Peptide synthesis method using adenylation enyzme

Abstract

The present invention relates to a method for synthesizing a peptide, the method including: activating a carboxy group of an amino acid that is a substrate for an adenylation enzyme by an adenylation reaction in a reaction solution containing the adenylation enzyme, the amino acid, and an amino compound that is a nucleophile; and forming an amide bond between the activated amino acid and the amino compound. The adenylation reaction is initiated in the presence of the nucleophile.

Description

Peptide synthesis method using adenyltransferase

[0001] This invention relates to a peptide synthesis method using adenylating enzymes.

[0002] In recent years, a wide variety of peptides with useful physiological activities, such as antimicrobial peptides and blood pressure-lowering peptides, have been developed and are widely used in the pharmaceutical and cosmetic fields. Development of biosensors using peptides is also progressing. The peptide market is expected to expand further in the future.

[0003] Peptides are generally synthesized using organic synthesis methods, which involve sequentially condensing amino acids with protected amino acids (amino acids with a protected N-terminus) and coupling agents to extend the amino acid chain, often using automated peptide synthesizers. However, such protected amino acids, coupling agents, and automated peptide synthesizers are very expensive, making organic synthesis of peptides costly.

[0004] On the other hand, enzymatic peptide synthesis methods are less expensive because they do not necessarily use protective amino acids or condensing agents, nor do they require special synthesis equipment. Examples of such enzymatic peptide synthesis methods include those using amino acid ligases and non-ribosomal peptide synthases (NPRS). However, peptide synthesis using amino acid ligases is limited to a few types of peptides due to the high substrate specificity of the enzyme.

[0005] Natural peptide biosynthesis involves not only ribosome-mediated peptide biosynthesis but also biosynthesis by mechanisms that do not involve ribosomes (ribosome-independent peptide biosynthesis). In ribosome-independent peptide biosynthesis, non-ribosomal peptides are synthesized by non-ribosomal peptide synthetases. Non-ribosomal peptide synthetases synthesize peptides by adenylating substrate amino acids and then forming amide bonds with amino compounds through condensation reactions. Non-ribosomal peptide synthetases have numerous modular structures, and each module mainly has multiple functional domains, such as adenylation domains, thiolation domains, and condensation domains. Non-ribosomal peptide synthetases can incorporate not only natural L-amino acids but also D-amino acids and modified amino acids, and can also synthesize peptides with complex structures such as cyclic peptides and complex peptides, making them suitable for the synthesis of a wide variety of peptides.

[0006] Non-patent document 1 reports the isolation of the adenylation domains constituting tyrosidine synthases B (TycB) and C (TycC), which are non-ribosomal peptide synthases (NRPS) derived from Bacillus brevis, and that they independently exhibit substrate-specific adenylation activity.

[0007] Non-patent document 2 discloses the synthesis of dipeptides L-Phe-L-Phe, L-Phe-L-Ala, and L-Phe-L-Leu using the adenylation domain of tyrosidine synthase TY1, a non-ribosomal peptide synthase (NRPS) derived from Bacillus brevis, as an adenylating enzyme.

[0008] Non-patent document 3 discloses a method for evaluating amide bond formation using the adenylated domain of a non-ribosomal peptide synthase, such as tyrosidine synthase A (TycA) derived from Brevibacillus parabrevis, as an adenylating enzyme, by a colorimetric assay. Non-patent document 3 discloses the synthesis of the dipeptide L-Trp-L-Pro by amide bond formation using the adenylated domain. Non-patent document 3 also shows that in the adenylation reaction and amide bond formation using the adenylated domain (TycA-A) of tyrosidine synthase A derived from Brevibacillus parabrevis, the free substrate amino acid is activated (adenylated) in the presence of ATP to become aminoacyl AMP, which then forms an amide bond through a nucleophilic substitution reaction with a nucleophilic amine.

[0009] Non-patent document 4 shows that the adenylation domain (TycA-A) of tyrosidine synthase A derived from Brevibacillus parabrevis activates not only L-amino acids but also D-amino acids, and that dipeptides can be synthesized using either L-amino acids or D-amino acids as nucleophiles.

[0010] However, it is known that conventional peptide synthesis methods using non-ribosomal peptide synthases produce peptides other than the target peptide as by-products (Non-Patent Literature 4). In Non-Patent Literature 4, a considerable amount of the by-product D-Trp-D-Trp was detected even under conditions where the amount was minimized.

[0011] There is a need for the development of enzymatic peptide synthesis methods that can more effectively suppress the generation of by-products.

[0012] Mootz H. D. and Marahiel M. A. , Journal of Bacteriology, Vol. 179, No. 21, p. 6843-6850, (1997) Dieckmann R. , et al. , FEBS Letters, 498, p. 42-45, (2001) Hara R. , et al. , Analytical Biochemistry, 477, p. 89-91, (2015) Kano et al. , Appl. Environ. Microbiol. , 85:e00120-19, (2019)

[0013] The present invention aims to provide an enzymatic peptide synthesis technology that can more effectively suppress the generation of by-products.

[0014] As a result of diligent research to solve the above problems, the inventors of the present invention have discovered that in a peptide synthesis method using adenylating enzymes, the generation of by-products can be significantly suppressed by initiating the adenylation reaction in the presence of a nucleophile, and have completed the present invention.

[0015] In other words, the present invention encompasses the following:

[0016] [1] A method for synthesizing a peptide, comprising activating the carboxyl group of an amino acid by an adenylation reaction in a reaction solution containing an adenylating enzyme, an amino acid that is a substrate of the adenylating enzyme, and an amino compound that is a nucleophile, and forming an amide bond between the activated amino acid and the amino compound, wherein the adenylation reaction is initiated in the presence of the nucleophile.

