Chemoenzymatic synthesis of liraglutide, semaglutide, and GLP-1

Abstract

The present invention provides a method for producing a peptide having the sequence His-X-Glu-Gly-Thr-Phe-Thr, comprising enzymatically coupling (a) a peptide C-terminal ester or thioester comprising a first peptide fragment having the sequence His-X-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-(thio)ester with (b) a peptide nucleophile having an N-terminal unprotected amine comprising a second peptide fragment having the sequence H-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Y-Glu-Phe-Ile-Ala-Trp-Leu-Val-Z-Gly-Arg-Gly. a method for synthesizing a peptide comprising: -Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Y-Glu-Phe-Ile-Ala-Trp-Leu-Val-Z-Gly-Arg-Gly, wherein X is Ala or an α-amino-isobutyric acid (Aib) residue; Y is Lys having a free side chain ε-amino group or the side chain ε-amino group of Lys is protected by a protecting group or the side chain ε-amino group of Lys is functionalized with an amino acid or another functional group; and Z is Arg or Lys.

Description

[Technical Field]

[0001] The present invention relates to a method for enzymatically coupling peptide fragments in the presence of a ligase in order to synthesize a peptide containing the sequence His-X-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Y-Glu-Phe-Ile-Ala-Trp-Leu-Val-Z-Gly-Arg-Gly. [Background technology]

[0002] Several peptides containing the amino acid sequence H-His-X-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Y-Glu-Phe-Ile-Ala-Trp-Leu-Val-Z-Gly-Arg-Gly-OH are well known in the art as insulin-secreting peptides. These peptides include GLP-1, liraglutide, and semaglutide.

[0003] Human GLP-1 (glucagon-like peptide-1) has the formula H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly-OH.

[0004] Liraglutide is an Arg molecule in which the ε-amino group of lysine at position 20 of the above sequence is substituted with palmitic acid separated by Glu. 20It is a GLP-1 analog. Therefore, liraglutide has the formula H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Pal-γ-Glu)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH (see also Figure 1, all chiral amino acid residues are L-amino acid residues). In Lys(Pal-γ-Glu), the ε-amino group of the Lys residue is linked to the γ-Glu carboxyl side chain, and Glu is N-palmitoylated.

[0005] Semaglutide has the formula H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-17-carboxyheptadecanoyl)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH. In this specification, AEEA-AEEA-γ-Glu-17-carboxyheptadecanoyl is N-(17-carboxy-l-oxoheptadecyl)-L-γ-glutamyl-2-[2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl (see also Figure 2, all chiral amino acid residues are L-amino acid residues).

[0006] These peptides can be used, for example, in the treatment of type II diabetes. Furthermore, liraglutide, for example, can be used in the treatment of obesity as an injectable adjunct to low-calorie diets and increased physical activity for habitual weight management in adult patients.

[0007] Processes for synthesizing peptides, including oligopeptides such as GLP-1, liraglutide, and semaglutide, are known in the art. Methods for synthesizing insulin-secreting peptides such as GLP-1 and its analogs are described in International Publication No. 2007147816 and International Publication No. 2016 / 046753. The "Background of the Invention" section of International Publication No. 2016 / 046753 provides a detailed description of suitable preparation methods, particularly recombinant methodologies, sequential synthesis on a solid support, solid-phase synthesis of liraglutide including coupling of a peptide sequence containing amino acid residues (1-10) with a sequence containing amino acid residues (11-31), or solid-phase synthesis of liraglutide including preparation of peptide sequences containing amino acid residues (1-4), (15-16), and (17-31), coupling of a peptide containing amino acid residues (15-16) with (17-31), and sequential addition of amino acids prior to coupling with a peptide containing amino acid sequence (1-4).

[0008] According to International Publication No. 2016 / 046753, GLP-1 peptides are prepared in processes involving liquid-phase or solid-phase peptide synthesis or a combination thereof. This process includes a final coupling step in which at least two fragments are coupled at a terminal Gly residue, and at least one of the fragments is prepared by coupling at least two sub-fragments. Liraglutide is obtained, in particular, by coupling His-Ala-Glu-Gly with Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Pal-Glu-OX)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH. In this sequence, X represents H or a protecting group for the Glu α-carboxylic acid group.

[0009] As can be seen from the "Background of the Invention" in International Publication No. 2016 / 046753, there is still a need to discover novel methods for the synthesis of GLP-1 proteins such as liraglutide or semaglutide in order to provide a better, more efficient and / or less expensive process, or to provide products that can be more easily purified to achieve products with improved yield and purity. In particular, there is a need to provide a method for preparing GLP-1 and its analogues, such as liraglutide or semaglutide, especially on an industrial scale, in good yield, without requiring the use of toxic or otherwise undesirable reagents, and that can be easily purified to obtain products of high purity.

[0010] Enzymatic synthesis of GLP-1 or its analogs, such as liraglutide or semaglutide, is not suggested in International Publication No. 2007147816 or International Publication No. 2016 / 046753, both of which focus on chemical synthesis.

[0011] However, as similarly described in the prior art above, the entirely chemical synthesis of peptides has drawbacks. Furthermore, peptides longer than 15 amino acids are often very difficult to synthesize in solid phase due to side reactions. As a result, purification is cumbersome. Therefore, peptides longer than 10 amino acids are often synthesized by a combination of solid-phase synthesis of side-chain protected oligopeptide fragments and subsequent chemical condensation in solution, such as 10+10 condensation to produce peptides of 20 amino acids. The main drawback of condensation of chemically side-chain protected oligopeptide fragments is that racemization occurs upon activation of the C-terminal amino acid residue of the acyl donor. In contrast, enzyme-catalyzed peptide coupling has several other advantages over chemical peptide synthesis, such as complete absence of racemization and no side reactions at the side-chain functional groups. For industrial applications, the concept of enzyme peptide synthesis based on a kinetic approach, i.e., using the C-terminal ester of an acyl donor, is the most attractive (see, for example, N. Sewald and H.-D. Jakubke, in: “Peptides: Chemistry and Biology”, 1st reprint, Ed. Wiley-VCH Verlag GmbH, Weinheim 2002).

[0012] The problem with enzymatic coupling in aqueous solutions is that the presence of water tends to promote hydrolysis rather than coupling. Several reports have been published regarding the enzymatic condensation of oligopeptide fragments in aqueous solutions (Kumaran et al. Protein Science, 2000, 9, 734; Bjoerup et al. Bioorg. Med. Chem. 1998, 6, 891; Homandberg et al. Biochemistry, 1981, 21, 3387; Komoriya et al. Int. J. Pep. Prot. Res. 1980, 16, 433).

[0013] According to Wells et al. (U.S. Patent No. 5,403,737), the enzymatic condensation of oligopeptides in aqueous solution can be significantly improved by modifying the active site of subtilisin BPN’ (SEQ ID NO: 2), a subtilisin derived from B. amyloliquefaciens. A subtilisin BPN’ variant called subtiligase was obtained that had a 500-fold increased synthetic to hydrolytic ratio (S / H ratio) compared to wild-type subtilisin BPN’ when two mutations, namely S221C and P225A, were introduced. In further experiments, Wells et al. added five additional mutations to subtiligase to further stabilize the enzyme (Proc. Natl. Acad. Sci. USA, 1994, 91, 12544). The new mutant, called stabiligase, was found to be moderately more resistant to sodium dodecyl sulfate and guanidinium hydrochloride, but hydrolysis was still the major side reaction.

[0014] WO 2016 / 056913 provides solutions to the undesirable high hydrolytic activity encountered when enzymes such as subtiligase or stabiligase are used for (oligo)peptide synthesis in an aqueous environment by providing specific mutations to subtilisin BPN’ variants or homologs thereof. These variants or homologs are particularly suitable for catalyzing the synthesis of peptides by coupling a first peptide fragment and a second peptide fragment, where the first fragment is a peptide C-terminal ester or thioester and the second fragment is a peptide nucleophile having an N-terminal unprotected amine.

[0015] The inventors considered applying enzymatic fragment condensation to the synthesis of GLP-1, liraglutide, and semaglutide starting from peptide fragments, as described in International Publication No. 2007147816 or International Publication No. 2016 / 046753. In particular, the inventors considered coupling a 10-mer peptide having amino acid residues 1-10 of liraglutide, semaglutide, or GLP-1 with a 21-mer peptide containing amino acid residues 11-31, using the 10-mer as a (thio) ester and the 21-mer as a nucleophile, by enzymatic fragment condensation. It was found that the presence of serine in both P1' and P2' is unfavorable for the peptide nucleophile in order to couple the C-terminal (thio) ester of the peptide having amino acid residues 1-10 with the peptide nucleophile containing amino acid residues 11-31. A further possible reason for insufficient effective coupling may be the presence of a non-hydrophobic amino acid (threonine) at P4 of the peptide C-terminal (thio) ester. Furthermore, the inventors attempted to couple peptide C-terminal (thio) esters having amino acid residues 1-4 with peptide nucleophiles containing amino acid residues 5-31, but were unsuccessful. The inventors concluded that the presence of histidine at P4 and / or glycine at P1 of the peptide C-terminal (thio) ester is particularly detrimental to effective coupling. While these peptides can be prepared by enzymatic coupling in aqueous reaction media in the presence of ligases, as shown in Examples 1 and 2 of this disclosure, some processes designed based on scientific considerations, such as the idea that ligases like subtilisine variants or their homologs prefer coupling of C-terminal peptide (thio) esters having a hydrophobic amino acid residue at the P4 position (the fourth amino acid from the C-terminus) of the peptide C-terminal ester or thio ester, have been found to have unexpectedly low yields.In particular, a method was employed to prepare a peptide containing the liraglutide amino acid sequence ("H-liraglutide-1~31-OH") by enzymatically coupling the 13-mer C-terminal ester His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-OCam-Leu-OH with the 18-mer peptide nucleophile H-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly in the presence of the subtilisin BPN' mutant. While the presence of the hydrophobic amino acid residue Val at the P4 position of the C-terminal ester suggested that these fragments might be particularly good for producing the liraglutide amino acid sequence in high yield, this peptide was obtained in very low yield (see Example 1). As shown in Example 2, the yields of H-liraglutide-1~31-OH from the corresponding 9-mer C-terminal esters and 22-mer peptide nucleophiles were even lower than expected, despite the fact that the presence of the hydrophobic Phe residue at the P4 position would make these fragments particularly good for the enzymatic condensation reaction. [Overview of the project] [Problems that the invention aims to solve]

[0016] The object of the present invention is to provide a novel method for the enzymatic synthesis of GLP-1 or its analogues, particularly liraglutide or semaglutide. Generally, alternative enzymatic peptide synthesis processes are needed for these peptides, and especially to broaden the range of means for producing them. In particular, the objective is to provide a process that overcomes one or more of the aforementioned problems or problems discussed in the prior art, more specifically, to improve overall yield or selectivity.

[0017] One or more other objects that may be the subject matter of the present invention can be derived from the following description. [Means for solving the problem]

[0018] Surprisingly, it was found that one or more of these objectives are fulfilled by a method of preparing GLP-1 or its analogues, which involves the enzymatic synthesis of peptides by fragment condensation, in which two specific fragments of the peptide are coupled in the presence of a ligase, particularly a subtilisin variant or homolog.

[0019] Therefore, the present invention relates to a method for synthesizing a peptide comprising the sequence His-X-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Y-Glu-Phe-Ile-Ala-Trp-Leu-Val-Z-Gly-Arg-Gly, wherein, - X is either Ala or an α-amino-isobutyric acid residue (Aib), - Y is Lys, and this Lys has a free side chain ε-amino group (i.e., a non-derivativeized lysine residue), or its side chain ε-amino group is protected by a protecting group, or it is functionalized by an amino acid or another functional group. - Z is either Arg or Lys, This method, (a) A peptide C-terminal ester or thioester comprising a first peptide fragment containing the sequence His-X-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-(thio)ester, (b) Enzymatic coupling of a peptide nucleophile having an N-terminal unprotected amine containing a second peptide fragment having the sequence H-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Y-Glu-Phe-Ile-Ala-Trp-Leu-Val-Z-Gly-Arg-Gly, wherein the enzymatic coupling is catalyzed by a ligase. [Modes for carrying out the invention]

[0020] It is particularly surprising that the method according to the present invention enables the synthesis of the target peptide (ligation product) in high yield even when carried out in an aqueous reaction medium. After all, the Ser at the P4 position of the C-terminal (thio) ester fragment is polar, and from a scientific standpoint, this was expected to be undesirable for the enzyme coupling reaction.

[0021] This was achieved without requiring any side-chain protecting groups on the peptide fragments, and without requiring functional groups to enhance solubility on one or both fragments (e.g., a 2-hydroxy-4-methoxybenzylamide group on the amide functional group of the peptide skeleton, or peptide tags of polar amino acids at the terminals of each fragment that do not participate in the coupling reaction). However, in certain embodiments, protecting groups or solubility-enhancing groups may be used. Since peptide nucleophiles have low solubility, a high S / H ratio without the need for solubility-enhancing groups is remarkable.

[0022] In the method according to the present invention, Y of the peptide nucleophile may be Lys having a free side-chain ε-amino group (i.e., a non-derivativeized lysine residue). However, the present invention also enables the coupling of peptide nucleophiles in which Y is Lys and its side-chain ε-amino group contains a functional group, in particular, peptide nucleophiles in which Y is Lysγ-Glu, Lys(AEEA-AEEA-γ-Glu, Lys(Pal-γ-Glu-OH) or Lys(AEEA-AEEA-γ-Glu-17-carboxyheptadecanoyl-OH). It is particularly surprising that efficient enzymatic coupling using such peptide nucleophiles is possible despite the presence of such sterically demanding groups. Even more surprisingly in this embodiment, the peptide nucleophile is highly hydrophobic (this is increased by the presence of a hydrophobic group (e.g., a fatty acid tail) on the side-chain ε-amino group of Lys), but also exhibits a high synthesis-to-hydrolysis ratio (S) even in aqueous reaction media. A S / H ratio is achieved. Generally, it is our knowledge that the S / H ratio is directly related to the concentration of the nucleophile and therefore directly related to the solubility of the nucleophile in aqueous solution. The presence of hydrophobic functional groups makes the solubility of the resulting molecules very low, so it is therefore very surprising that the S / H ratio is very high even in the case of coupling of (thio)esters and peptide nucleophiles, such as the 11-mer peptide (thio)ester and 20-mer peptide nucleophile as defined in the present invention. Reference methods that attempted to obtain the same ligation product by coupling different (thio)esters and peptide nucleophiles (with or without a side chain functional group provided at the Y position), for example, the corresponding 9-mer (thio)ester and 22-mer peptide nucleophile, were unsuccessful.

