JP2012167018A - Method for synthesizing glycopeptide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method capable of producing easily and effectively an objective designed glycopeptide.SOLUTION: This method for producing a modified peptide in which an amino acid residue having at least one sugar binding capacity is modified by a modifying group includes: (A) a step of obtaining a protected peptide containing two or more amino acid residues having a sugar binding capacity, wherein at least one of the amino acid residues having the sugar binding capacity is protected by a protecting group, and at least another of the amino acid residues having the sugar binding capacity is not protected; (B) a step of binding a modifying group to the unprotected amino acid residue having at least one sugar binding capacity in the protected peptide obtained in the step (A); and (C) a step of releasing the protection of the protecting group of the protected peptide having the modifying group obtained in the step (B).

Description

  The present invention relates to a method for synthesizing glycopeptides.

  As canceration progresses, it is well known that the sugar chain structure of mucin glycoproteins on the cell surface changes (see Non-Patent Document 1). Research and development of new therapeutic methods such as antibody drugs and cancer vaccines are underway (see Non-Patent Document 2). Currently, antigens of monoclonal antibodies such as CA15-3 and CA125 that are widely used in clinical settings are also expected to have a specific partial structure of the above mucin glycoprotein. Peptides are a group of target compounds that are extremely important in the drug discovery and medical industries. Although it has been reported that these compounds can be prepared by using organic chemical synthesis methods and enzyme synthesis methods in a complementary manner (see Patent Literature 1 and Non-Patent Literature 3), a plurality of complex sugar chains having different structures are reported. It is necessary to synthesize multiple types of amino acid derivatives including individual core structures (basic skeletons) in advance, and there are limitations on the types of sugar chain structures that can be imparted to the same peptide molecule. Therefore, it was difficult to synthesize these glycopeptides in large quantities.

  On the other hand, mucin-type glycoproteins are originally biosynthesized by many glycosyltransferase reactions in vivo, and an enzyme that transfers N-acetylgalactosamine, the first sugar, to the hydroxyl groups of serine and threonine present in polypeptide chains ( More than a dozen types of UDP-GalNAc: polypeptide N-acetylgalactosamine transferase, hereinafter abbreviated as ppGalNAcTs) have been discovered. However, with regard to the substrate specificity of ppGalNAcTs for peptide chains or glycopeptide chains, there are many experimental results that confirm that there is no general rule (consensus sequence) at all, although there is a certain tendency. Non-Patent Document 2 or Non-Patent Document 4). In other words, it is difficult for any ppGalNAcTs to arbitrarily modify GalNAc to any residue among serine and threonine present in the peptide chain, and the order (order of modification reaction) is controlled. It is also impossible to do.

  Japanese Patent Laid-Open No. 2003-2899 (Patent Document 2) discloses a glycosyltransferase in which a sugar chain is added to a peptide using a glycosyltransferase and the sugar chain cannot be introduced with the first glycosyltransferase. Discloses a method of introducing a sugar chain using However, this document is based on the premise that it depends only on the substrate specificity of glycosyltransferase, and by designing an artificially designed glycopeptide by blocking amino acids that can be artificially introduced into a sugar chain. It is not intended to produce effectively.

International Application Publication 2006/030840 Pamphlet JP 2003-2899 A

Nature Rev. Cancer (2004) 4, 45-60. Biochim. Biophys. Acta (2008) 1780, 564-563. J. Am. Chem. Soc. (2005) 127, 11804-11818. Chem. Biol. (2004) 11, 1009-1016.

  The subject of this invention is providing the method which can manufacture the target designed glycopeptide simply and effectively.

  As a result of intensive studies, the present inventors have previously linked the amino acid to which sugar is to be added with a sugar different from the sugar introduced by glycosyltransferase or a substitute (protecting group) thereof, Block sugar chain introduction by transglycosylation reaction, add the target sugar chain (including monosaccharide) to the amino acid at the target location, and then introduce the sugar or substitute (protecting group) introduced to block, The above problems have been solved by finding that a glycopeptide having a sugar chain introduced at a target location can be produced by selective desorption by hydrolysis or the like. In addition to the above, the present inventors produce a more complex target glycopeptide by adding a glycosylation to the blocked amino acid using the same or different glycosyltransferase as the first time. As a result, the above-mentioned problems have been solved.

  In the present invention, as a specific example, the hydroxyl group of any serine residue (Ser) and threonine residue (Thr) contained in the peptide chain is converted to a sugar other than N-acetyl-α-galactose (α-GalNAc) in advance. When a glycopeptide protected with an alternative (protecting group) or the like is prepared and subjected to a glycosyltransferase reaction using N-acetylgalactosamine transferase (GalNAc transferase; ppGalNAcTs) using the glycopeptide as a substrate, other residues remain. Α-GalNAc can be transferred to Ser and Thr. A sugar other than α-GalNAc or its substitute (protecting group) contained in the product obtained by the above reaction is hydrolyzed with an enzyme (glycosidase) that selectively cleaves, whereby Ser and Thr. The side chain can be converted to a free hydroxyl group. In addition, since the stereochemistry of the glycosidic bond formed by ppGalNAcTs between GalNAc and Ser or Thr is only α-type, a GalNAc residue is introduced into the side chains of Ser and Thr in advance by β-type glycoside bonds. In this case, the carbohydrate can be selectively hydrolyzed by β-hexosaminidase. Like β-GalNAc, α- or β-glucose (α-Glc or β-Glc), α-fucose (α-Fuc), β-N-acetylglucosamine (β-GlcNAc), etc. are also α- or β, respectively. -Since it can be selectively cleaved using an enzyme such as glucosidase, α-fucosidase, β-hexosaminidase, it can be used for protection against hydroxyl groups of Ser and Thr. If the hydroxyl groups of any Ser and Thr in the peptide sequence are protected with a plurality of sugars or their substitutes (protecting groups), Ser and / or Thr other than the residue can transfer α-GalNAc by ppGalNAcTs. The modification order of ppGalNAcTs can be changed. In addition, since α-GalNAc transferred to a peptide chain can be further sugar chain-extended using other glycosyltransferases, the sugar to be introduced as a protecting group for Ser and / or Thr side chain or its sugar Designing a series of enzyme reaction protocols using peptide substrates considering the number, position, and type of substitutes (protecting groups) and ppGalNAcTs, glycosidases, and glycosyltransferases, different sugar chain structures can be selected from any serine structure. • Construction of glycopeptides placed on threonine residues is possible.

  As a specific example of the present invention, the permissible range (repertoire) of glycopeptide synthesis by the enzymatic method is greatly expanded, and at the same time, it has an advantage over the conventional method in the throughput of library construction. The search for glycopeptide antigen structures (cancer epitopes) and drug discovery research and development will be accelerated.

  For example, in a GalNAc transfer reaction using ppGalNAcTs, an industrially useful disease-related glycopeptide library can be provided by controlling the sugar chain modification position and order of peptide chains and proteins.

Accordingly, the present invention provides the following.
(1) The following steps:
(A) a step of obtaining a protected peptide containing two or more amino acid residues having sugar binding ability, wherein at least one of the amino acid residues having sugar binding ability is protected by a protecting group, At least one other amino acid residue having binding ability is not protected;
(B) a step of attaching a modifying group to at least one amino acid residue having a sugar-binding ability that is not protected of the protected peptide obtained in step (A); and (C) obtained in step (B). Deprotecting a protecting group of a protected peptide having a modified group.
A method for producing a modified peptide, wherein an amino acid residue having at least one sugar-binding ability is modified with the modifying group.
(2)
Item 2. The production method according to Item 1, wherein the amino acid residue having sugar binding ability is at least one selected from asparagine, serine, threonine, hydroxyproline, hydroxylysine and tyrosine.
(2A)
Item 3. The method according to Item 1 or 2, wherein the amino acid residue having sugar binding ability is serine or threonine.
(3)
Item 3. The method according to any one of Items 1-2 and 2A, wherein the protecting group is at least one substituent selected from the group consisting of a sugar chain, a phosphate group, and a sulfate group.
(4)
The protecting group is selected from the group consisting of α-glucose, β-glucose, β-galactose, α-mannose, β-GlcNAc, α-Fuc and β-GalNAc, and disaccharides and modified sugars thereof. The method in any one of 1-3.
(5)
The method according to any one of Items 1 to 4, wherein the modifying group is a sugar chain, and the step (B) is performed using a glycosyltransferase.
(6)
The protecting group is a sugar chain, the step (A) is carried out by a chemical synthesis method, a modifying group is bound using a glycosyltransferase in the step (B), and a sugar hydrolase in the step (C) 6. The method according to any one of items 1 to 5, wherein a deprotection reaction is performed using
(7)
The sugar chain is α-glucose, β-glucose, β-galactose, α-mannose, β-acetylglucosamine, α-fucose, β-acetylgalactosamine or galactosyl-β1,3-N-acetylgalactosamine, In item 6, the sugar hydrolase is α-glucosidase, β-glucosidase, β-galactosidase, α-mannosidase, β-hexosaminidase, α-fucosidase, β-hexosaminidase or O-glucosidase, respectively. The method described.
(8)
The glycosyltransferases are β1,4-galactosyltransferase, α-1,3-galactosetransferase, β1,4-galactosetransferase, β1,3-galactosetransferase, β1,6-galactosetransferase, α2, 6-sialyltransferase, α1,4-galactose transferase, ceramide galactose transferase, α1,2-fucose transferase, α1,3-fucose transferase, α1,4-fucose transferase, α1,6-fucose transferase Enzyme, α1,3-N-acetylgalactosamine transferase, α1,6-N-acetylgalactosamine transferase, β1,4-N-acetylgalactosamine transferase, polypeptide N-acetylgalactosamine transferase, β1,4-Nacetyl Glucosamine transferase, β1,2-N acetylglucosamine transferase, β1,3-N acetylglucose Saminyltransferase, β1,6-N acetylglucosaminyltransferase, α1,4-N acetylglucosaminyltransferase, β1,4-mannosetransferase, α1,2-mannosetransferase, α1,3-mannosetransferase, α1, 4-mannose transferase, α1,6-mannose transferase, α1,2-glucose transferase, α1,3-glucose transferase, α2,3-sialyltransferase, α2,8-sialyltransferase, α1, 6-glucosamine transferase, α1,6-xylose transferase, β-xylose transferase (proteoglycan core structure synthase), β1,3-glucuronic acid transferase, hyaluronic acid synthase; UDP-N-acetylglucosamine: polypeptide N -Acetylglucosaminyltransferase, protein O-fucose transferase, protein O-mannose transfer A group consisting of an enzyme, protein O-glucosyltransferase; O-fucosyl peptide 3-β-acetylglucosaminyltransferase; and UDP-N-acetylglucosamine protein O-mannose β1,2-N-acetylglucosaminyltransferase 8. The method according to item 6 or 7, wherein the method is more selected.
(9)
The step (B) is performed by a chemical method, and when the protecting group and the modifying group have a hydroxyl group, the hydroxyl group is protected, and after the step (B) is finished, as the step (B2), Item 9. The method according to any one of Items 1 to 8, wherein a deprotecting step of the protecting group for the hydroxyl group is performed.
(10)
The following steps:
(D) a step of obtaining a protected peptide comprising 3 or more amino acid residues having sugar binding ability, wherein at least two of the amino acid residues having sugar binding ability are protected by a protecting group, At least one other amino acid residue having binding ability is not protected;
(E) a step of attaching a modifying group to at least one amino acid residue having a sugar-binding ability that is not protected of the protected peptide obtained in the step (D);
(F) a step of deprotecting one of the protecting groups of the protected peptide having a modifying group obtained in the step (E);
(G) Modification of the protected peptide having a modifying group obtained in the step (F) to at least one amino acid residue having sugar-binding ability selected from unprotected amino acid residues having sugar-binding ability A step of bonding a group; and (H) a step of deprotecting another type of the protected peptide having a modifying group obtained in (G), which is different from the one in the step (F),
Including
A method for producing a modified peptide, wherein at least two amino acid residues having sugar-binding ability are modified with a modifying group.
(11)
Item 11. The method according to Item 10, wherein the modifying group in the step (E) is different from the modifying group in the step (G).
(12)
The step (E) and the step (G) are carried out by a chemical method. When the protecting group and the modifying group have a hydroxyl group, the hydroxyl group is protected, and the step (E) and the step (G), respectively. ) The method according to any one of Items 10 to 11, wherein after the step is completed, a deprotecting step of the protecting group of the hydroxyl group is performed as the step (E2) and the step (G2), respectively.
(13)
The manufacturing method in any one of the items 10-12 which has at least 1 characteristic of the items 2-9.
(14)
Furthermore, the method in any one of the items 10-13 including the process of making a modification group couple | bond with the amino acid residue which has at least 1 sugar bonding ability which is not protected.
(15)
15. A method for producing a library of peptides to which a modifying group is bound, comprising the step of performing the method according to any of items 1-14.
(16)
A peptide to which a modifying group produced by the method according to any one of Items 1 to 14 is bound.
(17)
The library of the peptide which the modification group produced by the method of item 15 couple | bonded.

  In all these aspects, it is understood that each embodiment described herein can be applied in other aspects, as long as applicable.

  The development of cancer vaccines and antibody drugs using new glycopeptide compounds, which are cancer-specific antigens, will enable tailor-made medical treatment that adapts not only to the type of cancer but also to detailed symptoms, progression, metastasis, etc. This leads to the development of innovative medical technology that contributes to reducing the high medical costs that are borne.

  As described above, according to the present invention, an objectively designed glycopeptide can be obtained by artificially blocking an amino acid capable of introducing a sugar chain, not depending only on the substrate specificity of glycosyltransferase. An excellent method for producing glycopeptides or glycoproteins has been provided in that they can be produced effectively.

