WO2024128195A1 - 特定位置に酸素原子を有するアミノ酸を翻訳する工程を含む、ペプチドの製造方法 - Google Patents

特定位置に酸素原子を有するアミノ酸を翻訳する工程を含む、ペプチドの製造方法 Download PDF

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WO2024128195A1
WO2024128195A1 PCT/JP2023/044244 JP2023044244W WO2024128195A1 WO 2024128195 A1 WO2024128195 A1 WO 2024128195A1 JP 2023044244 W JP2023044244 W JP 2023044244W WO 2024128195 A1 WO2024128195 A1 WO 2024128195A1
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amino acid
unnatural amino
group
formula
atom
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French (fr)
Japanese (ja)
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翔太 田中
正次郎 篠原
効彦 中野
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Chugai Pharmaceutical Co Ltd
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Chugai Pharmaceutical Co Ltd
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Priority to EP23903481.2A priority Critical patent/EP4628592A1/en
Priority to CN202380084750.9A priority patent/CN120380162A/zh
Priority to JP2024564375A priority patent/JPWO2024128195A1/ja
Priority to KR1020257021523A priority patent/KR20250124834A/ko
Publication of WO2024128195A1 publication Critical patent/WO2024128195A1/ja
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    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1062—Isolating an individual clone by screening libraries mRNA-Display, e.g. polypeptide and encoding template are connected covalently
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
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    • C40B40/04—Libraries containing only organic compounds
    • C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof

Definitions

  • the present invention relates to a method for producing a peptide, which includes a step of translating an amino acid having an oxygen atom at a specific position.
  • Non-Patent Documents 1 and 4 it has been reported that ribosome modification improves the translation efficiency of D-amino acids or ⁇ -amino acids. It has also been reported that EF-Tu modification enables the translational incorporation of unnatural amino acids with bulky aromatic rings in the side chains that cannot be translated in unmodified form (see Non-Patent Document 2). In addition, it has been reported that modification of aminoacyl-tRNA synthetase enables the translational incorporation of N-methyl amino acids (see Patent Document 1).
  • modified tRNA Pro1E2 has achieved continuous translational incorporation of D-amino acids or ⁇ -amino acids (see Non-Patent Documents 7, 8, 14, and 15, and Patent Document 2). It has also been reported that the use of modified tRNA Pro1E2 was effective for continuous translational incorporation of cyclic ⁇ -amino acids (see Non-Patent Document 10) and for the sole translational incorporation of cyclic ⁇ -amino acids or aminobenzoic acids (see Non-Patent Documents 11 and 12).
  • the present invention is based on such findings, and specifically relates to [1] to [123].
  • [1] translating a nucleic acid that includes a codon encoding a first unnatural amino acid contiguous with a codon encoding a second unnatural amino acid;
  • (A) a nitrogen atom (nitrogen atom N1) constituting a main chain amino group and an oxygen atom (oxygen atom O1) other than the oxygen atom constituting a main chain carboxy group are bonded via two atoms;
  • the oxygen atom O1 constitutes an alkoxy group or an aryloxy group;
  • the nitrogen atom N1 is bonded to a linear or branched C 1
  • R 1 is a group represented by formula (II), a hydrogen atom or a linear or branched C 1 -C 6 alkyl group
  • R 2 and R 3 are each independently (a) a group represented by formula (II), (b) a hydrogen atom, or (c) a linear or branched C 1 -C 4 alkyl group
  • R 2b and R 3b are each independently a hydrogen atom or a linear or branched C 1 -C 4 alkyl group
  • R 2 and R 2b or R 3 and R 3b may form a ring together with the carbon atoms to which they are attached
  • At least one of R 1 , R 2 and R 3 is a group represented by formula (II), N 1 (nitro
  • R 1 is a group represented by the formula (II) or a linear or branched C 1 -C 6 alkyl group; The method according to any one of [21] to [28], wherein in the formula (II), R 5 is a linear or branched C 1 to C 6 alkyl group, a C 3 to C 10 cycloalkyl group, a C 6 to C 10 aryl group, a C 7 to C 12 arylalkyl group, or a C 7 to C 12 alkylaryl group. [32] The method according to any one of [21] to [31], wherein R 1 is a group represented by formula (II).
  • the first unnatural amino acid is the unnatural amino acid U1 and the second unnatural amino acid is the unnatural amino acid U2;
  • the unnatural amino acid U1 satisfies requirements (A) and (B),
  • the oxygen atom O1 is included in a side chain of the unnatural amino acid U1, the oxygen atom O1 constitutes a methoxy group or a phenoxy group, and the nitrogen atom N1 constitutes an unsubstituted amino group;
  • the first unnatural amino acid is the unnatural amino acid U1 and the second unnatural amino acid is the unnatural amino acid U2;
  • the unnatural amino acid U1 satisfies requirements (A) and (C)
  • the oxygen atom O1 is included in the side chain of the unnatural amino acid U1, the oxygen atom O1 constitutes a hydroxyl group, and the nitrogen atom N1 is substituted with a methyl group or an ethyl group;
  • the first unnatural amino acid is the unnatural amino acid U1 and the second unnatural amino acid is the unnatural amino acid U2;
  • the unnatural amino acid U1 satisfies the requirements (A) and (B), in which the nitrogen atom N1, the carbon atoms constituting the main chain of the unnatural amino acid U1, and the atoms constituting the side chain of the unnatural amino acid U1 together form a ring, and the oxygen atom O1 forms a methoxy group or a phenoxy group;
  • the unnatural amino acid U1 is an ⁇ -amino acid, in which a nitrogen atom (nitrogen atom N1) constituting a main chain amino group in the unnatural amino acid U1 is bonded to an oxygen atom (oxygen atom O1) other than the oxygen atom constituting a main chain carboxyl group via two or three atoms, the oxygen atom O1 is included in the side chain of the unnatural amino acid U1, the oxygen atom O1 constitutes a hydroxyl group, and the nitrogen atom N1 is substituted with a methyl group or an ethyl group;
  • the first unnatural amino acid is the unnatural amino acid U1 and the second unnatural amino acid is the unnatural amino acid U2;
  • the unnatural amino acid U1 satisfies the requirements (A) and (B), the oxygen atom O1 is present in a substituent substituted with the nitrogen atom N1, and the oxygen atom O1 constitutes a methoxy group or a phenoxy group;
  • the first unnatural amino acid is the unnatural amino acid U1 and the second unnatural amino acid is the unnatural amino acid U2;
  • the unnatural amino acid U1 satisfies requirements (A) and (B),
  • the oxygen atom O1 is included in a side chain of the unnatural amino acid U1, the oxygen atom O1 constitutes a methoxy group or a phenoxy group, and the nitrogen atom N1 constitutes an unsubstituted amino group;
  • the first unnatural amino acid is the unnatural amino acid U1 and the second unnatural amino acid is the unnatural amino acid U2;
  • the unnatural amino acid U1 satisfies requirements (A) and (C)
  • the oxygen atom O1 is included in the side chain of the unnatural amino acid U1, the oxygen atom O1 constitutes a hydroxyl group, and the nitrogen atom N1 is substituted with a methyl group or an ethyl group;
  • the first unnatural amino acid is the unnatural amino acid U1 and the second unnatural amino acid is the unnatural amino acid U2;
  • the unnatural amino acid U1 satisfies the requirements (A) and (B), in which the nitrogen atom N1, the carbon atoms constituting the main chain of the unnatural amino acid U1, and the atoms constituting the side chain of the unnatural amino acid U1 together form a ring, and the oxygen atom O1 forms a methoxy group or a phenoxy group;
  • the unnatural amino acid U1 is an ⁇ -amino acid, in which a nitrogen atom (nitrogen atom N1) constituting a main chain amino group in the unnatural amino acid U1 is bonded to an oxygen atom (oxygen atom O1) other than the oxygen atom constituting a main chain carboxyl group via two or three atoms, the oxygen atom O1 is included in the side chain of the unnatural amino acid U1, the oxygen atom O1 constitutes a hydroxyl group, and the nitrogen atom N1 is substituted with a methyl group or an ethyl group;
  • the first unnatural amino acid is the unnatural amino acid U1 and the second unnatural amino acid is the unnatural amino acid U2;
  • the unnatural amino acid U1 is an unnatural amino acid represented by formula (III), in which R5 is a methyl group or a phenyl group; The method of any one of [21], [23], [29], [32], [37], [44]-[47], [49], and [50], wherein a codon encoding the first unnatural amino acid is translated before a codon encoding the second unnatural amino acid.
  • the first unnatural amino acid is the unnatural amino acid U1 and the second unnatural amino acid is the unnatural amino acid U2;
  • the unnatural amino acid U1 is an unnatural amino acid represented by formula (IV), in which R5 is a methyl group or a phenyl group, and R1b is a hydrogen atom; The method of any one of [21], [24], [29], [33], [37], [46], [47], [49], and [50], wherein a codon encoding the first unnatural amino acid is translated before a codon encoding the second unnatural amino acid.
  • the first unnatural amino acid is the unnatural amino acid U1 and the second unnatural amino acid is the unnatural amino acid U2;
  • the unnatural amino acid U1 is an unnatural amino acid represented by formula (IV), in which R5 is a hydrogen atom and R1b is a methyl group or an ethyl group; The method of any one of [21], [24], [30], [33], [43], [44], [47], [49], and [50], wherein a codon encoding the first unnatural amino acid is translated before a codon encoding the second unnatural amino acid.
  • the first unnatural amino acid is the unnatural amino acid U1 and the second unnatural amino acid is the unnatural amino acid U2;
  • the unnatural amino acid U1 is an unnatural amino acid represented by formula (V), in which R5 is a phenyl group; The method of any one of [21], [25], [29], [34], [37], [44]-[46], [47], [49], and [50], wherein a codon encoding the first unnatural amino acid is translated before a codon encoding the second unnatural amino acid.
  • a method for producing a peptide wherein the first unnatural amino acid is the unnatural amino acid U1, the second unnatural amino acid is the unnatural amino acid U2, and the unnatural amino acid U1 is an unnatural amino acid represented by formula (I):
  • R1 is a methyl group or an ethyl group
  • R2 is a group represented by formula (II)
  • R 2b is a hydrogen atom or a linear or branched C 1 -C 4 alkyl group
  • N 1 (nitrogen atom N1) in formula (I) and O 1 (oxygen atom O1) in formula (II) are bonded via two or three atoms
  • N 1 (nitrogen atom N1) in formula (I) and O 1 (oxygen atom O1) in formula (II) are bonded via two or three atoms
  • In formula (II) represents the bonding position with N1
  • R4 is a C1 - C2 alkylene group
  • R5 is a hydrogen atom.
  • the first unnatural amino acid is the unnatural amino acid U1 and the second unnatural amino acid is the unnatural amino acid U2;
  • the unnatural amino acid U1 is an unnatural amino acid represented by formula (III), in which R5 is a methyl group or a phenyl group; The method of any one of [21], [23], [29], [32], [37], [44]-[47], [49], and [51], wherein a codon encoding the first unnatural amino acid is translated after a codon encoding the second unnatural amino acid.