[0023] Coupling with a peptide nucleophile in which Y is Lys and its side-chain ε-amino group is functionalized with an amino acid or another functional group has been found to be possible, in particular, using the subtilisin BPN' variant, as will be described in more detail elsewhere herein. Preferred embodiments of how coupling is carried out using a peptide nucleophile in which Y is Lys and its side-chain ε-amino group is functionalized will also be described in more detail below.

[0024] For the purposes of this invention, the "synthesis-to-hydrolysis ratio" (S / H ratio) refers to the amount of enzymatically synthesized (oligo)peptide product divided by the amount of the C-terminal ester or thioester of the (oligo)peptide obtained by hydrolysis of the ester or thioester group. For further details regarding the determination of the S / H ratio, refer to International Publication No. 2016 / 056913.

[0025] As used herein, the term "or" is defined as "and / or" unless otherwise specified or the context makes it clear that it means "either... or...".

[0026] As used herein, the terms “a” or “an” are defined as “at least one” unless otherwise specified or the context indicates that only the singular form should be used.

[0027] When referring to a singular noun (e.g., compound, additive), it implies that plural forms are also included unless the context indicates that only singular forms should be referred to.

[0028] In this specification, the term "pH" refers to apparent pH, i.e., pH measured using a standard calibrated pH electrode.

[0029] For the purposes of this invention, “peptide” means any chain composed of two or more amino acids. Thus, a peptide is generally an amide composed of two or more aminocarboxylic acid molecules (i.e., amino acids) by the formation of a covalent bond from the carbonyl carbon of one molecule to the nitrogen atom of another molecule, with the loss of conventional water. Although the term “peptide” is usually applied to structures formed from α-amino acids, a peptide may also contain other amino acids such as one or more beta-amino acids and / or one or more γ-amino acids.

[0030] The amino acid sequence of a peptide is called its primary structure. In the embodiment, the peptide essentially does not contain a secondary structure and essentially does not contain a tertiary structure.

[0031] In embodiments, the peptide synthesized or to be coupled by the method according to the present invention consists essentially of amino acid residues. For example, GLP-1 consists of amino acid residues. In further embodiments, the peptide consists essentially of amino acid units and protecting groups.

[0032] In further embodiments, the peptides synthesized or to be coupled by the method according to the present invention are conjugates of a peptide chain with another residue, such as a fatty acid. These peptides are called lipopeptides. Fatty acids can be used, for example, to alter solubility. Suitable examples of fatty acids are C8-C24 saturated fatty acids and C8-C24 unsaturated fatty acids.Optionally, a polar linker is provided between the peptide and the fatty acid to increase solubility, for example, in an aqueous environment. Liraglutides and semaglutides are peptides that are conjugates of a peptide chain with a fatty acid. Semaglutides contain a polar linker between the peptide and the fatty acid residue.

[0033] Typically, peptides (this term includes oligopeptides, proteins, and chimeric peptides) contain up to about 35,000 amino acid units, particularly 3 to 20,000, more specifically 4 to 1,000 or 5 to 500 amino acid units. The ligase according to the present invention may be used for the synthesis of peptides other than His-X-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Y-Glu-Phe-Ile-Ala-Trp-Leu-Val-Z-Gly-Arg-Gly. Such peptides preferably contain 500 or fewer amino acid units, particularly 200 or fewer, more specifically 100 or fewer amino acid units. In a particularly preferred embodiment, the synthesized peptide contains at least 10 amino acid units, more specifically at least 15 amino acids, at least 25 amino acids, or at least 40 amino acids. Such peptide fragments can be selected from a wide range, and the length of the fragments may be at least 2, particularly at least 5, and more specifically at least 10, with the upper limit determined by the length of the synthesized peptide.

[0034] In relation to the present invention, "oligopeptide" means a peptide composed of 2 to 200 amino acid units, particularly composed of 5 to 100 amino acid units, and more specifically, composed of 10 to 50 amino acid units.

[0035] For the purposes of the present invention, "peptide bond" means an amide bond between (i) the α-amino terminus of one α-amino acid or the beta-amino terminus of one beta-amino acid and (ii) the α-carboxyl terminus of one other α-amino acid or the beta-carboxyl terminus of one other beta-amino acid. Preferably, the peptide bond is between the α-amino terminus of one α-amino acid and the α-carboxyl terminus of another α-amino acid.

[0036] In relation to the present invention, "amino acid side chain" means any proteinogenic or non-proteinogenic amino acid side chain.

[0037] Proteinogenic amino acids are amino acids encoded by the genetic code. Proteinogenic amino acids include alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), serine (Ser), threonine (Thr), methionine (Met), cysteine ​​(Cys), asparagine (Asn), glutamine (Gln), tyrosine (Tyr), tryptophan (Trp), glycine (Gly), aspartic acid (Asp), glutamic acid (Glu), histidine (His), lysine (Lys), arginine (Arg), proline (Pro), and phenylalanine (Phe). Selenocysteine ​​(Sec, U) is an amino acid whose structure corresponds to cysteine, but which contains selenium instead of a sulfur atom. Proteinogenic amino acids are L-stereoisomers of the above amino acids (with the exception of glycine, which does not have stereoisomers).

[0038] The non-proteinogenic amino acid of a specific target in the method according to the present invention is 2-aminoisobutyric acid (Aib), which forms part of the peptide chain of semaglutide.

[0039] The term "(thio)ester" is used herein as an abbreviation of the phrase "ester or thioester."

[0040] The term "N-terminal protection" is used herein to indicate that a protecting group is provided to the N-terminal amine group of a peptide, typically the N-terminal α-amine group, which at least substantially protects the N-terminal amine group from coupling to the C-terminal carboxylic acid group of another peptide or the same peptide molecule.

[0041] The term "C-terminal protection" is used herein to indicate that a protecting group is provided to the C-terminal carboxylic acid group, typically the C-terminal α-carboxylic acid group, of a peptide that substantially protects the carboxylic acid group from coupling to the N-terminal amine group of another peptide or the same peptide molecule.

[0042] As used herein with respect to proteins or polypeptides (in particular enzymes such as ligases), the terms “mutated” or “mutation” mean that at least one amino acid in a wild-type or naturally occurring protein or polypeptide sequence is replaced by a different amino acid, inserted into the sequence, added to the sequence, or deleted from the sequence by mutagenesis of the nucleic acid encoding those amino acids. Mutagenesis is a well-known method in the art and includes, for example, site-directed mutagenesis by PCR or oligonucleotide-mediated mutagenesis, as described in Sambrook et al., Molecular Cloning—A Laboratory Manual, 2nd ed., Vol. 1-3 (1989). As used herein with respect to genes, the terms “mutated” or “mutation” mean that at least one nucleotide in the nucleic acid sequence of that gene or its regulatory sequence is replaced by a different nucleotide, inserted into the sequence, added to the sequence, or deleted from the sequence by mutagenesis, resulting in the transcription of a protein sequence with qualitatively or quantitatively altered function, or in the case of knockout of that gene.

[0043] In this specification, abbreviations for indicating amino acid substitutions use the single-letter amino acid code of the amino acid to be substituted, followed by a number indicating the location in the protein amino acid sequence where the substitution occurs. This number is the amino acid position in the wild-type amino acid sequence. Therefore, for mutated amino acid sequences, it is the amino acid position corresponding to the position where that number is located in the wild-type enzyme. Due to one or more other mutations (additions, insertions, deletions, etc.) at positions with smaller numbers, the actual positions do not need to be the same. Those skilled in the art can determine the corresponding positions using commonly known alignment techniques such as NEEDLE. This number is followed by the single-letter code of the amino acid that substitutes the wild-type amino acid within it. For example, S221C indicates the substitution of serine for cysteine ​​at the position corresponding to 221. X is used to indicate any proteinogenic amino acid other than the amino acid being substituted. For example, S221X indicates the substitution of serine for any other proteinogenic amino acid at the position corresponding to 221.

[0044] The term "ligase" is used herein to refer to an enzyme that has catalytic activity in the coupling of two peptides by catalyzing the formation of a peptide bond by coupling the C-terminus of one peptide with the N-terminus of another peptide. Generally, the ligases used in the methods according to the present invention have ligase activity for the coupling of an 11-mer His-X-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-(thio)ester with a 20-mer peptide nucleophile H-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Y-Glu-Phe-Ile-Ala-Trp-Leu-Val-Z-Gly-Arg-Gly.

[0045] As defined by Schechter and Berger, the active site residues in proteases, including ligases, are composed of adjacent pockets called subsites. Each subsite pocket binds to a corresponding residue in the peptide substrate sequence, referred to herein as a sequence position. According to this definition, amino acid residues in the substrate sequence are numbered sequentially from the cleavage site outward, such as -P4-P3-P2-P1-P1'-P2'-P3'-P4'- (the cleavable bond is located between the P1 and P1' positions), while the subsites (pockets) of the active site are labeled accordingly, such as -S4-S3-S2-S1-S1'-S2'-S3'-S4'- (Schechter and Berger, Biochem Biophys Res Commun. 1967 Apr 20;27(2):157-62). It should be noted that not all proteases possess all of the aforementioned subsites. For example, the S3' and / or S4' pockets may not be present in the subtilisin BPN' variant or its homologue according to the present invention.

[0046] For the purposes of this invention, "S1, S2, S3, and S4 pockets" refer to the amino acids of a protease (particularly a ligase) that interact with the amino acids of the peptide acyl donor. The C-terminal amino acid (first amino acid; P1) of the acyl donor peptide interacts with the amino acid in the S1 pocket of the protease. The second-to-last amino acid (second amino acid from the C-terminus; P2) of the acyl donor peptide interacts with the amino acid in the S2 pocket of the protease, the third amino acid (P3) interacts with S3, and the fourth amino acid (P4) interacts with the S4 pocket. The S1-S4 binding pockets of the protease may be far apart in the primary structure of the protease, but in three-dimensional space they are defined by several adjacent amino acids. For the purposes of this invention, "S1' and S2' pockets" refer to the amino acids of a protease that interact with the N-terminal amino acid of the peptide nucleophile. The N-terminal amino acid of the peptide nucleophile interacts with the amino acid in the S1' pocket of the protease. The second-to-last amino acid at the N-terminus of a peptide nucleophile interacts with an amino acid in the S2' pocket of the protease. While the S1' and S2' binding pockets of the protease may be far apart in the protease's primary structure, they are defined in three-dimensional space by several closely located amino acids.

[0047] When an enzyme is referred to in relation to an enzyme class (EC) in parentheses, the enzyme class is the class to which the enzyme is classified or may be classified according to the Enzyme Nomenclature provided by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB), which can be found at http: / / www.chem.qmul.ac.uk / iubmb / enzyme / . This implies that it also includes other suitable enzymes that have not yet been classified into a particular class but may be classified as such.

[0048] Homogenies typically have a common intended function with the peptide or enzyme they are homologous to, and can, for example, catalyze the same reaction, particularly enzyme coupling according to the method of this invention.

[0049] Amino acid or nucleotide sequences are said to be homologous if they exhibit a certain level of similarity. Whether two homologous sequences are closely related or more distantly related is indicated by a high or low "identity percentage" or "similarity percentage," respectively.

[0050] The terms “homology,” “homology percentage,” “identity percentage,” or “similarity percentage” are used interchangeably herein. For the purposes of the present invention, it is defined herein that complete sequences are aligned for the purpose of optimal comparison in order to determine the identity percentage of two amino acid sequences. To optimize the alignment between the two sequences, gaps may be introduced in either of the two sequences being compared. Such alignment is performed over the entire length of the sequences being compared. Alternatively, the alignment may be performed over a shorter length, for example, over about 20, about 50, about 100 or more nucleic acids or amino acids. The identity percentage is the percentage of identical matches between the two sequences across the reported alignment region.

[0051] The comparison of sequences and the determination of the degree of identity between two sequences can be achieved using mathematical algorithms. Those skilled in the art will recognize that several different computer programs are available for aligning two sequences and determining the homology between them (Kruskal, JB (1983) An overview of sequence comparison In D. Sankoff and JB Kruskal, (ed.), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, pp. 1-44 Addison Wesley). The degree of identity between two amino acid sequences can be determined using the Needleman-Wunsch algorithm for the alignment of two sequences (Needleman, SB and Wunsch, CD (1970) J. Mol. Biol. 48, pp. 443-453). The Needleman-Wunsch algorithm is implemented in the computer program NEEDLE. For the purposes of this invention, the NEEDLE program from the EMBOSS package was used (version 2.8.0 or later, EMBOSS: The European Molecular Biology Open Software Suite (2000), Rice, P. Longden, I. and Bleasby, A. Trends in Genetics 16, (6) pp276-277, http: / / emboss.bioinformatics.nl / ). For protein sequences, EBLOSUM62 is used as the substitution matrix. Other matrices can also be specified. The optional parameters used for amino acid sequence alignment are a gap-open penalty of 10 and a gap-extension penalty of 0.5. Those skilled in the art will recognize that all these different parameters yield slightly different results, but the overall percentage of identity between the two sequences does not change significantly when different algorithms are used.

[0052] Homology or identity between two aligned sequences is calculated as follows: the number of corresponding positions in the alignment that show the same amino acid in both sequences is divided by the total length of the alignment after subtracting the total number of gaps in the alignment. Identity as defined herein can be obtained from NEEDLE using the NOBRIEF option and is labeled as “Longest Identity” in the program output. For the purposes of the present invention, the level of identity (homology) between two sequences is calculated according to the definition of “Longest Identity” which can be performed using the NEEDLE program.

[0053] Polypeptide sequences, particularly enzyme sequences, can be further used as "query sequences" to perform searches against sequence databases, for example, to identify other family members or related sequences. Such searches can be performed using the BLAST program. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov). BLAST is used for amino acid sequences. The BLAST program uses the following as faults: - Cost of open gap: default = 11 for protein - Cost for elongation gap: default = 1 for protein - Expected value: Default = 10 - Word size: Default = 28 for megablast / 3 for protein.

[0054] Furthermore, the degree of local identity (homology) between an amino acid sequence query and the homologous sequence being searched is determined by the BLAST program. However, only sequence segments that give a match exceeding a certain threshold are compared. Therefore, identity is calculated by the program only for these matching segments. Thus, the identity calculated is referred to as local identity.

[0055] The term “homologous” is used herein, in particular, to refer to a peptide, more specifically an enzyme, that has at least 50%, preferably at least 60%, more preferably at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the peptide, particularly the enzyme, compared to the homologous peptide or enzyme. Obviously, sequence identity will be less than 100%. The percentage of sequence identity depends on the number of mutations and the length of the peptide (enzyme) compared to the homologous peptide. Deletions are not considered in “longest identity” alignment.

[0056] For the purposes of the present invention, "condensation" means the formation of a new amide bond between the C-terminal carboxyl functional group of a peptide and the N-terminal amine functional group of a nucleophile of a particular other peptide.