FIG. 1 shows a reaction step (a) when performing glycopeptide synthesis using an EA2 peptide having N-acetyl-β-galactose (β-GalNAc) in Thr7 as a substrate, and laser desorption ionization time-of-flight mass measured in each step. It is the figure which showed the spectrum (b) of the analyzer (MALDI-TOFMS). Black squares indicate β-GalNAc, and white squares indicate α-GalNAc. (C-1), (c-2) is the figure which showed the spectrum of ECD-MS / MS measured about two types of products produced | generated by the transglycosylation reaction using ppGalNAcT2. (C-1) is a measurement result for α-GalNAc mono-transfer glycopeptide, (c-2) is a measurement result for α-GalNAc bi-transfer glycopeptide, the mono-transfer glycopeptide is Thr11, and the bi-transfer glycopeptide is Thr2. It became clear that α-GalNAc was modified in Thr11. In these figures, an asterisk (*) given to T (Thr) indicates that Thr is bound to a carbohydrate. FIG. 2 shows a reaction step (a) when a glycopeptide was synthesized using an EA2 peptide having α-mannose (α-Man) in Thr7 as a substrate, and a spectrum (b) of MALDI-TOFMS measured in each step. FIG. White circles indicate α-Man, and white squares indicate α-GalNAc. FIG. 3 shows the reaction step (a) when a glycopeptide was synthesized using an EA2 peptide having β-galactose (β-Gal) in Thr7 as a substrate and the spectrum (b) of MALDI-TOFMS measured in each step. FIG. Black circles indicate β-Gal, and white squares indicate α-GalNAc. FIG. 4 shows a reaction step (a) when a glycopeptide was synthesized using an EA2 peptide having α-fucose (α-Fuc) in Thr7 as a substrate and a spectrum (b) of MALDI-TOFMS measured in each step. FIG. The black triangle indicates α-Fuc, and the white square indicates α-GalNAc. FIG. 5 shows the reaction step (a) when the glycopeptide synthesis was performed using MUC1 peptide having α-mannose (α-Man) in Thr17 as a substrate, and the spectrum (b) of MALDI-TOFMS measured in each step. FIG. White circles indicate α-Man, and white squares indicate α-GalNAc. FIG. 6 shows a reaction step (a) when a glycopeptide was synthesized using MUC1 peptide having β-galactose (β-Gal) in Thr17 as a substrate, and a spectrum (b) of MALDI-TOFMS measured in each step. FIG. Black circles indicate β-Gal, and white squares indicate α-GalNAc. FIG. 7 shows the reaction step (a) when a glycopeptide was synthesized using MUC1 peptide having α-fucose (α-Fuc) in Thr7 as a substrate, and the spectrum (b) of MALDI-TOFMS measured in each step. FIG. The black triangle indicates α-Fuc, and the white square indicates α-GalNAc. FIG. 8 shows a reaction step (a) when a glycopeptide was synthesized using a MUC5AC peptide having α-mannose (α-Man) in Thr9 as a substrate, and a spectrum (b) of MALDI-TOFMS measured in each step. FIG. White circles indicate α-Man, and white squares indicate α-GalNAc. FIG. 9 shows the reaction step (a) when a glycopeptide was synthesized using a MUC5AC peptide having β-galactose (β-Gal) in Thr9 as a substrate, and the spectrum (b) of MALDI-TOFMS measured in each step. FIG. Black circles indicate β-Gal, and white squares indicate α-GalNAc. FIG. 10 shows the reaction step (a) when the glycopeptide synthesis was carried out using MUC5AC peptide having α-fucose (α-Fuc) in Thr9 as a substrate and the spectrum (b) of MALDI-TOFMS measured in each step. FIG. The black triangle indicates α-Fuc, and the white square indicates α-GalNAc. FIG. 11 is a diagram showing a reaction process when synthesizing a glycopeptide using an EA2 peptide having no carbohydrate as a substrate and four types of glycopeptides obtained. Transfer of N-acetyl-α-galactose (α-GalNAc) to the substrate occurs in the order of Thr7, Thr11, Thr2, Thr3. Open squares indicate α-GalNAc. FIG. 12 shows a reaction step (a) when glycopeptide synthesis was performed using MUC5AC peptide having α-mannose (α-Man) and N-acetyl-β-galactose (β-GalNAc) in Thr7 and Thr11 as substrates, It is the figure which showed the spectrum (b) of MALDI-TOFMS measured in each process. White circles indicate α-Man, white squares indicate α-GalNAc, and black squares indicate β-GalNAc. FIG. 13 is a production scheme of a glycopeptide library according to the prior art. FIG. 14 is an example of a production scheme for a glycopeptide library according to the present invention. It is an application example of the present invention. By using a combination of an enzyme that transfers the first modification group (sugar) to the peptide chain and an enzyme that transfers the second modification group (sugar) using the modification group as a substrate, a complex modified peptide can be produced. FIG. 16 shows a reaction step (a) when synthesizing a glycopeptide using EA2 peptide having α-mannose (α-Man) and α-fucose (α-Fuc) in Thr7 and Thr11 as substrates, respectively. It is the figure which showed the spectrum (b) of the measured MALDI-TOFMS. White circles indicate α-Man, white squares indicate α-GalNAc, and black triangles indicate α-Fuc. FIG. 17 shows a reaction step (a) when a glycopeptide was synthesized using a MUC1-like peptide having α-mannose (α-Man) in Thr16 as a substrate, and a spectrum (b) of MALDI-TOFMS measured in each step. It is a figure. After transferring the modifying group to the peptide chain by the first enzyme, a glycopeptide having the extended modifying group can be produced by allowing the second enzyme to act. White circles indicate α-Man, white squares indicate α-GalNAc, and black diamonds indicate α-Neu5Ac.

  The present invention will be described below. Throughout this specification, it should be understood that the singular forms also include the plural concept unless specifically stated otherwise. Therefore, it should be understood that the expression of the singular includes the concept of the plural unless specifically stated otherwise. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

(Definition of terms)
Listed below are definitions of terms particularly used in the present specification.

  In the present specification, the “sugar chain” refers to a compound in which one or more unit sugars (monosaccharides and / or derivatives thereof) are connected (therefore, monosaccharides are also within the scope of “sugar chains”). When two or more unit sugars are connected, each unit sugar is bound by dehydration condensation by a glycosidic bond. Examples of such sugar chains include sugars (glucose, galactose, mannose, fucose, xylose, N-acetylglucosamine, N-acetylgalactosamine, sialic acid, and complexes and derivatives thereof) contained in the living body. Examples include, but are not limited to, a wide range of sugar chains that are decomposed or derived from complex biomolecules such as degraded polysaccharides, glycoproteins, proteoglycans, glycosaminoglycans, and glycolipids. Therefore, in the present specification, the sugar chain can be used interchangeably with “sugar”, “sugar”, and “carbohydrate”. Further, unless otherwise specified, the “sugar chain” in the present specification may include both sugar chains and sugar chain-containing substances.

The "monosaccharide" as used herein, especially when referring is not hydrolyzed to simpler molecules which refers to a compound represented by the general formula C _n H _2n O _n. Here, those in which n = 2, 3, 4, 5, 6, 7, 8, 9 and 10 are referred to as diose, triose, tetrose, pentose, hexose, heptose, octose, nonose and decourse, respectively. Generally, it corresponds to an aldehyde or ketone of a chain polyhydric alcohol. The former is called aldose and the latter is called ketose.

  In the present specification, the “monosaccharide derivative” refers to a substance in which one or more hydroxyl groups on the monosaccharide are substituted with another substituent and the resulting substance is not within the range of the monosaccharide. Examples of such monosaccharide derivatives include sugars having a carboxyl group (for example, aldonic acid in which C-1 position is oxidized to carboxylic acid (for example, D-gluconic acid in which D-glucose is oxidized), terminal Uronic acid in which C atom of carboxylic acid becomes carboxylic acid (D-glucuronic acid in which D-glucose is oxidized), sugar having amino group or amino group derivative (for example, acetylated amino group) (for example, N- Acetyl-D-glucosamine, N-acetyl-D-galactosamine, etc.), sugars having both amino groups and carboxyl groups (for example, N-acetylneuraminic acid (sialic acid), N-acetylmuramic acid, etc.), deoxy Such as, but not limited to, glycated sugars (eg, 2-deoxy-D-ribose), sulfated sugars containing sulfate groups, phosphorylated sugars containing phosphate groups, etc. NOT. Alternatively, the sugars form a hemiacetal structure, glycosides also reacted with an acetal structure and an alcohol, in the range of monosaccharide derivatives.

  The nomenclature and abbreviations used to describe sugars in the present invention follow conventional nomenclature.

  In this specification, galactose refers to any isomer, but is typically β-D-galactose, and is used to refer to β-D-galactose unless otherwise specified.

  In this specification, acetylgalactosamine refers to any isomer, but is typically N-acetyl-α-D-galactosamine, and refers to N-acetyl-α-D-galactosamine unless otherwise specified. Used as a thing.

  In this specification, mannose refers to any isomer, but is typically α-D-mannose, and is used to refer to α-D-mannose unless otherwise specified.

  In this specification, glucose refers to any isomer, but is typically β-D-glucose, and is used to refer to β-D-glucose unless otherwise specified.

  In this specification, acetylglucosamine refers to any isomer, but is typically N-acetyl-β-D-glucosamine, and unless otherwise specified, refers to N-acetyl-β-D-glucosamine. Used as a thing.

  In this specification, fucose refers to any isomer, but is typically α-L-fucose, and is used to refer to α-L-fucose unless otherwise specified.

  In this specification, acetylneuraminic acid refers to any isomer, but is typically α-N-acetylneuraminic acid, and unless otherwise specified refers to α-N-acetylneuraminic acid. Used as.

  For example, β-D-galactose is β-Gal; N-acetyl-α-D-galactosamine is α-GalNAc; α-D-mannose is α-Man; β-D-glucose is β-Glc; N-acetyl-β-D-glucosamine is expressed as β-GlcNAc; α-L-fucose as α-Fuc; α-N-acetylneuraminic acid as α-Neu5Ac and the like. Monosaccharides are generally joined by glycosidic bonds to form disaccharides and polysaccharides. The bond orientation with respect to the plane of the ring is indicated by α and β. Specific carbon atoms that form a bond between two carbons are also described. Thus, the two cyclic anomers are represented by α and β. For reasons of display, it may be expressed as a or b. Therefore, in the present specification, α and a, and β and b are used interchangeably for the anomeric notation.

  In the present specification, it is noted that the symbol, designation, abbreviation (Glc, etc.), etc. of sugar may be different when representing a monosaccharide and when used in a sugar chain. In the sugar chain, the unit sugar exists in a form in which hydrogen or a hydroxyl group is removed from the other side because dehydration condensation occurs between the unit sugar and another unit sugar to be bound. Therefore, when these sugar abbreviations are used to represent monosaccharides, all hydroxyl groups are present, but when used in a sugar chain, the hydroxyl groups of the sugars to which the hydroxyl groups are bound are dehydrated. It is understood that only oxygen remains after condensation.

  When a sugar is covalently bonded to albumin, the reducing end of the sugar is aminated and can be bound to other components such as albumin via its amine group, in which case the hydroxyl group at the reducing end is an amine group. Note that it has been replaced with.

  In the present specification, the “sugar chain-containing substance” refers to a substance containing sugar chains and substances other than sugar chains. Many such sugar chain-containing substances are found in the living body. For example, in addition to polysaccharides contained in the living body, complex polysaccharides such as degraded polysaccharides, glycoproteins, proteoglycans, glycosaminoglycans, glycolipids, etc. Examples include, but are not limited to, a wide range of sugar chains that are decomposed or derived from biomolecules.

  In this specification, “glycoprotein”, “glycopolypeptide” or “glycopeptide” is used interchangeably and refers to a protein, polypeptide or peptide containing a sugar chain. Such glycoproteins include various useful functional proteins such as, but not limited to, enzymes, hormones, cytokines, antibodies, vaccines, receptors, serum proteins and the like.

  As used herein, the terms “protein”, “polypeptide”, “oligopeptide” and “peptide” are used interchangeably herein and refer to a polymer of amino acids of any length. This polymer may be linear, branched, or cyclic. The amino acid contained in the polypeptide may be a natural amino acid or a non-natural amino acid, and may be a modified amino acid (for example, an amino acid containing a functional group capable of binding a sugar chain). As the scope to which the present invention is applied, there is no particular reason for limiting the length of the peptide, so there is no meaning to distinguish these terms from each other, so the terms should also be used interchangeably. . The term can also encompass one assembled into a complex of multiple polypeptide chains. The term also encompasses natural or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification (eg, conjugation with a labeling component). This definition also includes, for example, polypeptides containing one or more analogs of amino acids (eg, including unnatural amino acids, etc.), peptide-like compounds (eg, peptoids) and other modifications known in the art. Is included.

  In the present specification, the “amino acid” is used in the meaning usually used in the art, and refers to an organic compound having a carboxyl group and an amino group. In the present specification, the amino acid may be a natural amino acid or a non-natural amino acid. The term “natural amino acid” means the L-isomer of a natural amino acid present in vivo. Natural amino acids include glycine, alanine, valine, leucine, isoleucine, serine, homoserine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, γ-carboxyglutamic acid, Examples include arginine, ornithine, and lysine. Unless otherwise indicated, all amino acids referred to herein are L-forms, but forms using D-form amino acids are also within the scope of the present invention. The term “unnatural amino acid” refers to an amino acid that is not normally found in proteins in nature. Examples of non-natural amino acids include norleucine, para-nitrophenylalanine, homophenylalanine, para-fluorophenylalanine, 3-amino-2-benzylpropionic acid, homo-arginine D-form or L-form and D-phenylalanine. In the present invention, an amino acid modified based on a natural amino acid falls within the range of non-natural amino acids if it is not a natural amino acid. “Amino acid analog” refers to a molecule that is not an amino acid but is similar to the physical properties and / or function of an amino acid. Examples of amino acid analogs include ethionine, canavanine, 2-methylglutamine and the like. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. As used herein, amino acid analogs or amino acid mimetics can be used in place of amino acids.

  Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may also be referred to by a generally recognized one letter code. In the present specification, “amino acid” is intended to mean any of “amino acid residue”, “N-terminal amino acid residue”, and “C-terminal amino acid residue” unless otherwise specified. The Similarly, “amino acid residue” is intended to mean either “N-terminal amino acid residue” or “C-terminal amino acid residue” unless there is a particular intention.

  As used herein, the term “amino acid residue having a sugar binding ability” refers to an amino acid residue to which a sugar (modifying group) may be transferred by an enzyme, for example, serine that undergoes transfer of GalNAc by ppGalNAcT, Threonine is exemplified. Preferably, it is as follows. (1) the modifying group (sugar) can be transferred (by enzymatic method), (2) the protecting group can be introduced (by chemical method), (3) (by enzymatic method) It is advantageous that only the protecting group can be selectively removed (deprotected), and (4) the protecting group is of a different type from the modifying group. As what satisfies these, for example, asparagine (Asn), serine (Ser), threonine (Thr), hydroxyproline (Hyp), hydroxylysine and tyrosine can be mentioned. Examples of sugars (chains) that bind to these amino acids include N-acetylglucosamine (GlcNAc) for Asn; mannose (Man), N-acetylgalactosamine (GalNAc), GlcNAc, for Ser / Thr. Galactose (Gal), fucose (Fuc), glucose (Glc); Ara (arabinose), Gal, GlcNAc for Hyp; Xyl (xylose) for Ser; Glc for Tyr.