[0017] [2] The method according to [1] above, comprising adding ATP to a reaction solution containing an adenylating enzyme, an amino acid which is a substrate of the adenylating enzyme, and an amino compound which is a nucleophile, thereby initiating the adenylation reaction in the presence of the nucleophile.

[0018] [3] The method according to [1] or [2] above, wherein the adenylating enzyme is the adenylating domain of a non-ribosomal peptide synthase.

[0019] [4] The method according to [3] above, wherein the non-ribosomal peptide synthase is a tyrosidine synthase.

[0020] [5] The method according to [3] or [4] above, wherein the adenylation domain of the non-ribosomal peptide synthase is the adenylation domain of tyrosidine synthase A derived from Brevibacillus parabrevis.

[0021] [6] The method according to [3] or [4] above, wherein the adenylation domain of the non-ribosomal peptide synthase is the adenylation domain of module 2 of tyrosidine synthase B derived from Brevibacillus parabrevis.

[0022] [7] The method according to any one of [1] to [6] above, wherein the amino compound is an amino acid or oligopeptide with a protected carboxyl group.

[0023] [8] The method according to any one of [1] to [6] above, wherein the amino compound is an L-amino acid or D-amino acid with a protected carboxyl group.

[0024] [9] The method according to [1] to [8] above, wherein the amino compound is an ester or amide of an amino acid, or an inorganic or organic salt thereof.

[0025]

[10] The method according to [1] to [9] above, wherein the amino compound is a hydrochloride salt of an ester of an amino acid.

[0026]

[11] The method according to

[10] , wherein the hydrochloride salt of the amino acid ester is selected from the group consisting of glycine ethyl ester hydrochloride, glycine tert-butyl ester hydrochloride, and phenylalanine ethyl ester hydrochloride.

[0027]

[12] The method according to [5], wherein the amino acid that is the substrate of the adenylating enzyme is phenylalanine, tyrosine, tryptophan, alanine, leucine, or methionine.

[0028]

[13] The method according to [6], wherein the amino acid that is the substrate of the adenylase is proline.

[0029]

[14] The method according to [1] to

[13] above, wherein the amino compound is contained in the reaction solution in an amount such that, at the final concentration, its molar concentration is five times or more that of the amino acid which is the substrate of the adenylase.

[0030] This specification includes the disclosures of Japanese Patent Application No. 2025-055865, which forms the basis of the priority claim of this application.

[0031] According to the present invention, the generation of by-products in peptide synthesis using adenylating enzymes can be suppressed.

[0032] Figure 1 shows the mass spectrum of a dipeptide produced by a conventional method (comparative control test) in which the adenylation reaction is carried out in the absence of a nucleophile. Figure 2 shows the mass spectrum of a dipeptide produced by the peptide synthesis method of the present invention (synthesis test 1) in which the adenylation reaction is initiated in the presence of a nucleophile. Figure 3 shows the mass spectrum of a dipeptide produced by the peptide synthesis method of the present invention (synthesis test 2) in which the adenylation reaction is initiated in the presence of a nucleophile. Figure 4 shows the mass spectrum of a dipeptide produced by the peptide synthesis method of the present invention (synthesis test 3) in which the adenylation reaction is initiated in the presence of a nucleophile.

[0033] The present invention will be described in detail below.

[0034] This invention relates to a peptide synthesis method using an adenylating enzyme. In particular, this invention relates to a peptide synthesis method using an adenylating enzyme that can suppress the generation of by-products.

[0035] Peptide synthesis using adenylating enzymes is a method that utilizes the adenylating enzyme to activate an amino acid substrate (substrate amino acid) by adenylating its carboxyl group, and then synthesizes a peptide by attaching a nucleophilic amino compound to the activated amino acid via a nucleophilic substitution reaction (forming an amide bond). It is known that the substrate amino acid activated by adenylation is subjected to nucleophilic attack by the amino group of the nucleophilic amino compound, forming an amide bond. In this amide bond formation by nucleophilic substitution reaction, any amino compound can function as a nucleophile.

[0036] In the present invention, in a peptide synthesis method using an adenylating enzyme, the production of by-products can be suppressed by initiating the adenylation reaction in the presence of a nucleophile.

[0037] Specifically, the present invention provides a method for peptide synthesis comprising activating a carboxy group of an amino acid, which is a substrate for an adenylating enzyme, by adenylation (adenylation reaction) using the adenylating enzyme in a reaction solution containing the adenylating enzyme, the amino acid serving as the substrate of the adenylating enzyme, and an amino compound serving as a nucleophile, and forming an amide bond between the activated amino acid and the amino compound, wherein the method is characterized in that the adenylation reaction is initiated in the presence of the nucleophile.