[0057] The term "analog" of a peptide is used in particular for peptides that are structural and / or functional analogues of the aforementioned peptide. Functional analogues have the same target in vivo (e.g., the same target receptor on the cell membrane), while structural analogues have high similarity in amino acid sequence. Functional analogues of a peptide may have relatively low amino acid sequence identity across the entire amino acid sequence, e.g., about 50% or less, but may have high sequence identity (and therefore high structural similarity) with the peptide from which it is an analogue in segments of the amino acid sequence, such as near the N-terminus or C-terminus. Structural analogues include amino acid sequences in which the peptide has at least 60%, more specifically at least 70%, preferably at least 80%, more preferably at least 90%, and more preferably at least 95% sequence identity with the amino acid sequence of the peptide from which it is an analogue. For the sake of clarity and conciseness, features are described herein as being the same or as part of different embodiments, but it will be recognized that the scope of the invention may include embodiments having all or some combinations of the features described. Terms used herein that are not expressly defined herein shall be as defined in International Publication No. 2016 / 056913, or, if not defined therein, shall be used in accordance with common general knowledge.

[0058] The peptide C-terminal ester or thioester comprises a first peptide fragment containing the amino acid sequence His-X-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-(thio)ester, where X is Ala or α-amino-isobutyric acid unit (Aib). Particularly good results have been achieved using a first peptide fragment, where this is the amino acid sequence of the first peptide fragment. In certain embodiments, the N-terminus contains at least one amino acid (W v(See below) For example, the peptide may be extended by Gly or Phe, and the N-terminus of the extended peptide may be provided with a protecting group, usually an Edman-type protecting group (see below). If desired, a peptide tag may be provided at the N-terminus, for example, to change the solubility in the reaction medium. However, this is generally not necessary, especially in non-aqueous reaction mediums.

[0059] Peptide C-terminal esters or thioesters are typically activated (thio)esters, i.e., they contain a carboxyester or carboxythioester group that can participate in enzyme coupling reactions. In principle, any (substituted or unsubstituted) alkyl or (substituted or unsubstituted) aryl (thio)ester can be used. Typical examples of (thio)esters that can participate in enzyme coupling reactions are methyl-, ethyl-, propyl-, isopropyl-, phenyl-, benzyl- (e.g., p-carboxybenzyl-), 2,2,2-trichloroethyl-, 2,2,2-trifluoroethyl-, cyanomethyl-, and carboxyamidomethyl-(thio)esters.

[0060] Particularly favorable results were obtained using carboxyamide methyl type esters (Cam-esters) represented by the formula peptide-(C=O)-O-CX1X2-C(=O)N-R1R2. In this specification, each X1 and X2 independently represents a hydrogen atom or an alkyl group. Favorable results were achieved when both X1 and X2 are hydrogen atoms (peptide-(C=O)-O-CH2-C(=O)N-R1R2). In this specification, R1 represents a hydrogen atom or an alkyl group, and R2 represents a hydrogen atom or alkyl group, or an amino acid or peptide residue having a C-terminal carboxyamide or carboxylic acid functional group optionally protected on or on one or more of the side chain functional groups of an amino acid. In this specification, each alkyl group independently can represent a (substituted or unsubstituted) C1-C7 alkyl group, preferably a (substituted or unsubstituted) linear C1-C6 alkyl group, more preferably a (substituted or unsubstituted) linear C1-C3 alkyl group, and most preferably a methyl group. Particularly favorable results have been achieved in the method of the present invention in which both R1 and R2 represent a hydrogen atom, or in which R1 represents a hydrogen atom and R2 represents an amino acid or peptide residue having a C-terminal carboxyamide or carboxylic acid functional group optionally protected on or on one or more of the side chain functional groups of the amino acid.

[0061] It is particularly advantageous to use Cam-AA1-AA2-esters, where AA1 is the first amino acid residue and AA2 is the second amino acid residue. In this specification, AA1 is a hydrophobic amino acid residue such as alanine, valine, leucine, isoleucine, phenylalanine, methionine, or tryptophan unit. AA2 is a basic amino acid residue such as arginine or lysine unit. Particularly preferred are Cam-Phe-Arg and Cam-Phe-Lys. AA1 and AA2 usually have free side-chain functional groups, i.e., side-chain functional groups that do not have a protecting group or another residue.

[0062] Furthermore, particularly favorable results were also obtained using p-carboxyl-substituted benzyl esters represented by the formula peptide-(C=O)-O-CH2-C6H4-CO2E (wherein E represents a hydrogen atom, a positively charged salt ion such as an ammonium ion, or an amino acid or peptide residue having a C-terminal carboxyamide or carboxylic acid functional group optionally protected on or on one or more of the side-chain functional groups of an amino acid). Furthermore, favorable results were also obtained using p-carboxyl-substituted benzyl esters represented by the formula peptide-(C=O)-O-CH2-C6H4-CO2E (wherein E is defined as above, and one or more hydrogen atoms in the phenyl ring (C6H4 in the above formula) are replaced by substituents such as hydroxyl, alkoxy, aryloxy, or halogen).

[0063] The peptide C-terminal (thio) ester may be N-terminally unprotected or N-terminally protected.

[0064] A suitable N-terminal protecting group is an N-protecting group that can be used for peptide synthesis. Such groups are known to those skilled in the art. Examples of suitable N-terminal protecting groups include carbamate or acyl protecting groups, such as "Cbz" (benzyloxycarbonyl), "Boc" (tert-butyloxycarbonyl), "For" (formyl), "Fmoc" (9-fluorenylmethoxycarbonyl), "PhAc" (phenacetyl), and "Ac" (acetyl). The groups For, PhAc, and Ac can be enzymatically introduced and cleaved using the enzyme peptide deformylase, PenG acylase, or acylase, respectively.

[0065] The inventors have further found that substituted thiocarbamoyl groups, such as phenylthiocarbamoyl (PTC) groups, are useful protecting groups for the N-terminal α-amine functional group of a C-terminal (thio) ester in enzymatic synthesis of peptides by fragment condensation. The use of such groups is well known in itself, for example, from the Edman degradation process. Protecting agents usable for Edman-type amino acid sequencing are also referred herein as Edman-type protecting agents, and similar agents coupled to an amine group (particularly the N-terminal amine) of a peptide are referred herein as Edman-type protecting groups. The inventors have found that Edman-type protecting agents (bonded to the N-terminal α-amino functional group via an additional linked amino acid, e.g., glycine) form effective protecting groups when it is necessary for the peptide obtained after an enzymatic coupling reaction to obtain liraglutide or semaglutide to have a functional group provided at the Y position, for example, when coupling Pal to Lys-γ-Glu(Y) or when coupling 17-carboxyheptadecanoic acid to Lys-AEEA-AEEA-γ-Glu(Y). Substituting thiocarbamoyl groups can be provided to the N-terminal α-amino functional group by reacting the amine functional group with the corresponding isothiocyanate under (slightly) alkaline conditions. Therefore, a phenylthiocarbamoyl (PTC) group can be introduced using phenyl isothiocyanate (PITC), and a methylthiocarbamoyl (MTC) group can be introduced using methyl isothiocyanate (MITC). Under acidic conditions, such substituted thiocarbamoyl groups are cleaved from the peptide together with the α-amino acid bound in the form of a thiazolinone derivative.

[0066] This novel method for providing N-terminal protection has been found to be advantageous in terms of solubility in aqueous reaction systems compared to, for example, Fmoc. This has also been found to be advantageous compared to, for example, Boc in terms of suitability when using solid-phase synthesis. Edman-type protecting groups, such as substituted thiocarbamoyl moieties, function particularly well as protecting groups at neutral or alkaline pH and can be readily removed at acidic pH. Therefore, such groups are typically used in coupling reactions at neutral or alkaline pH using ligases such as subtilisin BPN' variants or homologs that have a good S / H ratio at such pH, ​​as described in more detail elsewhere herein.

[0067] Appropriate protection / deprotection conditions when using Edman-type protected moieties include conditions commonly known in the art when using such moieties in Edman-type decomposition methods. Substituted thiocarbamoyl groups have been found to be particularly effective in combination with their contribution to good solubility even in aqueous reaction media. Substituted thiocarbamoyl groups can be aromatic or aliphatic. Preferably, the substituted thiocarbamoyl groups are aryl-substituted thiocarbamoyl groups or alkyl-substituted thiocarbamoyl groups. Particularly preferred aryl-substituted thiocarbamoyl groups are C6-C12-aryl-substituted thiocarbamoyl groups, more specifically phenylthiocarbamoyl (PTC). Particularly preferred alkyl-substituted thiocarbamoyl groups are C1-C6-alkyl-substituted thiocarbamoyl groups, more specifically methylthiocarbamoyl (MTC). Further examples of preferred isothiocyanates used for the introduction of substituted thiocarbamoyl groups are mentioned in H. Matsunaga, T. Santa, K. Hagiwara, H. Homma, K. Imai, S. Uzu, K. Nakashima, S. Akiyama, Anal. Chem. 1995, 67, 4276, such as FITC, BAMPITC, DNTC, DNSAPITC, dansylamino-PITC, 3-POPIC, 4-POPIC, CIPIC, and 7-[(N,N-dimethylamino)sulfonyl]-2,1,3-benzoxadiazolyl-4-yl isothiocyanate (DBD-NCS). See the paragraph spanning the left and right columns on page 4276, referenced by the reference. Yet another preferred example is 7-aminosulfonyl-4-(2,1,3-benzoxadiazolyl)-isothiocyanate (ABD-NCS).

[0068] As an alternative to the substituted thiocarbamoyl moiety, another moiety suitable for sequencing amino acids in the peptide by Edman-type degradation can be used as a protecting group, similarly by labeling the N-terminus of the peptide C-terminal ester with the moiety via a linked amino acid, and then cleaving the moiety together with the linked amino acid residue from the rest of the coupling product after enzymatic coupling with a peptide nucleophile. Thus, a suitable protecting moiety that is labeled to the N-terminus of the peptide via a linked amino acid residue and can be cleaved and removed together with the linked amino acid residue is also referred to herein as an "Edman-type protecting group."

[0069] Furthermore, it is possible to link an Edman-type protecting group to the peptide C-terminal (thio) ester via two or more amino acids (i.e., via the peptide chain). The linked amino acids can then be removed by labeling the portion and cleaving the portion and the amino acids in a manner similar to that used in peptide sequencing. While the use of additional linked amino acids is not required, they can, if desired, be used, for example, to alter the solubility of the peptide C-terminal (thio) ester in a reaction medium of choice.

[0070] Therefore, in certain preferred embodiments, the method according to the present invention is (a) formula PW v The method comprises (b) enzymatically coupling a peptide C-terminal ester or thioester represented by -His-X-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-(thio)ester with a peptide nucleophile. In this specification, P represents an Edman-type protecting group, preferably a phenylthiocarbamoyl (PTC) or methylthiocarbamoyl (MTC) moiety. In this specification, v is an integer of at least 1, usually 1 to 10, preferably 1 to 4, more preferably 1, 2 or 3, and most preferably 1, where v represents the number of amino acid residues W. Each W may be the same or different. Typically, each W is selected from the group of proteinogenic amino acids, but in principle, other amino acids may be used if they can be cleaved and removed as PW under Edman-type cleavage conditions.

[0071] This N-terminally protected peptide C-terminal (thio) ester is coupled with a nucleophile (b), thereby forming the N-terminally protected peptide PW v -His-X-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Y-Glu-Phe-Ile-Ala-Trp-Leu-Val-Z-Gly-Arg-Gly is formed. Subsequently, this peptide undergoes a cleavage reaction, and here peptide W is formed. v-1 -His-X-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Y-Glu-Phe-Ile-Ala-Trp-Leu-Val-Z-Gly-Arg-Gly (this peptide can be extended at the C-terminus) is formed.

[0072] If v-1 > 0, group P is coupled to the N-terminal α-amino functional group of W in the peptide, and then PW is removed by cleavage. The coupling and cleavage cycle then continues until the peptide 1 The process is repeated until His-X-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Y-Glu-Phe-Ile-Ala-Trp-Leu-Val-Z-Gly-Arg-Gly is obtained.

[0073] Labeling of the peptide's N-terminus with P is typically achieved under weakly alkaline conditions, e.g., at about pH 8, using methods known to the Edman type for P. Cleavage of PW from the peptide's N-terminus is typically achieved under acidic conditions, usually at a pH of about 4 or less, particularly about 3 or less, e.g., in the range of 0 to 2, using methods known to the Edman type for P. For example, trifluoroacetic acid (TFA) may be used.

[0074] N-terminal protection of peptide (thio) esters is particularly useful in methods in which Y comprises a Lys(γ-Glu-OH) moiety having a free α-amino functional group that needs to be coupled with palmitic acid, or in cases where Y comprises a Lys(AEEA-AEEA-γ-Glu-OH) moiety having a free α-amino functional group that needs to be coupled with 17-carboxy-heptadecanoic acid.

[0075] In particular, good results have been achieved using peptide C-terminal (thio) esters that do not have a side-chain functional group to be protected. However, in embodiments, one or more side-chain functional groups, for example all side-chain functional groups, are provided with protecting groups. Suitable protecting groups are known to those skilled in the art. Carboxylic acid groups can be protected, for example, by cyclohexyl, benzyl, or allyl groups.

[0076] The activated C-terminal (thio) ester group of peptide C-terminal (thio) esters can be synthesized in high yield and purity using solid-phase synthesis without racemization. An additional advantage of using carboxyamidomethyl-type (thio) esters (where R1 represents a hydrogen atom and R2 represents an amino acid or peptide residue having a C-terminal carboxylic acid functional group optionally protected on or on one or more of the side-chain functional groups of an amino acid) is that their activated C-terminal ester or thio ester group can be synthesized using inexpensive and industrially available 2-chlorotrityl chloride.

[0077] The activated C-terminal (thio) ester group of peptide C-terminal (thio) esters can also be synthesized by solution-phase synthesis or by fermentation, i.e., by microorganisms. As is generally known in the art, the fermentation process involves the production of compounds, i.e., peptides, under aerobic or anaerobic conditions. A reliable method for obtaining peptide (thio) esters using fermentation is by so-called intein expression (see, e.g., EKLee, Journal of Chemical Technology and Biotechnology, 2010, 9, 11-18). Various intein expression kits are commercially available (e.g., IMPACT).TM (Kit). Other methods for the fermentation production of peptide (thio) esters are known in the art.

[0078] The peptide nucleophile having an N-terminal unprotected amine contains the amino acid sequence H-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Y-Glu-Phe-Ile-Ala-Trp-Leu-Val-Z-Gly-Arg-Gly ("second peptide fragment"). Particularly favorable results were achieved using the peptide nucleophile with this amino acid sequence. A particularly important advantage of the present invention is that enzyme coupling functions well even in aqueous solutions without the need to extend the C-terminus with a peptide tag or another derivative to increase the solubility or reactivity of the peptide nucleophile.