  In the present specification, “protected peptide”, “protected polypeptide” and “protected protein” are used interchangeably to represent the same concept, and one or more amino acid residues, particularly an amino acid residue having a sugar-binding ability. A polymer of amino acids (eg, a peptide) in which at least one group is protected. Such protection may be any treatment as long as it does not substantially occur at the amino acid residue to be protected when a reaction intended for modification is performed, and preferably a protecting group. This is achieved by binding (for example, a sugar chain different from the sugar chain to be modified).

  In the present specification, the “protecting group” is used in the same meaning as commonly used in the art, and refers to a group that protects a certain functional group. When carrying out a chemical reaction, if there are functional groups (for example, amino acid residues) that do not want to interfere with or change under the conditions, such functional groups are pre-reacted with the first reagent. Thus, after the target reaction is completed, the first functional group is restored again by the third chemical reaction. The atom or group introduced at this time is called a protecting group. Specific examples of preferred protecting groups are described herein in the examples and elsewhere. For example, typically, halogen (I, Br, Cl, F, etc.), lower (here, typically, C1-C6 is shown, but not limited thereto) alkoxy, lower alkylthio, lower alkylsulfonyloxy, aryl Represents sulfonyloxy and the like. ), An appropriate substituent can be protected by a method known in the art. Examples of such protecting groups include Protective Groups in Organic Synthesis, T., such as ethoxycarbonyl, t-butoxycarbonyl, acetyl, and benzyl. W. By Green, John Wiley & Sons Inc. (Second Edition, 1991) and the like can be mentioned. Moreover, the conversion of the functional group contained in each substituent such as a protecting group may be carried out by a known method other than the above production method [for example, Comprehensive Organic Transformations, R.A. C. Larock (1989), etc.] may be used as a target protecting group.

  As the protective group in “protected peptide” or the like, it is preferable to use a protective group that does not leave by a reaction mediated by a modifying group (such as an enzyme reaction). Therefore, a group different from the modifying group is usually used. Examples of the protecting group in “protected peptide” include α-glucose, β-glucose, β-galactose, α-mannose, β-acetylglucosamine, α-fucose, β-acetylgalactosamine or galactosyl-β1,3-N-acetyl. Galactosamine is preferred and these are deprotected by α-glucosidase, β-glucosidase, β-galactosidase, α-mannosidase, β-hexosaminidase, α-fucosidase, β-hexosaminidase or O-glucosidase, respectively. .

  “Hydroxyl protecting group” refers to a group that protects a hydroxyl group that can be reacted with the hydroxyl group of the protecting group and the modifying group in the “protecting peptide” and the like when the modifying group is introduced. Examples of such protecting groups include acyl-type protecting groups such as acetyl group and benzoyl group, ether-type protecting groups such as benzyl group, methyl group and allyl group, acetal-type protecting groups such as isopropylidene group and benzylidene group, etc. Can be mentioned.

  In the present specification, the term “unprotected” is used in the same meaning as commonly used in the field, and a protective group or the like is not bound, and the intended reaction (for example, glycosyltransferase) is performed. The state that can be received. Usually, a functional group such as —OH (hydroxyl group) is in a state capable of reacting, or in a state capable of being transferred by a glycosyltransferase or the like. When sugar or the like is used as a protecting group, the hydroxyl group may be further protected.

  In the present specification, the “modifying group” refers to a target substituent to be bonded to a side chain of an amino acid residue in a peptide or the like, for example, a sugar chain (monosaccharide, polysaccharide, complex sugar, etc.). Take as an example.

  In this specification, “deprotection” means to release a protected state and return to a reactive state, and typically means to remove a protecting group. Methods of deprotection are known in the art, for example, “Protective Groups in Organic Synthesis”, Theodora W., et al. Referring to Greene (John Wiley & Sons, Inc., New York, 2nd edition, 1991), it can be appropriately carried out by hydrolysis or the like.

  In the present specification, “modified peptide”, “modified polypeptide” and “modified protein” are used interchangeably and refer to a polymer of amino acids (peptide, polypeptide or protein, etc.) to which a modifying group is bonded.

  In the present specification, “glycosyltransferase” refers to a sugar acceptor such as a protein, a carbohydrate, a lipid, a steroid, or an alcohol, a sugar donor, that is, a sugar nucleotide or a lipid intermediate (sugar-linked dolichol). ) Is a general term for enzymes that synthesize sugars and synthesize complex carbohydrates, glycoproteins, and polysaccharides. In the present invention, the term refers to those which transfer sugars using peptides or proteins as sugar receptors. As a specific example, the glycosyltransferase that can be used in the present invention can be implemented in consideration of the target glycopeptide. As glycosyltransferases, in addition to those having the function of transferring sugar chains to amino acid residues having sugar-binding ability, those having the function of sugar chain elongation (functional sugar chain formation), and formation of sugar chains or sugar chains of mother nucleus Those responsible for the function of antigen formation are contemplated in the present invention. The distinction between glycosyltransferases is roughly divided into two types depending on the substrate that is the acceptor in the enzyme reaction. The first one uses a peptide as a substrate, and the other uses a sugar chain (including monosaccharides and disaccharides or more oligosaccharides) contained in a glycopeptide as a substrate. “Those having a sugar chain elongation function” correspond to the latter. Among the sugar chain structures of glycopeptides that can be produced by allowing the latter enzyme to act after the former enzyme, the sugar chain structure that is considered to form part of the sugar chain structure is the mother sugar chain, It is a chain antigen and is called a functional sugar chain. Some of the mother sugar chains (basic structures of sugar chains widely present in the living body) and sugar chain antigens (sugar chains known to have antigenicity) have overlapping structures. For example, Galβ1 → 3GalNAcα1 (→ Ser / Thr) is a sugar chain antigen known as T antigen, although it is a mother sugar chain called core type 1. A functional sugar chain refers to a sugar chain structure other than the mother nucleus sugar chain, such as a polylactosamine sugar chain that acts as a ligand that binds to a lectin, and that is known to exhibit certain activities and functions. However, some functional sugar chains and sugar chain antigens have overlapping structures, and examples thereof include sialyl Lewis X antigen having a tetrasaccharide structure. Specific examples of such glycosyltransferases are described in detail herein in the Examples and elsewhere. It is understood that the present invention can be synthesized for any of these types.

  In the present specification, the “chemical synthesis method” means that a substance is produced by a chemical reaction, and in the present invention, in particular, this means that a peptide or a peptide to which a protecting group is bound is produced by a chemical reaction. Examples of chemical synthesis methods of peptides that can be used in the present invention include liquid phase synthesis and solid phase synthesis. Specific examples of preferred ones that can be used in the present invention are described herein in the Examples and elsewhere.

  As used herein, “sugar hydrolase” is an enzyme that releases sugar from a substance to which sugar is bound. 3.2. Enzyme, particularly EC. 3.2.1. Glycoside-binding hydrolase or sugar hydrolase). Specific examples of preferred ones that can be used in the present invention are described herein in the Examples and elsewhere.

  As used herein, “deprotection reaction” refers to a reaction for removing a protecting group. In the present invention, when the protecting group is a sugar chain, it can be carried out by a biochemical reaction using a sugar hydrolase or the like.

  As used herein, “library” refers to a population comprising a plurality of similar members within a category. For example, in the present invention, a library of glycopeptides refers to a population of multiple, preferably different types of glycopeptides.

  In the present specification, the “amino acid having sugar chain binding ability” typically represents

(Wherein R ₁ is a hydrogen atom removed from the side chain of the amino acid, R ₂ is a hydroxyl group, —OR ₇ , —NR ₈ R ₉ or a protecting group, and R ₃ to R ₄ and R ₆ are each independently a modifying group such as a protecting group, hydrogen, or a fluorescent group (for example, fluorescent (for detection) or biotin (for binding with avidin or the like) PEG chain (water-soluble) at the N-terminus of the peptide. Including a case where a modifying group having a certain function such as for improvement) is bonded, R ₅ represents a group capable of binding to a sugar chain, R _{7 to} R ₉ are each independently hydrogen, It represents a modifying group such as a protecting group or a fluorescent group.

In this specification, the side chain of an amino acid refers to a group corresponding to R when the amino acid is represented by RCH (NH ₂ ) COOH. In this specification, the amino acid is represented by R _a R _b CH (NH) COOH (R _a and R _b are arbitrary groups, and may form a cyclic group together). Contains imino acid. Therefore, in the case of imino acid, a group corresponding to R _a or R _b or both can correspond to a side chain. Examples of individual amino acids include glycine, alanine, valine, leucine, isoleucine, serine, homoserine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, Examples include, but are not limited to, γ-carboxyglutamic acid, arginine, ornithine, and lysine. Here, in the case of cyclic imino acids such as proline, R ₁ and R ₃ or R ₄ , and when the amino acid is glycine, R ₁ may mean a single bond.

Here, R _{2 to} R ₄ and R ₆ are each independently hydrogen (when not protected), or Fmoc group, acetyl group, benzyl group, benzoyl group, t-butoxycarbonyl group, t-butyl. Dimethyl group, N-phthalimidyl group, silyl group, trimethylsilylethyl group, trimethylsilylethyloxycarbonyl group, 2-nitro-4,5-dimethoxybenzyl group, 2-nitro-4,5-dimethoxybenzylethyleneoxycarbonyl group, alkylenoyl group (For example, a pentenoyl group), an oxyalkylenoyl group, and the like. Preferably, an oxycarbonyl group is attached as a linker. This is because the oxycarbonyl group intervenes to facilitate the linkage with the amino group.

Here, R ₅ may be an aminooxy group, an N-alkylaminooxy group, a hydrazide group, an azide group, a thiosemicarbazide group, a cysteine residue, or the like.

  Preferred examples of the “amino acid having sugar chain binding ability” include, for example, asparagine (Asn), serine (Ser), threonine (Thr), hydroxyproline (Hyp), hydroxylysine and tyrosine (Tyr). . In particular, serine (Ser) and threonine (Thr) are preferable.

(General techniques used in this specification)
The techniques used herein are organic chemistry, biochemistry, genetic engineering, molecular biology, microbiology, genetics and related that are within the skill of the art unless otherwise indicated. Well-known and conventional techniques in the field are used. Such techniques are well described, for example, in the documents listed below and in references cited elsewhere herein.

  Molecular biological techniques, biochemical techniques, and microbiological techniques used herein are well known and commonly used in the art, and are described in, for example, Sambrook Maniatis, T. et al. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, F .; M.M. , Et al. eds, Current Protocols in Molecular Biology, John Wiley & Sons Inc. NY, 10158 (2000); Innis, M .; A. (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; A. et al. (1995). PCR Strategies, Academic Press; Sinsky, J. et al. J. et al. et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press; Gait, M .; J. et al. (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, M .; J. et al. (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F .; (1991). Oligonucleotides and Analogues: A Practical Approc, IRL Press; Adams, R .; L. et al. (1992). The Biochemistry of the Nucleic Acids, Chapman &Hall; Shabarova, Z. et al. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, G .; M.M. et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, G .; T.A. (1996). Bioconjugate Technologies, Academic Press; Method in Enzymology 230, 242, 247, Academic Press, 1994; Separate Experimental Medicine “Methods for Gene Introduction & Expression Analysis” Yodosha, 1997; Hatanaka, Nishimura et al., Carbohydrate Science and Engineering, Kodansha Scientific, 1997; Design and Physiological Function of Glycomolecules, is described in the Chemical Society of Japan, Japan Society for Publication Press, 2001, etc., and these are relevant for reference (may be all) in this specification. Incorporated.

(Medicine, cosmetics, etc., and treatment and prevention using the same)
In another aspect, the present invention relates to pharmaceuticals (eg, pharmaceuticals such as vaccines, health foods, residual proteins or lipids having reduced antigenicity) and cosmetics. The medicament and cosmetic may further comprise a pharmaceutically acceptable carrier and the like. Examples of the pharmaceutically acceptable carrier contained in the medicament of the present invention include any substance known in the art.

  Such suitable formulation materials or pharmaceutically acceptable carriers include antioxidants, preservatives, colorants, flavors, and diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffers. Include, but are not limited to, agents, delivery vehicles, diluents, excipients and / or pharmaceutical adjuvants. Typically, the medicament of the present invention is a composition comprising isolated pluripotent stem cells, or a variant or derivative thereof, together with one or more physiologically acceptable carriers, excipients or diluents. It is administered in the form of For example, a suitable vehicle can be water for injection, physiological solution, or artificial cerebrospinal fluid, which can be supplemented with other materials common to compositions for parenteral delivery. .

  Acceptable carriers, excipients or stabilizers used herein are non-toxic to the recipient and are preferably inert at the dosages and concentrations used and Phosphate, citrate, or other organic acids; ascorbic acid, α-tocopherol; low molecular weight polypeptides; proteins (eg, serum albumin, gelatin or immunoglobulin); hydrophilic polymers (eg, polyvinyl Pyrrolidone); amino acids (eg, glycine, glutamine, asparagine, arginine or lysine); monosaccharides, disaccharides and other carbohydrates (including glucose, mannose, or dextrin); chelating agents (eg, EDTA); sugar alcohols (eg, EDTA) Mannitol or sorbitol ; Salt-forming counterions (such as sodium); and / or non-ionic surface-active agents (e.g., Tween, Pluronic (pluronic) or polyethylene glycol (PEG)).

  Exemplary suitable carriers include neutral buffered saline or saline mixed with serum albumin. Preferably, the product is formulated as a lyophilizer using a suitable excipient (eg, sucrose). Other standard carriers, diluents and excipients may be included as desired. Other exemplary compositions include pH 7.0-8.5 Tris buffer or pH 4.0-5.5 acetate buffer, which may further include sorbitol or a suitable substitute thereof. .

  The medicament of the present invention can be administered orally or parenterally. Alternatively, the medicament of the present invention can be administered intravenously or subcutaneously. When administered systemically, the medicament used in the present invention may be in the form of a pharmaceutically acceptable aqueous solution free of pyrogens. Such a pharmaceutically acceptable composition can be easily prepared by those skilled in the art by considering pH, isotonicity, stability and the like. In this specification, the administration method includes oral administration, parenteral administration (for example, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, mucosal administration, intrarectal administration, intravaginal administration, local administration to the affected area, Skin administration, etc.). Formulations for such administration can be provided in any dosage form. Examples of such a pharmaceutical form include a liquid, an injection, and a sustained release agent.

  The medicament of the present invention may be prepared by using a physiologically acceptable carrier, excipient or stabilizer (Japanese Pharmacopoeia 15th edition or the latest edition, Remington's Pharmaceutical Sciences, 18th Edition, AR, as required. Gennaro, ed., Mack Publishing Company, 1990, etc.) can be prepared and stored in the form of a lyophilized cake or aqueous solution by mixing a sugar chain composition having a desired degree of purity.