[0038] The adenylating enzyme used in the present invention is not particularly limited, as long as it is an enzyme having the activity to adenylate the carboxyl group of a substrate amino acid (adenylation activity). In a preferred embodiment, the adenylating enzyme used in the present invention is the adenylation domain of a non-ribosomal peptide synthase (NPRS). In one embodiment, the adenylating enzyme used in the present invention is the adenylation domain of a tyrosidine synthase, which is a non-ribosomal peptide synthase. In one embodiment, the adenylating enzyme used in the present invention is the adenylation domain of a tyrosidine synthase (non-ribosomal peptide synthase) derived from Brevibacillus parabrevis. Examples of tyrosidine synthases (non-ribosomal peptide synthases) derived from Brevibacillus parabrevis include tyrosidine synthase A (TycA), tyrosidine synthase B (TycB), and tyrosidine synthase C (TycC). Tyrosidine synthase A from Brevibacillus parabrevis consists of one module (module 1), tyrosidine synthase B consists of three modules (modules 2-4), and tyrosidine synthase C consists of six modules (modules 5-10). Each of these modules is known to contain one adenylation domain, one thiolation domain, and one condensation domain and / or one epimerization domain. In one embodiment, the adenylating enzyme used in the present invention is the adenylating domain (TycA-A) of tyrosidine synthase A derived from Brevibacillus parabrevis. TycA-A derived from Brevibacillus parabrevis is typically a protein containing or consisting of the amino acid sequence shown in SEQ ID NO: 2, but is not limited to the following. The protein containing or consisting of the amino acid sequence shown in SEQ ID NO: 2 is encoded by a gene / polynucleotide containing or consisting of the nucleotide sequence shown in SEQ ID NO: 1, for example.TycA-A derived from Brevibacillus parabrevis may consist of an amino acid sequence having one or more, for example, 1 to 15 amino acid deletions, substitutions, insertions, and / or additions in the amino acid sequence shown in SEQ ID NO: 2, or it may consist of an amino acid sequence having 80% or more, preferably 90% or more, more preferably 97% or more, for example 98% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2. TycA-A derived from Brevibacillus parabrevis may have any additional amino acid sequences, such as a linker sequence or a tag sequence (e.g., a histidine tag), at its N-terminus and / or C-terminus, and such TycA-A may be a protein consisting of the amino acid sequence shown in SEQ ID NO: 4. The protein consisting of the amino acid sequence shown in SEQ ID NO: 4 has an additional sequence containing a histidine tag at the C-terminus of the amino acid sequence shown in SEQ ID NO: 2, and is encoded by a gene / polynucleotide consisting of the base sequence shown in SEQ ID NO: 3. TycA-A derived from Brevibacillus parabrevis can be genetically engineered by inducing protein expression using recombinant methods, etc., with nucleic acid constructs such as expression vectors or expression cassettes containing the gene / polynucleotide sequence that encodes it. The nucleotide sequence encoding TycA-A derived from Brevibacillus parabrevis may be the natural gene sequence or may be a modified sequence that has been optimized for codons or otherwise. More specifically, the nucleotide sequence encoding TycA-A from Brevibacillus parabrevis may include, for example, the protein-coding sequence of the natural TycAA gene from Brevibacillus parabrevis (e.g., the nucleotide sequence shown in SEQ ID NO: 5) or a nucleotide sequence that has been modified, such as by codon optimization, for recombinant expression (e.g., the nucleotide sequence shown in SEQ ID NO: 1 or 3, which is codon-optimized for recombinant expression in E. coli) or a nucleotide sequence that has been modified, but is not limited to, these.

[0039] In one embodiment, the adenylating enzyme used in the present invention is the adenylating domain (TycB-A) of module 2 of tyrosidine synthase B derived from Brevibacillus parabrevis. TycB-A derived from Brevibacillus parabrevis is typically a protein containing or consisting of the amino acid sequence shown in SEQ ID NO: 7, but is not limited to the following. The protein containing or consisting of the amino acid sequence shown in SEQ ID NO: 7 is encoded by a gene / polynucleotide containing or consisting of the nucleotide sequence shown in SEQ ID NO: 6, for example. TycB-A derived from Brevibacillus parabrevis may consist of an amino acid sequence having one or more, for example, 1 to 15 amino acid deletions, substitutions, insertions, and / or additions in the amino acid sequence shown in SEQ ID NO: 7, or it may consist of an amino acid sequence having 80% or more, preferably 90% or more, more preferably 97% or more, for example 98% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 7. TycB-A derived from Brevibacillus parabrevis may have any additional amino acid sequences, such as a linker sequence or a tag sequence (e.g., a histidine tag), at its N-terminus and / or C-terminus, and such TycB-A may be a protein consisting of the amino acid sequence shown in SEQ ID NO: 9. The protein consisting of the amino acid sequence shown in SEQ ID NO: 9 has an additional sequence containing a histidine tag at the C-terminus of the amino acid sequence shown in SEQ ID NO: 7, and is encoded by a gene / polynucleotide consisting of the base sequence shown in SEQ ID NO: 8. TycB-A derived from Brevibacillus parabrevis can be genetically engineered by inducing protein expression using recombinant methods, etc., with nucleic acid constructs such as expression vectors or expression cassettes containing the gene / polynucleotide sequence that encodes it. The nucleotide sequence encoding TycB-A derived from Brevibacillus parabrevis may be the natural gene sequence or may be a modified sequence that has been optimized for codons or otherwise.More specifically, the nucleotide sequence encoding TycB-A from Brevibacillus parabrevis may include, for example, the protein coding sequence of the natural TycBA gene from Brevibacillus parabrevis (e.g., the nucleotide sequence shown in SEQ ID NO: 10) or a nucleotide sequence that has been modified, such as by codon optimization, for recombinant expression (e.g., the nucleotide sequence shown in SEQ ID NO: 6 or 8, which is codon optimized for recombinant expression in E. coli) or a nucleotide sequence that has been modified, but is not limited to, these.