[0079] In one embodiment, the peptide nucleophile is C-terminally protected. In another embodiment, C-terminal protection is not included.

[0080] In particular, favorable results were achieved using peptide nucleophiles that do not have protected side-chain functional groups.

[0081] In embodiments, protecting groups are provided for one or more side-chain functional groups (particularly one or more hydroxyl, carboxyl, or amine groups) of the peptide nucleophile. Suitable protecting groups are known to those skilled in the art. Carboxylic acid groups can be protected, for example, by cyclohexyl, benzyl, or allyl groups, and amine functional groups can be protected, for example, by allyloxycarbonyl or trifluoroacetyl groups.

[0082] Peptide nucleophiles can be synthesized using methods known in the art, such as solid-phase synthesis, solution-phase synthesis, or fermentation.

[0083] As described above, Y is Lys, and the ε-amino group of the side chain of this Lys can be protected by a protecting group. However, protecting the side chain ε-amino group is generally not necessary for satisfactory coupling yield and rate, and is especially not necessary when subtilisin or its homologue is used as a ligase. In particular, the subtilisin BPN' variants or homologues described herein are suitable for coupling both fragments even when the ε-amino group of Lys at position Y does not contain a protecting group.

[0084] Therefore, typically, Y in a peptide nucleophile is a lysine residue having a free ε-amino side chain or a functionalized ε-amino side chain. For example, if GLP-1 is the peptide to be synthesized, or if Y in the peptide nucleophile already contains the functionalization required to obtain liraglutide or semaglutide, the product obtained by enzymatic coupling may be the target peptide (optionally, after removing any protecting groups). Alternatively, the product obtained by enzymatic coupling may subsequently undergo further reactions to be functionalized with an amino acid or another functional group, more specifically, a functional group selected from the group consisting of Pal-γ-Glu-OH and AEEA-AEEA-γ-Glu-N-17-carboxyheptadecanoyl-OH (wherein Pal is palmitoyl and AEEA-AEEA is 2-[2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl). Methods for functionalizing the free amino acid side chain of Y to obtain liraglutide or semaglutide, or to provide a peptide nucleophile suitable for the synthesis of liraglutide or semaglutide, may be based on methodologies generally known in the art, or on techniques described in the examples of the present invention or in literature cited in the references herein. In particular, the functionalization protocol may be used based on U.S. Patent No. 6,451,974B1.

[0085] In a particularly preferred embodiment, the peptide synthesized by the method according to the present invention is liraglutide.

[0086] The present invention is advantageous in that it enables the enzymatic coupling of a peptide C-terminal ester or thioester comprising (a) a first peptide fragment comprising the sequence His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-(thio)ester and a peptide nucleophile comprising (b) a second peptide fragment comprising the sequence H-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Y-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly (wherein Y is Lys(Pal-γ-Glu-OH)). Thus, enzymatic coupling can be performed to couple the corresponding 11-mer peptide C-terminal (thio)ester and 20-mer N-terminal nucleophile to obtain liraglutide. The N-terminal α-amino functional group of the peptide (thio)ester is protected by PW as defined elsewhere in this specification when describing Edman-type protecting groups. v Protecting groups such as the one represented by can be provided, but particularly good results were achieved by using a method in which a peptide nucleophile having Y=Lys(Pal-γ-Glu-OH) is coupled to a peptide C-terminal ester or thioester represented by the formula His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-(thio)ester, without using N-terminal protection of the peptide (thio)ester and without requiring any further protecting groups.

[0087] In a more preferred embodiment, liraglutide is prepared by enzymatically coupling a peptide C-terminal ester or thioester comprising (a) a first peptide fragment containing the sequence His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-(thio)ester with a peptide nucleophile comprising (b) a second peptide fragment containing the sequence H-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(γ-Glu-OH)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly, and then providing a palmitoyl group (Pal) to the Lys(γ-Glu-OH) residue, thereby obtaining liraglutide. In this embodiment, the N-terminal α-amino functional group of the peptide C-terminal ester or thioester is usually protected, preferably by an Edman-type protecting group, during the enzymatic coupling.

[0088] In a particularly preferred embodiment, liraglutide is prepared by enzymatically coupling a peptide C-terminal ester or thioester comprising (a) a first peptide fragment containing the sequence His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-(thio)ester with a peptide nucleophile comprising (b) a second peptide fragment containing the sequence H-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Y-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly (wherein Y is a lysine residue having a free ε-amino side chain), and then providing Pal-γ-Glu-OH to the ε-amino side chain, thereby obtaining liraglutide. In this embodiment, particularly good results were achieved without using any protecting group on the N-terminal α-amino functional group of the peptide (thio)ester.

[0089] In an alternative embodiment, the method according to the present invention includes the synthesis of semaglutide.

[0090] In a preferred embodiment, the synthesis of semaglutide comprises enzymatically coupling (a) a peptide C-terminal ester or thioester comprising a first peptide fragment comprising the sequence His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-(thio)ester with (b) a peptide nucleophile comprising a second peptide fragment comprising the sequence H-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Y-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly, wherein Y is Lys(AEEA-AEEA-γ-Glu-N-17-carboxyheptadecanoyl-OH), whereby semaglutide can be obtained directly. The N-terminal α-amino functional group of the peptide (thio)ester is provided with a protecting group such as the group represented by P-W as defined elsewhere herein when describing Edman-type protecting groups. v Protecting groups such as those represented by v can be provided, but in particular, without using protection of the N-terminal α-amino functional group of the peptide (thio)ester and without the need for any further protecting groups, a peptide nucleophile having Y = Lys(AEEA-AEEA-γ-Glu-N-17-carboxyheptadecanoyl-OH) was coupled to a peptide C-terminal ester or thioester represented by the formula His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-(thio)ester using a method that achieved particularly good results.

[0091] In further embodiments, the method for synthesizing semaglutide involves enzymatically coupling a peptide C-terminal ester or thioester comprising (a) a first peptide fragment containing the sequence His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-(thio)ester with a peptide nucleophile comprising (b) a second peptide fragment containing the sequence H-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Y-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly (wherein Y is Lys(AEEA-AEEA-γ-Glu-OH)), and then providing a 17-carboxyheptadecanoyl group to the Lys(AEEA-AEEA-γ-Glu-OH) portion. Thus, semaglutide can be obtained by further functionalization after enzymatic coupling. In this embodiment, the N-terminal α-amino functional group of the peptide C-terminal ester or thioester is typically protected, preferably by an Edman-type protecting group, during enzyme coupling.

[0092] In further embodiments, semaglutide is obtained by a method comprising enzymatically coupling a peptide C-terminal ester or thioester comprising (a) a first peptide fragment containing the sequence His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-(thio)ester with a peptide nucleophile comprising (b) a second peptide fragment containing the sequence H-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Y-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly (wherein Y is a lysine residue having a free ε-amino side chain), and then providing an AEEA-AEEA-γ-Glu-N-17-carboxyheptadecanoyl-OH group to the ε-amino side chain, thereby obtaining semaglutide. Thus, semaglutide can be obtained by functionalization after enzymatic coupling. In this embodiment, particularly good results were achieved without using any protecting group (or any side-chain functional group) at the N-terminal α-amino functional group of the peptide (thio) ester.

[0093] In yet another alternative embodiment, the synthesized peptide is GLP-1.

[0094] The ligase used to catalyze the coupling of a peptide C-terminal (thio) ester and a peptide nucleophile may be any ligase that has catalytic activity in the coupling of both peptides by catalyzing the formation of a peptide bond between the C-terminus of the peptide C-terminal (thio) ester and the N-terminus of the peptide nucleophile, where the S / H ratio for coupling to hydrolysis of the coupling product in the reaction medium used is greater than 1. Typically, the ligase can be classified as a serine protease, which can generally be classified under EC3.4.21. Generally, it has three catalytic residues in the order Asp, His, and Ser.

[0095] In particular, the ligase used in the method according to the present invention is an isolated enzyme. Therefore, if the ligase is produced in a living organism, it is isolated from the organism in which it is expressed, usually a recombinant organism, or from the reaction medium in which it is synthesized. In particular, the enzyme of the present invention is considered to have been isolated for the purposes of the present invention either in a crude form or in a form substantially purified by any suitable technique, such as the single-step purification method disclosed in Smith and Johnson, Gene 67:31-40 (1988).

[0096] In particular, the ligase may be a serine endoprotease. The ligase typically has an S / H ratio greater than 1, preferably 2 or more, and especially 5 or more, in the reaction medium used, especially in a reaction medium containing water, more specifically in an aqueous medium. The upper limit of this ratio is not important, and in practice it may be, for example, 100 or less, and especially 20 or less. The ligase used in the method according to the present invention generally has an improved "synthesis-to-hydrolysis ratio" (S / H ratio) compared to at least subtilisine BPN'.

[0097] The S / H ratio of the ligase used in the present invention (as used in the method), divided by the S / H ratio of subtilisin BPN', is usually greater than 100, preferably 250 or more, more preferably 500 or more, and particularly 1000 or more, under at least the conditions described in the examples. The upper limit of this ratio is not significant and may be almost infinite.

[0098] In particular, very favorable results were achieved using the subtilisin BPN' mutant or its homologue.

[0099] In particular, when performing enzyme coupling in a reaction medium containing water as the primary solvent (e.g., 50-100 wt.%) based on the total liquid, the subtilisin BPN' variant or its homologue, as described in International Publication No. 2016 / 056913, has been found to be particularly suitable. The contents of this publication are incorporated by reference, in particular, with respect to details concerning the subtilisin BPN' variant or homologue as set forth in its claims.

[0100] Therefore, the ligase typically used for coupling reactions has the following mutations compared to the subtilisin BPN' or its homologous sequence represented by SEQ ID NO: 2: - Deletion of amino acids corresponding to positions 75-83; - A mutation at the amino acid position corresponding to S221 (this mutation is S221C or S221 selenocysteine); - Preferably, a mutation at the amino acid position corresponding to P225; It is a subtilisin BPN' variant or homolog thereof that contains, - The amino acid positions are defined according to the sequence of the subtilisin BPN' represented by Sequence ID No. 2.

[0101] A more preferred ligase for use in the method according to the present invention may include one or more additional mutations, in particular one or more further mutations as specified elsewhere in this specification or in International Publication No. 2016 / 056913, which is incorporated herein by reference.

[0102] The mutation in the ligase, particularly at the amino acid position corresponding to S221 in the subtilisin BPN' variant or its homologue, is preferably S221C.

[0103] Mutations at the amino acid position corresponding to P225 are usually favorable for the S / H ratio in enzyme coupling. Mutations are usually selected from the group P225N, P225D, P225S, P225C, P225G, P225A, P225T, P225V, P225I, P225L, P225H, and P225Q, preferably from the group P225N, P225D, P225S, P225C, and P225G, more preferably P225N or P225D, and most preferably P225N.

[0104] For good enzyme stability, the ligase, particularly the subtilisin BPN' variant or its homolog, preferably contains one or more mutations selected from the group of mutations at amino acid positions corresponding to Q2, S3, P5, S9, I31, K43, M50, A73, S188, Q206, N212, N218, T254 and Q271 of SEQ ID NO: 2.

[0105] The preferred mutation at the position corresponding to Q2 corresponds to Q2K.

[0106] The preferred mutation at the S3 position corresponds to S3C.

[0107] The preferred mutation at the position corresponding to P5 corresponds to P5S.

[0108] The preferred mutation at the position corresponding to S9 corresponds to S9A.

[0109] The preferred mutation at the position corresponding to I31 corresponds to I31L.

[0110] The preferred mutation at the K43 location corresponds to K43N.

[0111] The preferred mutation at the location corresponding to M50 corresponds to M50F.

[0112] The preferred mutation at the position corresponding to A73 corresponds to A73L.

[0113] The preferred mutation at the location corresponding to S188 corresponds to S188P.

[0114] The preferred mutation at the position corresponding to Q206 corresponds to Q206C.

[0115] The preferred mutation at the position corresponding to N212 corresponds to N212G.

[0116] The preferred mutation at the position corresponding to T254 corresponds to T254A.

[0117] The preferred mutation at the position corresponding to Q271 corresponds to Q271E.

[0118] In particularly preferred embodiments, the ligase, especially the subtilisin BPN' variant or its homologue, contains at least six, preferably at least eight, more preferably at least ten, especially twelve, thirteen, or fourteen, of the mutations selected from the group of mutations at positions corresponding to Q2, S3, P5, S9, I31, K43, M50, A73, S188, Q206, N212, T254, and Q271. This is particularly preferred for the stability of the enzyme in reaction media containing water as the primary or sole solvent. The ligase may have further mutations compared to subtilisin BPN', in particular one or more further mutations as described in the references cited herein, provided that it has enzyme fragment condensation activity (coupling activity) in the preparation of peptides according to the present invention.

[0119] The enzymes according to the present invention, in particular, substitutes for subtilisin BPN' as template enzymes capable of inducing homologs of the subtilisin BPN' variant of the present invention by mutagenesis, are other subtilisins, especially subtilisins having at least 50% homology to subtilisin BPN'.

[0120] A suitable subtilisin sequence can be retrieved from the UNIPROT sequence database (http: / / www.uniprot.org / ), available as of August 11, 2014, by BLAST processing the database using 'subtilisin BPN' (SEQ ID NO: 2) as the query. However, sequence searches are not limited to UNIPROT or date. Those skilled in the art will know how to search for alternative sequence depositories or how to collect additional homologous sequences by sequencing (see, for example, Zooming in on metagenomics: molecular microdiversity of Subtilisin Carlsberg in soil. Gabor E, Niehaus F, Aehle W, Eck JJ Mol Biol. 2012 Apr 20;418(1-2):16-20).

[0121] In particular, the present invention further relates to a mutant having at least the deletion of an amino acid corresponding to L75 to G83 (including G83) of subtilisin BPN', a cysteine ​​or selenocysteine ​​at the position corresponding to position 221 of subtilisin BPN', and at least one of the further mutations of claim 1.

[0122] The sequence of subtilisin BPN' is shown in Sequence ID No. 2 (mature form). The gene encoding amino acids 107 to 275 of subtilisin BPN' is shown in Sequence ID No. 1. Subtilisin BPN' variants or homologs may be based on the enzyme as described in International Publication No. 2016 / 056913, provided that they have the aforementioned mutations.

[0123] In a favorable embodiment, the ligase is a subtilisin BPN' mutant having a deletion of an amino acid corresponding to positions 75-83, the mutation S221C, and one or more further mutations, preferably at least three further mutations, and particularly five-eight further mutations, at amino acid positions corresponding to M222, Y217, P225, F189, N218, E156, G166, and N62 of wild-type subtilisin BPN' (mature).

