hexanoate (compound ST03, F-Pnaz-Nle-OCH2CN). Stir at room temperature for 90 minutes. After cooling the reaction solution to 0 °C, add trifluoroacetic acid (0.45 mL). After stirring the reaction solution at room temperature for 30 minutes, purify the reaction solution by reverse-phase silica gel column chromatography (0.05% aqueous trifluoroacetic acid / 0.05% trifluoroacetic acid acetonitrile) to obtain the title compound (compound ST04, F-Pnaz-Nle-pCpA) (5.7 mg, 27%). LCMS (ESI) m / z = 1049.7 (M-H)- Retention time: 0.54 minutes (analysis conditions SQDFA05_02)

[0271] Buffer A was prepared as follows. Add acetic acid to an aqueous solution of N,N,N-trimethylhexadecane-1-aminium chloride (6.40 g, 20 mmol) and imidazole (6.81 g, 100 mmol) to obtain buffer A (1 L) with pH 8, 20 mM N,N,N-trimethylhexadecane-1-aminium, and 100 mM imidazole.

[0272] (2S)-2-Amino-3-[(5-fluoropyridin-3-yl)methoxy]propanoic acid (Compound ST05, S3F5MePyr-OH) Synthesis

Chemical Structure

[0273] Under a nitrogen atmosphere, DCM (0.2 ml) and H2O (0.2 ml) were added to (2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-[(5-fluoropyridin-3-yl)methoxy]propanoic acid (43.6 mg, 0.10 mmol) synthesized by the method described in Patent Document (WO2018225864A1) at room temperature. Then, 4-(3-phenylpropyl)piperidine (63.5 ul, 0.3 mmol) was added at room temperature and stirred for 1 hour. The reaction mixture was allowed to stand, and the aqueous layer was purified by reverse-phase silica gel column chromatography (0.1% aqueous formic acid solution / 0.1% formic acid acetonitrile solution) to obtain (2S)-2-amino-3-[(5-fluoropyridin-3-yl)methoxy]propanoic acid (Compound ST05, S3F5MePyr-OH) (14 mg, 66%). LCMS (ESI) m / z = 213.0 (M-H)- Retention time: 0.19 minutes (analysis condition SQDFA05_02)

[0274] (2S)-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonylamino]-3-[(5-fluoropyridin-3-yl)methoxy]propanoic acid (Compound ST06, F-Pnaz-S3F5MePyr-OH) Synthesis

Chemical formula

[0275] Under a nitrogen atmosphere, DMSO (250 uL) and triethylamine (16.0 ul, 0.12 mmol) were added to a mixture of (2S)-2-amino-3-[(5-fluoropyridin-3-yl)methoxy]propanoic acid (Compound ST05, S3F5MePyr-OH) (10.7 mg, 0.05 mmol) and (4-nitrophenyl) 4-(2-(4-fluorophenyl)acetamido)benzyl carbonate (21.2 mg, 0.05 mmol) synthesized by the method described in Patent Document (WO2018143145A1) at room temperature. The reaction mixture was stirred at room temperature for 1 hour and then purified by reverse-phase silica gel column chromatography (0.1% aqueous formic acid solution / 0.1% formic acid acetonitrile solution) to obtain (2S)-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonylamino]hexanoic acid (Compound ST06, F-Pnaz-S3F5MePyr-OH) (23 mg, 92%). LCMS(ESI) m / z = 498.4 (M-H)- Retention time: 0.66 min (analysis condition SQDFA05_02)

[0276] Cyanomethyl (2S)-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonylamino]-3-[(5-fluoropyridin-3-yl)methoxy]propanoate (Compound ST07, F-Pnaz-S3F5MePyr-OCH 2 CN) Synthesis

Chem.

[0277] Under a nitrogen atmosphere, to an acetonitrile solution (0.1 ml) of (2S)-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonylamino]-3-[(5-fluoropyridin-3-yl)methoxy]propanoic acid (Compound ST06, F-Pnaz-S3F5MePyr-OH) (9.99 mg, 0.02 mmol), 2-bromoacetonitrile (2.0 μL, 0.03 mmol) and N-ethyl-isopropylpropan-2-amine (DIPEA) (7.0 μL, 0.04 mmol) were added in sequence at room temperature. The reaction mixture was stirred at room temperature for 16 hours and then the reaction solution was concentrated to obtain crude product cyanomethyl (2S)-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonylamino]-3-[(5-fluoropyridin-3-yl)methoxy]propanoate (Compound ST07, F-Pnaz-S3F5MePyr-OCH2CN). The obtained crude product was dissolved in acetonitrile (0.45 mL) and used as it was in the next step. LCMS(ESI) m / z = 537.3 (M-H)- Retention time: 0.74 min (analysis condition SQDFA05_02)

[0278] [(2R,3S,4R,5R)-2-[[[(2R,3S,4R,5R)-5-(4-amino-2-oxopyrimidin-1-yl)-4-hydroxy-2-(phosphonooxymethyl)oxolan-3-yl]oxy-hydroxyphosphoryl]oxymethyl]-5-(6-aminopurin-9-yl)-4-hydroxyoxolan-3-yl] (2S)-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonylamino]-3-[(5-fluoropyridin-3-yl)methoxy]propanoate (Compound ST08, F-Pnaz-S3F5MePyr-pCpA) Synthesis

Chem.

[0279] Dissolve dihydrogen phosphate ((2R,3R,4R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-3-(((((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-4-((tetrahydrofuran-2-yl)oxy)tetrahydrofuran-2-yl)methyl (22 mg, 0.03 mmol), synthesized by the method described in the literature (Helv. Chim. Acta, 90, 297 - 310), in buffer A (9 mL), and administer an acetonitrile solution (0.45 mL, 0.02 mmol) of cyanomethyl (2S)-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonylamino]-3-[(5-fluoropyridin-3-yl)methoxy]propanoate (Compound ST07, F-Pnaz-S3F5MePyr-OCH2CN). Stir the reaction mixture at room temperature for 40 minutes. After cooling the reaction solution to 0 °C, add trifluoroacetic acid (0.45 mL). Stir the reaction solution at 0 °C for 1 hour, and then purify the reaction solution by reverse-phase silica gel column chromatography (0.05% aqueous trifluoroacetic acid / 0.05% trifluoroacetic acid acetonitrile) to obtain the title compound (Compound ST08, F-Pnaz-S3F5MePyr-pCpA) (6.5 mg, 29%). LCMS(ESI) m / z = 1132.7 (M-H)- Retention time: 0.51 minutes (analysis condition SQDFA05_02)