  The amount of the glycan composition used in the treatment method of the present invention takes into consideration the purpose of use, target disease (type, severity, etc.), patient age, weight, sex, medical history, cell morphology or type, etc. Thus, it can be easily determined by those skilled in the art. The frequency with which the treatment method of the present invention is applied to a subject (or patient) also depends on the purpose of use, target disease (type, severity, etc.), patient age, weight, sex, medical history, treatment course, etc. In view of this, it can be easily determined by those skilled in the art. Examples of the frequency include administration every day to once every several months (for example, once a week to once a month). It is preferable to administer once a week to once a month while observing the course.

  When the present invention is used as a cosmetic product, the cosmetic product can also be prepared while complying with the regulations stipulated by the authorities.

(Pesticide)
The composition of the present invention can also be used as an agrochemical component. When formulated as an agrochemical composition, it may contain agriculturally acceptable carriers, excipients, stabilizers and the like, if desired.

  When the composition of the present invention is used as an agrochemical, herbicide (such as pyrazolate), insecticide / acaricide (such as diazinon), fungicide (such as probenazole), plant growth regulator (eg, paclobutrazole) ), Nematicides (eg, benomyl), synergists (eg, piperonyl butoxide), attractants (eg, eugenol), repellents (eg, creosote), pigments (eg, , Food Blue No. 1, etc.), fertilizers (eg, urea, etc.), etc. can also be mixed as required.

(Health and food)
The present invention can also be used in the health / food field. In such a case, the points to be noted when used as an oral medicine as described above should be considered as necessary. In particular, when used as a functional food / health food such as a specific health food, it is preferable to treat it according to a medicine. Preferably, the sugar chain composition of the present invention can also be used as a low allergen food.

(Specific embodiment)
(Method of introducing a single modifying group)
In one aspect, the present invention provides the following step: (A) a step of obtaining a protected peptide containing two or more amino acid residues having sugar binding ability, and at least one of the amino acid residues having sugar binding ability One of the amino acid residues having a sugar-binding ability is protected by a protecting group, and at least one other amino acid residue is not protected; (B) of the protected peptide obtained in the step (A); A step of attaching a modifying group to an unprotected amino acid residue having at least one sugar-binding ability; and a step of deprotecting the protecting group of the protected peptide having the modifying group obtained in steps (C) and (B). And a method for producing a modified peptide in which at least one amino acid residue having a sugar-binding ability is modified with the modifying group. Thus, in the present invention, an amino acid to which a sugar is added is bound to a sugar different from a sugar introduced in advance by a glycosyltransferase or an alternative thereof (protecting group, preferably a monosaccharide). In this way, sugar chain introduction by sugar transfer reaction is blocked, the target sugar chain (including monosaccharide) is added to the amino acid at the target location, and then the sugar or substitute (blocking) introduced to block Group) is selectively removed by hydrolysis or the like. Each procedure will be described below.

  In the method of the present invention, first, as step (A), a step of obtaining a protected peptide containing two or more amino acid residues having sugar binding ability is performed.

  In the protected peptide produced in this step, at least one of the amino acid residues having sugar binding ability is protected by a protecting group, and at least one of the other amino acid residues having sugar binding ability is protected. Is not protected. In the process of producing a protected peptide, when the step of introducing a modifying group is performed by a chemical reaction, the protecting group is a sugar other than the modifying group, and the hydroxyl group is protected (for example, an acyl type such as an acetyl group or a benzoyl group). A protecting group, an ether-type protecting group such as a benzyl group, a methyl group or an allyl group, or an acetal-type protecting group such as an isopropylidene group or a benzylidene group). Such a protective peptide can be synthesized by any appropriate method, and can be performed, for example, by chemical synthesis (for example, peptide solid phase synthesis) or enzymatic method. For peptide synthesis, any method known in the prior art can be used, and the following can be exemplified: liquid phase method, Merrifield method (solid phase method). For these implementations, literature well known in the art, for example, Merrifield, RB: J. Am. Chem. Soc., 1963, 85, 2149-2154 on solid phase synthesis, as a textbook, for example, Methodsin Enzymology, Volume 289 , Solid-Phase Peptide Synthesis, Edited By Gregg B. Fields, ACADEMIC PRESS, etc.

  In this case, α-glucose, β-glucose, α-mannose, N-acetyl-β-glucosamine, α-fucose, N-acetyl-β-galactosamine and the like can be introduced, but are not limited thereto.

  In addition, if it can be made enzymatically, this can also be used. When a protecting group carbohydrate is introduced into a peptide chain by an enzymatic reaction, a sugar other than the glycosyltransferase used in the introduction of the following modifying group is used. It is preferred to use a transferase.

  Next, in the method of the present invention, as the step (B), a step of attaching a modifying group to at least one amino acid residue having an unprotected sugar-binding ability of the protected peptide obtained in the step (A). Is implemented. In this step, adding a modifying group to the protective peptide means that when the step of introducing the modifying group is performed by a chemical reaction, for example, as a modifying group, any sugar (natural type or non-natural type is not required), and The hydroxyl group is protected (for example, an acyl-type protective group such as an acetyl group or a benzoyl group, an ether-type protective group such as a benzyl group, a methyl group or an allyl group, or an acetal-type protective group such as an isopropylidene group or a benzylidene group). Can be implemented using As such a method, in addition to a biochemical method using a glycosyltransferase, it is preferable to use a glycosyltransferase that can use a glycosylation reaction by a chemical method. This is because a desired sugar chain can be specifically transferred to a specific location.

  Glycosyltransferase: β1,4-galactosyltransferase, α-1,3-galactosyltransferase, β1,4-galactosetransferase, β1,3-galactosyltransferase, β1,6-galactosetransferase, α2,6- Sialyltransferase, α1,4-galactose transferase, ceramide galactose transferase, α1,2-fucose transferase, α1,3-fucose transferase, α1,4-fucose transferase, α1,6-fucose transferase, α1,3-N-acetylgalactosamine transferase, α1,6-N-acetylgalactosamine transferase, β1,4-N-acetylgalactosamine transferase, polypeptide N-acetylgalactosamine transferase, β1,4-N acetylglucosamine transferase Enzyme, β1,2-N acetylglucosamine transferase, β1,3-N acetylglucosamine Transferase, β1,6-N acetylglucosamine transferase, α1,4-N acetylglucosamine transferase, β1,4-mannose transferase, α1,2-mannose transferase, α1,3-mannose transferase, α1,4 -Mannose transferase, α1,6-mannose transferase, α1,2-glucose transferase, α1,3-glucose transferase, α2,3-sialyltransferase, α2,8-sialyltransferase, α1,6 -Glucosamine transferase, α1,6-xylose transferase, β-xylose transferase (proteoglycan core structure synthase), β1,3-glucuronic acid transferase, hyaluronic acid synthase; UDP-N-acetylglucosamine: polypeptide N- Acetylglucosaminyltransferase, protein O-fucose transferase, protein O-mannose transferase Protein O-glucosyltransferase; O-fucosyl peptide 3-β-acetylglucosaminyltransferase; and UDP-N-acetylglucosamine protein O-mannose β1,2-N-acetylglucosaminyltransferase, or ppGalNAc-Ts (GalNAc transferase; ppGaNTase (see Glycobiology vol. 13 no. 1 pp. 1R ± 16R, 2003) can include the following: ppGaNTase-T1 (bovine; GenBank number (hereinafter the same)): L07780 / L17437 ), PpGaNTase-T1 (rat, Rattus norvegicus U35890), ppGaNTase-T1 (human Homo sapiensX85018), ppGaNTase-T1 (mouse Mus musculus U73820), ppGaNTase-T1 (pig, D85389), ppGaNTase-T2 (human, Homo sapiens X85019) ), PpGaNTase-T3 (human, Homo sapiens X92689), ppGaNTase-T3 (mouse, Musmusculus U70538), ppGaNTase-T4 (mouse, Mus musculus), ppGaNTase-T4 (human) Homo sapiensY08564), ppGaNTase-T5 (rat, Rattus noregicus AF049344), ppGaNTase-T6 (human, Homosapiens Y08565), ppGaNTase-T7 (rat, Rattus norvegicus AF076167), ppGaNTase-T7 (human, Homosapiens AJ002744-pp) Human, Homo sapiens AB040672), ppGaNTase-T10 (rat, Rattusnorvegicus AF241241), ppGaNTase-T11 (human, Homo sapiens Y12434), ppGaNTase-T12 (human, Homosapiens AB078146) ppGaNTase-T13 (human Homo sapiens AB078142). ); POFUT1 (fucose transferase) and the like. In a preferred embodiment, ppGalNAc-Ts can be used, but is not limited to this.

  Since each glycosyltransferase that can be used in the present invention has specificity or preference for the transfer position (eg, Chem. Biol. 2004, 11, 1009-1016), the position of the sugar in the target glycopeptide. Accordingly, those skilled in the art can appropriately determine the binding position and type of the protecting group, and use a suitable glycosyltransferase to produce a glycopeptide having a modifying group at the target position.

  In the method of the present invention, when the modifying group is formed by a chemical method, the protecting group for the hydroxyl group contained in the sugar (the protecting group and the sugar used as the modifying group) is removed as necessary in the next step. To do. Examples of such a method for deprotecting a sugar hydroxyl group include a chemical method (for example, a base treatment such as NaOH / MeOH).

  Next, as the step (C), a step of deprotecting the protective group of the protected peptide having the modifying group obtained in the step (B) is performed. Here, a representative example is removal of the protecting group (sugar) (deprotection). As a method for deprotecting such a protected peptide, a protective group can be selectively removed with a sugar hydrolase. Such selective removal has been achieved for the first time in the present invention, and such a result could not be achieved by the prior art because selective removal was not possible by the chemical method of the prior art. It can be said that. Examples of the sugar hydrolase corresponding to the protecting group carbohydrate that can be used in this step include α-mannosidase, β-hexosaminidase, α-fucosidase, α- / β-glucosidase, and the like. Depending on the protecting group, those skilled in the art can select as appropriate.

  For example, the following table can be given as an example of a combination of a protecting group and a sugar hydrolase (used in deprotection).

  Thus, as one of the features of the present invention, it can be said that a large number of sugar / cleaving enzyme variations are advantageous as a protecting group.

It can be said that the chemical synthesis method and the novel glycopeptide synthesis method have a complementary relationship. That is, in the chemical synthesis method, GalNAc is introduced into an amino acid residue (Ser / Thr residue, etc.) having an arbitrary sugar binding ability. On the other hand, in the synthesis method of the present invention, an arbitrary sugar binding ability is obtained. Any peptide substrate can be used in the present invention in which the side chain of an amino acid residue (such as a Ser / Thr residue) is a free hydroxyl group. For example, EA2, MUC1, MUC5Ac, etc. can be used. As examples of protecting groups, α-Man, β-Gal, α-Fuc and the like can be used. As the sugar chain to be transferred, α-GalNAc or the like can be used.

  In the present invention, the glycopeptides that can be produced using a method of protecting / deprotecting with a sugar can be roughly divided into the following two groups according to the combination of “modifying group-amino acid residue”. it can.

Group 1: Glycopeptides in which the combination of modifying group and amino acid residue is a natural type
(Eg GalNAcα1 → -Ser / Thr-)
Group 2: Glycopeptides whose modification group-amino acid residue combination is non-natural
(Eg Galα1 → -Asn-)
It is understood that any pattern can be applied in the present invention.

  A preferred embodiment is the case of Group 1, in which case it is usually a natural type, and it can be said that the enzyme can be used, so that the enzyme can be effectively used for the production of the glycopeptide.

In one embodiment, the amino acid residue having a sugar-binding ability that can be used in the present invention preferably satisfies the following conditions: (1) <Modified by enzymatic method> 2) <By chemical method> A protecting group can be introduced. (3) <By enzymatic method> Only the protecting group can be selectively removed. (4) The protecting group is of a different type from the modifying group. Amino acids satisfying one, preferably two, more preferably three, and most preferably all four are preferred, and specific preferred examples include: asparagine, serine, threonine, hydroxyproline, Hydroxylysine and tyrosine. In the case of the natural type, it is preferable to satisfy the requirements in <> for the requirements required for the “amino acid residue having sugar binding ability”. Examples include asparagine (Asn), serine (Ser), threonine (Thr), hydroxyproline (Hyp), hydroxylysine and tyrosine. Examples of sugars (chains) that bind to these amino acids include N-acetylglucosamine (GlcNAc) for Asn; mannose (Man), N-acetylgalactosamine (GalNAc), GlcNAc, for Ser / Thr. Galactose (Gal), fucose (Fuc), glucose (Glc); Ara (arabinose), Gal, GlcNAc for Hyp; Xyl (xylose) for Ser; Glc for Tyr.

  In consideration of the four requirements (1) to (4), serine or threonine is convenient, but is not limited thereto. For other amino acids, it can be carried out in consideration of the constraints. It can be said that being natural is possible because (1) is established and (2) may be synthesized (Angew. Chem. Int. Ed. 2004, 43, 1516-1520; Angew. Chem. Int. Ed. 2004, 42, 5186-5189, etc.). With regard to (3), those skilled in the art can implement it by taking into account known techniques in this field.

  Examples of the length of the peptide produced in the present invention include those having an arbitrary length. That is, a protected peptide can be produced by a chemical synthesis method or a method using an enzyme. In principle, there is no upper limit to the length of a peptide that can be produced by these methods. In reality, it is understood that modified peptides of any length can be produced. When using a chemical synthesis method, one having 100 residues is usually provided, and in order to produce a peptide having more than this, a peptide having 100 residues or more is constructed using a technique called ligation that connects the peptides. be able to. When a large number of types are provided as a library, it is preferable to provide them in a single method, and in such a case, a library of modified peptides having a length of up to 100 residues is provided.

  If the above conditions are considered by item, those skilled in the art can in principle find cases where they can be carried out without being bound by the conditions in <>. For example, the addition reaction of a modifying group can be performed from an enzymatic method to a chemical method. It is understood that can be substituted. Preferably, it can be said that it is reasonable to carry out under the conditions of <> in order to complete the protection peptide → modification group introduction → deprotection. For example, when the addition reaction of a modifying group is performed by a chemical method, first, after protecting the hydroxyl groups of the protecting group (sugar) and the modifying group (sugar) with an acetyl group or the like and chemically adding the modifying group to the peptide, Since the step of removing them may occur, the efficiency may be reduced. However, the modification by the enzyme method does not require those complicated steps, and is preferable in this respect because it is efficient.

  In another embodiment, the protecting group that can be used in the present invention can include at least one substituent selected from the group consisting of a sugar chain, a phosphate group, and a sulfate group. One example is a sugar chain, and it is preferable to use a sugar chain different from the modified chain.