  • the first unnatural amino acid is the unnatural amino acid U1 and the second unnatural amino acid is the unnatural amino acid U2;
  • the unnatural amino acid U1 is an unnatural amino acid represented by formula (IV), in which R5 is a methyl group or a phenyl group, and R1b is a hydrogen atom; The method of any one of [21], [24], [29], [33], [37], [46], [47], [49], and [51], wherein a codon encoding the first unnatural amino acid is translated after a codon encoding the second unnatural amino acid.
  • the first unnatural amino acid is the unnatural amino acid U1 and the second unnatural amino acid is the unnatural amino acid U2;
  • the unnatural amino acid U1 is an unnatural amino acid represented by formula (IV), in which R5 is a hydrogen atom and R1b is a methyl group or an ethyl group; The method of any one of [21], [24], [30], [33], [43], [44], [47], [49], and [51], wherein a codon encoding the first unnatural amino acid is translated after a codon encoding the second unnatural amino acid.
  • the first unnatural amino acid is the unnatural amino acid U1 and the second unnatural amino acid is the unnatural amino acid U2;
  • the unnatural amino acid U1 is an unnatural amino acid represented by formula (V), in which R5 is a phenyl group; The method of any one of [21], [25], [29], [34], [37], [44]-[47], [49], and [51], wherein a codon encoding the first unnatural amino acid is translated after a codon encoding the second unnatural amino acid.
  • a method for producing a peptide wherein the first unnatural amino acid is the unnatural amino acid U1, the second unnatural amino acid is the unnatural amino acid U2, and the unnatural amino acid U1 is an unnatural amino acid represented by formula (I):
  • R1 is a methyl group or an ethyl group
  • R2 is a group represented by formula (II)
  • R 2b is a hydrogen atom or a linear or branched C 1 -C 4 alkyl group
  • N 1 (nitrogen atom N1) in formula (I) and O 1 (oxygen atom O1) in formula (II) are bonded via two or three atoms
  • N 1 (nitrogen atom N1) in formula (I) and O 1 (oxygen atom O1) in formula (II) are bonded via two or three atoms
  • In formula (II) represents the bonding position with N1
  • R4 is a C1 - C2 alkylene group
  • R5 is a hydrogen atom.
  • [102] The method according to any one of [1] to [101], wherein the number of amino acid residues constituting the peptide is 2 or more and 100 or less.
  • [103] The method according to any one of [1] to [102], wherein the peptide is a cyclic peptide.
  • [104] The method according to [103], wherein the number of amino acid residues constituting the cyclic portion of the cyclic peptide is 5 to 30.
  • the peptide contains one or more N-substituted amino acid residues.
  • the method according to any one of [1] to [105], wherein the ClogP of the peptide is 4 or more and 25 or less.
  • [122] Obtaining a peptide by the method according to any one of [1] to [119]; and binding to the peptide one or more selected from the group consisting of a nucleic acid, a ribosome, a spacer, and a linker.
  • [123] Obtaining a peptide by the method according to any one of [1] to [119]; and binding a nucleic acid to the peptide.
  • 1 is a graph showing the amount of translation when peptides are translated using aminoacylated tRNA.
  • amino acids constituting a peptide include "natural amino acids” such as ⁇ -amino acids, and "unnatural amino acids” such as ⁇ -amino acids and ⁇ -amino acids.
  • the three-dimensional structure of an amino acid may be either L-amino acid or D-amino acid.
  • amino acids “natural amino acids”, and “unnatural amino acids” may be referred to as “amino acid residues”, “natural amino acid residues", and “unnatural amino acid residues", respectively.
  • natural amino acid refers to an ⁇ -amino carboxylic acid ( ⁇ -amino acid) and includes 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).
  • unnatural amino acid refers to an amino carboxylic acid not included in the above-mentioned "natural amino acid”.
  • unnatural amino acids include ⁇ -amino acids (including D-type and L-type), ⁇ -amino acids (including D-type and L-type), D- ⁇ -amino acids, L- ⁇ -amino acids having a side chain different from that of natural amino acids, ⁇ , ⁇ -disubstituted amino acids, and amino acids in which the amino group of the main chain is substituted (N-substituted amino acids).
  • the side chain of an unnatural amino acid is not particularly limited, and may have, in addition to a hydrogen atom, for example, an alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, cycloalkyl, etc.
  • the two side chains may form a ring.
  • these side chains may have one or more substituents.
  • the substituent can be selected from any functional group including a halogen atom, an O atom, a S atom, a N atom, a B atom, a Si atom, or a P atom.
  • C 1 -C 6 alkyl having a halogen as a substituent means “C 1 -C 6 alkyl" in which at least one hydrogen atom in the alkyl is replaced with a halogen atom, and specifically includes, for example, trifluoromethyl, difluoromethyl, fluoromethyl, pentafluoroethyl, tetrafluoroethyl, trifluoroethyl, difluoroethyl, fluoroethyl, trichloromethyl, dichloromethyl, chloromethyl, pentachloroethyl, tetrachloroethyl, trichloroethyl, dichloroethyl, chloroethyl, etc.
  • C 5 -C 10 aryl C 1 -C 6 alkyl having a substituent means “C 5 -C 10 aryl C 1 -C 6 alkyl” in which at least one hydrogen atom in the aryl and/or alkyl is replaced with a substituent.
  • “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 the functional group further having another substituent (for example, a substituent such as amino or halogen).
  • 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).
  • R represents an alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, or cycloalkyl group, which may have a substituent.
  • the carbon chain bonded to the N atom of the amino group in the main chain and the carbon atom at the ⁇ -position may form a ring, as in proline.
  • alkyl substitution of amino groups include N-methylation, N-ethylation, N-propylation, and N-butylation, and examples of aralkyl substitution include N-benzylation.
  • N-methyl amino acids include N-methylalanine, N-methylglycine, N-methylphenylalanine, N-methyltyrosine, N-methyl-3-chlorophenylalanine, N-methyl-4-chlorophenylalanine, N-methyl-4-methoxyphenylalanine, N-methyl-4-thiazolealanine, N-methylhistidine, N-methylserine, and N-methylaspartic acid.
  • substituents containing halogen include alkyl groups, cycloalkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, and aralkyl groups each having a halogen as a substituent, and more specific examples include fluoroalkyl, difluoroalkyl, and trifluoroalkyl.
  • oxy examples include alkoxy, cycloalkoxy, alkenyloxy, alkynyloxy, aryloxy, heteroaryloxy, and aralkyloxy.
  • Examples of oxycarbonyl include alkyloxycarbonyl, cycloalkyloxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, and aralkyloxycarbonyl.
  • Examples of carbonyloxy include alkylcarbonyloxy, cycloalkylcarbonyloxy, alkenylcarbonyloxy, alkynylcarbonyloxy, arylcarbonyloxy, heteroarylcarbonyloxy, and aralkylcarbonyloxy.
  • thiocarbonyl examples include alkylthiocarbonyl, cycloalkylthiocarbonyl, alkenylthiocarbonyl, alkynylthiocarbonyl, arylthiocarbonyl, heteroarylthiocarbonyl, and aralkylthiocarbonyl.
  • Examples of carbonylthio include alkylcarbonylthio, cycloalkylcarbonylthio, alkenylcarbonylthio, alkynylcarbonylthio, arylcarbonylthio, heteroarylcarbonylthio, and aralkylcarbonylthio.
  • aminocarbonyl examples include alkylaminocarbonyl, cycloalkylaminocarbonyl, alkenylaminocarbonyl, alkynylaminocarbonyl, arylaminocarbonyl, heteroarylaminocarbonyl, and aralkylaminocarbonyl.
  • Examples of carbonylamino include alkylcarbonylamino, cycloalkylcarbonylamino, alkenylcarbonylamino, alkynylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, and aralkylcarbonylamino.
  • there are groups in which the H atom bonded to the N atom in -NH-C( O)-R is further substituted with an alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl.
  • Examples of oxycarbonylamino include alkoxycarbonylamino, cycloalkoxycarbonylamino, alkenyloxycarbonylamino, alkynyloxycarbonylamino, aryloxycarbonylamino, heteroaryloxycarbonylamino, and aralkyloxycarbonylamino.
  • sulfonylamino examples include alkylsulfonylamino, cycloalkylsulfonylamino, alkenylsulfonylamino, alkynylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino, aralkylsulfonylamino, etc.
  • H atom bonded to the N atom in -NH-SO 2 -R is further substituted with an alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl.
  • aminosulfonyl examples include alkylaminosulfonyl, cycloalkylaminosulfonyl, alkenylaminosulfonyl, alkynylaminosulfonyl, arylaminosulfonyl, heteroarylaminosulfonyl, aralkylaminosulfonyl, etc.
  • sulfamoylamino examples include alkylsulfamoylamino, cycloalkylsulfamoylamino, alkenylsulfamoylamino, alkynylsulfamoylamino, arylsulfamoylamino, heteroarylsulfamoylamino, aralkylsulfamoylamino, etc.
  • the two H atoms bonded to the N atom in -NH-SO 2 -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.
  • thio examples include alkylthio, cycloalkylthio, alkenylthio, alkynylthio, arylthio, heteroarylthio, and aralkylthio.
  • sulfinyl examples include alkylsulfinyl, cycloalkylsulfinyl, alkenylsulfinyl, alkynylsulfinyl, arylsulfinyl, heteroarylsulfinyl, and aralkylsulfinyl.
  • sulfonyl examples include alkylsulfonyl, cycloalkylsulfonyl, alkenylsulfonyl, alkynylsulfonyl, arylsulfonyl, heteroarylsulfonyl, aralkylsulfonyl, and the like.
  • secondary amino examples include alkylamino, cycloalkylamino, alkenylamino, alkynylamino, arylamino, heteroarylamino, and aralkylamino.
  • tertiary amino examples include alkyl(aralkyl)amino, which is an amino group having any two substituents independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, etc., and these two substituents may form a ring.
  • substituted amidino examples 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, and aralkyl, such as alkyl(aralkyl)(aryl)amidino.
  • substituted guanidino examples include groups in which R, R', R'', and R''' are each independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl, or groups in which these groups form a ring.
  • aminocarbonylamino examples include groups in which R, R', and R'' are each independently selected from a hydrogen atom, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl, or groups in which these form a ring.
  • Substituents containing a B atom include boryl (-BR(R')) and dioxyboryl (-B(OR)(OR')). These two substituents R and R' are each independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, etc., or they may form a ring.
  • At least one atom constituting the "amino acid” constituting the peptide may be an atom (isotope) having the same atomic number (number of protons) but different mass number (sum of the number of protons and neutrons).
  • isotopes contained in the "amino acid" constituting the peptide include hydrogen atom, carbon atom, nitrogen atom, oxygen atom, phosphorus atom, sulfur atom, fluorine atom, and chlorine atom, each of which includes 2H , 3H , 13C , 14C , 15N , 17O , 18O , 31P , 32P, 35S , 18F , and 36Cl .
  • halogen atoms include F, Cl, Br, and I.
  • alkyl refers to a monovalent group derived by removing any one hydrogen atom from an aliphatic hydrocarbon, does not contain heteroatoms (atoms other than carbon and hydrogen atoms) or unsaturated carbon-carbon bonds in the skeleton, and has a subset of hydrocarbyl or hydrocarbon group structures containing hydrogen and carbon atoms.