[0280] (2S)-1-[[4-[[2-(4-Fluorophenyl)acetyl]amino]phenyl]methoxycarbonyl]pyrrolidine-2-carboxylic acid (Compound ST09, F-Pnaz-Pro-OH) Synthesis

Chemical Structure

[0281] Under a nitrogen atmosphere, DMSO (2.00 mL) and triethylamine (128 μL, 0.92 mmol) were added to a mixture of L-proline (46.1 mg, 0.40 mmol) and 4-(4-nitrophenyl) 4-(2-(4-fluorophenyl)acetamido)benzyl carbonate (178 mg, 0.42 mmol) synthesized by the method described in Patent Document (WO2018143145A1) at room temperature. After stirring the reaction mixture at room temperature for 2 days, it was purified by reverse-phase silica gel column chromatography (0.1% aqueous formic acid solution / 0.1% formic acid acetonitrile solution) to obtain (2S)-1-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonyl]pyrrolidine-2-carboxylic acid (Compound ST09, F-Pnaz-Pro-OH) (157 mg, 98%). LCMS (ESI) m / z = 399.2 (M-H)- Retention time: 0.66 minutes (analysis conditions SQDFA05_02)

[0282] 2-O-(Cyanomethyl) 1-O-[[4-[[2-(4-Fluorophenyl)acetyl]amino]phenyl]methyl] (2S)-pyrrolidine-1,2-dicarboxylate (Compound ST10, F-Pnaz-Pro-OCH 2 CN) Synthesis

Chemical Structure

[0283] Under a nitrogen atmosphere, to an acetonitrile solution (0.5 ml) of (2S)-1-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonyl]pyrrolidine-2-carboxylic acid (Compound ST09, F-Pnaz-Pro-OH) (40.0 mg, 0.10 mmol), 2-bromoacetonitrile (13.0 μL, 0.20 mmol) and N-ethyl-isopropylpropan-2-amine (DIPEA) (35.0 μL, 0.20 mmol) were added in sequence at room temperature. After the reaction mixture was stirred at room temperature for 16 hours, the reaction solution was concentrated to obtain the crude product 2-O-(cyanomethyl) 1-O-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methyl] (2S)-pyrrolidine-1,2-dicarboxylate (Compound ST10, F-Pnaz-Pro-OCH2CN). The obtained crude product was dissolved in acetonitrile (3.00 mL) and used as it was in the next step. LCMS(ESI) m / z = 438.3 (M-H)- Retention time: 0.76 minutes (analysis conditions SQDFA05_02)

[0284] 2-O-[(2R,3S,4R,5R)-2-[[[(2R,3S,4R,5R)-5-(4-Amino-2-oxopyrimidin-1-yl)-4-hydroxy-2-(phosphonooxymethyl)oxolan-3-yl]oxy-hydroxyphosphoryl]oxymethyl]-5-(6-Aminopurin-9-yl)-4-hydroxyoxolan-3-yl] 1-O-[[4-[[2-(4-Fluorophenyl)acetyl]amino]phenyl]methyl] (2S)-pyrrolidine-1,2-dicarboxylate (Compound ST11, F-Pnaz-Pro-pCpA) Synthesis

Chemical formula

[0285] To buffer A (60 mL), dihydrogen phosphate ((2R,3R,4R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-3-(((((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxyoxolan-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-4-((tetrahydrofuran-2-yl)oxy)oxolan-2-yl)methyl (72.2 mg, 0.10 mmol) synthesized by the method described in the literature (Helv. Chim. Acta, 90, 297 - 310) was dissolved, and an acetonitrile solution (3.00 mL, 0.10 mmol) of 2-O-(cyanomethyl) 1-O-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methyl] (2S)-pyrrolidine-1,2-dicarboxylate (Compound ST10, F-Pnaz-Pro-OCH2CN) was added, and the mixture was stirred at room temperature for 20 hours. After cooling the reaction solution to 0 °C, trifluoroacetic acid (3.00 mL) was added. After stirring the reaction solution at room temperature for 30 minutes, the reaction solution was purified by reverse-phase silica gel column chromatography (0.05% aqueous trifluoroacetic acid / 0.05% trifluoroacetic acid acetonitrile) to obtain the title compound (Compound ST11, F-Pnaz-Pro-pCpA) (11 mg, 11%). LCMS(ESI) m / z = 1033.4 (M-H)- Retention time: 0.49 minutes (analysis conditions SQDFA05_02)

[0286] 2-[[4-[[2-(4-Fluorophenyl)acetyl]amino]phenyl]methoxycarbonylamino]acetic acid (Compound ST12, F-Pnaz-Gly-OH) Synthesis

Chem.

[0287] Under a nitrogen atmosphere, to a mixture of glycine (30.0 mg, 0.40 mmol) and carbonic acid-(4-nitrophenyl)-4-(2-(4-fluorophenyl)acetamido)benzyl (178 mg, 0.42 mmol) synthesized by the method described in Patent Document (WO2018143145A1), DMSO (2.00 mL) and triethylamine (128 μL, 0.92 mmol) were added at room temperature. After the reaction mixture was stirred at room temperature for 2 days, it was purified by reverse-phase silica gel column chromatography (0.1% aqueous formic acid / 0.1% formic acid acetonitrile solution) to obtain 2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonylamino]acetic acid (Compound ST12, F-Pnaz-Gly-OH) (57 mg, 40%). LCMS(ESI) m / z = 359.2 (M-H)- Retention time: 0.60 minutes (analysis condition SQDFA05_02)

[0288] Cyanomethyl 2-[[4-[[2-(4-Fluorophenyl)acetyl]amino]phenyl]methoxycarbonylamino]acetate (Compound ST13, F-Pnaz-Gly-OCH 2 CN) Synthesis

Chemical Structure

[0289] Under a nitrogen atmosphere, to an acetonitrile solution (0.5 ml) of 2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonylamino]acetic acid (Compound ST12, F-Pnaz-Gly-OH) (36.0 mg, 0.10 mmol), 2-bromoacetonitrile (13.0 μL, 0.20 mmol) and N-ethyl-isopropylpropan-2-amine (DIPEA) (35.0 μL, 0.20 mmol) were added in sequence at room temperature. After the reaction mixture was stirred at room temperature for 16 hours, the reaction solution was concentrated to obtain the crude product cyanomethyl 2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonylamino]acetate (Compound ST13, F-Pnaz-Gly-OCH2CN). The obtained crude product was dissolved in acetonitrile (3.00 mL) and used as it was in the next step. LCMS(ESI) m / z = 398.3 (M-H)- Retention time: 0.69 minutes (analysis condition SQDFA05_02)