  In one embodiment, the protecting groups utilized in the present invention are α-glucose, β-glucose, β-galactose, α-mannose, N-acetyl-β-glucose, α-fucose and N-acetyl-β-. Examples thereof include galactose and disaccharides and modified sugars thereof. Preferably, the protecting group is a monosaccharide. In one embodiment, it is advantageous to use a monosaccharide such as α-glucose, β-glucose, β-galactose, α-mannose, N-acetyl-β-glucose, α-fucose or N-acetyl-β-galactose. It is. This is because it is not necessary to perform a complicated reaction and an easily available enzyme can be used.

  In one embodiment, the modifying group used in the present invention is a sugar chain (monosaccharide, polysaccharide, complex sugar, etc.), and step (B) is performed using a glycosyltransferase. Preferably, the modifying group is a monosaccharide. Here, as the glycosyltransferase used, an enzyme capable of transferring a sugar chain corresponding to a target modifying group is used.

  In one embodiment, the protecting group used in the present invention is a sugar chain, the step (A) is performed by a chemical synthesis method, the modifying group is bound using a glycosyltransferase in the step (B), ( In step C), the present invention can be carried out by performing a deprotection reaction using a sugar hydrolase. By performing the A step by a chemical synthesis method, a protecting group can be bonded to any position.

  In another embodiment, sugar chains used as protecting groups in the present invention include α-glucose, β-glucose, β-galactose, α-mannose, β-acetylglucosamine, α-fucose and β-acetylgalactosamine and Disaccharides can be used, and the sugar hydrolases used in the present invention include α-glucosidase, β-glucosidase, β-galactosidase, α-mannosidase, β-hexosaminidase, α-fucosidase, respectively. And β-hexosaminidase as well as O-glucosidase can be used.

  In one embodiment, the glycosyltransferase used in the present invention is β1,4-galactosyltransferase, α-1,3-galactosyltransferase, β1,4-galactosetransferase, β1,3-galactosetransferase, β1,6-galactose transferase, α2,6-sialyltransferase, α1,4-galactose transferase, ceramide galactose transferase, α1,2-fucose transferase, α1,3-fucose transferase, α1,4- Fucose transferase, α1,6-fucose transferase, α1,3-N-acetylgalactosamine transferase, α1,6-N-acetylgalactosamine transferase, β1,4-N-acetylgalactosamine transferase, polypeptide N-acetyl Galactosamine transferase, β1,4-N acetylglucosamine transferase, β1,2-N acetylglucose Minotransferase, β1,3-N acetylglucosaminetransferase, β1,6-N acetylglucosaminetransferase, α1,4-N acetylglucosaminyltransferase, β1,4-mannosetransferase, α1,2-mannosetransferase, α1,3-mannosetransferase, α1,4-mannosetransferase, α1,6-mannosetransferase, α1,2-glucosetransferase, α1,3-glucosetransferase, α2,3-sialyltransferase, α2 , 8-sialyltransferase, α1,6-glucosaminetransferase, α1,6-xylosetransferase, βxylosetransferase (proteoglycan core structure synthase), β1,3-glucuronic acid transferase, hyaluronic acid synthase; UDP-N-acetylglucosamine: polypeptide-N-acetylglucosaminyltransferase, protein O-fuco S-transferase, protein O-mannose transferase, protein O-glucose transferase; O-fucosyl peptide 3-β-acetylglucosaminyltransferase; UDP-N-acetylglucosamine protein O-mannose β1,2-N-acetyl Examples include glucosaminyl transferase. A person skilled in the art can transfer another or the same sugar using other glycosyltransferases by appropriately applying techniques known in the art and referring to the examples described in Examples and the like.

  In another embodiment, the step (B) is performed by a chemical method, and when the protecting group and the modifying group have a hydroxyl group, the hydroxyl group is protected, and after the completion of the step (B), the step (B2) As described above, the present invention can be carried out by performing a deprotection step of the hydroxyl group. Such a method of transferring a modifying group by a so-called chemical method is preferable when a sugar chain is used as a protecting group and a modifying group, because the specificity is reduced, and thus these are further protected with an acetyl group or the like. .

  In the present invention, when considering a glycopeptide in which the group 2, that is, the modification group-amino acid residue combination is a non-natural type, the case where the group 1 modification group-amino acid residue combination is a natural type glycopeptide In the step of introducing the modifying group in (2), a group 2 non-natural bond can be technically produced by replacing the method using an enzyme with a chemical method. This will be specifically described below.

  In group 2, that is, a glycopeptide in which the combination of a modifying group and an amino acid residue is a non-natural type, as the step (A), in the step of producing a protected peptide, a protecting group: a sugar other than the modifying group and its hydroxyl group Is preferably protected (for example, acetyl group). And as a manufacturing method of such a protection peptide, chemical synthesis (for example, peptide solid-phase synthesis) can be used.

  (B) In the step of adding a modifying group to the protected peptide as the step, the modifying group is any sugar (both natural and non-natural types can be used), and the hydroxyl group is protected. (For example, an acetyl group) can be used. When such a modifying group is introduced, a glycosylation reaction by a chemical method or the like can be used. Such chemical methods can be carried out with reference to the following: for example, glycosylation method using halogenated sugar, glycosylation method using trichloroacetimidate, glycosylation method using acetylated sugar, thiosaccharide And the like.

  In group 2, that is, a glycopeptide in which the combination of a modifying group and an amino acid residue is a non-natural type, the protecting group and the modifying group can be deprotected by performing the following step after step (B). . That is, when a sugar is used as a protecting group and a modifying group, the hydroxyl protecting group contained in the sugar is removed. Examples of such a method for deprotecting a sugar hydroxyl group include a chemical method (for example, a reaction using sodium methoxide or anhydrous ammonia in methanol).

  In group 2, that is, a glycopeptide in which the combination of a modifying group and an amino acid residue is a non-natural type, as the step (C), a protecting group (for example, sugar, phosphate group) is removed (deprotection) step. be able to. A method for deprotecting such a protected peptide is not limited thereto, although the protective group can be selectively removed by a sugar hydrolase.

  In one embodiment, a disaccharide (Galβ1-3GalNAc) is chemically introduced into the peptide chain as a protecting group, and O-glycanase (O-glycosidase) (activity that specifically recognizes and cleaves this disaccharide). ) Can be deprotected. In this case, a combination of the modifying group GalNAc and the protecting group Galβ1-3GalNAc can also be used. The technology using such a disaccharide is a combination of various protective groups (monosaccharides) in constructing a single protected peptide, and there are no longer any monosaccharide candidates as protective groups. Is particularly effective. Alternatively, it can also be used when GalNAc is added as a modifying group and then further modified with a glycosyltransferase (sialyltransferase etc.) based on GalNAc.

That is, in this case, the following combinations can be implemented:
Modifying group: N-acetyl-α-galactosamine modifying group-introducing enzyme: polypeptide N-acetylgalactosamine transferase protecting group: galactosyl-β1,3-N-acetylgalactosamine deprotecting enzyme: O-glycosidase.

  Such types of glycosyltransferases, glycosyl hydrolases, protecting groups, modifying groups, etc. are one of those that those skilled in the art appropriately incorporate into the production method depending on the design, production efficiency, cost, etc. of the glycopeptide to be produced. From this it is understood that any technique can be used.

(Method of introducing the modifying group multiple times)
In another aspect, the present invention provides a method for producing a glycopeptide, glycopolypeptide or glycoprotein into which two or more kinds of modifying groups are freely introduced. That is, the present invention provides a method for producing a modified peptide in which at least two amino acid residues having sugar-binding ability are modified with a modifying group.

  Such a method of the present invention is a step of (D) obtaining a protected peptide containing 3 or more amino acid residues having sugar binding ability, wherein at least two of the amino acid residues having sugar binding ability are protected. An amino acid residue that is protected by a group, and at least one other amino acid residue having the sugar-binding ability is not protected; the steps; (E) and (D) the protected peptide obtained in steps (D) is not protected; A step of attaching a modifying group to at least one amino acid residue having a sugar-binding ability; a step of deprotecting one of the protecting groups of the protected peptide having a modifying group obtained in steps (F) and (E) (G) at least one amino acid residue having a sugar binding ability selected from amino acid residues having a sugar binding ability of the protected peptide having a modifying group obtained in the step (F); Bond the modifying group ; And (H) deprotecting the another kind different from the ones of the protecting group of the protected peptide having a modified group obtained in (G) of the step (F) including,.

  Here, the modifying group in the step (E) and the modifying group in the step (G) may be the same or different. When different modifying groups are used, glycopeptides into which a plurality of types of modifying groups are introduced are produced.

  In the method for producing a modified peptide in which the amino acid residues having at least two sugar-binding ability are modified with a modifying group, any specific embodiment described in (Method of introducing a single modifying group) is used alone. Or they can be applied in combination.

  In a preferred embodiment of the present invention, the method may further include a step of attaching a modifying group to at least one amino acid residue having sugar-binding ability that is not protected. Such a process provides a method for producing a glycopeptide having a variety of modification groups.

(Method for producing a library of peptides to which a modifying group is attached)
In another aspect, the present invention provides a method of producing a library of peptides with attached modifying groups. This method is a method for producing a modified peptide in which at least one amino acid residue having sugar binding ability of the present invention is modified with the modifying group, or at least two amino acid residues having sugar binding ability are modified with a modifying group. This can be realized by including the step of carrying out the method for producing the modified peptide one or more times.

  For example, the prior art will be described with reference to FIG. In this figure, a method for synthesizing eight types of glycopeptides in the prior art is described. First, as shown in the leftmost column, by a solid phase synthesis method using a peptide synthesis resin (in the figure, a resin carrying an amino acid in which an amino group is protected by a protecting group (PG) is illustrated). It is necessary to elongate the peptide chain and bind a sugar (protecting group) to an appropriate amino acid (amino acid having a sugar binding ability). However, in this case, 8 batches are necessary and complicated.

  On the other hand, if it is this invention, as FIG. 14 describes, many types of glycopeptides can be produced using one type of synthetic substrate. That is, in this example, as shown in the left column, a protected peptide (indicated as a substrate in the figure) in which two different types of protecting groups are introduced into the Fmoc-amino acid is provided. In Step 1, this is divided into two groups, and a group in which a glycosyltransferase that mediates glycosyltransferase to mediate amino acid having the first sugar-binding ability is reacted and a group that is not treated are prepared. In step 2, a group in which hydrolase A (which hydrolyzes black circles) and hydrolase B (which hydrolyzes gray) is made to act on the glycosyltransferase-treated group (top). . For the group not subjected to enzyme treatment, hydrolase A (which hydrolyzes black circles) and hydrolase B (which hydrolyzes gray), and in addition, A group in which both degradation enzymes A and B are allowed to act is created. Further, in step 3, a group in which a glycosyltransferase is allowed to act and a group in which the glycosyltransferase is allowed to act are separately prepared for each group. Further, in the group that has been subjected to hydrolase A in step 2, hydrolase B is allowed to act in step 4, and in the step 2, hydrolase B is allowed to act on the group that is subject to hydrolase B in step 2. Act. As a result, three types of amino acids having sugar-binding ability at three positions are produced at once: three types, two types, two types, one type three, and one type non-bonded. be able to.

  Next, FIG. 15 shows a schematic diagram when the present invention is implemented with a combination of ppGalNAcT and other glycosyltransferases.

  As a substrate, a protected peptide into which two types of protecting groups have been introduced is prepared as in FIG. Thereafter, in step 1, saccharide is introduced using ppGalNAcT as glycosyltransferase 1. Thereafter, in Step 2, one of the protecting groups is removed using hydrolase A. Next, when going to step 3 with the arrow on the right side, the same enzyme is treated as glycosyltransferase 1. When this is treated with hydrolase B toward the right side, a glycopeptide having two modified right and left ends is provided.

  On the other hand, when treated with glycosyltransferase 2 (for example, SiaT) after step 2, the sugar is further introduced at the site where the sugar is introduced with ppGalNAcT, and the sugar chain is elongated. Thereafter, similarly, when performing step 3 and step 4, in step 3, a group treated with glycosyltransferase 1 and a group not treated are created. Thereafter, as a step 5, by creating a group in which the glycosyltransferase 1 is allowed to act and a group in which the glycosyltransferase 1 is not acted on the group into which the glycosyltransferase has been introduced, the group to which the modifying group is bound is not bound. Three types are produced: one with no modifying group at the right end. Further, when glycosyltransferase 2 is allowed to act after step 3, both the right end and left end sugars are elongated. Then, as Step 4 and Step 5, by creating a group that acts and does not act on glycosyltransferase 1, the extended sugar is bound to the left end and the right end, and the amino acid having sugar-binding ability therein is modified. Can produce what is not.

  Here, examples of the glycosyltransferase that can be used in the sugar chain elongation reaction include α1,3-FucT, α2,6-SiaT, α2,3-SiaT, β1,3-GlcNAcT, and β1,4-GalT. be able to.

  ST6GalNAc-III / IV (ST, d (ST)), ST6GalNAc-I (STn), C2Gn-T3 (Core2), C2 / 4Gn-T are responsible for the function of forming a mother nucleus sugar chain or sugar chain antigen. (Core2, 4), C2Gn-T1 (Core2), C1Gal-T1 (Core1), and the like.

  Among the transglycosylation reactions, the following are known for the formation of the mother nucleus sugar chain and the sugar chain antigen. Most of the mucin sugar chains are linked to the core protein by α-O-glycosidic linkages through the linkage of N-acetylgalactosamine with serine and threonine hydroxyl groups. In general, in addition to N-acetylgalactosamine, fucose, galactose, N-acetylglucosamine, and sialic acid are found in mucin, but the mother sugar chain (mother nucleus region) is directly linked to N-acetylgalactosamine. It consists of sugar.

  In addition, a sugar chain having antigenicity, represented by a cancer-related sugar chain antigen, is referred to as a sugar chain antigen. Those having antigenicity to peripheral sugar chains as typified by blood group antigens, mother nucleus structures such as Tn and T, and sialyl Tn, sialyl T, sialyl Lewis A antigen and isomers thereof having sialic acid bound thereto An antigen such as sialyl Lewis X antigen is exemplified.

  It is understood that the present invention is also used in the production of glycopeptides used for glycosyltransferases used for the formation of mother sugar chains and sugar antigens.

(Product)
In another aspect, the present invention also provides a method for producing a modified peptide in which at least one amino acid residue having sugar binding ability of the present invention is modified with the modifying group, or at least two amino acid residues having sugar binding ability. Provided is a peptide or a library of peptides to which a modification group produced by including a step of carrying out the method of producing a modified peptide modified with a modification group one or more times.

  References such as scientific literature, patents and patent applications cited herein are hereby incorporated by reference in their entirety to the same extent as if each was specifically described.

  Hereinafter, although an example explains the composition of the present invention in detail, the present invention is not limited to this. The reagents used in the following were commercially available unless otherwise specified.