  • Alkyl includes not only linear but also branched chain alkyls. Specific examples of alkyl include alkyls having 1 to 20 carbon atoms (C 1 to C 20 , hereinafter "C p to C q " means that the number of carbon atoms is p to q), preferably C 1 to C 10 alkyl, and more preferably C 1 to C 6 alkyl.
  • alkyl examples 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-dimethylmethylprop
  • alkylene refers to a divalent group derived by further removing one arbitrary hydrogen atom from the above-defined "alkyl".
  • specific examples of the alkylene include -CH2- , -( CH2 ) 2- , -( CH2 ) 3- , -CH( CH3 ) CH2- , -C( CH3 ) 2- , -( CH2 ) 4- , -CH ( CH3 ) CH2CH2- , -C( CH3 ) 2CH2- , -CH2CH( CH3 ) CH2- , -CH2C (CH3) 2- , -CH2CH2CH(CH3)-, -CH2CH ( CH2CH3 )-, -( CH2 ) 5- , -CH ( CH3 )CH ( CH2CH3 ) - , -( CH2 ) 6- , -( CH2 ) 7- , and -( CH2 ) 8 .
  • alkylene include C 1
  • alkenyl refers to a monovalent group having at least one double bond (two adjacent SP 2 carbon atoms). Depending on the arrangement of the double bond and the substituents (if present), the geometry of the double bond can be in an Entadel (E) or Entumble (Z), cis or trans configuration. Alkenyl includes not only linear but also branched chains.
  • Alkenyl is preferably C 2 -C 10 alkenyl, more preferably C 2 -C 6 alkenyl, and specifically includes, for example, vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl (including cis and trans), 3-butenyl, pentenyl, 3-methyl-2-butenyl, hexenyl, and the like.
  • alkynyl refers to a monovalent group having at least one triple bond (two adjacent SP carbon atoms). Alkynyl includes not only straight chain but also branched chain. Preferred examples of alkynyl include C 2 -C 10 alkynyl, more preferably C 2 -C 6 alkynyl, and specific examples thereof include ethynyl, 1-propynyl, propargyl, 3-butynyl, pentynyl, hexynyl, 3-phenyl-2-propynyl, 3-(2'-fluorophenyl)-2-propynyl, 2-hydroxy-2-propynyl, 3-(3-fluorophenyl)-2-propynyl, and 3-methyl-(5-phenyl)-4-pentynyl.
  • cycloalkyl refers to a saturated or partially saturated cyclic monovalent aliphatic hydrocarbon group, including monocyclic, bicyclic and spirocyclic rings.
  • Preferred examples of cycloalkyl include C3 - C8 cycloalkyl, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[2.2.1]heptyl and spiro[3.3]heptyl.
  • aryl refers to a monovalent aromatic hydrocarbon ring, preferably C 6 to C 10 aryl. Specific examples of aryl include phenyl and naphthyl (e.g., 1-naphthyl, 2-naphthyl).
  • heteroaryl refers to an aromatic cyclic monovalent group containing 1 to 5 heteroatoms in addition to carbon atoms.
  • the ring may be a single ring or a condensed ring with other rings, and may be partially saturated.
  • the number of atoms constituting the ring is preferably 5 to 10 (5- to 10-membered heteroaryl), and more preferably 5 to 7 (5- to 7-membered heteroaryl).
  • heteroaryl examples include 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, and imidazopyridyl
  • alkoxy refers to an oxy group bonded to an "alkyl” as defined above, and preferably includes C 1 to C 6 alkoxy. Specific examples of alkoxy include methoxy, ethoxy, 1-propoxy, 2-propoxy, n-butoxy, i-butoxy, s-butoxy, t-butoxy, pentyloxy, and 3-methylbutoxy.
  • alkenyloxy refers to an oxy group bonded to the above-defined “alkenyl”, and is preferably a C2 - C6 alkenyloxy.
  • alkenyloxy include vinyloxy, allyloxy, 1-propenyloxy, 2-propenyloxy, 1-butenyloxy, 2-butenyloxy (including cis and trans), 3-butenyloxy, pentenyloxy, and hexenyloxy.
  • cycloalkoxy refers to an oxy group bonded to the above-defined “cycloalkyl", and preferably includes C 3 -C 8 cycloalkoxy. Specific examples of cycloalkoxy include cyclopropoxy, cyclobutoxy, cyclopentyloxy, etc.
  • aryloxy refers to an oxy group bonded to the above-defined “aryl”, and is preferably a C 6 -C 10 aryloxy. Specific examples of aryloxy include phenoxy, 1-naphthyloxy, and 2-naphthyloxy.
  • arylalkoxy refers to an oxy group to which the above-defined “arylalkyl (aralkyl)" is bonded, and preferably includes a C 7 -C 12 arylalkoxy group. Specific examples of arylalkoxy include benzyloxy.
  • amino means -NH2 in a narrow sense, and -NRR' in a broad sense, where R and R' are independently selected from hydrogen atom, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, or R and R' form a ring together with the nitrogen atom to which they are attached.
  • R and R' are independently selected from hydrogen atom, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, or R and R' form a ring together with the nitrogen atom to which they are attached.
  • Preferred examples of amino include -NH2 , mono C1 - C6 alkylamino, di C1 - C6 alkylamino, 4- to 8-membered cyclic amino, etc.
  • monoalkylamino refers to a group in which R is a hydrogen atom and R' is an "alkyl” as defined above, among the “amino” as defined above, and preferably includes mono C 1 -C 6 alkylamino.
  • monoalkylamino include methylamino, ethylamino, n-propylamino, i-propylamino, n-butylamino, s-butylamino, t-butylamino, etc.
  • dialkylamino refers to a group in which R and R' are independently “alkyl” as defined above among “amino” as defined above, and preferably includes diC 1 -C 6 alkylamino. Specific examples of dialkylamino include dimethylamino and diethylamino.
  • aminoalkyl refers to a group in which one or more hydrogen atoms of an "alkyl” as defined above are substituted with an "amino” as defined above, and is preferably a C 1 -C 6 aminoalkyl.
  • Specific examples of aminoalkyl include 1-pyridylmethyl, 2-(1-piperidyl)ethyl, 3-(1-piperidyl)propyl, and 4-aminobutyl.
  • aralkyl refers to a group in which at least one hydrogen atom of an "alkyl” as defined above is substituted with an "aryl” as defined above, and is preferably a C 7 -C 14 aralkyl, more preferably a C 7 -C 10 aralkyl.
  • aralkyl include benzyl, phenethyl, and 3-phenylpropyl.
  • alkylaryl refers to a group in which at least one hydrogen atom of an "aryl” as defined above is replaced with an “alkyl” as defined above.
  • alkylaryl include ortho-tolyl, meta-tolyl, and para-tolyl.
  • alkylaryl examples include C 7 -C 12 alkylaryl, with C 7 -C 10 alkylaryl being preferred, C 7 -C 9 alkylaryl being more preferred, and C 7 -C 8 alkylaryl being most preferred.
  • unnatural amino acids can be found in International Publication No. WO 2013/100132 and International Publication No. WO 2018/143145.
  • Specific examples of unnatural amino acids include Ser(tBuOH), Ser(iPen), MeSer(iPen), Ser(nPr), Ser(NtBu-Aca), MeSer(tBuOH), MeSer(nPr), MeCys(StBu), MeGly, nBuGly, MeOEtGly, MeOPrGly, PhPrGly, PhOEtGly, Nle, Ser(3F5Me
  • Examples of unnatural amino acids include Pyr), Ser(Ph2Cl), MePhe, MeHph, MeAla, MeAla(3Pyr), Pic(2), D-Nva, D-Ser(Me), Hph, D-Hph, D-Ser(Ph), D-MeAbu, D-MeSer, Pro4RBn, Hyp(Ph), MePhe(3-C
  • At least one atom constituting the "amino acid” constituting the peptide may be an atom (isotope) having the same atomic number (number of protons) but different mass number (sum of the number of protons and neutrons).
  • isotopes contained in the "amino acid" constituting the peptide include hydrogen atom, carbon atom, nitrogen atom, oxygen atom, phosphorus atom, sulfur atom, fluorine atom, and chlorine atom, each of which includes 2H , 3H , 13C , 14C , 15N , 17O , 18O , 31P , 32P, 35S , 18F , and 36Cl .
  • One embodiment of the present invention is a method for producing a peptide , in which at least one of a first unnatural amino acid and a second unnatural amino acid satisfies the following requirement (A) and is an unnatural amino acid (unnatural amino acid U1) that satisfies at least one selected from the group consisting of the following requirements (B) and (C), and includes a step of translating a nucleic acid that includes adjacent codons encoding the first unnatural amino acid and codons encoding the second unnatural amino acid.
  • a nitrogen atom (nitrogen atom N1) constituting a main chain amino group and an oxygen atom (oxygen atom O1) other than the oxygen atom constituting a main chain carboxy group are bonded via two atoms.
  • the oxygen atom O1 constitutes an alkoxy group or an aryloxy group.
  • the nitrogen atom N1 is bonded to a linear or branched C 1 -C 6 alkyl group.
  • Peptide refers to two or more amino acids linked together by an amide bond. Peptides having an ester bond in a part of the main chain, such as depsipeptides, are also included in the term "peptide” as used herein. Without intending to be limiting, peptides in the present disclosure include linear peptides and cyclic peptides. In addition, peptides may be in the form of their pharma- ceutically acceptable salts.
  • a peptide has 2 to 100, 3 to 50, 4 to 30, or 5 to 30 amino acids linked by amide bonds and/or ester bonds.
  • the number of amino acids constituting the peptide is 30 or less, 20 or less, and 16 or less.
  • the number of amino acids constituting the peptide is 5 or more, 8 or more, and 9 or more.
  • the number of amino acids constituting the peptide is 5 to 30, 8 to 20, 8 to 16, and 9 to 16.
  • the number of amino acids constituting the peptide herein is, for example, 5 to 30, preferably 8 to 20, more preferably 8 to 16, and most preferably 9 to 16.
  • a "cyclic peptide” is a peptide having a cyclic structure composed of four or more amino acid residues. Such a cyclic structure is also referred to as a "cyclic portion.”
  • the cyclization of a cyclic peptide may be in any form, such as cyclization by a carbon-nitrogen bond such as an amide bond, cyclization by a carbon-oxygen bond such as an ester bond or an ether bond, cyclization by a carbon-sulfur bond such as a thioether bond, cyclization by a carbon-carbon bond, or cyclization by constructing a heterocyclic ring.
  • cyclization via a covalent bond such as an amide bond, a carbon-sulfur bond, or a carbon-carbon bond is preferred.
  • Cyclization via an amide bond is more particularly preferred, and the position of the carboxyl group or amino group used for cyclization may be on the main chain or on the side chain. More preferably, cyclization via an amide bond between a carboxyl group on the side chain and an amino group on the main chain at the N-terminus is preferred.
  • the cyclic structure is different from the cyclic structures (e.g., benzene ring, pyrrolidine ring) contained in phenylalanine, tyrosine, proline, etc.