[0290] [(2R,3S,4R,5R)-2-[[[(2R,3S,4R,5R)-5-(4-Amino-2-oxopyrimidin-1-yl)-4-hydroxy-2-(phosphonooxymethyl)oxolan-3-yl]oxy-hydroxyphosphoryl]oxymethyl]-5-(6-Aminopurin-9-yl)-4-hydroxyoxolan-3-yl] 2-[[4-[[2-(4-Fluorophenyl)acetyl]amino]phenyl]methoxycarbonylamino]acetate (Compound ST14, F-Pnaz-Gly-pCpA) Synthesis

Chem.

[0291] Dissolve dihydrogen phosphate ((2R,3R,4R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-3-(((((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-4-((tetrahydrofuran-2-yl)oxy)tetrahydrofuran-2-yl)methyl (72.2 mg, 0.10 mmol) synthesized by the method described in the literature (Helv. Chim. Acta, 90, 297 - 310) in buffer A (60 mL), and administer an acetonitrile solution (3.00 mL, 0.10 mmol) of cyanomethyl 2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonylamino]acetate (Compound ST13, F-Pnaz-Gly-OCH2CN). Stir at room temperature for 90 minutes. After cooling the reaction solution to 0 °C, add trifluoroacetic acid (3.00 mL). After stirring the reaction solution at room temperature for 30 minutes, purify the reaction solution by reverse-phase silica gel column chromatography (0.05% aqueous trifluoroacetic acid / 0.05% trifluoroacetic acid acetonitrile) to obtain the title compound (Compound ST14, F-Pnaz-Gly-pCpA) (16 mg, 16%). LCMS (ESI) m / z = 993.6 (M - H)- Retention time: 0.47 minutes (analysis condition SQDFA05_02)

[0292] (2S)-2-[[4-[[2-(4-Fluorophenyl)acetyl]amino]phenyl]methoxycarbonylamino]-3-hydroxybutanoic acid (Compound ST15, F-Pnaz-Thr-OH) Synthesis

Chem.

[0293] Under a nitrogen atmosphere, DMSO (2.00 mL) and triethylamine (128 μL, 0.92 mmol) were added to a mixture of L-threonine (47.6 mg, 0.40 mmol) and (4-nitrophenyl) 4-(2-(4-fluorophenyl)acetamido)benzyl carbonate (178 mg, 0.42 mmol) synthesized by the method described in Patent Document (WO2018143145A1) at room temperature. After the reaction mixture was stirred at room temperature for 2 days, it was purified by reverse-phase silica gel column chromatography (0.1% aqueous formic acid solution / 0.1% formic acid acetonitrile solution) to obtain (2S)-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonylamino]-3-hydroxybutanoic acid (Compound ST15, F-Pnaz-Thr-OH) (141 mg, 87%). LCMS(ESI) m / z = 403.3 (M-H)- Retention time: 0.59 minutes (analysis conditions SQDFA05_02)

[0294] Cyanomethyl (2S)-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonylamino]-3-hydroxybutanoate (Compound ST16, F-Pnaz-Thr-OCH 2 CN) Synthesis

Chemical Structure

[0295] Under a nitrogen atmosphere, to an acetonitrile solution (1.0 ml) of (2S)-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonylamino]-3-hydroxybutanoic acid (Compound ST15, F-Pnaz-Thr-OH) (81.0 mg, 0.20 mmol), 2-bromoacetonitrile (268 μL, 4.00 mmol) and N-ethyl-isopropylpropan-2-amine (DIPEA) (69.9 μL, 0.40 mmol) were added in sequence at room temperature. After the reaction mixture was stirred at room temperature for 3 hours, it was purified by reverse-phase silica gel column chromatography (0.1% aqueous formic acid solution / 0.1% formic acid acetonitrile solution) to obtain cyanomethyl (2S)-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonylamino]-3-hydroxybutanoate (Compound ST16, F-Pnaz-Thr-OCH2CN) (72.0 mg, 81%). LCMS(ESI) m / z = 442.3 (M-H)- Retention time: 0.68 minutes (analysis conditions SQDFA05_02)

[0296] [(2R,3S,4R,5R)-2-[[[(2R,3S,4R,5R)-5-(4-amino-2-oxopyrimidin-1-yl)-4-hydroxy-2-(phosphonooxymethyl)oxolan-3-yl]oxy-hydroxyphosphoryl]oxymethyl]-5-(6-aminopurin-9-yl)-4-hydroxyoxolan-3-yl] (2S,3R)-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonylamino]-3-hydroxybutanoate (Compound ST17, F-Pnaz-Thr-pCpA) Synthesis

Chemical Structure

[0297] To Buffer A (60 mL), dihydrogen phosphate ((2R,3R,4R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-3-(((((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxyoxolan-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-4-((tetrahydrofuran-2-yl)oxy)oxolan-2-yl)methyl (72.2 mg, 0.10 mmol) synthesized by the method described in the literature (Helv. Chim. Acta, 90, 297 - 310) was dissolved, and an acetonitrile solution (3.00 mL, 0.10 mmol) of cyanomethyl (2S)-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonylamino]-3-hydroxybutanoate (Compound ST16, F-Pnaz-Thr-OCH2CN) was added, and the mixture was stirred at room temperature for 90 minutes. After cooling the reaction solution to 0 °C, trifluoroacetic acid (3.00 mL) was added. After stirring the reaction solution at room temperature for 30 minutes, the reaction solution was purified by reverse-phase silica gel column chromatography (0.05% aqueous trifluoroacetic acid / 0.05% trifluoroacetic acid acetonitrile) to obtain the title compound (Compound ST17, F-Pnaz-Thr-pCpA) (7 mg, 7%). LCMS(ESI) m / z = 1037.4 (M-H)- Retention time: 0.48 minutes (analysis condition SQDFA05_02)

[0298] (2S)-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonylamino]-4-methylpentanoic acid (Compound ST18, F-Pnaz-Leu-OH) Synthesis