  Hereinafter, the present invention will be described in more detail with reference to examples. However, these are merely examples, and the scope of the present invention is not limited to the examples.

  In the Examples and the specification, amino acid residues may be represented by abbreviations with the amino acid position appended. For example, Thr9 means the 9th threonine.

(Experimental example A1)
When a glycan transfer reaction using ppGalNAcT2 is performed using the EA2 peptide (PTTDSTTPAPTTK (SEQ ID NO: 1)) as a substrate, α-GalNAc is transferred in preference to the seventh threonine (Thr7) from the N-terminal side (non-patented). References 4 and 5). Therefore, as long as ppGalNAcT2 is used, a glycopeptide in which α-GalNAc is preferentially transferred to a serine / threonine residue other than Thr7 cannot be obtained. Therefore, by applying the present invention, glycosyl transfer using ppGalNAcT2 as a substrate with a synthetic EA2 peptide (PTTDST T PAPTK (SEQ ID NO: 2) added with β-GalNAc in the underlined portion) in which β-GalNAc residue is added to Thr7 in advance It was decided to obtain a glycopeptide in which α-GalNAc was introduced into serine / threonine residues other than Thr7 by subjecting the reaction to subsequent hydrolysis reaction of α-GalNAc residue using Jack bean β-hexosaminidase . The reaction process is shown in FIG.

(Glucose transfer reaction using ppGalNAcT2 to β-GalNAc-added EA2 peptide)
A chemically synthesized β-GalNAc-added EA2 peptide (PTTDST T PAPTK (SEQ ID NO: 2), β-GalNAc added to the underlined portion) was used as a substrate and subjected to a glycosyl transfer reaction using ppGalNAcT2. The reaction was performed under a constant temperature of 37 ° C. using a total of 40 μl of a solution containing 125 μM substrate and 10 mM MnCl ₂ , 100 mM Tris-HCl buffer pH 7.5, 125 μM UDP-GalNAc, 120 nM ppGalNAcT2. After the reaction for 12 hours, 160 μl of acetonitrile was added to deactivate the enzyme, and the reaction was stopped. The product was analyzed by MALDI-TOFMS. The results are shown in the middle part of FIG. A molecular weight peak in which one or two α-GalNAc residues are transferred to the peptide chain appears, and the α-GalNAc is added to serine / threonine residues other than Thr7 by using the EA2 peptide added with β-GalNAc as a substrate. It was shown that glycopeptides introduced with can be prepared. In addition, the chemical structure and glycosylated amino acid residues of each of the produced glycopeptides to which the α-GalNAc residue was transferred at one place and two places were analyzed by ECD-MS method. The results are shown in FIGS. 1 (c-1) and 1 (c-2), respectively. The amino acid residues to which α-GalNAc was transferred were found to be Thr11 for a single transfer glycopeptide and Thr2 and Thr11 for a two transfer glycopeptide.

(Removal of β-GalNAc residue by β-hexosaminidase)
The enzyme reaction product was concentrated to dryness under reduced pressure, and then 20 μl of water was added to completely dissolve the residue to obtain a substrate solution. After purifying 1 μl of the substrate solution with ZipTipC18, 10 μl of 150 mM citrate buffer (pH 5.0) was added and incubated at 37 ° C. for 5 minutes, and 5 μl of Jack bean β-hexosaminidase (20 mU). Was added and reacted at 37 ° C. for 12 hours. After completion of the reaction, the product was analyzed by MALDI-TOFMS. The results are shown in the lower part of FIG. A molecular weight peak from which the β-GalNAc residue was removed appeared, and it was shown that β-GalNAc can be removed from the resulting glycopeptide using Jack bean β-hexosaminidase.

(Experimental example A2)
When glycan transfer reaction using ppGalNAcT2 is performed using EA2 peptide (PTTDSTTPAPTTK (SEQ ID NO: 1)) as a substrate, α-GalNAc is transferred in preference to the seventh threonine (Thr7) from the N-terminal side (J. Biol.Chem. (2006) 281,8613-8618). Therefore, as long as ppGalNAcT2 is used, a glycopeptide in which α-GalNAc is preferentially transferred to a serine / threonine residue other than Thr7 cannot be obtained. Therefore, a synthetic EA2 peptide in which an α-Man residue has been added to Thr7 in advance (PTTDST T PAPTK (SEQ ID NO: 3), with α-Man added to the underlined portion) as a substrate, a glycosyltransferase reaction using ppGalNAcT2, followed by Jack bean. The glycopeptide was obtained by subjecting it to a hydrolysis reaction of an α-Man residue using α-mannosidase. The reaction process is shown in FIG.

(Glucose transfer reaction using ppGalNAcT2 to α-Man-added EA2 peptide)
A chemically synthesized α-Man-added EA2 peptide (PTTDST T PAPTK (SEQ ID NO: 3), α-Man added to the underlined portion) was used as a substrate and subjected to a glycosyl transfer reaction using ppGalNAcT2. The reaction was carried out using a total of 80 μl of a solution containing 125 μM substrate, 10 mM MnCl ₂ , 100 mM Tris-HCl buffer pH 7.5, 125 μM UDP-GalNAc, 120 nM ppGalNAcT2 at 37 ° C. under constant temperature conditions. After the reaction for 6 hours, 160 μl of acetonitrile was added to inactivate the enzyme to stop the transglycosylation reaction. The product was analyzed by MALDI-TOFMS. The results are shown in the middle part of FIG. A molecular weight peak in which α-GalNAc residue is transferred to one or two positions on the peptide chain appears, and α-GalNAc is added to serine / threonine residues other than Thr7 by using EA2 peptide added with α-Man as a substrate. It was shown that glycopeptides introduced with can be prepared.

(Removal of α-Man residue by α-mannosidase)
The enzyme reaction product was concentrated to dryness under reduced pressure, and 30 μl of 150 mM sodium citrate buffer (pH 4.5) was added to completely dissolve the residue to obtain a substrate solution. After incubation for 5 minutes at 37 ° C. was added so that the ZnCl ₂ in 50mM said substrate solution, and reacted for 24 hours with the addition to 37 ° C. to 5μl of Jack bean alpha-mannosidase (20 mU). After completion of the reaction, the product was analyzed by MALDI-TOFMS. The results are shown in the lower part of FIG. A molecular weight peak from which the α-Man residue was removed appeared, and it was shown that α-Man can be removed from the above-described glycopeptide using Jack bean α-mannosidase.

(Experimental example A3)
Synthetic EA2 peptide (PTTDST T PAPTK (SEQ ID NO: 4), β-Gal added to the underlined portion) with Thr7 previously added with β-Gal residue as a substrate, glycosyltransferase reaction using ppGalNAcT2, followed by Aspergillus niger
A glycopeptide in which α-GalNAc was introduced into serine / threonine residues other than Thr7 was obtained by subjecting to β-Gal residue hydrolysis reaction using β-galactosidase. The reaction process is shown in FIG.

(Glucosyl transfer reaction using ppGalNAcT2 to β-Gal-added EA2 peptide)
A chemically synthesized β-Gal-added EA2 peptide (PTTDST T PAPTK (SEQ ID NO: 4), β-Gal added to the underlined portion) was used as a substrate and subjected to a glycosyl transfer reaction using ppGalNAcT2. The reaction uses 125 μM substrate and 10 mM MnCl ₂ , 100 mM Tris-HCl buffer pH 7.5, 125 μM UDP-GalNAc, 120 nM ppGalNAcT2) in a total of 80 μl under a constant temperature of 37 ° C. Carried out. After the reaction for 6 hours, 160 μl of acetonitrile was added to deactivate the enzyme, and the reaction was stopped. The product was analyzed by MALDI-TOFMS. The results are shown in the middle part of FIG. A molecular weight peak in which the α-GalNAc residue is transferred to the peptide chain appears, and α-GalNAc is introduced into serine / threonine residues other than Thr7 by using EA2 peptide with β-Gal added as a substrate. It was shown that glycopeptides can be prepared.

(Removal of β-Gal residue by β-galactosidase)
The enzyme reaction product was concentrated to dryness under reduced pressure, and 30 μl of 150 mM sodium citrate buffer (pH 4.5) was added to completely dissolve the residue to obtain a substrate solution. After incubating the substrate solution at 37 ° C. for 5 minutes, 10 μl of Aspergillus niger β-galactosidase (50 mU) was added and reacted at 37 ° C. for 24 hours. After completion of the reaction, the product was analyzed by MALDI-TOF-MS. The results are shown in the lower part of FIG. A molecular weight peak from which the β-Gal residue was removed appeared, indicating that β-Gal can be removed from the resulting glycopeptide using Aspergillus niger β-galactosidase.

(Experiment A4)
Synthetic EA2 peptide (PTTDST T PAPTTK (SEQ ID NO: 5), α-Fuc added to the underlined portion) with α-Fuc residue added in advance to Thr7 as a substrate, glycation reaction using ppGalNAcT2, followed by Bovine kidney α -A glycopeptide in which α-GalNAc was introduced into a serine / threonine residue other than Thr7 was subjected to a hydrolysis reaction of α-Fuc residue using fucosidase. The reaction process is shown in FIG.

(Glucose transfer reaction using ppGalNAcT2 to α-Fuc-added EA2 peptide)
The chemically-synthesized α-Fuc-added EA2 peptide (PTTDST T PAPTK (SEQ ID NO: 5), α-Fuc substituted in the underlined portion) was used as a substrate and subjected to a glycosyltransferase reaction using ppGalNAcT2. The reaction was carried out using a total of 40 μl of a solution containing 500 μM substrate and 10 mM MnCl ₂ , 100 mM Tris-HCl buffer pH 7.5, 500 μM UDP-GalNAc, 120 nM ppGalNAcT2 at a constant temperature of 37 ° C. After 6 hours, 160 μl of acetonitrile was added to inactivate the enzyme and the reaction was stopped. The product was analyzed by MALDI-TOFMS. The results are shown in the middle part of FIG. A molecular weight peak in which α-GalNAc residue is transferred to one or two sites appears, and α-GalNAc is introduced into serine / threonine residues other than Thr7 by using EA2 peptide with α-Fuc added as a substrate. It was shown that prepared glycopeptides can be prepared.

(Removal of α-Fuc residue by α-fucosidase)
The enzyme reaction product was concentrated to dryness under reduced pressure, and then 20 μl of 150 mM citrate phosphate buffer (pH 5.0) was added to completely dissolve the residue to obtain a substrate solution. After incubating the substrate solution at 37 ° C. for 5 minutes, 10 μl of Bovine kidney α-fucosidase (50 mU) was added and reacted at 37 ° C. for 20 hours. After completion of the reaction, the product was analyzed by MALDI-TOF-MS. The results are shown in the lower part of FIG. A molecular weight peak from which the α-Fuc residue was removed appeared, and it was shown that α-Fuc can be removed from the resulting glycopeptide using Bovine kidney α-fucosidase.

(Experimental example B1)
When a transglycosylation reaction using ppGalNAcT2 using MUC1 peptide (AHGVTSAPDTRPAPPGSTAPP (SEQ ID NO: 6)) as a substrate, α-GalNAc is transferred in preference to the 17th threonine (Thr17) from the N-terminal side (J. Biochem. (1999) 126, 975-985). Therefore, as long as ppGalNAcT2 is used, a glycopeptide in which α-GalNAc is preferentially transferred to serine / threonine residues other than Thr17 cannot be obtained. Therefore, a synthetic MUC1 peptide in which an α-Man residue was added to Thr17 in advance (AHGVTSAPDTRPAPGS T APP (SEQ ID NO: 7), α-Man added to the underlined portion) was used as a substrate, followed by a sugar transfer reaction using ppGalNAcT2, followed by α- The glycopeptide was obtained by subjecting it to a hydrolysis reaction of Man residues. The reaction process is shown in FIG.

(Glucose transfer reaction using ppGalNAcT2 to α-Man-added MUC1 peptide)
A chemically synthesized α-Man-added MUC1 peptide (AHGVTSAPDTRPAPPS T APP (SEQ ID NO: 7), α-Man added to the underlined portion) was used as a substrate and subjected to a transglycosylation reaction using ppGalNAcT2. The reaction was performed under a constant temperature of 37 ° C. using a total of 80 μl of a solution containing 125 μM substrate and 10 mM MnCl ₂ , 100 mM Tris-HCl buffer pH 7.5, 125 μM UDP-GalNAc, 120 nM ppGalNAcT2. . After the reaction for 6 hours, 160 μl of acetonitrile was added to inactivate the enzyme to stop the transglycosylation reaction. The product was analyzed by MALDI-TOFMS. The results are shown in the middle part of FIG. A molecular weight peak in which the α-GalNAc residue is transferred to the peptide chain at one place appears, and α-GalNAc is introduced into serine / threonine residues other than Thr17 by using MUC1 peptide with α-Man added as a substrate. It was shown that glycopeptides can be prepared.

(Removal of α-Man residue by α-mannosidase)
The enzyme reaction product was concentrated to dryness under reduced pressure, and 30 μl of 150 mM sodium citrate buffer (pH 4.5) was added to completely dissolve the residue to obtain a substrate solution. ZnCl ₂ was added to the substrate solution to 50 mM and incubated at 37 ° C. for 5 minutes, 5 μl of Jack bean α-mannosidase (20 mU) was added and reacted at 37 ° C. for 24 hours. After completion of the reaction, the product was analyzed by MALDI-TOFMS. The results are shown in the lower part of FIG. A molecular weight peak from which the α-Man residue was removed appeared, and it was shown that α-Man can be removed from the above-described glycopeptide using Jack bean α-mannosidase.

(Experiment B2)
Synthetic MUC1 peptide (AHGVTSAPDTRPAPGS T APP (SEQ ID NO: 8), β-Gal added to the underlined portion) with Thr17 previously added with β-Gal residue as a substrate, transglycosylation using ppGalNAcT2, followed by Aspergillus niger β -A glycopeptide in which α-GalNAc was introduced into serine / threonine residues other than Thr17 was obtained by subjecting it to a hydrolysis reaction of β-Gal residues using galactosidase. The reaction process is shown in FIG.

(Glucose transfer reaction using ppGalNAcT2 to β-Gal-added MUC1 peptide)
A chemically synthesized β-Gal-added MUC1 peptide (AHGVTSAPPDTRPAPS T APP (SEQ ID NO: 8), β-Gal added to the underlined portion) was used as a substrate and subjected to a transglycosylation reaction using ppGalNAcT2. The reaction was performed under a constant temperature condition of 37 ° C. using 125 μM substrate and a total of 80 μl of a solution containing 10 mM MnCl ₂ , 100 mM Tris-HCl buffer pH 7.5, 125 μM UDP-GalNAc, 120 nM ppGalNAcT2. After the reaction for 6 hours, 160 μl of acetonitrile was added to deactivate the enzyme, and the reaction was stopped. The product was analyzed by MALDI-TOFMS. The results are shown in the middle part of FIG. A molecular weight peak in which the α-GalNAc residue is transferred to the peptide chain at one place appears, and α-GalNAc is introduced into serine / threonine residues other than Thr17 by using the MUC1 peptide added with β-Gal as a substrate. It was shown that glycopeptides can be prepared.