  • the number of amino acids constituting the cyclic portion of a cyclic peptide is not limited, but examples include 5 or more, 6 or more, 8 or more, and 9 or more. Other examples include 30 or less, 20 or less, and 16 to 11 or less.
  • the number of amino acid residues constituting the cyclic portion is preferably 5 to 20, more preferably 8 to 16, and most preferably 9 to 16. In one embodiment, the total number of amino acid residues constituting the cyclic portion of the cyclic peptide is 5 to 30, and of these, 5 to 15 amino acid residues form the cyclic structure.
  • the total number of amino acid residues constituting the cyclic peptide is, for example, 5 to 30, preferably 8 to 20, more preferably 8 to 16, and most preferably 9 to 16.
  • the cyclic peptide may have a linear portion.
  • the term "linear portion" used to refer to a partial structure of a cyclic peptide refers to a portion that is not included in the main chain structure of the cyclic portion and has at least one amide bond and/or ester bond on the chain of the portion.
  • the linear portion is preferably one in which two or more amino acid residues are linked by amide bonds. In this case, as in depsipeptides, an ester bond may be included in a part of the main chain.
  • the number of amino acid residues (number of units) in the linear portion is, for example, 0 to 8, preferably 0 to 8, more preferably 0 to 5, and most preferably 0 to 3.
  • the linear portion in this specification may include natural amino acids and non-natural amino acids (including amino acids that have been chemically modified or skeletal-converted).
  • the peptide includes a peptide having one or more N-substituted amino acid residues.
  • the number of N-substituted amino acid residues in the peptide is, for example, 1 to 13, preferably 3 to 12, more preferably 4 to 11, and most preferably 5 to 10.
  • the peptide has a ClogP of, for example, 4 to 25, preferably 6 to 23, more preferably 8 to 21, and most preferably 9 to 20.
  • ClogP is a computer-calculated partition coefficient and can be determined according to the principles described in the CLOGP Reference Manual Daylight Version 4.9 (Release Date: August 1, 2011, https://www.daylight.com/dayhtml/doc/clogp/).
  • One method for calculating ClogP is by using Daylight Chemical Information Systems, Inc.'s Daylight Version 4.95 (Release Date: August 1, 2011, ClogP algorithm version 5.4, database version 28, https://www.daylight.com/dayhtml/doc/release_notes/index.html).
  • the ClogP of the peptide is, for example, 28% to 174%, preferably 42% to 160%, more preferably 56% to 146%, and most preferably 63% to 139% of the ClogP value of cyclosporine A (ClogP: 14.36).
  • the ClogP (ClogP/number of amino acid residues) of the peptide per constituent amino acid residue is 1.0 or more, preferably 1.1 or more.
  • the upper limit of the ClogP/number of amino acid residues of the peptide according to this embodiment is preferably 1.8 or less, more preferably 1.7 or less, even more preferably 1.6 or less, and even more preferably 1.5 or less.
  • the range of the ClogP/number of amino acid residues of the peptide according to this embodiment is, for example, 1.0 to 1.8, preferably 1.0 to 1.7, more preferably 1.1 to 1.6, and most preferably 1.1 to 1.5.
  • the peptide has a molecular weight of, for example, 500 g/mol to 2000 g/mol, preferably 1000 g/mol to 1800 g/mol, more preferably 1300 g/mol to 1600 g/mol, and most preferably 1400 g/mol to 1500 g/mol.
  • the molecular weight in this specification means the sum of the atomic weights of the atoms constituting the compound molecule (unit: "g/mol"), and is obtained by calculating the sum of the atomic weights of the atoms included in the molecular structure (unit: "g/mol”). In this specification, the unit of molecular weight may be omitted.
  • the molecular weight of the peptide according to this embodiment can be measured by any method known in the art, preferably by liquid chromatography, and more preferably by liquid chromatography mass spectrometry (LC/MS) as described in the examples.
  • the number of unnatural amino acids contained in the peptide is 2 or more, 4 or more, 5 or more, 6 or more, 7 or more, or 8 or more. Also, 20 or less, 15 or less, 14 or less, 13 or less, 12 or less, 10 or less, or 9 or less.
  • the number of unnatural amino acids contained in the peptide in the present disclosure is 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, or 80% or more of the number of amino acids constituting the cyclic portion of the cyclic peptide.
  • the types of unnatural amino acids contained in the peptide in the present disclosure are 1 or more, 2 or more, 3 or more, 4 or more, 7 or more, or 8 or more, and also 20 or less, 15 or less, 14 or less, 13 or less, 12 or less, 10 or less, or 9 or less.
  • the number of unnatural amino acids contained in the peptide is, for example, 1 to 13, preferably 3 to 12, more preferably 4 to 11, and most preferably 5 to 10.
  • the sites containing the unnatural amino acid are not limited, but in one embodiment, the unnatural amino acid can be included at the position of the starting amino acid, and further, the unnatural amino acid can be included at the position of the second and/or third amino acid.
  • the peptide produced by the method according to this embodiment has an amino acid sequence including a first unnatural amino acid and a second unnatural amino acid adjacent to each other. Furthermore, at least one of the first unnatural amino acid and the second unnatural amino acid is an unnatural amino acid (hereinafter also referred to as "unnatural amino acid U1") that satisfies the above requirement (A) and at least one selected from the group consisting of the above requirements (B) and (C).
  • the peptide may include any unnatural amino acid in addition to the above first unnatural amino acid and second unnatural amino acid.
  • the nitrogen atom constituting the main chain amino group (hereinafter also referred to as “nitrogen atom N1”) and an oxygen atom other than the oxygen atom constituting the main chain carboxy group (hereinafter also referred to as “oxygen atom O1”) are bonded via two atoms” means that in the chemical structure of the amino acid, the oxygen atom O1 is bonded via two atoms counting from the nitrogen atom constituting the main chain amino group. In this case, the nitrogen atom constituting the main chain amino group is not included in the count.
  • the oxygen atom constituting the main chain carboxy group refers to the oxygen atom constituting the carboxy group corresponding to the C-terminus of the amino acid, and is not the carboxy group present in the side chain of glutamic acid, aspartic acid, etc.
  • General formulas (i) to (iii) are schematic diagrams for explaining requirement (A), in which X represents an oxygen atom O1, R A , R B , R C , and R D represent optional substituents, and p represents an optional integer of 1 or more.
  • the unnatural amino acid U1 is not limited to these general formulas, and can be understood with reference to these specific examples.
  • General formula (i) shows N-substituted glycine, in which the third atom X, counting from the nitrogen atom (indicated as NH) constituting the main chain amino group, corresponds to the oxygen atom O1.
  • General formula (ii) shows N,O-disubstituted-D-serine, in which the third atom X, counting from the nitrogen atom (indicated as NH) constituting the main chain amino group, corresponds to the oxygen atom O1.
  • the number of atoms between the nitrogen atom N1 and the oxygen atom O1 can be multiple, depending on the counting method, but in that case, the smallest number is used. In other words, the number of atoms is counted so that the nitrogen atom N1 and the oxygen atom O1 are at the shortest distance.
  • the oxygen atom O1 constitutes an alkoxy group or an aryloxy group
  • the oxygen atom O1 is an oxy group.
  • the oxygen atom O1 is not an oxygen atom that constitutes a hydroxyl group, a carbonyl group, a carboxy group, a nitro group, etc.
  • the nitrogen atom N1 is bonded to a linear or branched C 1 -C 6 alkyl group
  • the amino group at the N-terminus is not an unsubstituted amino group (-NH 2 ) but is an amino group substituted with a linear or branched C 1 -C 6 alkyl group.
  • R A and R B are linear or branched C 1 -C 6 alkyl groups.
  • the nitrogen atom N1 is an unsubstituted amino group or is substituted with a methyl or ethyl group. In a preferred embodiment, the nitrogen atom N1 is substituted with a methyl group.
  • one of the first unnatural amino acid and the second unnatural amino acid is the unnatural amino acid U1, and the other is an unnatural amino acid different from the unnatural amino acid U1 (hereinafter also referred to as "unnatural amino acid U2").
  • the unnatural amino acid U2 may be any unnatural amino acid that is not identical to the unnatural amino acid U1.
  • both the first unnatural amino acid and the second unnatural amino acid may be the unnatural amino acid U1.
  • the unnatural amino acid U1 may satisfy both of the above requirements (A) and (B), may satisfy both of the above requirements (A) and (C), or may satisfy all of the above requirements (A), (B), and (C).
  • the unnatural amino acid U1 may be a D- ⁇ -amino acid, a D-amino acid, or an L-amino acid (but not a natural amino acid).
  • the oxygen atom O1 may be present in a substituent bonded to the nitrogen atom N1.
  • An example of this case is an amino acid represented by the above general formula (i).
  • the oxygen atom O1 may be present in the side chain of the unnatural amino acid U1.
  • An example of this case is an amino acid represented by the general formula (ii) above.
  • the nitrogen atom N1, the carbon atoms constituting the main chain of the unnatural amino acid U1, and the atoms constituting the side chain of the unnatural amino acid U1 may together form a ring.
  • An example of this case is an amino acid represented by the above general formula (iii).
  • the oxygen atom O1 may be combined with a linear or branched C 1 -C 6 alkyl group, a C 3 -C 10 cycloalkyl group, a C 6 -C 10 aryl group, a C 7 -C 12 arylalkyl group, or a C 7 -C 12 alkylaryl group to form a corresponding C 1 -C 6 alkoxy group, a C 3 -C 10 cycloalkoxy group, a C 6 -C 10 aryloxy group, a C 7 -C 12 arylalkyloxy group, or a C 7 -C 12 alkylaryloxy group.
  • the oxygen atom O1 may be combined with a linear or branched C 1 -C 6 alkyl group, a C 3 -C 10 cycloalkoxy group, a C 6 -C 10 aryloxy group, a C 7 -C 12 arylalkyloxy group, or a C 7 -C 12 alkylaryloxy group.
  • the oxygen atom O1 may be combined with a methoxy group, an ethoxy group, a phenoxy group, or a benzyloxy group.
  • the oxygen atom O1 may be combined with a methoxy group or a phenoxy group.
  • the oxygen atom O1 may form, together with a linear or branched C 1 -C 6 alkyl group, a corresponding C 1 -C 6 alkoxy group.
  • the oxygen atom O1 may form a methoxy or ethoxy group. In a more preferred embodiment, it may form a methoxy group.
  • the oxygen atom O1 may be combined with a C 6 -C 10 aryl group to form a corresponding C 6 -C 10 aryloxy group. In a preferred embodiment, the oxygen atom O1 may be combined with a hydrogen atom to form a corresponding hydroxy group.
  • the unnatural amino acid U1 does not contain heteroatoms other than nitrogen and oxygen atoms.
  • heteroatoms include sulfur atoms.
  • one of the first and second unnatural amino acids is an unnatural amino acid U1
  • the other is an unnatural amino acid U2 in which the nitrogen atom constituting the main chain amino group of the unnatural amino acid U2 is substituted with a linear or branched C 1 -C 6 alkyl group, preferably a methyl or ethyl group, and more preferably a methyl group.