[0124] Of these mutations, particularly those corresponding to M222P, Y217H, P225N, F189W, N218D, E156N, G166E, and N62A, yielded favorable results. Sequence ID 3 is Ca 2+ The present invention shows a subtilisin BPN' variant (for use) having a deletion of the binding loop, S221C, and the aforementioned further mutation. The His tag is included to facilitate purification and is not necessary for ligase activity. Further preferred enzymes may include one or more additional mutations, in particular one or more further mutations as specified elsewhere in this specification or in International Publication No. 2016 / 056913, which is incorporated herein by reference.

[0125] In the method of the present invention, the enzymatic reaction is usually carried out in a fluid containing water. Preferably, the reaction is carried out in a buffer fluid. The water content is usually 10 to 100 vol%, preferably 20 vol.% or more, preferably 40 vol.% or more, particularly 50 vol.% or more, and more specifically 60 vol.% or more, based on the total liquid. In particular, good results have been achieved in reaction media containing 70 to 100 vol% water, more specifically 90 to 100 vol.%, 95 to 100 vol.%, or 98 to 100 vol.% water. The term "aqueous" is used for media consisting of at least substantially water.

[0126] In principle, any buffer solution is suitable. Good buffer solutions are known to those skilled in the art. For example, David Sheehan in Physical Biochemistry, 2nd See Ed. Wiley-VCH Verlag GmbH, Weinheim 2009; http: / / www.sigmaaldrich.com / life-science / core-bioreagents / biological-buffers / learning-center / buffer-calculator.html. Particularly good results were achieved using Good buffers such as Tricin. The buffer concentration can be selected over a wide range, e.g., 10–1000 mM, especially 25–500 mM, more specifically 50–250 mM. Relatively low molar concentrations of buffers were found to be advantageous for coupling peptide nucleophiles (where Y is Lys(Pal-γ-Glu-OH), etc.).

[0127] The pH of the buffer solution for the coupling reaction in the method according to the present invention may be at least 5, particularly at least 6, and preferably at least 7. The desired pH is usually less than 11, particularly less than 10, and even more preferably less than 9. Typically, the optimal pH for enzyme coupling is between 7 and 9.

[0128] Due to the high S / H ratio, a significantly excess peptide C-terminal ester or thioester or peptide nucleophile is generally not required to achieve high yields in the condensation reaction. Generally, these are contacted in nearly stoichiometric ratios, or with an excess of the peptide C-terminal ester, particularly in a molar ratio of (a) peptide C-terminal ester or thioester to (b) peptide nucleophile in the range of 1:1 to 5:1. While satisfactory results are achieved for stoichiometric ratios, an excess of peptide C-terminal (thio)ester has been found to be advantageous for reaction rate. Therefore, preferably, the molar ratio of (a) peptide C-terminal ester or thioester to (b) peptide nucleophile is in the range of 1.05:1.0 to 4:1, more preferably in the range of 1.1:1.0 to 3:1, even more preferably in the range of 1.2:1.0 to 2.5:1.0, and particularly in the range of 1.2:1.0 to 2.0:1.0.

[0129] In the method of the present invention, it may be advantageous to add an additive to the fluid in which the reaction is carried out in order to improve the solubility of the peptide fragment or to improve the reaction yield. Such additives may be salts or organic molecules, such as guanidium hydrochloride, urea, sodium dodecasulfate, or Tween. However, in embodiments where Y is, for example, Lys(Pal-γ-Glu-OH), good results have been achieved without the use of such additives even in a completely aqueous reaction medium.

[0130] The reaction may be carried out in a completely aqueous liquid, or in a mixture of water and a water-miscible cosolvent, such as N,N-dimethylformamide (DMF), N-methylpyrrolidinone (NMP), N,N-dimethylacetamide (DMA), dimethyl sulfoxide (DMSO), acetonitrile, ether, such as tetrahydrofuran (THF), 2-methyltetrahydrofuran (Me-THF), or 1,2-dimethoxyethane, or (halogenated) alcohol, such as methanol, ethanol, isopropanol, tert-butanol, 2,2,2-trifluoroethanol (TFE), 1,1,1,3,3,3-hexafluoroisopropanol, or a mixture of these organic solvents. Depending on the stability of the subtilisin BPN' variant and the solubility of the peptide substrate, the amount of cosolvent is preferably less than 70 vol%, more preferably less than 60 vol%, even more preferably less than 50 vol%, and most preferably less than 40%.

[0131] In principle, the temperature during enzyme fragment condensation is not critical, as long as a temperature is selected that allows the ligase used to exhibit sufficient activity and stability. Such temperatures can be determined routinely. Generally, the temperature may be at least -10°C, particularly at least 0°C, or at least 10°C. Generally, the temperature may be 70°C or less, particularly 60°C or less, or 50°C or less. Optimal temperature conditions can be readily determined by those skilled in the art for specific ligases for specific enzyme fragment condensation through routine experiments based on common general knowledge and the information disclosed herein. Generally, a temperature in the range of 20–50°C is advantageous.

[0132] The present invention further relates to the use of Edman-type agents to provide protecting groups during peptide synthesis in methods including enzymatic coupling of peptides by fragment condensation. Accordingly, the present invention further relates to (a) formula PW v -AA n -A peptide C-terminal ester or thioester represented by a (thio) ester, and formula AA m The present invention relates to a method for synthesizing peptides, comprising enzymatically coupling a peptide nucleophile represented by , wherein the coupling is catalyzed by a ligase, preferably a subtilisin BPN' variant or homolog, as described elsewhere in this specification.

[0133] In this specification, P represents an Edman-type protecting group, preferably a thiocarbamoyl group, as defined above. Coupling of P to the N-terminus of a peptide is typically achieved under weakly alkaline conditions, e.g., at about pH 8, in a manner known to the Edman-type methodology for P. In this specification, v is an integer of at least 1, usually preferably 1 to 10, preferably 1 to 5, more preferably 1, 2 or 3, most preferably 1, where v represents the number of amino acid residues W, where each W may be the same or different, preferably as defined above. Each AA represents an amino acid residue, n is an integer representing the number of amino acid residues of the peptide C-terminal ester or thioester, and m is an integer representing the number of amino acid residues of the peptide nucleophile. Typically, the sum of n and v is at least 4 to enable recognition by ligase. Preferably, n is in the range of 3 to 200, particularly in the range of 3 to 50, more specifically in the range of 3 to 25. In certain embodiments, n is at least 4, at least 6, at least 8, at least 10, at least 15, or at least 20. Preferably, m is in the range of 3 to 200, particularly in the range of 5 to 50, more specifically in the range of 8 to 30. In certain embodiments, m is at least 4, at least 10, at least 15, or at least 20.

[0134] Coupling product PW v-AA n -AA m It undergoes a cleavage reaction, and peptide W v-1 -AA n -AA m A peptide W is formed. Typically, cleavage is achieved under acidic conditions. If v-1 > 0, then peptide W is formed. v-1 -AA n -AA m At the N-terminal position, the base P is coupled to W, and PW v-1 -AA n -AA m This is formed, and then PW is cut. This then becomes formula AA n -AA m This process is repeated until a peptide represented by [formula] is obtained.

[0135] Therefore, the present invention relates to a method for synthesizing a peptide comprising the sequence His-X-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Y-Glu-Phe-Ile-Ala-Trp-Leu-Val-Z-Gly-Arg-Gly, wherein, - X is an Ala or α-aminoisobutyric acid (Aib) residue, - Y is Lys, and this Lys has a free side chain ε-amino group (i.e., a non-derivativeized lysine residue), or its side chain ε-amino group is protected by a protecting group, or its side chain ε-amino group is functionalized by an amino acid or another functional group. - Z is either Arg or Lys, This method, (c) A first peptide C-terminal ester or thioester fragment comprising the sequence His-X-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-(thio)ester, (d) A second peptide nucleophile fragment having an N-terminal unprotected amine containing the sequence H-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Y-Glu-Phe-Ile-Ala-Trp-Leu-Val-Z-Gly-Arg-Gly The process involves enzymatic coupling of the two molecules, which is catalyzed by a ligase, the ligase being a subtilisin BPN' variant or a homolog thereof having at least 80%, 85%, or 90%, preferably 95%, sequence identity. Preferably, the ligase is a subtilisin BPN' variant having a deletion of an amino acid corresponding to positions 75-83, a mutation S221C, and one or more further mutations at amino acid positions corresponding to M222, Y217, P225, F189, N218, E156, G166, and N62 of wild-type subtilisin BPN' (mature), preferably at least three further mutations, particularly five to eight further mutations (most preferably one of which is at P225).

[0136] In particularly advantageous embodiments, the ligase used in the method according to the present invention is a subtilisin BPN' variant, or a homolog thereof having at least 80%, 85%, or 90%, preferably 95%, sequence identity with SEQ ID NO: 14, and includes mutants Q2K, S3C, P5S, S9A, I31L, K43N, M50F, A73L, Δ75-83, E156S, G166S, G169A, S188P, Q206C, N212G, Y217H, S221C, M222P, P225N, T254A, and Q271E, and optionally includes a His tag.

[0137] In another preferred embodiment, the ligase used in the method according to the present invention is a subtilisin BPN' variant having SEQ ID NO: 3, or a homolog thereof having at least 80%, 85%, or 90%, preferably 95%, sequence identity, and includes mutants Q2K, S3C, P5S, S9A, I31L, K43N, M50F, N62A, A73L, Δ75-83, E156N, G166E, G169A, S188P, F189W, Q206C, N212G, Y217H, N218D, S221C, M222P, P225N, T254A, Q271E, and optionally includes a His tag.

[0138] Sequence ID 14 is Ca2+ The present invention shows a subtilisin BPN' variant (for use) having a deletion of the binding loop, S221C, and further mutations. The His tag is included to facilitate purification and is not necessary for ligase activity.

[0139] Furthermore, all of the ligases described, preferably those specified by SEQ ID NO: 14 and SEQ ID NO: 3, as well as homologs thereof having at least 80%, 85%, or 90%, preferably 95%, sequence identity, are also embodiments of the present invention.

[0140] In a preferred embodiment, the method is further characterized in that Y is Lys, and its side chain ε-amino group is functionalized with a functional group selected from the group consisting of γ-Glu-OH, Pal-γ-Glu-OH, AEEA-AEEA-γ-Glu-OH, and AEEA-AEEA-γ-Glu-N-17-carboxyheptadecanoyl-OH (wherein Pal is palmitoyl and AEEA-AEEA is -2-[2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl).

[0141] In a more preferred embodiment, Y is Lys(γ-Glu-OH), Lys(AEEA-AEEA-γ-Glu-OH), Lys(Pal-γ-Glu-OH), or Lys(AEEA-AEEA-γ-Glu-17-carboxyheptadecanoyl-OH).

[0142] The terms "peptide fragment" or "fragment" refer to a peptide having a partial amino acid sequence, with respect to a longer peptide having a defined sequence.

[0143] The present invention will be described below by the following embodiments, but will not be limited thereto. [Examples]

[0144] Ligase production Mutagenesis, cloning, and expression Sequence ID 1 shows the wild-type gene encoding amino acids -107 to -275 of subtilisin BPN'. Here, codons encoding amino acids -107 to -1 are present. These amino acids include signal, pre, and pro sequences that are cleaved and removed during complete maturation. Sequence ID 2 shows mature wild-type subtilisin BPN' (i.e., lacking amino acids -107 to -1). The ligase used for the examples was as shown in Sequence ID 3. Compared to mature wild-type subtilisin BPN', this ligase contained the mutants Q2K, S3C, P5S, S9A, I31L, K43N, M50F, N62A, A73L, Δ75-83, E156N, G166E, G169A, S188P, F189W, Q206C, N212G, Y217H, N218D, S221C, M222P, P225N, T254A, and Q271E. Furthermore, to facilitate rapid and efficient purification, a C-terminal His tag is attached after amino acid 275, as shown in Sequence ID No. 3. The corresponding amino acid sequences are numbered according to the subtilisin BPN' numbering scheme. Therefore, in order to maintain the subtilisin BPN' numbering for the ligase used, the numbering is skipped from 74 to 83.

[0145] The gene encoding the ligase used in the following synthesis examples was obtained from GenScript. The gene was cloned into the pUB-110 Escherichia coli (E. coli)-Bacillus subtilis (B. subtilis) shuttle vector (pBES) using MluI and BamHI site-based vectors (by GenScript). In the shuttle vector, gene expression is regulated by the aprE promoter. The vector contained the pUB ori for Bacillus replication and a kanamycin resistance marker. The vector also contained the ColE1 ori for replication and an ampicillin resistance marker for maintenance in Escherichia coli (E. coli). The resulting plasmid pBES-ligase HIS was grown in Escherichia coli (E. coli) TOP10 and transformed into Bacillus subtilis (B. subtilis) GX4935 (trpC2 metB10 lys-3 ΔnprE ΔaprE).

[0146] Ligase production and purification Single microbial colonies of Bacillus subtilis containing a plasmid with the target subtilisin mutant gene were seeded in 5 mL of LB containing kanamycin (10 μg / mL) in a shaking incubator at 37°C. 0.6 mL of the overnight culture was added to 30 mL of Terrific Broth supplemented with antibiotics (kanamycin 10 μg / mL) and amino acids (100 mg / L Trp, 100 mg / L Met, and 100 mg / L Lys). The cells were grown in a shaking incubator (200 rpm) at 37°C for 48 hours. Cells were harvested by centrifugation (15 min, 4,000 rpm, 4°C). The medium (30 mL) was decanted and concentrated in a Sartorius Vivaspin 15R unit (15 mL, 10 kDa MW cutoff) using two centrifugation steps (15 min, 4,000 rpm, 4°C). Next, the concentrated medium (0.5 mL) was replaced with buffer A (25 mM tricine, pH 7.5, 0.5 M NaCl) in three washing / concentration steps (14 mL of buffer A, 10 minutes, 4,000 rpm, 4°C). For His tag purification, Talon resin (2.5 mL, Clonetech) was added to a plastic column cartridge. The resin was washed with 20 mL of MilliQ water and equilibrated with 20 mL of buffer A. The crude enzyme was loaded onto the column and washed with 5 mL of buffer A. The enzyme was eluted using 15 mL of buffer B (25 mM tricine, pH 7.5, 0.5 M NaCl, 500 mM imidazole). The eluate was concentrated by centrifugation (15 minutes, 4000 rpm, 4°C) in Sartorius Vivaspin 15R (15 mL, 10 kDa MW cutoff), and the buffer was replaced with 25 mM tricine, pH 7.5, through three washing / concentration steps (15 mL buffer, 10 minutes, 4,000 rpm, 4°C).

[0147] The purity of the protein was analyzed by SDS-PAGE, and the enzyme concentration was determined as described in the international publication pamphlet No. 2016056913(A1). The purity was over 90%. The resulting aqueous solution (25 mM tricine, pH 7.5) containing approximately 2 mg / mL of the obtained enzyme was used directly for oligopeptide fragment condensation.