Chemical Structure

[0299] Under a nitrogen atmosphere, to a mixture of L-leucine (13.1 mg, 0.10 mmol) and carbonic acid-(4-nitrophenyl)-4-(2-(4-fluorophenyl)acetamido)benzyl (42.4 mg, 0.10 mmol) synthesized by the method described in Patent Document (WO2018143145A1), DMSO (0.50 mL) and triethylamine (32.1 μL, 0.23 mmol) were added at room temperature. After the reaction mixture was stirred at room temperature for 1 hour, it was purified by reverse-phase silica gel column chromatography (0.1% aqueous formic acid / 0.1% formic acid acetonitrile solution) to obtain (2S)-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonylamino]-4-methylpentanoic acid (Compound ST18, F-Pnaz-Leu-OH) (28 mg, 67%). LCMS (ESI) m / z = 415.3 (M-H)- Retention time: 0.73 minutes (analysis condition SQDFA05_02)

[0300] Cyanomethyl (2S)-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonylamino]-4-methylpentanoate (Compound ST19, F-Pnaz-Leu-OCH 2 CN) Synthesis

Chemical formula

[0301] Under a nitrogen atmosphere, to an acetonitrile solution (75.0 μL) of (2S)-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonylamino]-4-methylpentanoic acid (Compound ST18, F-Pnaz-Leu-OH) (12.0 mg, 0.03 mmol), 2-bromoacetonitrile (20.0 μL, 0.30 mmol) and N-ethyl-isopropylpropan-2-amine (DIPEA) (7.86 μL, 0.05 mmol) were added in sequence at room temperature. After the reaction mixture was stirred at room temperature for 1 hour, the reaction solution was concentrated to obtain the crude product cyanomethyl (2S)-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonylamino]-4-methylpentanoate (Compound ST19, F-Pnaz-Leu-OCH2CN). The obtained crude product was dissolved in acetonitrile (0.75 mL) and used as it was in the next step. LCMS(ESI) m / z = 454.3 (M-H)- Retention time: 0.82 minutes (analysis condition SQDFA05_02)

[0302] [(2R,3S,4R,5R)-2-[[[(2R,3S,4R,5R)-5-(4-amino-2-oxopyrimidin-1-yl)-4-hydroxy-2-(phosphonooxymethyl)oxolan-3-yl]oxy-hydroxyphosphoryl]oxymethyl]-5-(6-aminopurin-9-yl)-4-hydroxyoxolan-3-yl] (2S)-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonylamino]-4-methylpentanoate (Compound ST20, F-Pnaz-Leu-pCpA) Synthesis

Chem.

[0303] Dissolve dihydrogen phosphate ((2R,3R,4R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-3-(((((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-4-((tetrahydrofuran-2-yl)oxy)tetrahydrofuran-2-yl)methyl (72.2 mg, 0.10 mmol) synthesized by the method described in the literature (Helv. Chim. Acta, 90, 297-310) in buffer A (15 mL), and add an acetonitrile solution (0.75 mL, 0.10 mmol) of cyanomethyl (2S)-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonylamino]-4-methylpentanoate (compound ST19, F-Pnaz-Leu-OCH2CN). Stir at room temperature for 60 minutes. After cooling the reaction solution to 0 °C, add trifluoroacetic acid (0.75 mL). After stirring the reaction solution at room temperature for 30 minutes, purify the reaction solution by reverse-phase silica gel column chromatography (0.05% aqueous trifluoroacetic acid / 0.05% trifluoroacetic acid acetonitrile) to obtain the title compound (compound ST20, F-Pnaz-Leu-pCpA) (9.6 mg, 31%). LCMS(ESI) m / z = 1049.6 (M-H)- Retention time: 0.54 minutes (analysis condition SQDFA05_02)

[0304] 2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonyl-methylamino]acetic acid (Compound ST21, F-Pnaz-MeG-OH) Synthesis

Chem.

[0305] Under a nitrogen atmosphere, DMSO (15 mL) and triethylamine (953.4 mg, 9.42 mmol) were added to a mixture of sarcosine (483 mg, 5.42 mmol) and (4-nitrophenyl) 4-((2-(4-fluorophenyl)acetamido)benzyl) carbonate (2.0 g, 4.71 mmol) synthesized by the method described in Patent Document (WO2018143145A1) at room temperature. After the reaction mixture was stirred at room temperature for 16 hours, it was purified by reverse-phase silica gel column chromatography (0.1% aqueous formic acid solution / 0.1% formic acid acetonitrile solution) to obtain 2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonyl-methylamino]acetic acid (Compound ST21, F-Pnaz-MeG-OH) (1.4 g, 79%). LCMS (ESI) m / z = 397 (M+Na)+ Retention time: 0.88 minutes (analysis condition SMD method3)

[0306] Cyanomethyl 2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonyl-methylamino]acetate (Compound ST22, F-Pnaz-MeG-OCH 2 CN) Synthesis

Chemical Structure

[0307] Under a nitrogen atmosphere, 2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonyl-methylamino]acetic acid (Compound ST21, F-Pnaz-MeG-OH) (1.38 g, 3.69 mmol) and N-ethyl-isopropylpropan-2-amine (DIPEA) (0.95 g, 7.38 mmol) were dissolved in DMF (28 mL), 2-bromoacetonitrile (1.74 g, 14.75 mmol) was added at room temperature, and the mixture was stirred at room temperature for 16 hours. The reaction solution was concentrated and purified by normal-phase silica gel column chromatography (ethyl acetate / petroleum ether) to obtain cyanomethyl 2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonyl-methylamino]acetate (Compound ST22, F-Pnaz-MeG-OCH2CN) (1.2 g, 79%). LCMS (ESI) m / z = 436 (M+Na)+ Retention time: 0.70 minutes (analysis condition SMD method4)

[0308] Synthesis of [(2R,3S,4R,5R)-2-[[[(2R,3S,4R,5R)-5-(4-amino-2-oxopyrimidin-1-yl)-4-hydroxy-2-(phosphonooxymethyl)oxolan-3-yl]oxy-hydroxyphosphoryl]oxymethyl]-5-(6-aminopurin-9-yl)-4-hydroxyoxolan-3-yl] 2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonyl-methylamino]acetate compound ST23, F-Pnaz-MeG-pCpA