  • one of the first and second unnatural amino acids is an unnatural amino acid U1 and the other is an unnatural amino acid U2, where the unnatural amino acid U2 has as its side chain a linear or branched C 1 -C 6 alkyl group optionally substituted with a 5-10 membered aryl group, or a 5-10 membered aryl group optionally substituted with a C 1 -C 6 linear or branched alkyl group, preferably the unnatural amino acid U2 has as its side chain a 5-10 membered aryl group substituted with a linear or branched C 1 -C 4 alkyl group, or a C 1 -C 3 linear alkyl group, more preferably the unnatural amino acid U2 has as its side chain a methyl group or a benzyl group.
  • one of the first unnatural amino acid and the second unnatural amino acid is an unnatural amino acid U1, and the other is an unnatural amino acid U2, where the unnatural amino acid U2 is an alpha amino acid.
  • the unnatural amino acid U2 is an alpha amino acid in which the nitrogen atom constituting the main chain amino group is substituted with a methyl group and has a methyl group as a side chain.
  • the unnatural amino acid U2 is an alpha amino acid in which the nitrogen atom constituting the main chain amino group is substituted with a methyl group and has a benzyl group as a side chain.
  • the first unnatural amino acid and the second unnatural amino acid are adjacent to each other, and the first unnatural amino acid may be at the N-terminus and the second unnatural amino acid at the C-terminus, or the second unnatural amino acid may be at the N-terminus and the first unnatural amino acid at the C-terminus.
  • the unnatural amino acid U1 may be at the N-terminus and the unnatural amino acid U2 may be at the C-terminus, or the unnatural amino acid U2 may be at the N-terminus and the unnatural amino acid U1 may be at the C-terminus.
  • the first unnatural amino acid is an unnatural amino acid U1
  • the second unnatural amino acid is an unnatural amino acid U2
  • the unnatural amino acid U1 satisfies requirements (A) and (B)
  • the oxygen atom O1 is present in a substituent substituted with a nitrogen atom N1
  • the oxygen atom O1 constitutes a methoxy group or a phenoxy group
  • the codon encoding the first unnatural amino acid is translated before the codon encoding the second unnatural amino acid.
  • the method for producing a peptide according to this embodiment is such that the first unnatural amino acid is an unnatural amino acid U1, the second unnatural amino acid is an unnatural amino acid U2, the unnatural amino acid U1 satisfies requirements (A) and (B), the oxygen atom O1 is contained in the side chain of the unnatural amino acid U1, the oxygen atom O1 constitutes a methoxy group or a phenoxy group, the nitrogen atom N1 constitutes an unsubstituted amino group, and the codon encoding the first unnatural amino acid is translated before the codon encoding the second unnatural amino acid.
  • the method for producing a peptide according to this embodiment is such that the first unnatural amino acid is an unnatural amino acid U1, the second unnatural amino acid is an unnatural amino acid U2, the unnatural amino acid U1 satisfies requirements (A) and (C), the oxygen atom O1 is included in the side chain of the unnatural amino acid U1, the oxygen atom O1 constitutes a hydroxyl group, the nitrogen atom N1 is substituted with a methyl group or an ethyl group, and the codon encoding the first unnatural amino acid is translated before the codon encoding the second unnatural amino acid.
  • the method for producing a peptide according to this embodiment is such that the first unnatural amino acid is an unnatural amino acid U1, the second unnatural amino acid is an unnatural amino acid U2, the unnatural amino acid U1 satisfies requirements (A) and (B), the nitrogen atom N1, the carbon atoms constituting the main chain of the unnatural amino acid U1, and the atoms constituting the side chain of the unnatural amino acid U1 together form a ring, the oxygen atom O1 forms a methoxy group or a phenoxy group, and the codon encoding the first unnatural amino acid is translated before the codon encoding the second unnatural amino acid.
  • the method for producing a peptide includes a step of translating a nucleic acid containing adjacent codons encoding a first unnatural amino acid and a second unnatural amino acid, the first unnatural amino acid being unnatural amino acid U1, the second unnatural amino acid being unnatural amino acid U2, the unnatural amino acid U1 being an ⁇ -amino acid, the nitrogen atom constituting the main chain amino group (nitrogen atom N1) being bonded to an oxygen atom other than the oxygen atom constituting the main chain carboxyl group (oxygen atom O1) via two or three atoms, the oxygen atom O1 being included in the side chain of the unnatural amino acid U1, the oxygen atom O1 being a hydroxyl group, the nitrogen atom N1 being substituted with a methyl group or an ethyl group, and the codon encoding the first unnatural amino acid being translated before the codon encoding the second unnatural amino acid.
  • the first unnatural amino acid is an unnatural amino acid U1
  • the second unnatural amino acid is an unnatural amino acid U2
  • the unnatural amino acid U1 satisfies requirements (A) and (B)
  • the oxygen atom O1 is present in a substituent substituted with a nitrogen atom N1
  • the oxygen atom O1 constitutes a methoxy group or a phenoxy group
  • the codon encoding the first unnatural amino acid is translated after the codon encoding the second unnatural amino acid.
  • the method for producing a peptide according to this embodiment is such that the first unnatural amino acid is an unnatural amino acid U1, the second unnatural amino acid is an unnatural amino acid U2, the unnatural amino acid U1 satisfies requirements (A) and (B), the oxygen atom O1 is contained in the side chain of the unnatural amino acid U1, the oxygen atom O1 constitutes a methoxy group or a phenoxy group, the nitrogen atom N1 constitutes an unsubstituted amino group, and the codon encoding the first unnatural amino acid is translated after the codon encoding the second unnatural amino acid.
  • the method for producing a peptide according to this embodiment is such that the first unnatural amino acid is an unnatural amino acid U1, the second unnatural amino acid is an unnatural amino acid U2, the unnatural amino acid U1 satisfies requirements (A) and (C), the oxygen atom O1 is included in the side chain of the unnatural amino acid U1, the oxygen atom O1 constitutes a hydroxyl group, the nitrogen atom N1 is substituted with a methyl group or an ethyl group, and the codon encoding the first unnatural amino acid is translated after the codon encoding the second unnatural amino acid.
  • the first unnatural amino acid is an unnatural amino acid U1
  • the second unnatural amino acid is an unnatural amino acid U2
  • the unnatural amino acid U1 satisfies requirements (A) and (B)
  • the nitrogen atom N1 the carbon atoms constituting the main chain of the unnatural amino acid U1
  • the atoms constituting the side chain of the unnatural amino acid U1 together form a ring
  • the oxygen atom O1 forms a methoxy group or a phenoxy group
  • the codon encoding the first unnatural amino acid is translated after the codon encoding the second unnatural amino acid.
  • At least one of the first unnatural amino acid and the second unnatural amino acid is the unnatural amino acid U1, where the unnatural amino acid U1 is an unnatural amino acid of formula (I):
  • n is an integer of 0 or more and 2 or less, preferably an integer of 0 or more and 2 or less, more preferably an integer of 0 or more and 1 or less, and most preferably 0.
  • R 1 is (a) a group represented by formula (II), (b) a hydrogen atom, or (c) a linear or branched C 1 -C 6 alkyl group. In certain embodiments, R 1 is not a hydrogen atom.
  • R 4 is a C 1 -C 2 alkylene group.
  • R 5 is a hydrogen atom, a linear or branched C 1 -C 6 alkyl group, a C 3 -C 10 cycloalkyl group, a C 6 -C 10 aryl group, a C 7 -C 12 arylalkyl group, or a C 7 -C 12 alkylaryl group, preferably a methyl group, an ethyl group, a phenyl group, or a benzyl group, and more preferably a methyl group or a phenyl group.
  • R5 is a linear or branched C1-C6 alkyl group, preferably a methyl or ethyl group, more preferably a methyl group.
  • R5 is a C6 - C10 aryl group, preferably a phenyl group.
  • R5 is a hydrogen atom.
  • R2 and R3 are each independently a group represented by formula (II), a hydrogen atom, or a linear or branched C1 - C4 alkyl group, preferably a group represented by formula (II) or a hydrogen atom. In one embodiment, at least one of R2 and R3 may be a group represented by formula (II).
  • R 2b and R 3b each independently represent a hydrogen atom or a linear or branched C 1 -C 4 alkyl group, preferably a hydrogen atom.
  • R2 and R2b or R3 and R3b may form a ring together with the carbon atoms to which they are attached.
  • At least one of R 1 , R 2 and R 3 is a group represented by formula (II), and N 1 (nitrogen atom N1) in formula (I) and O 1 (oxygen atom O1) in formula (II) are bonded via two atoms, where N 1 represents a nitrogen atom and O 1 represents an oxygen atom.
  • R4 and R2 may form a ring together with the carbon atom to which R2 is bonded and N1 .
  • An example of this case is an amino acid represented by the above general formula (iii).
  • R2 is a substituent represented by formula (II)
  • at least one of R1 in formula (I) and R5 in formula (II) is not a hydrogen atom.
  • R1 in formula (I) is not a hydrogen atom.
  • R5 in formula (II) is not a hydrogen atom.
  • neither R1 in formula (I) nor R5 in formula (II) is a hydrogen atom.
  • n 2 or more, a plurality of R3 and R3b may be present.
  • each may be independently the same or different from each other, and when a plurality of R3b are present, each may be independently the same or different from each other.
  • the unnatural amino acid U1 is an amino acid represented by the following formula (III), (IV) or (V). In one embodiment, the unnatural amino acid U1 is an amino acid represented by the following formula (III). In one embodiment, the unnatural amino acid U1 is an amino acid represented by the following formula (IV). In one embodiment, the unnatural amino acid U1 is an amino acid represented by the following formula (V). In formulas (III), (IV) and (V), R 1b is a hydrogen atom or a linear or branched C 1 -C 6 alkyl, and R 5 has the same meaning as R 5 in formula (II).
  • the unnatural amino acid U1 is an amino acid represented by formula (III), (IVb) or (Vb). In one embodiment, the unnatural amino acid U1 is an amino acid represented by the following formula (IVb). In one embodiment, the unnatural amino acid U1 is an amino acid represented by the following formula (Vb). In formulas (III), (IVb) and (Vb), R 1b is a hydrogen atom or a linear or branched C 1 -C 6 alkyl, and R 5 has the same meaning as R 5 in formula (II).
  • R1 and R1b are methyl or ethyl groups, preferably methyl groups. In one embodiment, R1 and R1b are hydrogen atoms.
  • R 5 is a linear or branched C 1 -C 6 alkyl group, a C 3 -C 10 cycloalkyl group, a C 6 -C 10 aryl group, a C 7 -C 12 arylalkyl group, or a C 7 -C 12 alkylaryl group, and R 1 and R 1b are hydrogen atoms.
  • R 5 is a linear or branched C 1 -C 6 alkyl group, a C 6 -C 10 aryl group, or a C 7 -C 12 arylalkyl group, and R 1 and R 1b are hydrogen atoms.
  • R 5 is a methyl group, an ethyl group, a phenyl group, or a benzyl group, and R 1 and R 1b are hydrogen atoms. In a further preferred embodiment, R 5 is a methyl group or a phenyl group, and R 1 and R 1b are hydrogen atoms.