[0148] Examples of enzyme fragment condensation Materials and methods Unless otherwise noted, chemicals were obtained from commercial sources and used without further purification. Ligase SEQ ID NO: 3 was used in all enzyme fragment condensations. Analytical HPLC was performed at 40°C using an Agilent 1260 infinity liquid chromatograph with a reversed-phase column (Phenomenex, C18, particle size 5 μm, 250 x 4.6 mm). UV detection was performed at 220 nm using a UV-VIS204 linear spectrometer. The gradient program consisted of a linear gradient slope of eluent B from 0–25 / 5% to 98% and eluent B from 25.1–30 / 5% (eluent A: 0.5 mL / L methanesulfonic acid (MSA) in H2O, eluent B: 0.5 mL / L MSA in acetonitrile). The flow rate was 1 mL / min from 0 to 25.1 minutes, 2 mL / min from 25.2 to 29.8 minutes, and then returned to 1 mL / min until stopped at 30 minutes. The injection volume was 10 μL. Preparative HPLC was performed using a Varian PrepStar system with a stationary phase column (Phenomenex, C18, particle size 10 μm, 250 x 50 mm). LC-MS was performed at 40°C using an Agilent 1200 series liquid chromatograph with a reversed phase column (Phenomenex, C18, particle size 5 μm, 150 x 4.6 mm). UV detection and gradient programming were as described for analytical HPLC. Molecular weight was determined using an Agilent 6130 quadrupole LC / MS system.

[0149] Protocol 1: Synthesis of Fmoc-glycolic acid 2.5 g of tert-butyl 2-hydroxyacetate was dissolved in a mixture of pyridine (15 ml) and dichloromethane (DCM, 30 ml). Next, 5 g of Fmoc-chloride in 15 ml of dry DCM was added dropwise at 0°C. The reaction mixture was stirred at room temperature for 24 hours. The solvent was removed under vacuum, the residue was redissolved in 40 ml of DCM, washed twice with 20 ml of 1 M sodium bicarbonate solution, washed twice with 20 ml of aqueous salt solution, dried on anhydrous magnesium sulfate, and concentrated. The resulting tert-butyl ester of Fmoc-glycolate (4 g) was dissolved in trifluoroacetic acid (TFA), triisopropylsilane (TIS), and water (95 / 2.5 / 2.5, v / v / v, 15 mL) and stirred for 120 minutes. The solvent was removed under vacuum, and the viscous residue was redissolved in a 5% sodium bicarbonate solution (150 ml) and washed three times with diethyl ether (75 ml). Next, the aqueous solution was mixed with ethyl acetate (45 mL) and acidified to pH=2 at 0°C with 40% phosphoric acid. The organic layer was collected and dried over anhydrous magnesium sulfate. The solvent was removed under vacuum to obtain the final product, Fmoc-glycolic acid (Fmoc-GA).

[0150] Protocol 2: Synthesis of oligopeptide-OCam-Leu-OH ester One gram of pre-loaded Fmoc-Leu-Wang resin (loaded at 0.81 mmol / gram) was washed with DCM (2 x 2 min, 10 mL) and N,N'-dimethylformamide (DMF, 2 x 2 min, 10 mL), and Fmoc deprotection was performed using piperidine / DMF (1 / 5, v / v, 2 x 8 min, 10 mL). After washing with DMF (6 x 2 min, 10 mL), Fmoc-GA (4 equivalents) was coupled to the resin using 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU, 4 equivalents), OxymaPure (4 equivalents), and di-isopropylethylamine (DIPEA, 8 equivalents) in DMF (45 min, 10 mL). After washing with DMF (2x2 min, 10 mL), the resin was Fmoc-deprotected using piperidine / DMF (1 / 5, v / v, 2x8 min, 10 mL). Cam-Leu-OH esters were formed by coupling a first Fmoc-protecting amino acid with 4 equivalents of Fmoc-Xxx-OH, 4 equivalents of N,N'-diisopropylcarbodiimide (DIC), and 0.1 equivalents of 4-dimethylaminopyridine (DMAP) in DMF (2x60 min, 10 mL). Herein, and in other parts of this disclosure, "Xxx" represents a single amino acid (a variable as shown in the sequence in the following examples). A commercially available Fmoc-Aib-OH component was used for the semaglutide starting material.

[0151] After washing with DMF (6 x 2 mins, 10 mL), the peptide was extended according to the standard SPPS protocol (Weng C. Chan and Peter White, OUP Oxford, 2000). Cleavage from the resin and side-chain deprotection were performed for 120 minutes using a mixture of TFA / TIS / water (95 / 2.5 / 2.5, v / v / v, 15 mL). The crude peptide was precipitated using methyl tert-butyl ether (MTBE) / n-heptane (1 / 1, v / v, 50 mL). The precipitated peptide was recovered by centrifugation, washed twice with MTBE / n-heptane (1 / 1, v / v, 50 mL), and then lyophilized in acetonitrile / water (1 / 1, v / v, 50 mL). The crude product was purified by preparative HPLC, and the pure fraction was lyophilized.

[0152] As described above, however, in the pre-loaded Fmoc-Arg(Pbf)-Rink resin (loaded at 0.62 mmol / gram), several alternative peptide Cam-esters, namely H- 1 His- 2 Ala- 3 Glu- 4 Gly- 5 Thr- 6 Phe- 7 Thr- 8 Ser- 9 Asp- 10 Val- 11 Ser-OCam-Phe-Arg-NH2,H- 1 His- 2 Ala- 3 Glu- 4 Gly- 5 Thr- 6 Phe- 7 Thr- 8 Ser- 9 Asp- 10 Val- 11 Ser-OCam-Leu-Arg-NH2, and H- 1 His- 2 Ala- 3 Glu- 4 Gly- 5 Thr- 6 Phe- 7 Thr- 8 Ser- 9 Asp- 10 Val- 11 Ser-OCam-Trp-Arg-NH2 was prepared.

[0153] Protocol 3: Synthesis of C-terminal acid nucleophiles of oligopeptides One gram of pre-loaded Fmoc-Gly-Wang resin (loaded at 0.30 mmol / gram) was washed with DCM (2x2 min, 10 mL) and DMF (2x2 min, 10 mL), and Fmoc deprotection was performed using piperidine / DMF (1 / 5, v / v, 2x8 min, 10 mL). The peptide was extended according to the standard SPPS protocol (Weng C. Chan and Peter White, OUP Oxford, 2000). Cleavage from the resin and side chain deprotection were performed for 120 minutes using a mixture of TFA / TIS / water (95 / 2.5 / 2.5, v / v / v, 15 mL). The crude peptide was precipitated using MTBE / n-heptane (1 / 1, v / v, 50 mL). The precipitated peptide was recovered by centrifugation, washed twice with MTBE / n-heptane (1 / 1, v / v, 50 mL), and then lyophilized in acetonitrile / water (1 / 1, v / v, 50 mL). The crude product was purified by preparative HPLC, and the pure fraction was lyophilized.

[0154] Protocol 4: H-Gly- 1 His- 2 Ala- 3 Glu- 4 Gly- 5 Thr- 6 Phe- 7 Thr- 8 Ser- 9 Asp- 10 Val- 11 PTC protection of Ser-OCam-Leu-OH 100mg H-Gly- 1 His- 2 Ala- 3 Glu- 4 Gly- 5 Thr- 6 Phe- 7 Thr- 8 Ser- 9 Asp- 10 Val- 11Ser-OCam-Leu-OH was dissolved in 10 mL of pyridine / water (1 / 1, v / v). To this mixture, 25 mg of phenyl isothiocyanate was added and the solution was stirred at ambient temperature for 14 h. The crude reaction mixture was diluted with 50 mL of water and washed three times with 50 mL of dichloromethane (DCM). The aqueous layer was purified by preparative HPLC and the pure fractions were lyophilized to afford the PTC-Gly protected peptide.

[0155] Protocol 5: Synthesis of γ-Glu or Pal-γ-Glu containing peptides Commercially available Fmoc-Lys(Boc-γ-Glu-O t Bu)-OH or Fmoc-Lys(Pal-γ-Glu-O t Bu)-OH components were used according to the general protocol 3.

[0156] Protocol 6: Synthesis of semaglutide fragment H- 12 Ser- 13 Tyr- 14 Leu- 15 Glu- 16 Gly- 17 Gln- 18 Ala- 19 Ala- 20 Lys(AEEA-AEEA-γ-Glu-N-17-carboxyheptadecanoyl-OH)- 21 Glu- 22 Phe- 23 Ile- 24 Ala- 25 Trp- 26 Leu- 27 Val- 28 Arg- 29 Gly- 30 Arg- 31 Synthesis of Gly-OH Commercially available Fmoc- 20 Lys(Mtt)-OH and Boc- 12 Ser( tUsing the Bu)-OH component, we followed general protocol 3. After SPPS of the Boc-12-31-Wang fragment, the Mtt protecting group was removed using 10 mL of TIS / TFA / DCM (1 / 1 / 48, v / v / v, 3 x 15 mins). Fmoc-AEEA-OH (twice), Fmoc-Glu-O t Bu, and 17-carboxyheptadecanoyl-O t Standard SPPS procedure was used for Bu coupling. Cleavage from the resin and side-chain deprotection were performed for 120 minutes using a mixture of TFA / TIS / water (95 / 2.5 / 2.5, v / v / v, 15 mL). The crude peptide was precipitated using MTBE / n-heptane (1 / 1, v / v, 50 mL). The precipitated peptide was recovered by centrifugation, washed twice with MTBE / n-heptane (1 / 1, v / v, 50 mL), and then lyophilized in acetonitrile / water (1 / 1, v / v, 50 mL). The crude product was purified by preparative HPLC, and the pure fraction was lyophilized.

[0157] Example 1 (see reference): Enzymatic synthesis of liraglutide precursor H-liraglutide-1~31-OH using a 13-mer + 18-mer approach In an HPLC vial, 6 mg of H- 1 His- 2 Ala- 3 Glu- 4 Gly- 5 Thr- 6 Phe- 7 Thr- 8 Ser- 9 Asp- 10 Val- 11 Ser- 12 Ser- 13 Tyr-OCam-Leu-OH and 9 mg of H- 14 Leu- 15 Glu- 16 Gly- 17 Gln- 18 Ala- 19 Ala- 20 Lys- 21 Glu- 22 Phe- 23 Ile-24 Ala- 25 Trp- 26 Leu- 27 Val- 28 Arg- 29 Gly- 30 Arg- 31 Gly-OH was dissolved in 800 μL of 2 M guanidia chloride water. To this mixture, 50 μL of 1 M tricine buffer pH 9.0 was added, and the pH was adjusted to 8.3 with 4 M NaOH solution. Subsequently, 10 μL of TCEP (tris(2-carboxyethyl)phosphine) solution (100 mg / mL in water) and 100 μL of ligase solution (10 mg / mL) were added. The mixture was reacted at ambient temperature. Every 15 minutes, 10 μL of the reaction mixture was taken out, quenched in 980 μL of 5 vol% MSA in acetonitrile / water (2 / 1, v / v), and analyzed by LC-MS.

[0158] After 90 minutes, all Cam ester starting material was consumed, and the product and hydrolysis peaks were integrated. Ligation product H- 1 His- 2 Ala- 3 Glu- 4 Gly- 5 Thr- 6 Phe- 7 Thr- 8 Ser- 9 Asp- 10 Val- 11 Ser- 12 Ser- 13 Tyr- 14 Leu- 15 Glu- 16 Gly- 17 Gln- 18 Ala- 19 Ala- 20 Lys- 21 Glu- 22 Phe- 23 Ile- 24 Ala- 25 Trp- 26 Leu- 27 Val- 28 Arg- 29 Gly- 30 Arg-31 Gly-OH is present at 14 area%, and hydrolyzed Cam-ester H- 1 His- 2 Ala- 3 Glu- 4 Gly- 5 Thr- 6 Phe- 7 Thr- 8 Ser- 9 Asp- 10 Val- 11 Ser- 12 Ser- 13 Tyr-OH accounted for 86% of the area.

[0159] The product H-liraglutide-1~31-OH could be obtained by preparative HPLC and subsequent lyophilization of the pure fraction.

[0160] Example 2 (see reference): Enzymatic synthesis of liraglutide precursor H-liraglutide-1~31-OH using a 9-mer + 22-mer approach In an HPLC vial, 6 mg of H- 1 His- 2 Ala- 3 Glu- 4 Gly- 5 Thr- 6 Phe- 7 Thr- 8 Ser- 9 Asp-OCam-Leu-OH and 10 mg of H- 10 Val- 11 Ser- 12 Ser- 13 Tyr- 14 Leu- 15 Glu- 16 Gly- 17 Gln- 18 Ala- 19 Ala- 20 Lys- 21 Glu- 22 Phe- 23 Ile- 24 Ala- 25 Trp- 26 Leu- 27 Val-28 Arg- 29 Gly- 30 Arg- 31 Gly-OH was dissolved in 800 μL of 2 M guanidia chloride water. To this mixture, 50 μL of 1 M tricine buffer pH 9.0 was added, and the pH was adjusted to 8.3 with 4 M NaOH solution. Subsequently, 10 μL of TCEP solution (100 mg / mL in water) and 100 μL of ligase solution (10 mg / mL) were added. The mixture was reacted at ambient temperature. Every 15 minutes, 10 μL of the reaction mixture was quenched in 980 μL of 5 vol% MSA in acetonitrile / water (2 / 1, v / v), and analyzed by LC-MS.

[0161] After 300 minutes, all Cam-ester starting material was consumed, and the product and hydrolysis peaks were integrated. Ligation product H- 1 His- 2 Ala- 3 Glu- 4 Gly- 5 Thr- 6 Phe- 7 Thr- 8 Ser- 9 Asp- 10 Val- 11 Ser- 12 Ser- 13 Tyr- 14 Leu- 15 Glu- 16 Gly- 17 Gln- 18 Ala- 19 Ala- 20 Lys- 21 Glu- 22 Phe- 23 Ile- 24 Ala- 25 Trp- 26 Leu- 27 Val- 28 Arg- 29 Gly- 30 Arg- 31 Gly-OH is present at 8 area%, and hydrolyzed Cam-ester H- 1 His- 2 Ala- 3 Glu-4 Gly- 5 Thr- 6 Phe- 7 Thr- 8 Ser- 9 Asp-OH accounted for 92% of the area.

[0162] The product H-liraglutide-1~31-OH could be obtained by preparative HPLC and subsequent lyophilization of the pure fraction.