Chemical Structure

[0309] Dissolve ((2R,3R,4R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-3-(((((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-4-((tetrahydrofuran-2-yl)oxy)tetrahydrofuran-2-yl)methyl dihydrogen phosphate (422 mg, 0.58 mmol), synthesized by the method described in the literature (Helv. Chim. Acta, 90, 297 - 310), in buffer A (100 mL). Dropwise add a solution of cyanomethyl 2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonyl-methylamino]acetate (Compound ST22, F-Pnaz-MeG-OCH2CN) (120.7 mg, 0.29 mmol) in acetonitrile (5 mL) over 15 minutes or more using a syringe pump, and stir at room temperature for 5 hours. Add trifluoroacetic acid (2.3 mL) to the reaction solution. After freeze-drying the reaction solution, purify it by reverse-phase silica gel column chromatography (0.05% aqueous trifluoroacetic acid / 0.05% trifluoroacetic acid acetonitrile) to obtain the title compound (Compound ST23, F-Pnaz-MeG-pCpA) (76.7 mg, 26%). LCMS(ESI) m / z = 1007.5 (M-H)- Retention time: 0.48 minutes (analysis condition SQDFA05_02)

[0310] The synthetic intermediate of Compound ST28 was synthesized according to the following scheme.

Chemical formula

[0311] Synthesis of (2S)-2-[9H-fluoren-9-ylmethoxycarbonyl(methyl)amino]-3-pyridin-3-ylpropanoic acid 2,2,2-trifluoroacetic acid (compound ST24, Fmoc-MeA3Pyr-OH·TFA)

Chemical formula

[0312] (2S)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-3-pyridin-3-ylpropanoic acid (15 g, 38.62 mmol), trifluoroacetic acid (27 mL, 348 mmol) and paraformaldehyde ((CH2O) n )(3.48 g, 116 mmol) were suspended in toluene (50 mL) and stirred at 40 °C for 16 h under a nitrogen atmosphere. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure. The residue was dissolved in DCM and washed with a saturated aqueous sodium hydrogen carbonate solution. The organic layer was dried over anhydrous sodium sulfate, filtered, and the solvent was removed by concentration under reduced pressure to obtain (S)-5-oxo-4-(pyridin-3-ylmethyl)oxazolidine-3-carboxylic acid (9H-fluoren-9-yl)methyl as a crude product.

[0313] The obtained crude product, (S)-5-oxo-4-(pyridin-3-ylmethyl)oxazolidine-3-carboxylic acid (9H-fluoren-9-yl) (18 g, 44.95 mmol), was dissolved in dichloroethane (100 mL), and triethylsilane (Et3SiH) (47 g, 404.20 mmol) and trifluoroacetic acid (100 mL) were added at room temperature. The reaction mixture was stirred at 70 °C for 16 h under a nitrogen atmosphere, and then the reaction mixture was concentrated under reduced pressure. The obtained residue was dissolved in isopropyl acetate, and a mixed solution of t-butyl methyl ether and hexane (9:1) was added. The solution was stirred at room temperature for 20 min and then allowed to stand at 4 °C for 1 h. The resulting precipitate was collected by filtration and washed with a cooled mixed solution of t-butyl methyl ether and hexane (9:1) to obtain (2S)-2-[9H-fluoren-9-ylmethoxycarbonyl(methyl)amino]-3-pyridin-3-ylpropanoic acid 2,2,2-trifluoroacetic acid (Compound ST24, Fmoc-MeA3Pyr-OH·TFA) (19 g, 95%). LCMS(ESI) m / z = 403 (M+H)+ Retention time: 0.77 min (analysis condition SMD method1)

[0314] Synthesis of (2S)-2-(methylamino)-3-pyridin-3-ylpropanoic acid (compound ST25, MeA3Pyr-OH)

Chemical formula

[0315] (2S)-2-[9H-Fluoren-9-ylmethoxycarbonyl(methyl)amino]-3-pyridin-3-ylpropanoic acid; 2,2,2-Trifluoroacetic acid (Compound ST24, Fmoc-MeA3Pyr-OH·TFA) (19 g, 47.21 mmol) was added with DMF (72 mL) and piperidine (28.5 mL) at room temperature, and the reaction mixture was stirred at room temperature for 3 hours. Diethyl ether (140 mL) and hexane (280 mL) were added, and the mixture was further stirred at room temperature for 3 hours. The resulting precipitate was collected by filtration to quantitatively obtain (2S)-2-(methylamino)-3-pyridin-3-ylpropanoic acid (Compound ST25, MeA3Pyr-OH) (7 g). LCMS(ESI) m / z = 181 (M+H)+ Retention time: 0.15 minutes (analysis condition SMD method2)

[0316] Synthesis of (2S)-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonyl-methylamino]-3-pyridin-3-ylpropanoic acid (compound ST26, F-Pnaz-MeA3Pyr-OH)

Chemical formula

[0317] Under a nitrogen atmosphere, a mixture of (2S)-2-(methylamino)-3-pyridin-3-ylpropanoic acid (Compound ST25, MeA3Pyr-OH) (970 mg, 5.38 mmol) and (4-nitrophenyl) 4-(2-(4-fluorophenyl)acetamido)benzyl carbonate synthesized by the method described in Patent Document (WO2018143145A1) (2 g, 4.71 mmol) was added with DMSO (15 mL) and triethylamine (950 mg, 9.43 mmol) at room temperature. After the reaction mixture was stirred at 40 °C for 16 hours, it was purified by reverse-phase silica gel column chromatography (0.1% aqueous formic acid solution / 0.1% formic acid acetonitrile solution) to obtain (2S)-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonyl-methylamino]-3-pyridin-3-ylpropane Acid(Compound ST26, F-Pnaz-MeA3Pyr-OH) (1.0 g, 46%) was obtained. LCMS (ESI) m / z = 466 (M+H)+ Retention time: 0.98 min (analysis condition SMD method3)

[0318] Synthesis of cyanomethyl (2S)-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonyl-methylamino]-3-pyridin-3-ylpropanoate (compound ST27, F-Pnaz-MeA3Pyr-OCH 2 CN)