[0040] Adenylating enzymes, such as the adenylating domain of a non-ribosomal peptide synthase (e.g., tyrosidine synthase), including the adenylating domain of a tyrosidine synthase (non-ribosomal peptide synthase) derived from Brevibacillus parabrevis, can be genetically engineered, for example, using the gene encoding them. Alternatively, commercially available adenylating enzymes may be used. In this invention, the reaction solution is prepared using an adenylating enzyme. In one embodiment, the reaction solution can be prepared using the adenylating domain alone (i.e., detached from other modules or domains of the non-ribosomal peptide synthase).

[0041] The amino acid that is a substrate for an adenylating enzyme (substrate amino acid) used in the present invention has its carboxyl group adenylated by the action of the adenylating enzyme to form an adenylated product. The adenylated product is in an activated state, that is, in a state susceptible to nucleophilic attack (nucleophilic substitution reaction) by a nucleophile. The substrate amino acid used in the present invention may be a proteinogenic amino acid (20 types of amino acids that constitute natural proteins), or may be a non-proteinogenic amino acid (an amino acid that does not constitute natural proteins). The substrate amino acid used in the present invention may be a proteinogenic amino acid: glycine (Gly), alanine (Ala), serine (Ser), threonine (Thr), asparagine (Asn), glutamine (Gln), aspartic acid (Asp), glutamic acid (Glu), lysine (Lys), arginine (Arg), histidine (His), valine (Val), leucine (Leu), isoleucine (Ile), tyrosine (Tyr), phenylalanine (Phe), tryptophan (Pro), methionine (Met), or cysteine (Cys), or may be modified amino acids or derivatives thereof. The substrate amino acid used in the present invention may be L-form (L-amino acid) or D-form (D-amino acid). The substrate amino acid used in the present invention may be a natural amino acid or an unnatural amino acid. The substrate amino acid used in the present invention has a free carboxyl group, and typically has an unprotected free carboxyl group. The free amino group of the substrate amino acid used in the present invention may be protected or unprotected (unprotected). In one embodiment, the substrate amino acid used in the present invention may be a free amino acid. In one embodiment, the substrate amino acid used in the present invention may be a peptide-constituting amino acid having a free carboxyl group. When the substrate amino acid used in the present invention is a peptide-constituting amino acid having a free carboxyl group, the substrate amino acid is contained as the constituent amino acid at the carboxy terminus (C-terminus) of a peptide contained in the reaction solution.

[0042] The substrate amino acid used in the present invention can be selected depending on the adenylating enzyme used. For example, if the adenylating enzyme is the adenylation domain of tyrosidine synthase A derived from Brevibacillus parabrevis, the substrate amino acid may be phenylalanine, tyrosine, tryptophan, alanine, leucine, or methionine, or their homologs or derivatives. The adenylation domain of tyrosidine synthase A derived from Brevibacillus parabrevis is known to use phenylalanine, as well as tyrosine, tryptophan, alanine, leucine, and methionine as substrates, and to use both the L and D forms of these as substrates. On the other hand, if the adenylating enzyme is, for example, the adenylation domain of module 2 of tyrosidine synthase B derived from Brevibacillus parabrevis, the substrate amino acid may be proline, or its homologues or derivatives, preferably L-proline. Examples of proline homologues include, but are not limited to, azetidine carboxylic acid and pipecolic acid. Examples of proline derivatives include, but are not limited to, hydroxyprolines (typically cis-4-hydroxy-L-proline, trans-4-hydroxy-L-proline, cis-3-hydroxy-L-proline, and trans-3-hydroxy-L-proline, and their D-forms). In the present invention, an "amino acid homologue" is not limited to, but may be, for example, a compound in which the length and / or structure of the carbon chain of the side chain of the amino acid has been changed from the original amino acid. In the present invention, the "derivatives" of amino acids are not limited to the following, but may be compounds in which atoms in the side chain of an amino acid are substituted, or in which a functional group (e.g., a hydroxyl group), a protecting group, or a labeling substance (e.g., a fluorescent substance, a radioisotope) is introduced into the side chain.

[0043] The nucleophile used in the present invention is an amino compound. In the present invention, "amino compound" means a compound having an amino group. In a preferred embodiment, the amino compound that is the nucleophile used in the present invention is an amino acid (here, meaning a free amino acid; hereafter, the same applies to amino acids as nucleophiles unless otherwise specified in the context), or a compound containing an amino acid residue having a free amino group. In a more preferred embodiment, the amino compound that is the nucleophile used in the present invention is an amino acid, or a peptide such as an oligopeptide. The amino compound that is the nucleophile used in the present invention may be an amino acid or an oligopeptide, and preferably an amino acid. The amino acid residue at the amino terminus of the amino acid or peptide used as a nucleophile in the present invention may be a proteinogenic amino acid (one of the 20 amino acids that are components of natural proteins) or a non-proteinogenic amino acid (an amino acid that is not a component of natural proteins). The amino acid residue at the amino terminus of an amino acid or peptide used as a nucleophile in the present invention may be a protein amino acid such as glycine (Gly), alanine (Ala), serine (Ser), threonine (Thr), asparagine (Asn), glutamine (Gln), aspartic acid (Asp), glutamic acid (Glu), lysine (Lys), arginine (Arg), histidine (His), valine (Val), leucine (Leu), isoleucine (Ile), tyrosine (Tyr), phenylalanine (Phe), tryptophan (Pro), methionine (Met), or cysteine ​​(Cys), or a modified amino acid or derivative thereof. The amino compound that is the nucleophile used in the present invention may be an L-amino acid or a D-amino acid. The amino compound that is the nucleophile used in the present invention may be a peptide containing an L-amino acid and / or a D-amino acid at its amino terminus. The amino compound used as a nucleophile in this invention may be a natural or unnatural amino compound, and may be a natural or unnatural amino acid or peptide. The amino compound used as a nucleophile in this invention may have a protected carboxyl group or may be unprotected.The amino compound used as a nucleophile in this invention is typically one in which the amino group (terminal amino group) is not protected. Preferably, the amino compound used as a nucleophile in this invention is an amino acid different from the substrate amino acid. In cases where the substrate amino acid is an L-amino acid and the nucleophile is its corresponding D-amino acid, or vice versa, the substrate amino acid and the nucleophile are different amino acids.