  • one of the first unnatural amino acid and the second unnatural amino acid is an unnatural amino acid U1 and the other is an unnatural amino acid U2, wherein the unnatural amino acid U2 has formula (VI):
  • m is an integer of 0 to 2, preferably 0 or 1, and most preferably 0.
  • R 1c is a hydrogen atom or a linear or branched C 1 -C 6 alkyl group, preferably a linear or branched C 1 -C 6 alkyl group, more preferably a methyl group or an ethyl group, and particularly preferably a methyl group.
  • R 2c and R 3c are each independently a hydrogen atom, a linear or branched C 1 -C 6 alkyl group optionally substituted with a 5-10 membered aryl group, or a 5-10 membered aryl group optionally substituted with a C 1 -C 6 linear or branched alkyl group, preferably a linear or branched C 1 -C 6 alkyl group optionally substituted with a 5-10 membered aryl group, or a 5-10 membered aryl group optionally substituted with a C 1 -C 6 linear or branched alkyl group, more preferably a linear or branched C 1 -C 4 alkyl group, or a 5-10 membered aryl group substituted with a C 1 -C 3 linear alkyl group.
  • R 2c and R 3c are each independently a methyl group or a benzyl group. In one embodiment, R 2c and R 3c are a methyl group. In one embodiment, R 2c and R 3c are a benzyl group.
  • R 2bc and R 3bc are each independently a hydrogen atom or a linear or branched C 1 -C 4 alkyl group, preferably a hydrogen atom.
  • R 2c and R 2bc or R 3c and R 3bc may form a ring together with the carbon atoms to which they are attached.
  • the nitrogen atom to which R 1c is bonded and R 1c , and the carbon atom to which R 2c is bonded and R 2c may together form a ring.
  • R 3 and R 3b there may be two each of R 3 and R 3b .
  • R 3c When there are multiple R 3c , they may be independently the same or different from each other, and when there are multiple R 3bc , they may be independently the same or different from each other.
  • R 1c and R 2c are methyl groups and m is 0. In another embodiment, R 1c is a methyl group, R 2c is a benzyl group and m is 0.
  • the method for producing a peptide according to this embodiment includes a step of translating a nucleic acid that includes adjacent codons encoding a first unnatural amino acid and a second unnatural amino acid.
  • the codon encoding the first unnatural amino acid may be translated before the codon encoding the second unnatural amino acid, or the codon encoding the first unnatural amino acid may be translated after the codon encoding the second unnatural amino acid. That is, either the codon encoding the first unnatural amino acid or the codon encoding the second unnatural amino acid may be located at the 5' end of the mRNA.
  • the codon encoding the unnatural amino acid U1 may be translated before the codon encoding the unnatural amino acid U2, or the codon encoding the unnatural amino acid U1 may be translated after the codon encoding the unnatural amino acid U2. That is, either of the codons encoding the unnatural amino acid U1 and the unnatural amino acid U2 may be located on the 5' end of the mRNA.
  • the codon encoding the unnatural amino acid U1 is translated before the codon encoding the unnatural amino acid U2.
  • a peptide is produced by translating an mRNA that codes for a peptide containing a specific unnatural amino acid.
  • 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 (a sequence of three nucleotides), and these codons usually correspond to all 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 the like may also be used as initiation codons in addition to AUG.
  • AUG is a codon that codes for methionine (Met), and in eukaryotes and archaea, translation begins directly from methionine.
  • initiation codon AUG corresponds to N-formylmethionine (fMet), so translation begins from formylmethionine.
  • stop codons There are three types of stop codons: UAA (ochre), UAG (amber), and UGA (opal).
  • RF translation release factor
  • an mRNA that encodes a peptide containing a specific unnatural amino acid can be exemplified by one that contains a codon that encodes a peptide containing an unnatural amino acid at least at the position of the start amino acid.
  • iSP initiation suppression
  • methionine is generally translated as the N-terminal amino acid as the translation initiation amino acid.
  • a dedicated "initiator tRNA" is used to initiate translation, and translation is initiated when the initiator tRNA binds to methionine (formylmethionine in prokaryotes) and is transported to the ribosome, whereupon the N-terminal amino acid becomes methionine (formylmethionine in prokaryotes).
  • the initiation suppression method translates a peptide with a desired amino acid at its N-terminus by removing the initiator tRNA with methionine aminoacylated from the translation system (or preventing it from being produced) and adding an initiator tRNA with a previously prepared aminoacylated desired amino acid to the translation system instead.
  • the tolerance of unnatural amino acids is higher during introduction to the N-terminus than during amino acid elongation, and unnatural amino acids with structures significantly different from those of natural amino acids can be used as N-terminal amino acids (see non-patent literature: J Am Chem Soc. 2009 Apr 15;131(14):5040-1. Translation initiation with initiator tRNA charged with exotic peptides. Goto Y, Suga H.).
  • the initiator tRNA contained in the translation system may be acylated with an unnatural amino acid.
  • a “translation system” is defined as a composition for translating a peptide.
  • the “translation system” in this specification is preferably a combination of protein factors involved in translation, tRNA, amino acids, energy sources such as ATP, and their regeneration systems, and is capable of translating mRNA into protein.
  • a system during which translation is in progress may also be included in the “translation system” in this specification.
  • the translation system in this specification may include a nucleic acid that serves as a template for translating a peptide, and may also include an initiation factor, an elongation factor, a release factor, an aminoacyl-tRNA synthetase, and the like. These factors can be obtained by purifying them from extracts of various cells.
  • Examples of cells for purifying factors include prokaryotic cells or eukaryotic cells.
  • Examples of prokaryotic cells include Escherichia coli cells, extremely thermophilic bacteria cells, and Bacillus subtilis cells.
  • Examples of eukaryotic cells include those using yeast cells, wheat germ, rabbit reticulocytes, plant cells, insect cells, or animal cells as materials.
  • tRNA and aminoacyl-tRNA synthetase ARS
  • artificial tRNA and artificial aminoacyl-tRNA synthetase that recognizes unnatural amino acids can be used.
  • By using artificial tRNA and artificial aminoacyl-tRNA synthetase a peptide with an unnatural amino acid introduced site-specifically can be synthesized.
  • transcription from template DNA can be performed by adding an RNA polymerase such as T7 RNA polymerase to the translation system.
  • the translation process in this embodiment may be carried out in a cell-free translation system.
  • the cell-free translation system may be a reconstituted cell-free translation system, and specifically, a translation system composed of factors derived from E. coli may be used.
  • the cell-free translation system may also include ribosomes derived from E. coli.
  • the cell-free translation system preferably includes a tRNA having an anticodon complementary to a codon encoding a first unnatural amino acid, and also includes a tRNA having an anticodon complementary to a codon encoding a second unnatural amino acid.
  • the main types of translation systems include those that use living cells and those that use cell extracts (cell-free translation systems).
  • a translation system that uses living cells a system in which the desired aminoacyl-tRNA and mRNA are introduced into living cells such as Xenopus oocytes or mammalian cells by microinjection or lipofection to perform peptide translation (Nowak et al., Science (1995) 268: 439-442).
  • Examples of cell-free translation systems include 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), and rabbit reticulocytes (Jackson Known translation systems use extracts from human cells (Barton 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).
  • Cell-free translation systems can be prepared appropriately by methods known to those skilled in the art or methods equivalent thereto.
  • Cell-free translation systems also include translation systems constructed by isolating and purifying factors necessary for peptide translation and reconstituting them (reconstituted cell-free translation systems) (Shimizu et al., Nat Biotech (2001) 19: 751-755).
  • the reconstituted cell-free translation system may typically include ribosomes, amino acids, tRNA, aminoacyl-tRNA synthetase (aaRS), 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 factors (RRF), NTPs as an energy source, an energy regeneration system, and other factors necessary for translation.
  • 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.
  • a reconstituted cell-free translation system can be appropriately constructed using them.
  • commercially available reconstituted cell-free translation systems such as PUREfrex (registered trademark) from Gene Frontier and PURExpress (registered trademark) from New England BioLabs can also be used.
  • PUREfrex registered trademark
  • PURExpress registered trademark
  • the desired translation system can be constructed by reconstituting only the necessary components among the components of the translation system.
  • PURESYSTEM (registered trademark) (BioComber, Japan) is a reconstituted cell-free translation system in which protein factors, energy regeneration enzymes, and ribosomes required for translation in E. coli are extracted and purified, and then mixed with tRNA, amino acids, ATP, GTP, etc. Not only does it contain a small amount of impurities, but because it is a reconstituted system, it is easy to create a system that does not contain protein factors or amino acids that you want to eliminate ((i) Nat Biotechnol. 2001; 19: 751-5. Cell-free translation reconstituted with purified components.
  • translation synthesis involves, for example, the synthesis of protein factors necessary for translation in E. coli (methionyl-tRNA transformylase, EF-G, RF1, RF2, RF3, RRF, IF1, IF2, IF3, EF-Tu, EF-Ts, ARS (AlaRS, ArgRS, AsnRS, AspRS, CysRS, GlnRS, GluRS, GlyRS, HisRS, IleRS, LeuRS, LysRS, MetRS, PheRS, ProRS, SerRS, ThrRS, TrpRS, TyrRS, This can be done by adding mRNA to a known cell-free translation system such as the PURESYSTEM, which is a mixture of an appropriate selection of components such as ribosomes, amino acids, creatine kinase, myokinase, inorganic pyrophosphatase, nucleoside diphosphate kinase, E.
  • PURESYSTEM is a mixture of an appropriate selection of components such
  • coli-derived tRNA creatine phosphate, potassium glutamate, HEPES-KOH pH 7.6, magnesium acetate, spermidine, dithiothreitol, GTP, ATP, CTP, UTP, etc.
  • T7 RNA polymerase by adding T7 RNA polymerase, transcription and translation from a template DNA containing a T7 promoter can be coupled.
  • peptides containing unnatural amino acids can be translated and synthesized by adding to the system the desired aminoacyl-tRNA group or an unnatural amino acid group (e.g., F-Tyr) that is acceptable to aminoacyl-tRNA synthetase (ARS) (Kawakami T, et al.
  • F-Tyr unnatural amino acid group
  • mRNAs encoding peptides containing unnatural amino acids can be translated by including a modified ARS in the system in place of or in addition to the natural ARS and including an unnatural amino acid group in the system.
  • the efficiency of translation of mRNA encoding peptides containing unnatural amino acids and the associated incorporation of unnatural amino acids can be increased by using mutants of ribosomes such as EF-Tu (Dedkova LM, et al. Construction of modified ribosomes for incorporation of D-amino acids into proteins. Biochemistry 2006, 45, 15541-51., Doi Y, et al. 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.
  • the translation system may also include a ribosome containing the modified L31 protein described in WO 2021/117848.
  • the ribosome containing the modified L31 protein is preferably contained in a ratio of at least 50% or more, preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, and particularly preferably 90% or more (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more) in terms of the number of molecules relative to the total ribosomes contained in the translation system.
  • total ribosomes is not particularly limited as long as it is a ribosome contained in the translation system, but can be exemplified by the sum of ribosomes containing the modified L31 protein and other ribosomes (e.g., ribosomes containing Escherichia coli wild-type L31 and ribosomes functionally equivalent thereto).