Chemical Structure

[0319] Under a nitrogen atmosphere, (2S)-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonyl-methylamino]-3-pyridin-3-ylpropane Acid A mixture of (Compound ST26, F-Pnaz-MeA3Pyr-OH) (800 mg, 1.72 mmol) and N-ethyl-isopropylpropan-2-amine (DIPEA) (444 mg, 3.44 mmol) was dissolved in DCM (20 mL), 2-bromoacetonitrile (818 mg, 6.82 mmol) was added at room temperature, and the mixture was stirred at room temperature for 6 hours. The reaction solution was concentrated and purified by normal-phase silica gel column chromatography (ethyl acetate / petroleum ether) to obtain cyanomethyl (2S)-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonyl-methylamino]-3-pyridin-3-ylpropanoate (Compound ST27, F-Pnaz-MeA3Pyr-OCH2CN) (221 mg, 25%). LCMS (ESI) m / z = 505 (M+H)+ Retention time: 0.83 min (analysis condition SMD method4)

[0320] Synthesis of [(2R,3S,4R,5R)-2-[[[(2R,3S,4R,5R)-5-(4-amino-2-oxopyrimidin-1-yl)-4-hydroxy-2-(phosphonooxymethyl)oxolan-3-yl]oxy-hydroxyphosphoryl]oxymethyl]-5-(6-aminopurin-9-yl)-4-hydroxyoxolan-3-yl] (2S)-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonyl-methylamino]-3-pyridin-3-ylpropanoate (compound ST28, F-Pnaz-MeA3Pyr-pCpA)

Chemical Structure

[0321] Dissolve ((2R,3R,4R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-3-(((((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-4-((tetrahydrofuran-2-yl)oxy)tetrahydrofuran-2-yl)methyl dihydrogen phosphate (400 mg, 0.55 mmol), synthesized by the method described in the literature (Helv. Chim. Acta, 90, 297 - 310), in buffer A (100 mL). Add dropwise a solution of cyanomethyl (2S)-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonyl-methylamino]-3-pyridin-3-ylpropanoate (Compound ST27, F-Pnaz-MeA3Pyr-OCH2CN) (146 mg, 0.29 mmol) in acetonitrile (5 mL) over 15 minutes or more using a syringe pump, and stir at room temperature for 1 hour. Add trifluoroacetic acid (2.3 mL) to the reaction solution. After freeze-drying the reaction solution, purify it by reverse-phase silica gel column chromatography (0.05% trifluoroacetic acid aqueous solution / 0.05% trifluoroacetic acid acetonitrile) to obtain the title compound (Compound ST28, F-Pnaz-MeA3Pyr-pCpA) (64.4 mg, 5%). LCMS(ESI) m / z = 1098.5 (M-H)- Retention time: 0.39 minutes (analysis condition SQDFA05_01)

[0322] Synthesis of (2S,3S)-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonylamino]-3-methylpentanoic acid (compound ST29, F-Pnaz-Ile-OH)

Chemical Structure

[0323] Under a nitrogen atmosphere, a mixture of L-isoleucine (52.5 mg, 0.40 mmol) and carbonic acid-(4-nitrophenyl)-4-(2-(4-fluorophenyl)acetamido)benzyl (178 mg, 0.42 mmol) synthesized by the method described in Patent Document (WO2018143145A1) was added with DMSO (2 mL) and triethylamine (128 μL, 0.92 mmol) at room temperature. After the reaction mixture was stirred at room temperature for 2.5 days, it was purified by reverse-phase silica gel column chromatography (0.1% aqueous formic acid solution / 0.1% formic acid acetonitrile solution) to obtain (2S,3S)-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonylamino]-3-methylpentanoic acid (Compound ST29, F-Pnaz-Ile-OH) (125 mg, 75%). LCMS (ESI) m / z = 415.4 (M-H)- Retention time: 0.74 minutes (analysis condition SQDFA05_02)

[0324] Synthesis of cyanomethyl (2S,3S)-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonylamino]-3-methylpentanoate (Compound ST30, F-Pnaz-Ile-OCH 2 CN)

Chemical formula

[0325] Under a nitrogen atmosphere, (2S,3S)-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonylamino]-3-methylpentanoic acid (Compound ST29, F-Pnaz-Ile-OH) (42 mg, 0.1 mmol) and 2-bromoacetonitrile (13 μL, 0.200 mmol) were dissolved in acetonitrile (500 μL), and N-ethyl-isopropylpropan-2-amine (DIPEA) (35 μL, 0.200 mmol) was added at room temperature, followed by stirring at room temperature for 16 hours. The reaction solution was concentrated to obtain the crude product cyanomethyl (2S,3S)-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonylamino]-3-methylpentanoate (Compound ST30, F-Pnaz-Ile-OCH2CN). The obtained crude product was dissolved in acetonitrile (3.00 mL) and used as it was in the next step. LCMS(ESI) m / z = 454 (M-H)- Retention time: 0.83 minutes (analysis condition SQDFA05_02)

[0326] Synthesis of [(2R,3S,4R,5R)-2-[[[(2R,3S,4R,5R)-5-(4-amino-2-oxopyrimidin-1-yl)-4-hydroxy-2-(phosphonooxymethyl)oxolan-3-yl]oxy-hydroxyphosphoryl]oxymethyl]-5-(6-aminopurin-9-yl)-4-hydroxyoxolan-3-yl] (2S,3S)-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonylamino]-3-methylpentanoate (Compound ST31, F-Pnaz-Ile-pCpA)

Chem.

[0327] Dissolve dihydrogen phosphate ((2R,3R,4R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-3-(((((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-4-((tetrahydrofuran-2-yl)oxy)tetrahydrofuran-2-yl)methyl (72.2 mg, 0.100 mmol) synthesized by the method described in the literature (Helv. Chim. Acta, 90, 297-310) in buffer A (60 mL), and administer an acetonitrile solution (3.00 mL) of cyanomethyl (2S,3S)-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonylamino]-3-methylpentanoate (compound ST30, F-Pnaz-Ile-OCH2CN) (45.5 mg, 0.100 mmol), and stir at room temperature for 20 hours. After cooling the reaction solution to 0 °C, add trifluoroacetic acid (3.00 mL). After stirring the reaction solution at room temperature for 30 minutes, purify the reaction solution by reverse-phase silica gel column chromatography (0.05% aqueous trifluoroacetic acid / 0.05% trifluoroacetic acid acetonitrile) to obtain the title compound (compound ST31, F-Pnaz-Ile-pCpA) (12 mg, 11.4%). LCMS(ESI) m / z = 1049.4 (M-H)- Retention time: 0.54 minutes (analysis condition SQDFA05_02)

[0328] Synthesis of [(2R,3S,4R,5R)-2-[[[(2R,3S,4R,5R)-5-(4-amino-2-oxopyrimidin-1-yl)-4-hydroxy-2-(phosphonooxymethyl)oxolan-3-yl]oxy-hydroxyphosphoryl]oxymethyl]-5-(6-aminopurin-9-yl)-4-hydroxyoxolan-3-yl] (2S)-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonyl-methylamino]-3-phenylpropanoate (Compound ST32, F-Pnaz-MePhe-pCpA)

Chem.