[0163] Example 3: Enzymatic synthesis of H-liraglutide-1~31-OH using the 11-mer + 20-mer approach In an HPLC vial, 6 mg of H- 1 His- 2 Ala- 3 Glu- 4 Gly- 5 Thr- 6 Phe- 7 Thr- 8 Ser- 9 Asp- 10 Val- 11 Ser-OCam-Leu-OH and 10 mg of H- 12 Ser- 13 Tyr- 14 Leu- 15 Glu- 16 Gly- 17 Gln- 18 Ala- 19 Ala- 20 Lys- 21 Glu- 22 Phe- 23 Ile- 24 Ala- 25 Trp- 26 Leu- 27 Val- 28 Arg- 29 Gly- 30 Arg- 31Gly-OH was dissolved in 800 μL of 2 M guanidia chloride water. To this mixture, 50 μL of 1 M tricine buffer pH 9.0 was added, and the pH was adjusted to 8.3 with 4 M NaOH solution. Subsequently, 10 μL of TCEP solution (100 mg / mL in water) and 100 μL of ligase solution (10 mg / mL) were added. The mixture was reacted at ambient temperature. Every 15 minutes, 10 μL of the reaction mixture was taken out, quenched in 980 μL of 5 vol% MSA in acetonitrile / water (2 / 1, v / v), and analyzed by LC-MS.

[0164] After 180 minutes, all Cam-ester starting material was consumed, and the product and hydrolysis peaks were integrated. Ligation product H- 1 His- 2 Ala- 3 Glu- 4 Gly- 5 Thr- 6 Phe- 7 Thr- 8 Ser- 9 Asp- 10 Val- 11 Ser- 12 Ser- 13 Tyr- 14 Leu- 15 Glu- 16 Gly- 17 Gln- 18 Ala- 19 Ala- 20 Lys- 21 Glu- 22 Phe- 23 Ile- 24 Ala- 25 Trp- 26 Leu- 27 Val- 28 Arg- 29 Gly- 30 Arg- 31 Gly-OH accounts for 96 area, and hydrolyzed Cam-ester H- 1 His- 2 Ala- 3 Glu- 4 Gly- 5 Thr- 6 Phe-7 Thr- 8 Ser- 9 Asp- 10 Val- 11 Ser- 12 Ser- 13 The Tyr-OH concentration was 4% by area.

[0165] The product H-liraglutide-1~31-OH could be obtained by preparative HPLC and subsequent lyophilization of the pure fraction.

[0166] Therefore, the method according to the present invention yields, unexpectedly, much higher yields than in the comparative method in which the coupling site for the C-terminal (thio) ester and peptide nucleophile is two amide bonds to the C-terminus or N-terminus of H-liraglutide-1~31-OH, the desired coupling product (i.e., H-liraglutide-1~31-OH) 20 This invention provides a peptide having the amino acid sequence of liraglutide without derivatization in Lys.

[0167] Example 4: Using the 11-mer + 20-mer approach, PTC-Gly-liraglutide-1~31-[ 20 Enzymatic synthesis of Lys(γ-Glu)-OH In an HPLC vial, 6 mg of PTC-Gly- 1 His- 2 Ala- 3 Glu- 4 Gly- 5 Thr- 6 Phe- 7 Thr- 8 Ser- 9 Asp- 10 Val- 11 Ser-OCam-Leu-OH and 10 mg of H- 12 Ser- 13 Tyr- 14 Leu- 15 Glu- 16 Gly- 17 Gln- 18 Ala- 19 Ala- 20 Lys(γ-Glu)-21 Glu- 22 Phe- 23 Ile- 24 Ala- 25 Trp- 26 Leu- 27 Val- 28 Arg- 29 Gly- 30 Arg- 31 Gly-OH was dissolved in 800 μL of 2M guanidia chloride water.

[0168] To this mixture, 50 μL of 1 M tricine buffer pH 9.0 was added, and the pH was adjusted to 8.3 using 4 M NaOH solution. Subsequently, 10 μL of TCEP solution (100 mg / mL in water) and 100 μL of ligase solution (10 mg / mL) were added. The mixture was reacted at ambient temperature. Every 15 minutes, 10 μL of the reaction mixture was taken out, quenched in 980 μL of 5 vol% MSA in acetonitrile / water (2 / 1, v / v), and analyzed by LC-MS.

[0169] After 180 minutes, all Cam-ester starting materials were consumed, and the product and hydrolysis peaks were integrated. Ligation product PTC-Gly- 1 His- 2 Ala- 3 Glu- 4 Gly- 5 Thr- 6 Phe- 7 Thr- 8 Ser- 9 Asp- 10 Val- 11 Ser- 12 Ser- 13 Tyr- 14 Leu- 15 Glu- 16 Gly- 17 Gln- 18 Ala- 19 Ala- 20 Lys(γ-Glu)- 21 Glu- 22 Phe- 23 Ile- 24 Ala- 25 Trp-26 Leu- 27 Val- 28 Arg- 29 Gly- 30 Arg- 31 Gly-OH accounts for 93 area, and hydrolyzed Cam-ester PTC-Gly- 1 His- 2 Ala- 3 Glu- 4 Gly- 5 Thr- 6 Phe- 7 Thr- 8 Ser- 9 Asp- 10 Val- 11 The amount of Ser-OH was 7% by area.

[0170] Product PTC-Gly-liraglutide-1~31-[ 20 Lys(γ-Glu)-OH could be obtained by preparative HPLC and subsequent lyophilization of the pure fraction.

[0171] Example 5: PTC-Gly-liraglutide-1~31-[ from Example 4 20 Using the Lys(γ-Glu)-OH precursor, H-liraglutide-1~31-[ 20 Synthesis of Lys(Pal-γ-Glu)-OH 2mg PTC-Gly- 1 His- 2 Ala- 3 Glu- 4 Gly- 5 Thr- 6 Phe- 7 Thr- 8 Ser- 9 Asp- 10 Val- 11 Ser - 12 Ser- 13 Tyr- 14 Leu- 15 Glu- 16 Gly- 17 Gln- 18 Ala- 19 Ala- 20 Lys(γ-Glu)-21 Glu- 22 Phe- 23 Ile- 24 Ala- 25 Trp - 26 Leu- 27 Val- 28 Arg- 29 Gly- 30 Arg- 31 Gly-OH was dissolved in 500 μL of water and 500 μL of pyridine. 2 mg of N-hydroxysuccinimide palmitate (Pal-OSu) was added to this solution, and the mixture was reacted at ambient temperature for 5 hours, after which the solvent was evaporated under vacuum. To cleave (deprotect) the PTC-Gly group, the crude product PTC-Gly- 1 His- 2 Ala- 3 Glu- 4 Gly- 5 Thr- 6 Phe- 7 Thr- 8 Ser- 9 Asp- 10 Val- 11 Ser- 12 Ser- 13 Tyr- 14 Leu- 15 Glu- 16 Gly- 17 Gln- 18 Ala- 19 Ala- 20 Lys(Pal-γ-Glu)- 21 Glu- 22 Phe- 23 Ile- 24 Ala- 25 Trp- 26 Leu- 27 Val- 28 Arg- 29 Gly- 30 Arg- 31 Gly-OH was dissolved in 5 vol% trifluoroacetic acid in water.

[0172] After completion (15 minutes), the product H- 1 His- 2 Ala- 3 Glu-4 Gly- 5 Thr- 6 Phe- 7 Thr- 8 Ser- 9 Asp- 10 Val- 11 Ser - 12 Ser- 13 Tyr- 14 Leu- 15 Glu- 16 Gly- 17 Gln- 18 Ala- 19 Ala- 20 Lys(Pal-γ-Glu)- 21 Glu- 22 Phe- 23 Ile- 24 Ala- 25 Trp- 26 Leu- 27 Val- 28 Arg- 29 Gly- 30 Arg- 31 Gly-OH was obtained, purified by preparative HPLC, and the pure fraction was freeze-dried.

[0173] Example 6: Using the 11-mer + 20-mer approach, H-liraglutide-1~31-[ 20 Enzymatic synthesis of Lys(Pal-γ-Glu)-OH (Pal = palmitoyl) In an HPLC vial, 6 mg of H- 1 His- 2 Ala- 3 Glu- 4 Gly- 5 Thr- 6 Phe- 7 Thr- 8 Ser- 9 Asp- 10 Val- 11 Ser-OCam-Leu-OH and 10 mg of H- 12 Ser- 13 Tyr- 14 Leu- 15 Glu- 16 Gly- 17 Gln-18 Ala- 19 Ala- 20 Lys(Pal-γ-Glu)- 21 Glu- 22 Phe- 23 Ile- 24 Ala- 25 Trp- 26 Leu- 27 Val- 28 Arg- 29 Gly- 30 Arg- 31 Gly-OH was dissolved in 950 μL of water. To this mixture, 50 μL of 1 M tricine buffer pH 9.0 was added, and the pH was adjusted to 8.1 using 3 M NaOH solution. Subsequently, 10 μL of TCEP solution (100 mg / mL in water) and 100 μL of ligase solution (10 mg / mL) were added. The mixture was reacted at ambient temperature. Every 15 minutes, 10 μL of the reaction mixture was taken out, quenched in 980 μL of 5 vol% MSA in acetonitrile / water (2 / 1, v / v), and analyzed by LC-MS.

[0174] After 180 minutes, all Cam-ester starting material was consumed, and the product and hydrolysis peaks were integrated. Ligation product H- 1 His- 2 Ala- 3 Glu- 4 Gly- 5 Thr- 6 Phe- 7 Thr- 8 Ser- 9 Asp- 10 Val- 11 Ser- 12 Ser- 13 Tyr- 14 Leu- 15 Glu- 16 Gly- 17 Gln- 18 Ala- 19 Ala- 20 Lys(Pal-γ-Glu)- 21 Glu- 22 Phe- 23 Ile- 24 Ala- 25Trp- 26 Leu- 27 Val- 28 Arg- 29 Gly- 30 Arg- 31 Gly-OH accounts for 95 area, and hydrolyzed Cam-ester H- 1 His- 2 Ala- 3 Glu- 4 Gly- 5 Thr- 6 Phe- 7 Thr- 8 Ser- 9 Asp- 10 Val- 11 The amount of Ser-OH was 5% by area.

[0175] This example demonstrates that the present invention enables the direct synthesis of liraglutide in high yield in a single enzyme coupling step.

[0176] Several alternative peptide Cam-esters, namely (1)H- 1 His- 2 Ala- 3 Glu- 4 Gly- 5 Thr- 6 Phe- 7 Thr- 8 Ser- 9 Asp- 10 Val- 11 Ser-OCam-Phe-Arg-NH2, (2)H- 1 His- 2 Ala- 3 Glu- 4 Gly- 5 Thr- 6 Phe- 7 Thr- 8 Ser- 9 Asp- 10 Val- 11 Ser-OCam-Leu-Arg-NH2, and (3)H- 1 His- 2 Ala- 3 Glu- 4 Gly- 5Thr- 6 Phe- 7 Thr- 8 Ser- 9 Asp- 10 Val- 11 The same ligation reaction as above was carried out, except that Ser-OCam-Trp-Arg-NH2 was used. Generally, the reaction is H- 1 His- 2 Ala- 3 Glu- 4 Gly- 5 Thr- 6 Phe- 7 Thr- 8 Ser- 9 Asp- 10 Val- 11 The conversion proceeded faster than with the Ser-OCam-Leu-OH (180 min) ester, with complete conversion occurring after 80 minutes in case (1), 100 minutes in case (2), and 85 minutes in case (3).

[0177] Example 7: H-liraglutide-1~31-OH from the precursor of Example 3, synthesized enzymatically, is derived from H-liraglutide-1~31-[ 20 Synthesis of Lys(Pal-γ-Glu)-OH Using the protocol described in U.S. Patent No. 6451974B1, the precursor H- 1 His- 2 Ala- 3 Glu- 4 Gly- 5 Thr- 6 Phe- 7 Thr- 8 Ser- 9 Asp- 10 Val- 11 Ser- 12 Ser- 13 Tyr- 14 Leu- 15 Glu- 16 Gly- 17 Gln- 18 Ala- 19 Ala- 20 Lys- 21 Glu- 22 Phe- 23Ile- 24 Ala- 25 Trp- 26 Leu- 27 Val- 28 Arg- 29 Gly- 30 Arg- 31 The Pal-γ-Glu moiety is coupled to the Gly-OH, and the product H- 1 His- 2 Ala- 3 Glu- 4 Gly- 5 Thr- 6 Phe- 7 Thr- 8 Ser- 9 Asp- 10 Val- 11 Ser- 12 Ser- 13 Tyr- 14 Leu- 15 Glu- 16 Gly- 17 Gln- 18 Ala- 19 Ala- 20 Lys(Pal-γ-Glu)- 21 Glu- 22 Phe- 23 Ile- 24 Ala- 25 Trp- 26 Leu- 27 Val- 28 Arg- 29 Gly- 30 Arg- 31 Gly-OH was obtained.

[0178] Example 8: Using the 11-mer + 20-mer approach, H-semaglutide-1~31-[ 20 Synthesis of Lys(AEEA-AEEA-γ-Glu-N-17-carboxyheptadecanoyl-OH)]-OH In an HPLC vial, 6 mg of H- 1 His- 2 Aib- 3 Glu- 4 Gly- 5 Thr- 6 Phe-7 Thr- 8 Ser- 9 Asp- 10 Val- 11 Ser-OCam-Leu-OH and 10 mg of H- 12 Ser- 13 Tyr- 14 Leu- 15 Glu- 16 Gly- 17 Gln- 18 Ala- 19 Ala- 20 Lys(AEEA-AEEA-γ-Glu-N-17-carboxyheptadecanoyl-OH)- 21 Glu- 22 Phe- 23 Ile- 24 Ala- 25 Trp- 26 Leu- 27 Val- 28 Arg- 29 Gly- 30 Arg- 31 Gly-OH was dissolved in 950 μL of water. To this mixture, 50 μL of 1 M tricine buffer pH 9.0 was added, and the pH was adjusted to 8.1 using 3 M NaOH solution. Subsequently, 10 μL of TCEP solution (100 mg / mL in water) and 100 μL of ligase solution (10 mg / mL) were added. The mixture was reacted at ambient temperature. Every 15 minutes, 10 μL of the reaction mixture was taken out, quenched in 980 μL of 5 vol% MSA in acetonitrile / water (2 / 1, v / v), and analyzed by LC-MS.

[0179] After 180 minutes, all Cam-ester starting material was consumed, and the product and hydrolysis peaks were integrated. Ligation product H- 1 His- 2 Aib- 3 Glu- 4 Gly- 5 Thr- 6 Phe- 7 Thr- 8 Ser- 9 Asp- 10 Val- 11 Ser-12 Ser- 13 Tyr- 14 Leu- 15 Glu- 16 Gly- 17 Gln- 18 Ala- 19 Ala- 20 Lys(AEEA-AEEA-γ-Glu-N-17-carboxyheptadecanoyl-OH)- 21 Glu- 22 Phe- 23 Ile- 24 Ala- 25 Trp- 26 Leu- 27 Val- 28 Arg- 29 Gly- 30 Arg- 31 Gly-OH accounts for 96 area, and hydrolyzed Cam-ester H- 1 His- 2 Ala- 3 Glu- 4 Gly- 5 Thr- 6 Phe- 7 Thr- 8 Ser- 9 Asp- 10 Val- 11 The amount of Ser-OH was 4% by area.