  • the translation system contains magnesium ions. It is known that magnesium ions are necessary to maintain the association state of the small subunit and the large subunit of the ribosome.
  • the L31 protein is one of the proteins that form an interaction between the large subunit and the small subunit of the ribosome. It has been revealed that the magnesium ion concentration required to maintain the association state of ribosomes lacking the L31 protein or ribosomes containing shortL31 is higher than that of ribosomes containing intact L31.
  • the method of the present invention can further include a step of adding magnesium ions to the translation system of the present invention.
  • a translation system to which magnesium ions have been added in advance can be used.
  • the amount of magnesium to be added is not particularly limited, but can be exemplified 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 values of 9 mM or less, 8 mM or less, 7 mM or less, 6 mM or less, 5 mM or less, 4 mM or less, or 3 mM or less.
  • the translation system may be a reconstituted cell-free protein synthesis system derived from a prokaryote, and may contain, depending on the purpose, tRNA (e.g., any aminoacylated tRNA, an artificial aminoacylated tRNA corresponding to a non-natural amino acid), protein factors (e.g., GTP, ATP, creatine phosphate, pH buffer, potassium acetate, spermidine, dithiothreitol, enzymes (e.g., creatine kinase, myokinase, inorganic pyrophosphatase, nucleoside diphosphate kinase), and various amino acids).
  • tRNA e.g., any aminoacylated tRNA, an artificial aminoacylated tRNA corresponding to a non-natural amino acid
  • protein factors e.g., GTP, ATP, creatine phosphate, pH buffer, potassium acetate, spermidine, dithiothreitol
  • the cell-free translation system includes a first tRNA bound to a first unnatural amino acid and a second tRNA bound to a second unnatural amino acid.
  • the first tRNA includes an anticodon complementary to a codon encoding the first unnatural amino acid
  • the second tRNA includes an anticodon complementary to a codon encoding the second unnatural amino acid.
  • the nucleic acid sequence of the mRNA includes a codon encoding the first unnatural amino acid and a codon encoding the second unnatural amino acid adjacent to each other. In this way, when the mRNA is translated, the corresponding first unnatural amino acid and second unnatural amino acid are introduced into the translated peptide.
  • Either the codon encoding the first unnatural amino acid or the codon encoding the second unnatural amino acid may be on the 5' side.
  • tRNA In order to introduce an unnatural amino acid into a peptide by translating an mRNA encoding the peptide containing the unnatural amino acid, it is necessary to aminoacylate a tRNA that is orthogonal and efficiently incorporated into the ribosome ((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.). The following five methods can be used to aminoacylate tRNA.
  • aminoacyl-tRNA synthetases are prepared for each amino acid as an enzyme for aminoacylation of tRNA. Therefore, the first method is to utilize the fact that some ARSs can tolerate unnatural amino acids such as N-MeHis, or to prepare and use mutant aminoacyl-tRNA synthetases that can tolerate unnatural amino acids ((i) Proc Natl Acad Sci U S A. 2002; 99: 9715-20.
  • An engineered Escherichia coli tyrosyl-tRNA synthetase for site-specific incorporation of an unnatural amino acid into proteins in eukaryotic translation and its application in a wheat germ cell-free system.
  • aminoacyl-tRNA can be produced using the following method.
  • a template DNA encoding the desired tRNA sequence and with a T7, T3 or SP6 promoter located upstream is prepared, and RNA can be synthesized by transcription using an RNA polymerase suitable for the promoter, such as T7 RNA polymerase or T3, SP6 RNA polymerase.
  • the tRNA can be extracted and purified from cells, and the desired tRNA can be extracted using a probe with a complementary sequence to the tRNA sequence.
  • cells transformed with an expression vector for the desired tRNA can also be used as the source.
  • RNA of the desired sequence can also be synthesized by chemical synthesis.
  • aminoacyl-tRNA can be obtained by linking the thus obtained tRNA, in which CA has been removed from the CCA sequence at the 3' end, to separately prepared aminoacylated pdCpA or pCpA using RNA ligase (pdCpA method, pCpA method).
  • the tRNA is useful in the production of peptides.
  • aminoacylation can be performed by preparing a full-length tRNA and using flexizyme, a ribozyme that carries active esters of various unnatural amino acids on the tRNA.
  • aminoacyl-tRNA can be produced using a natural ARS or a modified version thereof.
  • aminoacyl-tRNA once consumed in the translation system can be regenerated by the natural ARS or a modified version thereof, so there is no need to have a large amount of pre-produced aminoacyl-tRNA present in the translation system.
  • modified ARSs are described in International Publication No. 2016/148044. These methods for producing aminoacyl-tRNA can also be combined as appropriate.
  • the first tRNA and the second tRNA may be artificial tRNA or transcribed tRNA.
  • the first tRNA and the second tRNA may have a body derived from a naturally occurring tRNA.
  • the "body” of a tRNA refers to the main body of the tRNA excluding the anticodon.
  • the "body derived from a naturally occurring tRNA” means that the body of the tRNA has the same polynucleotide sequence as the body of a naturally occurring tRNA. That is, in one embodiment, the first tRNA and the second tRNA may be a body of a naturally occurring tRNA to which a non-natural amino acid is bound.
  • the peptide is a cyclic peptide, and some or all of the amino acid residues form a cyclic structure.
  • the translation step alone is not necessarily sufficient, and a separate step of cyclizing the translated peptide may be further included.
  • Examples of cyclization include 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, and the like.
  • an amide bond is preferred because of its high metabolic stability.
  • the translation step of the peptide and the cyclization reaction step may be separate or may proceed continuously. Cyclization can be performed by a method known to those skilled in the art, for example, as described in WO 2013/100132, WO 2008/117833, WO 2012/074129, and the like.
  • the type of bond in the ring formation is not limited, but may be a bond between the N-terminus and C-terminus of the peptide, a bond between the N-terminus of the peptide and a side chain of another amino acid residue, a bond between the C-terminus of the peptide and a side chain of another amino acid residue, or a bond between the side chains of amino acid residues, or a combination of two or more of these may be used.
  • the peptide according to this embodiment may form a complex with another molecule such as a nucleic acid.
  • this complex is referred to as a peptide complex.
  • the peptide complex is obtained by further binding one or more selected from the group consisting of a nucleic acid, a ribosome, a spacer, and a linker to the peptide according to this embodiment.
  • a peptide-nucleic acid complex in which a peptide and a nucleic acid are linked is preferable.
  • one or more selected from the group consisting of a spacer and a linker can be appropriately present between the peptide and the nucleic acid.
  • a linker a polymer (e.g., a polymer of 5) of puromycin and hexaethylene glycol (spc18) is exemplified, and as an example of the spacer, an amino acid is exemplified.
  • the nucleic acid referred to here can be a nucleic acid encoding a peptide, which is a phenotype, that is, an RNA or DNA, which is a genotype of the peptide, more preferably an RNA, and most preferably an mRNA.
  • An example of a method for producing a peptide-nucleic acid complex is a method that utilizes the fact that an antibiotic puromycin, which is an analog of aminoacyl-tRNA, is non-specifically linked to a peptide during mRNA translation elongation by ribosomes.
  • a nucleic acid containing a base sequence encoding a spacer downstream of a base sequence encoding the peptide (target peptide) can be used for producing a peptide-nucleic acid complex.
  • the spacer examples include, but are not limited to, a sequence containing glycine or serine.
  • spc18 a polymer of hexaethylene glycol
  • a puromycin analogue can be used instead of puromycin to prepare a peptide-nucleic acid complex.
  • Peptide-nucleic acid complexes have been reported as mRNA display (Proc Natl Acad Sci USA. 1997; 94: 12297-302. RNA-peptide fusions for the in vitro selection of peptides and proteins.
  • the present invention relates to a method for producing a peptide or a library comprising peptides, comprising, for example, the steps of: (1) producing a non-cyclic peptide comprising one or more unnatural amino acids by the methods described herein, the non-cyclic peptide comprising an amino acid residue having a reactive site at its side chain on the C-terminus side and an amino acid residue having another reactive site on the N-terminus side; and (2) bonding the reactive site of the amino acid residue on the N-terminus side to the reactive site of an amino acid residue having a reactive site on its side chain on the C-terminus side to form an amide bond, a carbon-carbon bond or a thioether bond.
  • the library in this specification is a display library, which is a library of peptides tagged (i.e., encoded) by nucleic acids (e.g., a DNA-encoded library/DEL, an mRNA display library, a DNA display library; see, for example, Molecules. 2019 Apr; 24(8): 1629.), or a library of untagged peptides, preferably a display library, more preferably an mRNA display library, a DNA display library, or a ribosome display library, and most preferably an mRNA display library.
  • a display library which is a library of peptides tagged (i.e., encoded) by nucleic acids (e.g., a DNA-encoded library/DEL, an mRNA display library, a DNA display library; see, for example, Molecules. 2019 Apr; 24(8): 1629.), or a library of untagged peptides, preferably a display library, more preferably an m
  • cyclization methods for cyclizing peptides using amide bonds include a cyclization method in which the amino group of methionine at the N-terminus is cross-linked with the amino group of lysine located downstream (on the C-terminus side) using disuccinimidyl glutarate (DSG); a cyclization method in which an amino acid derivative having a chloroacetyl group is introduced as the translation initiation amino acid at the N-terminus and Cys is located downstream to form a thioether by an intramolecular cyclization reaction; and a cyclization method in which a peptide having cysteine or a cysteine analog at the N-terminus and an active ester in the side chain of the amino acid on the C-terminus is translated and then cyclized using native chemical ligation.
  • DSG disuccinimidyl glutarate
  • the C-terminal portion of the peptide of the present disclosure may be chemically modified rather than remaining a carboxylic acid.
  • the carboxylic acid portion may be reacted with piperidine or the like to convert it to a piperidine amide or the like.
  • Gly or G glycine
  • Ile or I isoleucine
  • Leu or L leucine
  • Phe or F phenylalanine
  • Pro or P proline
  • Thr or T threonine
  • Example 1 Synthesis of aminoacylated pCpA for use in a cell-free translation system
  • Aminoacylated pCpA (compounds ST01, ST04, ST07, ST10, ST13, ST16, ST19, ST22, ST25, ST28, ST31, ST34, ST37, ST40, ST41, and ST42) were synthesized according to the following scheme.
  • Compound ST01 was synthesized according to the method described in patent document (WO 2018/143145).
  • buffer solution A (60.0 mL) was dissolved ((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 dihydrogenphosphate (36 mg, 0.05 mmol) synthesized by the method described in the literature (Helv. Chim.
  • Buffer solution A was prepared 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 8, 20 mM N,N,N-trimethylhexadecane-1-aminium, and 100 mM imidazole.
  • buffer solution A (60.0 mL) was dissolved ((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 dihydrogenphosphate (36 mg, 0.05 mmol) synthesized by the method described in the literature (Helv. Chim.
  • 2-iodoacetic acid (37.2 mg, 0.2 mmol) was dissolved in water (0.5 mL) and stirred at 0° C. for 5 minutes.
  • an acetonitrile solution (0.5 mL) of 3-phenylpropan-1-amine (54.1 mg, 0.4 mmol) and DIPEA (70.0 ⁇ L, 0.4 mmol) at 0° C.