[0044] In this invention, the term "peptide" refers to a compound containing a polymer structure in which two or more amino acids are linked by amide bonds. The peptide in this invention is not limited to the following, but may be a compound containing a polymer structure in which two or more, for example, 2 to 100, or 2 to 50 amino acids are linked by amide bonds. In one embodiment, the peptide in this invention may be a dipeptide (a compound containing a polymer structure in which two amino acids are linked by amide bonds) or a tripeptide (a compound containing a polymer in which two amino acids are linked by amide bonds). In this invention, the term "oligopeptide" refers to a compound containing a polymer structure in which 2 to 10 amino acids are linked by amide bonds.

[0045] The amino compound used as a nucleophile in the present invention may be an amino acid or peptide in which the carboxyl group (terminal carboxyl group) is protected. In one embodiment, the amino compound used as a nucleophile in the present invention is an amino acid or oligopeptide with a protected carboxyl group. In one embodiment, the amino compound used as a nucleophile in the present invention is an L-amino acid or D-amino acid with a protected carboxyl group. Carboxyl group protection can be carried out, for example, by esterification or amidation.

[0046] In one embodiment, the amino compound that serves as the nucleophile in the present invention may be an ester of an amino acid, or an inorganic or organic acid salt of an amino acid ester. Examples of esters include, but are not limited to, methyl esters, ethyl esters, propyl esters, butyl esters (e.g., tert-butyl esters), and alkyl esters such as pentyl esters. In one embodiment, the amino compound that serves as the nucleophile in the present invention may be an amide of an amino acid, or an inorganic or organic acid salt of an amide of an amino acid. Examples of amides include, but are not limited to, compounds in which two hydrogen atoms are bonded to a nitrogen atom, compounds in which one hydrogen atom and a hydrocarbon are bonded to a nitrogen atom, compounds in which two carbon atoms or heteroatoms are bonded to a nitrogen atom, and compounds in which two carbon atoms or heteroatoms are cyclically formed on a nitrogen atom. Examples of inorganic acid salts include, but are not limited to, hydrochloride salts, sulfate salts, phosphate salts, etc. Examples of organic acid salts include, but are not limited to, acetate salts, succinate salts, citrate salts, tartrate salts, lactate salts, etc. In one embodiment, the amino compound that serves as the nucleophile in the present invention may be an ester hydrochloride of an amino acid, for example, an ethyl ester hydrochloride of an amino acid, a tertiary (tert-) butyl ester hydrochloride of an amino acid, etc. In one embodiment, the amino compound that serves as the nucleophile in the present invention may be an ethyl ester hydrochloride of an amino acid selected from the group consisting of ethyl glycine hydrochloride, tertiary (tert-) butyl ester hydrochloride of glycine, and ethyl phenylalanine hydrochloride.

[0047] In this invention, a reaction solution (peptide synthesis reaction solution) containing an adenylating enzyme, a substrate amino acid, and a nucleophilic amino compound is used to adenylate the substrate amino acid and form an amide bond with the nucleophilic amino compound.

[0048] The reaction solution may, but is not limited to, contain adenylating enzyme in a final concentration of 0.001 mg / mL to 50 mg / mL, preferably 0.05 mg / mL to 10 mg / mL, for example, 0.1 mg / mL to 2 mg / mL.

[0049] The reaction solution may, but is not limited to, contain the substrate amino acid at a final concentration of 0.1 to 100 mM, preferably 1 to 100 mM, for example, 1 to 50 mM, 1 to 30 mM, or 10 to 50 mM.

[0050] The reaction solution may contain a substrate amino acid and a nucleophilic amino compound in an amount such that the amino compound is preferably three times or more, more preferably five times or more, of the substrate amino acid at its final concentration, for example, 5 to 20 times its molar concentration. In relation to the reaction solution (peptide synthesis reaction solution) of the present invention, "final concentration" refers to the concentration in the reaction solution at the start of the adenylation reaction.

[0051] The reaction solution may further contain a buffer. Examples of buffers include, but are not limited to, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, 2-cyclohexylaminoethanesulfonic acid (CHES), phosphate buffer, phosphate-buffered saline (PBS), Tris-HCl solution, Tris-buffered saline (TBS), and sodium citrate buffer. The reaction solution may also further contain any components that do not affect the initiation of the adenylation reaction. For example, the reaction solution may contain sodium hydroxide added for pH adjustment.