[0329] Synthesized by the method described in WO2018 / 225864.

[0330] Synthesis of [(2R,3S,4R,5R)-2-[[[(2R,3S,4R,5R)-5-(4-amino-2-oxopyrimidin-1-yl)-4-hydroxy-2-(phosphonooxymethyl)oxolan-3-yl]oxy-hydroxyphosphoryl]oxymethyl]-5-(6-aminopurin-9-yl)-4-hydroxyoxolan-3-yl] (2R)-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonylamino]propanoate (Compound ST33, F-Pnaz-D-Ala-pCpA) [Chemical formula]

[0331] Synthesized by the method described in WO2018 / 143145.

[0332] Synthesis of [[(2R,3S,4R,5R)-2-[[[(2R,3S,4R,5R)-5-(4-amino-2-oxopyrimidin-1-yl)-4-hydroxy-2-(phosphonooxymethyl)oxolan-3-yl]oxy-hydroxyphosphoryl]oxymethyl]-5-(6-aminopurin-9-yl)-4-hydroxyoxolan-3-yl] 1-O-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methyl] (2S)-piperidine-1,2-dicarboxylate (Compound ST34, F-Pnaz-Pic(2)-pCpA)]] [Chemical formula]

[0333] Synthesized by the method described in WO2020 / 138336.

[0334] Synthesis of [[(2R,3S,4R,5R)-2-[[[(2R,3S,4R,5R)-5-(4-amino-2-oxopyrimidin-1-yl)-4-hydroxy-2-(phosphonooxymethyl)oxolan-3-yl]oxy-hydroxyphosphoryl]oxymethyl]-5-(6-aminopurin-9-yl)-4-hydroxyoxolan-3-yl] (2S)-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonyl-methylamino]-4-phenylbutanoate (Compound ST35, F-Pnaz-MeHph-pCpA)]] [Chemical formula]

[0335] Synthesized by the method described in Patent Document (WO2020138336A1).

[0336] Synthesis of [[(2R,3S,4R,5R)-2-[[[(2R,3S,4R,5R)-5-(4-amino-2-oxopyrimidin-1-yl)-4-hydroxy-2-(phosphonooxymethyl)oxolan-3-yl]oxy-hydroxyphosphoryl]oxymethyl]-5-(6-aminopurin-9-yl)-4-hydroxyoxolan-3-yl] (2S)-2-[[4-[[2-(4-fluorophenyl)acetyl]amino]phenyl]methoxycarbonyl-methylamino]-4-phenylbutanoate (Compound ST36, F-Pnaz-SPh2Cl-pCpA)]] [Chemical formula]

[0337] Synthesized by the method described in Patent Document (WO2020138336A1).

[0338] Example 13. Synthesis of Aminoacyl tRNA According to the method described in "Aminoacyl-tRNA synthesis using aminoacyl pCpA: Part 1" of Example 5, various aminoacyl-tRNAs were synthesized and recovered. Each of the recovered aminoacyl-tRNAs was dissolved in 1 mM sodium acetate. The correspondence between the names of the completed aminoacyl-tRNAs and the aminoacyl pCpA amino acids used is as shown in Table 18.

[0339] [Table 18]

[0340] Example 14. Preparation of Escherichia coli Strains Expressing Mutants Eight types of L31 mutant strains (L31(1-27), L31(1-32), L31(1-37), L31(1-42), L31(1-47), L31(1-52), L31(1-57), L31(1-67)) that express with various lengths from the N-terminal side of the L31 protein were constructed. According to the protocol attached to the Quick and Easy Conditional Knockout Kit (loxP / Cre) (Gene Bridges), an Escherichia coli strain was constructed.

[0341] Creation of a Functional Cassette with Added Homology Arms According to the method described in Example 1, a functional cassette was created. The correspondence between the length of the expression region of L31 in each strain and the primer used for PCR of the functional cassette used to prepare the strain is shown in Table 19.

[0342] [Table 19]

[0343] Creation of L31 Mutant Strains An L31 mutant strain was prepared according to the method described in Example 1.

[0344] Example 15. Purification of Ribosomes Preparation of Ribosomes Using L31 Mutant Strains, L31short Strains, L31intact Strains, and W3110 Strains According to the method described in Example 2, culturing of strains, disruption of Escherichia coli, purification of Escherichia coli, Butylsepharose purification, and ultracentrifugation purification were performed. For all strains, the description regarding the L31intact strain and L31short strain in Example 2 was followed. Mg10 Lysis Buffer described in Example 2 was used for the disruption of Escherichia coli. A total of 11 types of ribosomes, including 8 types of L31short ribosome, L31intact ribosome, WT ribosome, and L31 mutant ribosome, were prepared to have a final concentration of 10 to 20 μM.

Industrial Applicability

[0345] The present invention provides a method for producing a peptide and a peptide library containing a non-natural amino acid, and a modified L31 protein for use in the method. By using the production method of the present invention, an mRNA encoding a peptide containing a non-natural amino acid can be efficiently translated, and the peptide and a library containing the same can be efficiently produced.

Claims

1. A method for producing a peptide, comprising a step of translating an mRNA encoding a peptide containing one or more unnatural amino acids in a translation system containing ribosomes containing a modified L31 protein, wherein the modified L31 protein consists of a protein selected from the group consisting of the following (1) to (3): (1) A protein containing an amino acid sequence in which 6 or more and 45 or less amino acid residues are deleted from the C-terminus in the amino acid sequence represented by SEQ ID NO: 1; (2) A protein containing an amino acid sequence in which one or more amino acids are inserted, substituted, deleted and / or added in the amino acid sequence of the protein described in (1) above, and having an amino acid sequence having 90% or more sequence identity with the amino acid sequence of the protein described in (1) above; (3) A protein containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence of the protein described in (1) above is selected from, The method according to claim 1, wherein the ribosome containing the protein described in (2) and (3) above has higher translation activity of a peptide containing an unnatural amino acid as compared with a ribosome containing wild-type E. coli L31 containing the amino acid sequence of SEQ ID NO:

1.