(Deprotection of β-Gal residue by β-galactosidase)
The enzyme reaction product was concentrated to dryness under reduced pressure, and 30 μl of 150 mM sodium citrate buffer (pH 4.5) was added to completely dissolve the residue to obtain a substrate solution. After incubating the substrate solution at 37 ° C. for 5 minutes, 10 μl of Aspergillus niger β-galactosidase (50 mU) was added and reacted at 37 ° C. for 24 hours. After completion of the reaction, the product was analyzed by MALDI-TOFMS. The results are shown in the lower part of FIG. Glycopeptide in which α-GalNAc residue is transferred to one site and a molecular weight peak appears, and β-Gal added MUC1 peptide is used as a substrate and α-GalNAc is introduced into serine / threonine residues other than Thr17 It was shown that can be prepared.

(Experiment B3)
Synthetic MUC1 peptide (AHGVTSAPPDTRPAPS T APP (SEQ ID NO: 9), α-Fuc added to underlined portion) with α-Fuc residue added in advance to Thr17 as a substrate, followed by transglycosylation using ppGalNAcT2 and subsequent Bovine kidney α -A glycopeptide in which α-GalNAc was introduced into serine / threonine residues other than Thr17 was obtained by subjecting it to a hydrolysis reaction of α-Fuc residues using fucosidase. The reaction process is shown in FIG.

(Glucose transfer reaction using ppGalNAcT2 to α-Fuc-added MUC1 peptide)
A chemically synthesized α-Fuc-added MUC1 peptide (AHGVTSAPDTRPAPPS T APP (SEQ ID NO: 9), α-Fuc substituted in the underlined portion) was used as a substrate and subjected to a transglycosylation reaction using ppGalNAcT2. The reaction was carried out using a total of 80 μl of a solution containing 500 μM substrate and 10 mM MnCl ₂ , 100 mM Tris-HCl buffer pH 7.5, 500 μM UDP-GalNAc, 120 nM ppGalNAcT2 at a constant temperature of 37 ° C. After 6 hours, 160 μl of acetonitrile was added to inactivate the enzyme and the reaction was stopped. The product was analyzed by MALDI-TOFMS. The results are shown in the middle part of FIG. Glycopeptide in which α-GalNAc residue is transferred at one position and a glycopeptide in which α-GalNAc is introduced into serine / threonine residues other than Thr17 by using MUC1 peptide with α-Fuc added as a substrate It was shown that can be prepared.

(Removal of α-Fuc residue by α-fucosidase)
The enzyme reaction product was concentrated to dryness under reduced pressure, and then 20 μl of 150 mM citrate phosphate buffer (pH 5.0) was added to completely dissolve the residue to obtain a substrate solution. After incubating the substrate solution at 37 ° C. for 5 minutes, 10 μl of Bovine kidney α-fucosidase (50 mU) was added and reacted at 37 ° C. for 20 hours. After completion of the reaction, the product was analyzed by MALDI-TOF-MS. The results are shown in the lower part of FIG. A molecular weight peak from which the α-Fuc residue was removed appeared, and it was shown that α-Fuc can be removed from the resulting glycopeptide using Bovine kidney α-fucosidase.

(Experimental example C1)
When a transglycosylation reaction using ppGalNAcT2 with MUC5AC peptide (GTTPSPVPTTSTSAP (SEQ ID NO: 10)) as a substrate is performed, α-GalNAc is transferred in preference to the ninth threonine (Thr9) from the N-terminal side (J. Biol.Chem. (2006) 281,8613-8618). Therefore, as long as ppGalNAcT2 is used, a glycopeptide in which α-GalNAc is preferentially transferred to serine / threonine residues other than Thr9 cannot be obtained. Therefore, by applying the present invention, glycosyl transfer using ppGalNAcT2 as a substrate with a synthetic MUC5AC peptide (GTTPSPVP T TSTTSAP (SEQ ID NO: 11), with α-Man added to the underlined portion) obtained by adding an α-Man residue to Thr9 in advance The glycopeptide was obtained by subjecting to a reaction and subsequent hydrolysis reaction of α-Man residue using Jack bean α-mannosidase. The reaction process is shown in FIG.

(Glucose transfer reaction using ppGalNAcT2 to α-Man-added MUC5AC peptide)
A chemically synthesized α-Man-added MUC5AC peptide (GTTPSPVP T TSTSAP (SEQ ID NO: 11), α-Man added to the underlined portion) was used as a substrate, and the product was subjected to a glycosyl transfer reaction using ppGalNAcT2. The reaction was performed under a constant temperature condition of 37 ° C. using a total of 80 μl of a solution containing 125 μM substrate, 10 mM MnCl ₂ , 100 mM Tris-HCl buffer pH 7.5, 125 μM UDP-GalNAc, 120 nM ppGalNAcT2. After the reaction for 6 hours, 160 μl of acetonitrile was added to inactivate the enzyme to stop the transglycosylation reaction. The product was analyzed by MALDI-TOFMS. The results are shown in the middle part of FIG. Serine and threonine residues other than Thr9 can be obtained by using a MUC5AC peptide to which α-Man is added as a molecular weight peak where α-GalNAc residue is transferred to 1, 2 or 3 positions on the peptide chain. It was shown that a glycopeptide introduced with α-GalNAc can be prepared.

(Removal of α-Man residue by α-mannosidase)
The enzyme reaction product was concentrated to dryness under reduced pressure, and 30 μl of 150 mM sodium citrate buffer (pH 4.5) was added to completely dissolve the residue to obtain a substrate solution. After incubation for 5 minutes at 37 ° C. was added so that the ZnCl ₂ in 50mM said substrate solution, and reacted for 24 hours with the addition to 37 ° C. to 5μl of Jack bean alpha-mannosidase (20 mU). After completion of the reaction, the product was analyzed by MALDI-TOFMS. The results are shown in the lower part of FIG. A molecular weight peak from which the α-Man residue was removed appeared, and it was shown that α-Man can be removed from the resulting glycopeptide using Jack bean α-mannosidase.

(Experimental example C2)
Synthetic MUC5AC peptide (GTTPSPVP T TSTSAP (SEQ ID NO: 12), β-Gal added to the underlined portion) added with β-Gal residue in advance to Thr9 as a substrate, glycation reaction using ppGalNAcT2, followed by Aspergillus niger β -A glycopeptide in which α-GalNAc was introduced into serine / threonine residues other than Thr9 was subjected to a hydrolysis reaction of β-Gal residues using galactosidase. The reaction process is shown in FIG.

(Glucosyl transfer reaction using ppGalNAcT2 to β-Gal-added MUC5AC peptide)
A chemically synthesized β-Gal-added MUC5AC peptide (GTTPSPVP T TSTSAP (SEQ ID NO: 12), β-Gal added to the underlined portion) was used as a substrate and subjected to a transglycosylation reaction using ppGalNAcT2. The reaction was performed under constant temperature conditions of 37 ° C. using a total of 80 μl of a solution containing 125 μM substrate and 10 mM MnCl ₂ , 100 mM Tris-HCl buffer pH 7.5, 125 μM UDP-GalNAc, 120 nM ppGalNAcT2. After the reaction for 6 hours, 160 μl of acetonitrile was added to deactivate the enzyme, and the reaction was stopped. The product was analyzed by MALDI-TOFMS. The results are shown in the middle part of FIG. A molecular weight peak in which the α-GalNAc residue is transferred to one, two, or three places in the peptide chain appears, and the β-Gal-added MUC5AC peptide is used as a substrate for serine / threonine residues other than Thr9. It was shown that glycopeptides introduced with α-GalNAc can be prepared.

(Removal of β-Gal residue by β-galactosidase)
The enzyme reaction product was concentrated to dryness under reduced pressure, and 30 μl of 150 mM sodium citrate buffer (pH 4.5) was added to completely dissolve the residue to obtain a substrate solution. After incubating the substrate solution at 37 ° C. for 5 minutes, 10 μl of Aspergillus niger β-galactosidase (50 mU) was added and reacted at 37 ° C. for 24 hours. After completion of the reaction, the product was analyzed by MALDI-TOF-MS. The results are shown in the lower part of FIG. A molecular weight peak from which the β-Gal residue was removed appeared, indicating that β-Gal can be removed from the resulting glycopeptide using Aspergillus niger β-galactosidase.

(Experimental example C3)
Synthetic MUC5AC peptide in which α-Fuc residue is added to Thr9 in advance (GTTPSPVP T TSTSTAP (SEQ ID NO: 13), α-Fuc is added to the underlined portion) as a substrate, transglycosylation reaction using ppGalNAcT2, followed by Bovine kidneyy α -Glycopeptides in which α-GalNAc was introduced into serine / threonine residues other than Thr9 were subjected to hydrolysis reaction of α-Fuc residues using fucosidase. The reaction process is shown in FIG.

(Glucose transfer reaction using ppGalNAcT2 to α-Fuc-added MUC5AC peptide)
A chemically synthesized α-Fuc-added MUC5AC peptide (GTTPSPVP T TSTSAP (SEQ ID NO: 13), α-Fuc substituted in the underlined portion) was used as a substrate, and the product was subjected to a glycosyl transfer reaction using ppGalNAcT2. The reaction was carried out using a total of 80 μl of a solution containing 500 μM substrate, 10 mM MnCl ₂ , 100 mM Tris-HCl buffer pH 7.5, 500 μM UDP-GalNAc, 120 nM ppGalNAcT2 under constant temperature conditions of 37 ° C. After 6 hours, 160 μl of acetonitrile was added to inactivate the enzyme and the reaction was stopped. The product was analyzed by MALDI-TOFMS. The results are shown in the middle part of FIG. A molecular weight peak in which the α-GalNAc residue is transferred to one, two or three locations appears, and α-Fuc-added MUC5AC peptide is used as a substrate for serine / threonine residues other than Thr9. It was shown that a glycopeptide introduced with GalNAc can be prepared.

(Removal of α-Fuc residue by α-fucosidase)
The enzyme reaction product was concentrated to dryness under reduced pressure, and 20 μl of 150 mM citrate phosphate buffer (pH 5.0) was added to completely dissolve the residue to obtain a substrate solution. After incubating the substrate solution at 37 ° C. for 5 minutes, 10 μl of Bovine kidney α-fucosidase (50 mU) was added and reacted at 37 ° C. for 20 hours. After completion of the reaction, the product was analyzed by MALDI-TOF-MS. The results are shown in the lower part of FIG. A molecular weight peak from which the α-Fuc residue was removed appeared, and it was shown that α-Fuc can be removed from the resulting glycopeptide using Bovine kidney α-fucosidase.

(Experimental example D1)
When the EA2 peptide (PTTDSTTPAPTTK (SEQ ID NO: 1)) is used as a substrate for the transglycosylation reaction using ppGalNAcT2, α-GalNAc is first transferred in preference to the seventh threonine (Thr7) from the N-terminal side (non-patented). References 4 and 5), when the reaction is carried out using a large excess of sugar donor, the second, third and fourth α-GalNAc transitions occur in the order of Thr11, Thr2 and Thr3. Discovered (Figure 11). Therefore, as long as ppGalNAcT2 is used, a glycopeptide in which α-GalNAc is preferentially transferred to serine / threonine residues other than Thr7 and Thr11 cannot be obtained. Therefore, a synthetic EA2 peptide (PTTDST T ₇ PAP T ₁₁ TK (SEQ ID NO: 14)) in which α-Man residue and β-GalNAc residue (stereoisomers of α-GalNAc which is a natural type) are added in advance to Thr7 and Thr11, respectively. In addition, α-Man was added to [7-position] and β-GalNAc was added to [11-position] as a substrate to examine whether it was possible to prepare a glycopeptide to which the present invention was applied. The reaction process is shown in FIG.

(Glycotransfer reaction using ppGalNAcT2 to α-Man, β-GalNAc-added EA2 peptide)
Chemically synthesized α-Man, β-GalNAc-added EA2 peptide (PTTDST T ₇ PAP T ₁₁ TK (SEQ ID NO: 14), α-Man added at [position 7] and β-GalNAc added at [position 11]) as a substrate , And subjected to a glycosyl transfer reaction using ppGalNAcT2. The reaction was carried out using a total of 80 μl of a solution containing 125 μM substrate, 10 mM MnCl ₂ , 100 mM Tris-HCl buffer pH 7.5, 125 μM UDP-GalNAc, 120 nM ppGalNAcT2 at 37 ° C. under constant temperature conditions. After the reaction for 6 hours, 160 μl of acetonitrile was added to inactivate the enzyme to stop the transglycosylation reaction. The product was analyzed by MALDI-TOFMS. The results are shown in the second stage of FIG. A molecular weight peak in which an α-GalNAc residue is transferred to a peptide chain appears, and by using an EA2 peptide with two residues of α-Man and β-GalNAc as a substrate, serine other than Thr7 and Thr11 It was shown that a glycopeptide having α-GalNAc introduced into a threonine residue can be prepared.

(Removal of α-Man residue by α-mannosidase)
The enzyme reaction product was concentrated to dryness under reduced pressure, and 30 μl of 150 mM sodium citrate buffer (pH 4.5) was added to completely dissolve the residue to obtain a substrate solution. After incubation for 5 minutes at 37 ° C. was added to the said substrate solution 25μl consisting of ZnCl ₂ in 50 mM, and reacted for 24 hours by adding 15μl of Jack bean alpha-mannosidase (20 mU) and 37 ° C.. After completion of the reaction, the product was analyzed by MALDI-TOFMS. The results are shown in the third row of FIG. A molecular weight peak from which the α-Man residue was removed appeared, and it was shown that α-Man can be removed from the above-described glycopeptide using Jack bean α-mannosidase.

(Removal of β-GalNAc residue by β-hexosaminidase)
The enzyme reaction solution was concentrated to dryness under reduced pressure, and then the residue was completely dissolved with 30 μl of 150 mM citrate buffer (pH 5.0). After incubating 20 μl of the substrate solution at 37 ° C. for 5 minutes, 15 μl of Jack bean β-hexosaminidase (375 mU) was added and reacted at 37 ° C. for 18 hours. After completion of the reaction, the product was analyzed by MALDI-TOFMS. The results are shown in the fourth row of FIG. A molecular weight peak from which the β-GalNAc residue was removed appeared, and it was shown that β-GalNAc can be removed from the resulting glycopeptide using Jack bean β-hexosaminidase.