[0052] In this invention, in a peptide synthesis method using an adenylating enzyme, the adenylation reaction is initiated in the presence of a nucleophile in order to suppress the generation of by-products. In this invention, the adenylation reaction can be initiated in the presence of a nucleophile by adding the nucleophile to the reaction solution (reaction system) before the adenylation reaction begins. In a preferred embodiment, the adenylation reaction can be initiated in the presence of a nucleophile by adding the nucleophile to the reaction solution in addition to the adenylating enzyme and the substrate amino acid, and then adding at least one component necessary for adenylation to the reaction solution. Adenylation by an adenylating enzyme requires an adenylating enzyme and a substrate amino acid. Furthermore, since adenylation in this invention is a reaction in which adenosine monophosphate (AMP) derived from adenosine triphosphate (ATP) is covalently bonded to the carboxyl group of the substrate amino acid and pyrophosphate (PPi) is released, adenosine triphosphate (ATP) is usually also required for adenylation by an adenylating enzyme. In the present invention, the adenylation reaction is preferably carried out in the presence of ATP. At the start of the adenylation reaction, the reaction solution must contain ATP in addition to the adenylating enzyme, substrate amino acid, and nucleophilic amino compound. The adenylation reaction will not start in a reaction solution that does not contain adenosine triphosphate (ATP). In a preferred embodiment, it is preferable to prepare the reaction solution by mixing the adenylating enzyme, substrate amino acid, and nucleophilic amino compound, and then add ATP to the reaction solution afterward (i.e., after mixing the adenylating enzyme, substrate amino acid, and nucleophilic amino compound). This allows the adenylation reaction and the subsequent amide bond formation to be initiated in the presence of the nucleophile. The reaction solution may, but is not limited to, contain ATP at a final concentration of 0.1 to 100 mM, for example, 10 to 100 mM.

[0053] For adenylation by adenylase, the reaction solution contains magnesium ions (Mg ２＋ It is also preferable to further include, specifically, MgSO ４ It is also preferable to include magnesium salts such as the following. In one embodiment, together with the adenylase, substrate amino acid, and nucleophile amino compound, MgSO ４A reaction solution may be prepared by mixing magnesium salts such as or a solution thereof, and ATP may be added thereafter. In one embodiment, MgSO4 is added to a reaction solution containing an adenylating enzyme, a substrate amino acid, and a nucleophile amino compound, along with ATP. ４ Magnesium salts or solutions thereof may be added later. In one embodiment, MgSO4 is added to a reaction solution containing adenylase, a substrate amino acid, a nucleophile amino compound, and ATP. ４ Magnesium salts or solutions thereof may be added later. Examples of magnesium salts include, but are not limited to, magnesium sulfate (MgSO4). ４ ), magnesium hydrochloride (MgCl ２ Examples include magnesium citrate, magnesium malate, magnesium glycinate, magnesium gluconate, magnesium acetate, magnesium bromide, and magnesium carbonate. The concentration ratio of ATP to magnesium ions (magnesium salts) in the reaction solution may be 1:0.5 to 1:2, for example, 1:1 to 1:1.5, but is not limited thereto. The reaction solution may also be prepared by adding a buffer solution. The reaction solution may also be prepared by adding any other components that do not affect the initiation of the adenylation reaction.

[0054] The adenylation reaction by an adenylating enzyme is induced under temperature conditions in which the adenylating enzyme is active. To initiate the adenylation reaction in the presence of a nucleophile, the reaction solution of the present invention, in particular a reaction solution containing at least an adenylating enzyme, a substrate amino acid, a nucleophilic amino compound, and ATP, is reacted under temperature conditions in which the adenylating enzyme is active, preferably near its optimal temperature. In one embodiment, it is preferable to incubate the reaction solution containing at least an adenylating enzyme, a substrate amino acid, a nucleophilic amino compound, and ATP at 30°C to 45°C, for example, about 37°C (36.5°C to 37.5°C). In one embodiment, ATP may be added to the reaction solution containing the adenylating enzyme, the substrate amino acid of the adenylating enzyme, and the nucleophilic amino compound, and then the reaction solution may be incubated under temperature conditions in which the adenylating enzyme is active, preferably near its optimal temperature, to induce the adenylation reaction. The reaction time for the adenylating enzyme can be selected depending on the adenylating enzyme used. The reaction time for the adenylating enzyme is not limited to the following, but can be carried out for 6 to 120 hours, for example, 12 to 96 hours. After the enzymatic reaction is complete, it is preferable to inactivate the enzyme by heating or other means.

[0055] In this way, peptides can be produced by adenylating (activating) the substrate amino acid and forming an amide bond (peptide bond) with a nucleophile amino compound (particularly an amino acid or peptide).

[0056] In one embodiment, a dipeptide can be synthesized in the method of the present invention by using a free amino acid as a substrate amino acid and a free amino acid as a nucleophile. In one embodiment, a tripeptide can be synthesized in the method of the present invention by using a dipeptide as a substrate amino acid and a free amino acid as a nucleophile. In one embodiment, a tripeptide can be synthesized in the method of the present invention by using a free peptide as a substrate amino acid and a dipeptide as a nucleophile. In one embodiment, a peptide with a length of 4 amino acids or more can be synthesized in the method of the present invention by using a free peptide as a substrate amino acid and a peptide with a length of 3 amino acids or more as a nucleophile. In one embodiment, these peptide synthesis steps can also be combined in the method of the present invention. When a peptide synthesized using a carboxyl-protected amino compound as a nucleophile is used as a substrate amino acid for further extension, the synthesized peptide may be deprotected before being subjected to the next adenylation reaction. The peptide synthesized by the method of the present invention is also understood according to the above definition of peptide.

[0057] In the present invention, in a peptide synthesis method using an adenylating enzyme, by initiating the adenylation reaction in the presence of a nucleophile as described above, the adenylation (activation) of the substrate amino acid and the subsequent amide bond formation with the nucleophile are carried out with high efficiency, and as a result, the generation of by-products can be significantly suppressed.

[0058] The synthesized peptides can be separated or purified by conventional methods such as column chromatography.