2. The method according to claim 1, wherein the ratio of the ribosome containing the modified L31 protein to all ribosomes in the translation system is 50% or more.

3. The method according to claim 1 or 2, further comprising a step of cyclizing the peptide.

4. The method according to any one of claims 1 to 3, wherein the peptide contains an unnatural amino acid at the position of its starting amino acid.

5. The steps described in the following (a) to (c): (a) A step of producing a peptide by the method according to any one of claims 1 to 4, (b) A step of contacting a target substance with the peptide or a library containing the peptide, and (c) A step of selecting a peptide that binds to the target substance A method for screening a peptide that binds to a target substance, comprising.

6. The proteins described in the following (1) to (3) (provided that a protein containing an amino acid sequence in which 8 amino acid residues are deleted from the C-terminus in the amino acid sequence represented by SEQ ID NO: 1 and a protein having an amino acid sequence represented by MKKDIHPKYEEITASCSSCGNVMKIRSTVGHDLNLDVCSKVPPVLHWQTA are excluded): (1) A protein consisting of an amino acid sequence in which 9 to 45 amino acid residues from the C-terminus are deleted in the amino acid sequence represented by SEQ ID NO: 1, (2) A protein containing an amino acid sequence in which 1 to 9 amino acids are inserted, substituted, deleted and / or added in the amino acid sequence of the protein described in (1) above, and having an amino acid sequence having 90% or more sequence identity with the amino acid sequence of the protein described in (1) above, (3) A protein containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence of the protein described in (1) above A modified L31 protein selected from the group consisting of: A ribosome containing the proteins described in (1), (2) and (3) above has a higher translation activity of a peptide containing a non-natural amino acid as compared with a ribosome containing wild-type E. coli L31 having the amino acid sequence of SEQ ID NO:

1. A modified L31 protein.

7. The proteins described in the following (1) to (3) (provided that, in the amino acid sequence represented by SEQ ID NO: 1, a protein containing an amino acid sequence in which 8 amino acid residues are deleted from the C-terminus and a protein having the amino acid sequence represented by MKKDIHPKYEEITASCSSCGNVMKIRSTVGHDLNLDCSSKVPPVLHWQTA are excluded): (1) A protein consisting of an amino acid sequence represented by any one selected from the group consisting of SEQ ID NO: 2 and 42 to 48, (2) A protein containing an amino acid sequence in which 1 to 9 amino acids are inserted, substituted, deleted and / or added in the amino acid sequence of the protein described in (1) above, and having an amino acid sequence having 90% or more sequence identity with the amino acid sequence of the protein described in (1) above, (3) A protein containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence of the protein described in (1) above A modified L31 protein selected from the group consisting of: A ribosome containing the proteins described in (2) and (3) above has a higher translation activity of a peptide containing a non-natural amino acid as compared with a ribosome containing wild-type E. coli L31 having the amino acid sequence of SEQ ID NO:

1. A modified L31 protein.

8. An isolated nucleic acid encoding the modified L31 protein according to Claim 6 or 7.

9. A vector or cell comprising the nucleic acid according to claim 8.

10. The steps described in the following (a) to (c): (a) Culturing the cell according to claim 9, (b) Generating a lysate from the culture of the cell, and (c) Purifying ribosomes from the lysate A method for producing ribosomes comprising a modified L31 protein, comprising:

11. Ribosomes comprising the modified L31 protein according to claim 6 or 7.

12. A composition comprising the ribosomes according to claim 11.

13. A synthetic system for cell-free translation with high translational activity of mRNA encoding a peptide containing a non-natural amino acid, comprising a modified L31 protein, wherein the modified L31 protein is a protein described in the following (1) to (3): (1) A protein consisting of an amino acid sequence in which 6 to 45 amino acid residues from the C-terminus are deleted in the amino acid sequence represented by SEQ ID NO: 1, (2) A protein comprising an amino acid sequence in which 1 to 9 amino acids are inserted, substituted, deleted and / or added in the amino acid sequence of the protein described in (1) above, and having an amino acid sequence having 90% or more sequence identity with the amino acid sequence of the protein described in (1) above, (3) A protein comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence of the protein described in (1) above selected from the group consisting of: The synthetic system, wherein ribosomes comprising the proteins described in (2) and (3) above have higher translational activity of peptides containing non-natural amino acids as compared with ribosomes containing wild-type E. coli L31 comprising the amino acid sequence of SEQ ID NO:

1.

14. The following steps: (a) Producing ribosomes by the method according to claim 10, and (b) Mixing the ribosomes with an initiator tRNA acylated with a non-natural amino acid A method for producing a cell-free translation system, comprising:

15. The following steps: (a) Disrupting Escherichia coli to generate a lysate under conditions where the magnesium ion concentration is 5 mM or less, and (b) Purifying ribosomes from the lysate A method for producing a cell-free translation system, comprising:

16. The following steps: (a) Disrupting Escherichia coli to generate a lysate under conditions where the magnesium ion concentration is 5 mM or less, (b) Purifying ribosomes from the lysate, and Step (c): Mixing the ribosome with an initiation tRNA acylated with a non-natural amino acid A method for producing a cell-free translation system, comprising the above steps. **Claim 17** The method according to any one of claims 14 to 16, wherein the cell-free translation system further contains 2 to 8 mM of magnesium ions. **Claim 18** The method according to any one of claims 14 to 17, wherein the cell-free translation system is a reconstituted cell-free translation system. **Claim 19** The following steps: (i) A step of producing a cell-free translation system by the method according to any one of claims 14 to 18, and (ii) A step of translating an mRNA encoding a peptide containing one or more non-natural amino acids using the cell-free translation system A method for producing a peptide, comprising the above steps. **Claim 20** The method according to claim 19, wherein the peptide contains a non-natural amino acid at the position of its starting amino acid. **Claim 21** The method according to claim 19 or 20, wherein the translation is performed by initiation suppression. **Claim 22** The following steps: (I) A step of producing a peptide by the method according to any one of claims 19 to 21, (II) A step of contacting a target substance with the peptide or a library containing the peptide, and (III) A step of selecting a peptide that binds to the target substance A method for screening a peptide that binds to a target substance, comprising the above steps. **Claim 23** The method according to claim 22, wherein the peptide or the library containing the peptide is a library containing the peptide. **Claim 24** The method according to claim 23, wherein the library is a display library. **Claim 25** The method according to claim 24, wherein the library is an mRNA display library.

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