[0180] This example demonstrates that the present invention enables the direct synthesis of semaglutide in high yield in a single enzyme coupling step.

[0181] Example 9: Enzymatic synthesis of H-liraglutide-1~31-OH using the 11-mer+20-mer approach with the enzyme mutant of SEQ ID NO: 14 In an HPLC vial, 10 mg of H- 1 His- 2 Ala- 3 Glu- 4 Gly- 5 Thr- 6 Phe- 7 Thr- 8 Ser- 9 Asp- 10Val- 11 Ser-OCam-Leu-OH and 10 mg of H- 12 Ser- 13 Tyr- 14 Leu- 15 Glu- 16 Gly- 17 Gln- 18 Ala- 19 Ala- 20 Lys- 21 Glu- 22 Phe- 23 Ile- 24 Ala- 25 Trp- 26 Leu- 27 Val- 28 Arg- 29 Gly- 30 Arg- 31 Gly-OH was dissolved in 500 μL of 50 mM tricine buffer at pH 9.0. The pH was adjusted to 8.3 using 3 M NaOH solution. Subsequently, 10 μL of TCEP solution (100 mg / mL in water) and 100 μL of ligase solution (10 mg / mL) were added. The mixture was reacted at ambient temperature. Every 15 minutes, 10 μL of the reaction mixture was taken out, quenched in 980 μL of 5 vol% MSA in acetonitrile / water (2 / 1, v / v), and analyzed by LC-MS.

[0182] After 240 minutes, all Cam-ester starting material was consumed, and the product and hydrolysis peaks were integrated. Ligation product H- 1 His- 2 Ala- 3 Glu- 4 Gly- 5 Thr- 6 Phe- 7 Thr- 8 Ser- 9 Asp- 10 Val- 11 Ser- 12 Ser- 13 Tyr- 14 Leu- 15 Glu- 16 Gly- 17 Gln- 18 Ala-19 Ala- 20 Lys- 21 Glu- 22 Phe- 23 Ile- 24 Ala- 25 Trp- 26 Leu- 27 Val- 28 Arg- 29 Gly- 30 Arg- 31 Gly-OH accounts for 92 area, and hydrolyzed Cam-ester H- 1 His- 2 Ala- 3 Glu- 4 Gly- 5 Thr- 6 Phe- 7 Thr- 8 Ser- 9 Asp- 10 Val- 11 The Ser-OH concentration was 8% by area.

[0183] The product H-liraglutide-1~31-OH could be obtained by preparative HPLC and subsequent lyophilization of the pure fraction.

[0184] Example 10: Identification of a fragment suitable for enzymatic synthesis of liraglutide using serine endoprotease derived from a subtilisin BPN' mutant (C221S mutation) having SEQ ID NO: 3. 1 mg each of liraglutide 1-31 (with and without Pal-γ-Glu at the lysine position 20) was dissolved in 1 mL of trichine buffer (50 mM, pH=8.0). 1 μL of endoprotease solution (1 mg / mL) was added to this mixture, and the reaction mixture was stirred at room temperature.

[0185] Hydrolytic activity was monitored by analyzing samples every 30 minutes using LC-MS analysis.

[0186] For both peptides, the highest hydrolytic activity was obtained at the linkage between amino acids 25 and 26 (resulting in fragments 1-25 + 26-31), followed by hydrolytic activity at the linkage between amino acids 5 and 6 (resulting in fragments 1-5 + 6-31). The advantages of the 25-mer + 6-mer approach were tested in Comparative Example 11.

[0187] Example 11: Enzymatic synthesis of liraglutide precursor H-liraglutide-1~31-OH using the 25-mer+6-mer approach (reference), the 5-mer+26-mer approach (reference), or the 11-mer+20-mer approach (according to the present invention) in parallel. In an HPLC vial, 3 μmol of ester (1:H- 1 His- 2 Ala- 3 Glu- 4 Gly- 5 Thr- 6 Phe- 7 Thr- 8 Ser- 9 Asp- 10 Val- 11 -Ser 12 Ser- 13 Tyr- 14 Leu- 15 Glu- 16 Gly- 17 Gln- 18 Ala- 19 Ala- 20 Lys(Pal-γ-Glu)- 21 Glu- 22 Phe- 23 Ile- 24 Ala- 25 Trp-OCam-Leu-OH, 2:H- 1 His- 2 Ala- 3 Glu- 4 Gly- 5 Thr-OCam-Leu-OH, or 3:H- 1 His- 2 Ala- 3 Glu- 4 Gly- 5 Thr- 6Phe- 7 Thr- 8 Ser- 9 Asp- 10 Val- 11 (Ser-OCam-Leu-OH) and 2 μmol of amine (1:H- 26 Leu- 27 Val- 28 Arg- 29 Gly- 30 Arg- 31 Gly-OH, 2:H- 6 Phe- 7 Thr- 8 Ser- 9 Asp- 10 Val- 11 Ser- 12 Ser- 13 Tyr- 14 Leu- 15 Glu- 16 Gly- 17 Gln- 18 Ala- 19 Ala- 20 Lys(Pal-γ-Glu)- 21 Glu- 22 Phe- 23 Ile- 24 Ala- 25 Trp- 26 Leu- 27 Val- 28 Arg- 29 Gly- 30 Arg- 31 Gly-OH, or 3:H- 12 Ser- 13 Tyr- 14 Leu- 15 Glu- 16 Gly- 17 Gln- 18 Ala- 19 Ala- 20 Lys(Pal-γ-Glu)- 21 Glu- 22 Phe- 23 Ile- 24 Ala- 25 Trp- 26 Leu- 27 Val- 28 Arg- 29Gly- 30 Arg- 31 Gly-OH was dissolved in 950 μL of water. 50 μL of 1 M tricine buffer pH 9.0 was added to this mixture, and the pH was adjusted to 8.1 using 3 M NaOH solution. Subsequently, 10 μL of TCEP solution (100 mg / mL in water) and 100 μL of ligase solution (according to SEQ ID NO: 3) (10 mg / mL) were added. The mixture was allowed to react at ambient temperature. Every 15 minutes, 10 μL of the reaction mixture was taken out, quenched in 980 μL of 5 vol% MSA in acetonitrile / water (2 / 1, v / v), and analyzed by LC-MS.

[0188] After 180 minutes, the Cam-ester starting material was consumed for each of the three reactions. Samples of the mixture of the resulting products were analyzed, and the product and hydrolysis peaks were integrated. The ligation product of reaction 1 was 72 area%, the ligation product of reaction 2 was 53 area%, and the ligation product of reaction 3 was 95 area%.

[0189] array Sequence ID 1: Wild-type gene encoding subtilisin BPN' amino acids -107~275 ENA|K02496|K02496.1 B. Subtilisin BPN' Bacillus amyloliquefaciens [ka]

[0190] Sequence ID 2: Wild-type subtilisin BPN' (mature) >SUBT_BACAM Subtilisin BPN' Bacillus amyloliquefaciens Mature 1-275 >sp|P00782|108-382 [ka]

[0191] Sequence ID 3: Mutations of Q2K, S3C, P5S, S9A, I31L, K43N, M50F, N62A, A73L, Δ75-83, E156N, G166E, G169A, S188P, F189W, Q206C, N212G, Y217H, N218D, S221C, M222P, P225N, T254A, and Q271E, as well as subtilisin BPN' variants with His tag. [ka]

[0192] Sequence ID 14: Mutations of Q2K, S3C, P5S, S9A, I31L, K43N, M50F, A73L, Δ75-83, E156S, G166S, G169A, S188P, Q206C, N212G, Y217H, S221C, M222P, P225N, T254A, and Q271E, as well as subtilisin BPN' variants with His tag. [ka]

Claims

1. (a) A peptide C-terminal ester or thioester comprising a first peptide fragment containing the sequence His-X-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-(thio)ester, (b) A peptide nucleophile having an N-terminal unprotected amine comprising a second peptide fragment containing the sequence H-Ser-Tyr-Leu-Glu-Gly-Glun-Ala-Ala-Y-Glu-Phe-Ile-Ala-Trp-Leu-Val-Z-Gly-Arg-Gly A method for synthesizing a peptide comprising the sequence His-X-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Y-Glu-Phe-Ile-Ala-Trp-Leu-Val-Z-Gly-Arg-Gly, comprising enzymatic coupling of the following: - X is an Ala or α-aminoisobutyric acid (Aib) residue, - Y is Lys, and the Lys has a free side chain ε-amino group, or the side chain ε-amino group of the Lys is protected by a protecting group selected from an allyloxycarbonyl group and a trifluoroacetyl group, or the side chain ε-amino group of the Lys is functionalized by an amino acid or a functional group selected from the group consisting of γ-Glu-OH, Pal-γ-Glu-OH, AEEA-AEEA-γ-Glu-OH and AEEA-AEEA-γ-Glu-N-17-carboxyheptadecanoyl-OH [wherein Pal is palmitoyl and AEEA-AEEA is -2-[2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl], - Z is Arg or Lys, The aforementioned enzymatic coupling is catalyzed by a ligase. The ligase is a subtilisin BPN' mutant or its homolog. The aforementioned subtilisin BPN' variant contains only mutation (A) compared to the subtilisin BPN' represented by Sequence ID No.

2. The aforementioned mutation (A) - Deletion of amino acids corresponding to positions 75-83; and - A mutation at the amino acid position corresponding to S221, which is S221C or S221 selenocysteine; It consists of, The aforementioned amino acid positions are defined according to the sequence of subtilisin BPN' represented by Sequence ID No. 2, The homolog contains the mutation (A) and has at least 90% sequence identity with the subtilisin BPN' variant. A method comprising optionally attaching a His tag to the subtilisin BPN' mutant or its homologue.

2. The method according to claim 1, wherein the synthesized peptide is liraglutide.

3. (a) A peptide C-terminal ester or thioester containing the sequence His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-(thio)ester, (b) A peptide nucleophile containing the sequence H-Ser-Tyr-Leu-Glu-Gly-Glun-Ala-Ala-Y-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly (wherein Y is Lys(Pale-γ-Glu-OH)) and The method according to claim 2, comprising enzyme coupling catalyzed by a ligase.

4. (a) Formula P-W ｖ A peptide C-terminal ester or thioester represented by -His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-(thio)ester (wherein P is an Edman protecting group which is a carbamate protecting group, an acyl protecting group, or a substituted thiocarbamoyl group, v is an integer from 0 to 5, and each W independently represents the same or different amino acid residue), (b) A peptide nucleophile containing the sequence H-Ser-Tyr-Leu-Glu-Gly-Glun-Ala-Ala-Lys(γ-Glu-OH)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly and The method according to claim 2, comprising enzyme coupling catalyzed by a ligase, and subsequently providing a palmitoyl group (Pa) to the Lys(γ-Glu-OH).

5. (a) A peptide C-terminal ester or thioester containing the sequence His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-(thio)ester, (b) A peptide nucleophile comprising the sequence H-Ser-Tyr-Leu-Glu-Gly-Glun-Ala-Ala-Y-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly (wherein Y is a lysine residue having a free ε-amino side chain) and The method according to claim 2, comprising enzyme coupling catalyzed by a ligase, and subsequently providing Pal-γ-Glu-OH to the amino side chain.

6. The method according to claim 1, wherein the synthesized peptide is semaglutide.

7. (a) A peptide C-terminal ester or thioester containing the sequence His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-(thio)ester, (b) A peptide nucleophile containing the sequence H-Ser-Tyr-Leu-Glu-Gly-Glun-Ala-Ala-Y-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly (wherein Y is Lys(AEEA-AEEA-γ-Glu-N-17-carboxyheptadecanoyl-OH)) and The method according to claim 6, comprising enzyme coupling catalyzed by a ligase.

8. (a) Formula P-W ｖ A peptide C-terminal ester or thioester represented by -His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-(thio)ester (wherein P is an Edman protecting group which is a carbamate protecting group, an acyl protecting group, or a substituted thiocarbamoyl group, v is an integer from 0 to 5, and each W independently represents the same or different amino acid residue), (b) A peptide nucleophile containing the sequence H-Ser-Tyr-Leu-Glu-Gly-Glun-Ala-Ala-Y-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly (wherein Y is Lys(AEEA-AEEA-γ-Glu-OH)) and The method according to claim 6, comprising enzyme coupling catalyzed by a ligase, and subsequently providing a 17-carboxyheptadecanoyl group to the Lys(AEEA-AEEA-γ-Glu-OH).

9. (a) A peptide C-terminal ester or thioester containing the sequence His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-(thio)ester, (b) A peptide nucleophile comprising the sequence H-Ser-Tyr-Leu-Glu-Gly-Glun-Ala-Ala-Y-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly (wherein Y is a lysine residue having a free side chain ε-amino group) and The method according to claim 6, comprising enzyme coupling catalyzed by a ligase, and subsequently providing an AEEA-AEEA-γ-Glu-N-17-carboxyheptadecanoyl-OH group to the ε-amino group of Lys.

10. The method according to claim 1, wherein the synthesized peptide is GLP-1.

11. The method according to claim 4 or 8, wherein P is the Edman-type protecting group and v is 1.

12. The method according to any one of claims 1 to 11, wherein the homologue of the subtilisin BPN' variant is selected from the group P225N, P225D, P225S, P225C, P225G, P225A, P225T, P225V, P225I, P225L, P225H, and P225Q, and comprises a mutation at the amino acid position corresponding to P225.

13. The method according to any one of claims 1 to 12, wherein the homolog of the subtilisin BPN' variant comprises 1 to 13 mutations selected from the group consisting of Q2K, S3C, P5S, S9A, I31L, K43N, M50F, A73L, S188P, Q206C, N212G, T254A, and Q271E.

14. The C-terminus of the aforementioned peptide C-terminal ester or thioester is defined by the formula: -Ser-(C=O)-O-CX １ X ２ It is expressed as -C(=O)-NH-AA1-AA2, where X １ and X ２ The method according to any one of claims 1 to 13, wherein represents independently a hydrogen atom or an alkyl group, AA1 represents an alanine, valine, leucine, isoleucine, phenylalanine, methionine, or tryptophan unit having a free side-chain functional group, and AA2 represents an arginine or lysine unit having a free side-chain functional group.

15. The method according to any one of claims 1 to 14, wherein the homolog of the subtilisin BPN' variant is represented by SEQ ID NO: 3 without His tag or SEQ ID NO: 14 without His tag, or has at least 90% sequence identity with SEQ ID NO: 3 without His tag or SEQ ID NO: 14 without His tag.

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