[0059] The present invention will be described in more detail below using examples. However, the technical scope of the present invention is not limited to these examples.

[0060] [Example 1] Preparation of adenylating enzyme 1) Preparation of adenylating enzyme TycA-A A DNA comprising the TycAA gene encoding the adenylation domain TycA-A (SEQ ID NO: 4) of tyrocidine synthetase A, which is a non-ribosomal peptide synthetase (NRPS) derived from Brevibacillus parabrevis, (SEQ ID NO: 3) was inserted into the plasmid vector pUCSNT to prepare a TycA-A expression vector. The obtained TycA-A expression vector was transformed into NEB （Ｒ） 10-beta Competent E. coli cells (New England Biolabs, Inc.), followed by transformation, and the transformed cells were cultured overnight at 37°C.

[0061] A single colony was collected from a plate on which the transformed cells were cultured, inoculated into 3 mL of liquid medium, and pre-cultured at 37°C. 1 mL of the pre-culture was collected, inoculated into 100 mL of liquid medium, and cultured at 30°C for 20 hours. The culture was centrifuged at 3000×g at 4°C for 10 minutes, and the cell pellet was collected. 30 mL of lysis buffer (50 mmol / L Tris-HCl, DNase, pH 8.0) was added to the cell pellet and suspended. The obtained suspension was sonicated, then centrifuged at 20,000×g at 4°C for 30 minutes, and the supernatant was collected.

[0062] To the collected supernatant was added TALON （Ｒ） Metal Affinity Resin (Takara Bio USA, Inc.) in an amount of 1 mL, and the mixture was stirred for 2 hours, followed by purification using the chromatography system AKTA ＴＭ Protein purification was performed using Avant 25. The buffer was exchanged into 50 mmol / L Tris-HCl (pH 8.0) buffer, and concentrated to a protein concentration of 1 mg / mL using Amicon Ultra-0.5 mL centrifugal filter (Merck), which is a centrifugal ultrafiltration device. An adenylating enzyme TycA-A solution was thus prepared.

[0063] 2) Preparation of Adenylating Enzyme TycB-A The TycBA gene (SEQ ID NO: 8), which encodes the adenylating domain TycB-A (SEQ ID NO: 9) of module 2 of tyrosidine synthase B, a non-ribosomal peptide synthase (NRPS) derived from Brevibacillus parabrevis, was incorporated into the plasmid vector pET28a(+) to create a TycB-A expression vector. The obtained TycB-A expression vector was introduced into T7 Express Competent E. coli cells (New England Biolabs, Inc.) to transform them, and the transformed cells were cultured overnight at 37°C.

[0064] A single colony was taken from a plate of transformed cells, inoculated into 3 mL of liquid medium, and pre-cultured at 37°C. 0.5 mL of the pre-culture solution was taken, inoculated into 100 mL of liquid medium, and cultured at 37°C until the OD reached 0.4. Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 0.4 mM, and the cells were cultured at 15°C for 24 hours. The culture solution was centrifuged at 3000 × g at 4°C for 10 minutes, and the cell pellet was collected. 30 mL of lysis buffer (50 mmol / L Tris-HCl, DNase, pH 8.0) was added to the cell pellet, and the cells were suspended. The resulting suspension was sonicated, then centrifuged at 20,000 × g at 4°C for 30 minutes, and the supernatant (soluble fraction) was collected. The adenylating enzyme TycB-A soluble fraction was prepared in this manner.

[0065] [Example 2] Synthesis of dipeptides using adenylating enzyme 1) Synthesis test 1 L-phenylalanine (0.005 mmol) as substrate for adenylating enzyme, glycine ethyl ester hydrochloride (0.1 mmol) as nucleophile, adenylating enzyme TycA-A (0.05 mg) prepared in Example 1, MgSO ４ A mixture of (0.005 mmol) and Tris-HCl solution (50 mM, pH 8.0) was prepared to a final volume of 500 μL. Adenosine triphosphate (ATP) (0.005 mmol) was added to the mixture, and the mixture was shaken at 37°C for 24 hours. The enzymatic reaction was stopped by heating this peptide synthesis reaction solution at 95°C for 5 minutes.

[0066] 2) Synthesis Test 2: L-tyrosine (0.05 mmol) as a substrate for adenylating enzyme, glycine tert-butyl ester hydrochloride (0.25 mmol) as a nucleophile, adenylating enzyme TycA-A (1.06 mg) prepared in Example 1, MgSO ４ A mixture of (0.1 mmol) and Tris-HCl solution (50 mM, pH 8.0) was prepared to a final volume of 1.5 mL. ATP (0.1 mmol) was added to the mixture, and the mixture was shaken at 37°C for 90 hours. The enzymatic reaction was stopped by heating this peptide synthesis reaction solution at 95°C for 5 minutes.

[0067] 3) Synthesis Test 3: L-proline (0.05 mmol) as a substrate for adenylating enzyme, L-phenylalanine ethyl hydrochloride (0.25 mmol) as a nucleophile, soluble fraction of adenylating enzyme TycB-A prepared in Example 1 (1 mL; enzyme content approximately 8 mg), MgSO ４ A mixture of (0.05 mmol) and Tris-HCl solution (50 mM, pH 8.0) was prepared to a final volume of 1.5 mL. ATP (0.05 mmol) was added to the mixture, and the mixture was shaken at 37°C for 24 hours. The enzymatic reaction was stopped by heating this peptide synthesis reaction solution at 95°C for 5 minutes.