  • DMSO 2.0 mL was added, and the reaction solution was concentrated to distill off acetonitrile and water.
  • buffer solution A (30.0 mL) was dissolved ((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 dihydrogenphosphate (36 mg, 0.05 mmol) synthesized by the method described in the literature (Helv. Chim.
  • buffer solution A (30.0 mL) was dissolved ((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 dihydrogenphosphate (36 mg, 0.05 mmol) synthesized by the method described in the literature (Helv. Chim.
  • buffer solution A (30.0 mL) was dissolved ((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 dihydrogenphosphate (36 mg, 0.05 mmol) synthesized by the method described in the literature (Helv. Chim.
  • buffer solution A (30.0 mL) was dissolved ((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 dihydrogenphosphate (36 mg, 0.05 mmol) synthesized by the method described in the literature (Helv. Chim.
  • buffer solution A (30.0 mL) was dissolved ((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 dihydrogenphosphate (36 mg, 0.05 mmol) synthesized by the method described in the literature (Helv. Chim.
  • O-phenyl-D-serine (18.1 mg, 0.1 mmol) and carbonate-(4-nitrophenyl)-4-(2-(4-fluorophenyl)acetamido)benzyl (42.4 mg, 0.1 mmol) synthesized by the method described in the patent document (WO 2018/143145) were added to DMSO (0.5 mL) and stirred at room temperature for 5 minutes.
  • buffer solution A (30.0 mL) was dissolved ((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 dihydrogenphosphate (36 mg, 0.05 mmol) synthesized by the method described in the literature (Helv. Chim.
  • DBU 1,8-diazabicyclo[5.4.0]-7-undecene
  • methyl-D-serine (100.0 mg, 0.64 mmol) and carbonate-(4-nitrophenyl)-4-(2-(4-fluorophenyl)acetamido)benzyl (273.0 mg, 0.64 mmol) synthesized by the method described in the patent document (WO 2018/143145) were added to DMSO (2.14 mL) and stirred at room temperature for 5 minutes.
  • buffer solution A (45.0 mL) was dissolved ((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 dihydrogenphosphate (60 mg, 0.08 mmol) synthesized by the method described in the literature (Helv. Chim.
  • DBU 1,8-diazabicyclo[5.4.0]-7-undecene
  • DBU 1,8-diazabicyclo[5.4.0]-7-undecene
  • N-methyl-alanine (15.5 mg, 0.15 mmol) and carbonate-(4-nitrophenyl)-4-(2-(4-fluorophenyl)acetamido)benzyl (63.7 mg, 0.15 mmol) synthesized by the method described in the patent document (WO 2018/143145) were added to DMSO (0.38 mL) and stirred at room temperature for 5 minutes.
  • Triethylamine (46.0 ⁇ L, 0.33 mmol) was added at 0 ° C., and the reaction mixture was stirred at 30 ° C.
  • buffer solution A (50.0 mL) was dissolved ((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 dihydrogenphosphate (94 mg, 0.13 mmol) synthesized by the method described in the literature (Helv. Chim.
  • Compound ST41 was synthesized according to the method described in patent document (WO 2018/225864).
  • Compound ST42 was synthesized according to the method described in patent document (WO 2020/138336).
  • Compound LCT-12 was synthesized according to the method described in patent document (WO 2020/138336).
  • tRNAGluUAG(-CA) was prepared by a standard method.
  • SEQ ID NO: 1 TR-1) tRNA(Glu)uag-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCCU uag ACGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGC
  • SEQ ID NO: 2 TR-2) tRNA(Glu)cug-CA RNA sequence: GUCCCCUUCGUCUAGAGGCCCAGGACACCGCCCU cug ACGGCGGUAACAGGGGUUCGAAUCCCCUAGGGGACGC
  • Example 4 Preparation of aminoacyl-tRNA mixture using aminoacyl pCpA A reaction solution was adjusted to 25 ⁇ M transcription tRNA (Glu) uag-CA (SEQ ID NO: 1), 50 mM HEPES-KOH pH 7.5, 20 mM MgCl 2 , 1 mM ATP, 0.6 unit/ ⁇ L T4 RNA ligase (New England Bio Lab.), and 0.25 mM aminoacyl pCpA (DMSO solution) by diluting with nuclease free water, and a ligation reaction was carried out at 15° C. for 45 minutes. However, the reaction solution before adding T4 RNA ligase and aminoacyl pCpA was heated at 95° C. for 2 minutes and then left at room temperature for 5 minutes to perform refolding of tRNA in advance.
  • Glu transcription tRNA
  • SEQ ID NO: 1 50 mM HEPES-KOH pH 7.5
  • 20 mM MgCl 2 1
  • Example 5 Preparation of aminoacyl-tRNA mixture using aminoacyl pCpA
  • a reaction solution was prepared by diluting with nuclease free water to 25 ⁇ M transcription tRNA (Glu) cug-CA (SEQ ID NO: 2), 50 mM HEPES-KOH pH 7.5, 20 mM MgCl 2 , 1 mM ATP, 0.6 unit/ ⁇ L T4 RNA ligase (New England Bio Lab.), and 0.25 mM aminoacyl pCpA (DMSO solution), and ligation reaction was carried out at 15° C. for 45 minutes. However, the reaction solution before adding T4 RNA ligase and aminoacyl pCpA was heated at 95° C. for 2 minutes and then left at room temperature for 5 minutes to perform refolding of tRNA in advance.
  • aminoacyl-tRNA was recovered by ethanol precipitation.
  • the obtained aminoacyl-tRNA was dissolved in 1 mM sodium acetate immediately before being added to the cell-free translation system.
  • Example 6 Preparation of initiator aminoacyl-tRNA mixture using aminoacyl pCpA
  • a reaction solution was prepared by diluting with nuclease-free water to 25 ⁇ M transcription tRNA (fMet) cau-CA (SEQ ID NO: 3), 50 mM HEPES-KOH pH 7.5, 20 mM MgCl2, 1 mM ATP, 0.6 unit/ ⁇ L T4 RNA ligase (New England Bio Lab.), and 0.25 mM aminoacyl pCpA (DMSO solution of ST45), and ligation reaction was carried out at 15° C. for 45 minutes. However, the reaction solution before adding T4 RNA ligase and aminoacyl pCpA was heated at 95° C.
  • Example 7 Preparation of mRNA Template mRNA, sequence mR-1 or sequence mR-2, was synthesized from template DNA (SEQ ID NO: 4 or SEQ ID NO: 5) by in vitro transcription reaction using RiboMAX Large Scale RNA production System T7 (Promega, P1280) and purified using RNeasy Mini kit (Qiagen).
  • Template DNA D-1 (SEQ ID NO: 4) GTAATACGACTCACTATAGGGTTAACTTTAAGAAGGAGATATACATATGATTTTTATTGGTTTTATTATTctacagATTCCGGGTTAAGCTTCG Template DNA: D-2 (SEQ ID NO:5) GTAATACGACTCACTATAGGGTTAACTTTAAGAAGGAGATATACATATGATTTTTATTGGTTTTATTATTcagctaATTCCGGGTTAAGCTTCG Template mRNA: mR-1 (SEQ ID NO: 6) GGGUUAACUUUAAGAAGGAGAUAUACAUAUGAUUUUUAUUGGUUUUAUUAUUcuacagAUUCCGGGUUAAGCUUCG Template mRNA: mR-2 (SEQ ID NO: 7) GGGUUAACUUUAAGAAGGAGAUAUACAUAUGAUUUUUAUUGGUUUUAUUAUUcagcuaAUUCCGGGUUAAGCUUCG Template mRNA: mR-2 (SEQ
  • Example 8 Translation and synthesis of peptides
  • the translation system used was PURE SYSTEM (registered trademark) (BioComber), a reconstituted cell-free protein synthesis system derived from prokaryotes.
  • the translation system includes 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 E.
  • coli tRNA Ala1B 0.26 ⁇ M EF-G, 4 ⁇ g/ml creatine kinase, 3 ⁇ g/ml myokinase, 2 units/ml inorganic pyrophosphatase, 1.1 ⁇ g/ml nucleoside diphosphate kinase, 2.7 ⁇ M IF1, 0.4 ⁇ M IF2, 1.5 ⁇ M IF3, 40 ⁇ M EF-Tu, 35 ⁇ M EF-Ts, 1 ⁇ M EF-P-Lys, 0.4 unit/ ⁇ l RNasein Ribonuclease inhibitor (Promega, N2111), 0.4 to 0.5 ⁇ M Penicillin G Amidase (PGA), 2.7 ⁇ M AlaRS, 1 ⁇ M GlyRS, 0.4 ⁇ M IleRS, 0.5 ⁇ M mutant PheRS (WO 2016/148044), 0.16 ⁇ M ProRS, 0.09 ⁇ M ThrRS, 1 ⁇ M mutant ValRS (WO 2016
  • Magnesium acetate was added to the translation solution to give a magnesium acetate concentration of 4 mM to prepare a translation reaction mixture, and initiator aminoacylated tRNA (compound AAtR-16) was added at 25 ⁇ M, and aminoacylated tRNA (one of compounds AAtR-1 to AAtR-13, and one of compounds AAtR-14 and AAtR-15) was added at 10 ⁇ M each.
  • mRNA mR-1 or mR-2
  • ribosomes were added at 1.2 ⁇ M, and the mixture was allowed to stand at 37°C for 1 hour.
  • translation begins from codons 29 to 31 (AUG), generating a translated peptide represented by BdpPhe-Ile-MePhe-Ile-Gly-MePhe-Ile-Ile-X1-X2-Ile-Pro-Gly (the amino acids are expressed in single letter notation as BdpF-I-MeF-I-G-MeF-I-I-X1-X2-I-P-G, where X1 and X2 represent the unnatural amino acid residues corresponding to the aminoacylated tRNA used).
  • Example 9 Analysis Overview
  • the solution containing the translated peptide obtained in Example 8 was diluted 10-fold and analyzed using an LC-FLR-MS device.
  • the analytical data was evaluated by identifying the retention time of the target translated peptide based on the MS spectrum and calculating the peak area of the fluorescence absorption spectrum corresponding to the retention time.
  • the amount of translated peptide was evaluated as a relative content based on a calibration curve prepared using LCT12 synthesized in Example 2 as a standard.
  • LC-MS was performed under the analytical conditions shown in Table 4.
  • Example 10 Translation result (1) Using mR-1 as the template mRNA and one of the compounds AAtR-1 to AAtR-13 and compound AAtR-14 as the aminoacylated tRNA, each translated peptide and translation amount were evaluated. The results are shown in Table 5.
  • Example 11 Translation result (2) Using mR-1 as the template mRNA and one of the compounds AAtR-1 to AAtR-13 and compound AAtR-15 as the aminoacylated tRNA, each translated peptide and translation amount were evaluated. The results are shown in Table 6.
  • Example 12 Translation result (3) Using mR-2 as the template mRNA and compound AAtR-4 or AAtR-5 and compound AAtR-14 or compound AAtR-15 as the aminoacylated tRNA, each translated peptide and translation amount were evaluated. The results are shown in Table 7.

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