[0068] 4) Comparative test (conventional synthesis method) Adenylase TycA-A (0.05 mg), ATP (0.005 mmol), MgSO4 prepared in Example 1 ４ A mixture of (0.005 mmol) and Tris-HCl solution (50 mM, pH 8.0) was prepared to a final volume of 500 μL. L-phenylalanine (0.005 mmol) was added to the mixture, and the mixture was shaken at 37°C for 15 minutes. Next, glycine ethyl hydrochloride (0.1 mmol) was added as a nucleophile, and the mixture was shaken at 37°C for 24 hours. The enzymatic reaction was stopped by heating this peptide synthesis reaction solution at 95°C for 5 minutes.

[0069] [Example 3] LC-MS Analysis The peptide synthesis reaction solution obtained in Example 2 was subjected to liquid chromatography-mass spectrometry (LC-MS) to analyze the synthesized peptide. The results are shown in Table 1 and Figures 1-4.

[0070]

[0071] In a comparative control test following a conventional method, the mass spectrum of the peptide synthesis reaction solution obtained by adding a nucleophile after the adenylation reaction had started in the absence of a nucleophile to form an amide bond showed peaks (m / z 251, 273) of the target product (target dipeptide L-Phe-Gly ethyl ester; PheGlyOEt) at a retention time of 1.33 minutes, but peaks (m / z 313, 335) of the by-product (dipeptide L-Phe-L-Phe) were also observed at a retention time of 1.49 minutes (Figure 1, Table 1).

[0072] In contrast, in the mass spectrum of the peptide synthesis reaction solution obtained by the adenylation reaction and amide bond formation initiated in the presence of a nucleophile in synthesis experiment 1, a peak for the target product (target dipeptide L-Phe-Gly ethyl ester; PheGlyOEt) (m / z 251, 273) was observed at a retention time of 1.33 minutes, but no peak for the by-product (dipeptide L-Phe-L-Phe) was observed within the same retention time range as the comparative control experiment (Figure 2, Table 1).

[0073] Similarly, in synthesis experiment 2, the mass spectrum of the peptide synthesis reaction solution obtained by the adenylation reaction and amide bond formation initiated in the presence of a nucleophile showed a peak (m / z 239) of the target product (target dipeptide L-Tyr-Gly tert-butyl ester; TyrGlyOtBu) at a retention time of 1.44 minutes, but no peaks of by-products were observed (Figure 3, Table 1). L-Tyr-Gly tert-butyl ester is a peptide in which the C-terminus of the dipeptide L-Tyr-Gly is protected by a protecting group tert-butyl.

[0074] Furthermore, in synthesis experiment 3, the mass spectrum of the peptide synthesis reaction solution obtained by the adenylation reaction and amide bond formation initiated in the presence of a nucleophile showed a peak (m / z 291) of the target product (target dipeptide L-Pro-Phe ethyl ester; ProPheOEt) at a retention time of 1.63 minutes, but no peaks of by-products were observed (Figure 4, Table 1).

[0075] LC-MS analysis revealed that by performing adenylation and amide bond formation in the presence of a nucleophile, without prior adenylation in the absence of a nucleophile, the target peptide can be efficiently synthesized while suppressing the generation of by-products.

[0076] All publications, patents, and patent applications cited herein shall be incorporated herein by direct reference.

Claims

1. A method for synthesizing a peptide, comprising activating the carboxyl group of an amino acid by an adenylation reaction in a reaction solution containing an adenylating enzyme, an amino acid that is a substrate of the adenylating enzyme, and an amino compound that is a nucleophile, and forming an amide bond between the activated amino acid and the amino compound, wherein the adenylation reaction is initiated in the presence of the nucleophile.

2. The method according to claim 1, comprising adding ATP to a reaction solution containing an adenylating enzyme, an amino acid that is a substrate of the adenylating enzyme, and an amino compound that is a nucleophile, thereby initiating the adenylation reaction in the presence of the nucleophile.

3. The method according to claim 1, wherein the adenylating enzyme is the adenylating domain of a non-ribosomal peptide synthase.

4. The method according to claim 3, wherein the non-ribosomal peptide synthase is tyrosidine synthase.

5. The method according to claim 3, wherein the adenylation domain of the non-ribosomal peptide synthase is the adenylation domain of tyrosidine synthase A derived from Brevibacillus parabrevis.

6. The method according to claim 3, wherein the adenylation domain of the non-ribosomal peptide synthase is the adenylation domain of module 2 of tyrosidine synthase B derived from Brevibacillus parabrevis.

7. The method according to claim 1, wherein the amino compound is an amino acid or oligopeptide with a protected carboxyl group.

8. The method according to claim 1, wherein the amino compound is an L-amino acid or D-amino acid with a protected carboxyl group.

9. The method according to claim 1, wherein the amino compound is an ester or amide of an amino acid, or an inorganic or organic salt thereof.

10. The method according to claim 1, wherein the amino compound is a hydrochloride salt of an amino acid ester.

11. The method according to claim 10, wherein the hydrochloride salt of the amino acid ester is selected from the group consisting of glycine ethyl ester hydrochloride, glycine tert-butyl ester hydrochloride, and phenylalanine ethyl ester hydrochloride.

12. The method according to claim 5, wherein the amino acid that is the substrate of the adenylating enzyme is phenylalanine, tyrosine, tryptophan, alanine, leucine, or methionine.

13. The method according to claim 6, wherein the amino acid that is the substrate of the adenylase is proline.

14. The method according to claim 1, wherein the amino compound is contained in the reaction solution in an amount such that, at the final concentration, its molar concentration is five times or more that of the amino acid which is the substrate of the adenylase.

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