(Experimental example D2)
Synthetic EA2 peptide (PTTDST T ₇ PAP T ₁₁ TK (SEQ ID NO: 47)) in which α-Man and α-Fuc are respectively added in advance to Thr7 and Thr11, α-Man in [7th position] and α-Fuc in [11th position] Is used as a substrate for the glycosyltransferase reaction using ppGalNAcT2 followed by the hydrolysis reaction of α-Man using Jack Bean α-mannosidase, and again using the transglycosylation reaction using ppGalNAcT2 and α-fucosidase. It was decided to obtain a glycopeptide in which α-GalNAc was introduced into Thr2 and Thr7. The reaction process is shown in FIG.

(Glycotransfer reaction using ppGalNAcT2 to α-Man, α-Fuc-added EA2 peptide)
Using chemically synthesized α-Man and α-Fuc-added EA2 peptide (PTTDST T ₇ PAP T ₁₁ TK (SEQ ID NO: 47), α-Man added at [7th position] and α-Fuc added at [11th position]) as a substrate , And subjected to a glycosyl transfer reaction using ppGalNAcT2. The reaction was carried out using a total of 80 μl of a solution containing 125 μM substrate, 10 mM MnCl ₂ , 100 mM Tris-HCl buffer pH 7.5, 125 μM UDP-GalNAc, 120 nM ppGalNAcT2 at 37 ° C. under constant temperature conditions. After the reaction for 6 hours, 160 μl of acetonitrile was added to inactivate the enzyme to stop the transglycosylation reaction. The product was analyzed by MALDI-TOFMS. The results are shown in the second stage of FIG. A molecular weight peak in which the α-GalNAc residue is transferred to the peptide chain at one position appears. By using the EA2 peptide to which α-Man and α-Fuc are added as a substrate, serine / threonine residues other than Thr7 and 11 are used. It was shown that glycopeptides introduced with α-GalNAc can be prepared.

(Removal of α-Man residue by α-mannosidase)
The enzyme reaction product was concentrated to dryness under reduced pressure, and 30 μl of 150 mM sodium citrate buffer (pH 4.5) was added to completely dissolve the residue to obtain a substrate solution. After incubation for 5 minutes at 37 ° C. was added to the said substrate solution 25μl consisting of ZnCl ₂ in 50 mM, and reacted for 24 hours by adding 15μl of Jack bean alpha-mannosidase (20 mU) and 37 ° C.. After completion of the reaction, the product was analyzed by MALDI-TOFMS. The results are shown in the third row of FIG. A molecular weight peak from which the α-Man residue was removed appeared, and it was shown that α-Man can be removed from the above-described glycopeptide using Jack bean α-mannosidase.

(Glucose transfer reaction using ppGalNAcT2 to α-GalNAc, α-Fuc-added EA2 peptide)
The enzyme reaction solution was concentrated to dryness under reduced pressure, and then the residue was completely dissolved in 50 μl of 100 mM Tris-HCl buffer (pH 7.5) to obtain a substrate solution. The substrate solution was adjusted to a total of 80 μl solution containing 10 mM MnCl ₂ , 125 μM UDP-GalNAc, 120 nM ppGalNAcT2, and was carried out under a constant temperature condition of 37 ° C. After the reaction for 6 hours, 160 μl of acetonitrile was added to inactivate the enzyme to stop the transglycosylation reaction. The product was analyzed by MALDI-TOFMS. The results are shown in the fourth row of FIG. A molecular weight peak in which the α-GalNAc residue is transferred to the peptide chain appears, and α-GalNAc is introduced into serine / threonine residues other than Thr11 by using EA2 peptide added with α-Fuc as a substrate. It was shown that glycopeptides can be prepared.

(Removal of α-Fuc residue by α-fucosidase)
The enzyme reaction product was concentrated to dryness under reduced pressure, and then 20 μl of 150 mM citrate phosphate buffer (pH 5.0) was added to completely dissolve the residue to obtain a substrate solution. After incubating the substrate solution at 37 ° C. for 5 minutes, 10 μl of bovine kidney α-fucosidase (50 mU) was added and reacted at 37 ° C. for 20 hours. After completion of the reaction, the product was analyzed by MALDI-TOF-MS. The results are shown in the fifth row of FIG. A molecular weight peak from which the α-Fuc residue was removed appeared, indicating that α-Fuc can be removed from the resulting glycopeptide using bovine kidney α-fucosidase.

(Experimental example D3)
Glycosyltransferase using ppGalNAcT2 and sialyltransferase ST6GalNAc1 as a substrate with synthetic MUC1 peptide (AHGVTSAPDTTRAHGV T ₁₆ SAPD (SEQ ID NO: 52), α-Man added at [position 16]) with α-Man added to Thr16 in advance After being subjected to the reaction, it was subjected to a hydrolysis reaction of α-Man using Jack Bean α-mannosidase, and subjected to a transglycosylation reaction using ppGalNAcT2, and NeuAcα (2-6) GalNAcα1 and α-GalNAc were added to Thr7 and Thr16. It was decided to obtain each introduced glycopeptide. The reaction process is shown in FIG.

(Glucose transfer reaction using ppGalNAcT2 to α-Man-added MUC1 peptide)
A chemically synthesized α-Man-added EA2 peptide (AHGVTSAPDTRAHGV T ₁₆ SAPD (SEQ ID NO: 52), α-Man added at [position 16]) was used as a substrate and subjected to a transglycosylation reaction using ppGalNAcT2. The reaction was carried out using a total of 20 μl of a solution containing 125 μM substrate, 10 mM MnCl ₂ , 100 mM Tris-HCl buffer (pH 7.5), 125 μM UDP-GalNAc, 120 nM ppGalNAcT2 at a constant temperature of 37 ° C. . After the reaction for 6 hours, 160 μl of acetonitrile was added to inactivate the enzyme to stop the transglycosylation reaction. The product was analyzed by MALDI-TOFMS. The results are shown in the second row of FIG. A molecular weight peak in which the α-GalNAc residue is transferred to the peptide chain appears, and α-GalNAc is introduced into serine / threonine residues other than Thr16 by using EA2 peptide added with α-Man as a substrate. It was shown that glycopeptides can be prepared.

(Sialic acid transfer reaction using ST6GalNAcI)
The enzyme reaction product was purified by reversed-phase HPLC, and a peptide having one α-GalNAc residue introduced therein was used as a substrate for the reaction. The reaction uses 50 μM substrate, 10 mM MnCl ₂ , 100 mM Tris-HCl buffer (pH 6.5), 2.5 mM CMP-NANA, 2.87 μg ST6GalNAcI in total 20 μl solution, and constant temperature conditions at 20 ° C. Conducted below. After the reaction after 3 days, 160 μl of acetonitrile was added to inactivate the enzyme to stop the transglycosylation reaction. The product was analyzed by MALDI-TOFMS. The results are shown in the third row of FIG. A molecular weight peak in which a Neu5Ac residue is transferred to a peptide chain at one position appears, and Sialyl Tn [Neu5Acα (2-6) GalNAcα1-Thr / in which a Neu5Ac residue is introduced into an α-GalNAc residue present in the substrate. Ser] -containing glycopeptides have been shown to be prepared.

(Removal of α-Man residue by α-mannosidase)
The enzyme reaction solution was concentrated to dryness under reduced pressure, and then 20 μl of 150 mM sodium citrate buffer (pH 4.5) was added to completely dissolve the residue to obtain a substrate solution. After incubation for 5 minutes at 37 ° C. was added to the said substrate solution 25μl consisting of ZnCl ₂ in 50 mM, and reacted for 24 hours by adding 15μl of Jack bean alpha-mannosidase (20 mU) and 37 ° C.. After completion of the reaction, the product was analyzed by MALDI-TOFMS. The results are shown in the fourth row of FIG. A molecular weight peak from which the α-Man residue was removed appeared, and it was shown that α-Man can be removed from the above-described glycopeptide using Jack bean α-mannosidase.

(Glycotransfer reaction using ppGalNAcT2 to sialyl-Tn residue-added Muc1 peptide)
The enzyme reaction solution was concentrated to dryness under reduced pressure, and then the residue was completely dissolved with 10 μl of 100 mM Tris-HCl buffer (pH 7.5) to obtain a substrate solution. The substrate solution was adjusted to a total solution of 20 μl containing 10 mM MnCl ₂ , 125 μM UDP-GalNAc, 120 nM ppGalNAcT2, and the reaction was carried out under constant temperature conditions of 37 ° C. After the reaction for 12 hours, 160 μl of acetonitrile was added to inactivate the enzyme to stop the transglycosylation reaction. The product was analyzed by MALDI-TOFMS. The results are shown in the fifth row of FIG. A molecular weight peak in which an α-GalNAc residue is transferred to a peptide chain appears, and sialyl Tn sialyl Tn [Neu5Acα (2-6) GalNAcα1-Thr / Ser], Tn [GalNAcGalNAcα1-Thr / Ser] antigen structures are present. It was shown that mixed glycopeptides can be prepared.

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, this invention should not be limited and limited to this embodiment. It is understood that the scope of the present invention should be construed only by the claims. It is understood that those skilled in the art can implement an equivalent range based on the description of the present invention and the common general technical knowledge from the description of specific preferred embodiments of the present invention. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

  This is a method by which the intended designed glycopeptide can be produced simply and effectively, and can be applied in the fields of pharmaceuticals, agricultural chemicals, foods and the like.

Sequence number 1: PTTDSTTPAPTTK (EA2 peptide)
SEQ ID NO: 2: PTTDST T ₇ PAPTTK (synthetic EA2 peptide with β-GalNAc added to [position 7])
SEQ ID NO: 3: PTTDST T ₇ PAPTTK (synthetic EA2 peptide with α-Man added at [position 7])
SEQ ID NO: 4: PTTDST T ₇ PAPTTK (synthetic EA2 peptide with β-Gal added to [position 7])
SEQ ID NO: 5: PTTDST T ₇ PAPTTK (synthetic EA2 peptide with α-Fuc added to [position 7])
Sequence number 6: AHGVTSAPDTRPAPGSTAPP (MUC1 peptide)
SEQ ID NO: 7: AHGVTSAPDTRPAPGS T ₁₇ APP (synthetic MUC1 peptide with α-Man added to [position 17])
SEQ ID NO: 8: AHGVTSAPDTRPAPGS T ₁₇ APP (synthetic MUC1 peptide with β-Gal added to [position 17])
SEQ ID NO: 9: AHGVTSAPDTRPAPGS T ₁₇ APP (synthetic MUC1 peptide with α-Fuc added to [position 17])
Sequence number 10: GTTPSPVPTTSTTSAP (MUC5AC peptide)
SEQ ID NO: 11 GTTPSPVP T ₉ TSTTSAP (synthetic MUC5AC peptide with α-Man added at [position 9])
SEQ ID NO: 12 GTTPSPVP T ₉ TSTTSAP (synthetic MUC5AC peptide with β-Gal added to [position 9])
SEQ ID NO: 13 GTTPSPVP T ₉ TSTTSAP (synthetic MUC5AC peptide with α-Fuc added at [position 9])
SEQ ID NO: 14: PTTDST T ₇ PAP T ₁₁ TK (synthetic EA2 peptide with α-Man added at [position 7] and β-GalNAc added at position 11)
SEQ ID NO: 15: PTTDST T ₇ PAP T ₁₁ TK (synthetic EA2 peptide with β-GalNAc added at [position 7] and α-GalNAc added at [position 11]; FIG. 1)
SEQ ID NO: 16: P T ₂ TDST T ₇ PAP T ₁₁ TK (synthetic EA2 peptide with β-GalNAc added at [position 7] and α-GalNAc added at positions 2 and 11; FIG. 1)
SEQ ID NO: 17: PTTDSTTPAP T ₁₁ TK (synthetic EA2 peptide with α-GalNAc added to [position 11]; FIGS. 1 to 4)
SEQ ID NO: 18: P T ₂ TDSTTPAP T ₁₁ TK (synthetic EA2 peptide with α-GalNAc added to [position 2, 11]; FIGS. 1, 2 and 4)
SEQ ID NO: 19: P T ₂ TDST T ₇ PAP T ₁₁ TK (synthetic EA2 peptide with α-GalNAc added at [position 7] and α-Man added at positions 2 and 11; FIG. 2)
SEQ ID NO: 20 PTTDST T ₇ PAP T ₁₁ TK (synthetic EA2 peptide with β-Gal added at [position 7] and α-GalNAc added at [position 11]; FIG. 3)
SEQ ID NO: 21: PTTDST T ₇ PAP T ₁₁ TK (synthetic EA2 peptide with α-Fuc added at [position 7] and α-GalNAc added at position 11; FIG. 4)
SEQ ID NO: 22: P T ₂ TDST T ₇ PAP T ₁₁ TK (synthetic EA2 peptide with α-Fuc added at [position 7] and α-GalNAc added at positions 2 and 11; FIG. 4)
SEQ ID NO: 23: AHGV T ₅ SAPDTRPAPGS T ₁₇ APP (synthetic MUC1 peptide with α-GalNAc added to [position 5] and α-Man added to [position 17]; FIG. 5)
SEQ ID NO: 24: AHGV T ₅ SAPDTRPAPGSTAPP (synthetic MUC1 peptide with α-GalNAc added to [position 5]; FIGS. 5 to 7)
SEQ ID NO: 25: AHGV T ₅ SAPDTRPAPGS T ₁₇ APP (synthetic MUC1 peptide with α-GalNAc added at [position 5] and β-Gal added at [position 17]; FIG. 6)
SEQ ID NO: 26: AHGV T ₅ SAPDTRPAPGS T ₁₇ APP (synthetic MUC1 peptide with α-GalNAc added to [position 5] and α-Fuc added to [position 17]; FIG. 7)
SEQ ID NO: 27: GT T ₃ PSPVP T ₉ TSTTSAP (synthetic MUC5AC peptide with α-GalNAc added at [9th position] and α-Man added at [3rd position]; FIG. 8)
SEQ ID NO: 28: GT T ₃ PSPVP T ₉ TST T ₁₃ SAP (synthetic MUC5AC peptide in which α-Man is added to [position 9] and α-GalNAc is added to [positions 3, 13]; FIG. 8)
SEQ ID NO _{29: GT T 3 P S 5} PVP T 9 TST T 13 SAP ([9 -position] to alpha-Man, [3, 5, 13 of Synthesis was added alpha-GalNAc in MUC5AC peptide; Figure 8 )
SEQ ID NO: 30: GT T ₃ PSPVPTTSTTSAP (synthetic MUC5AC peptide with α-GalNAc added to [position 3]; FIGS. 8 to 10)
SEQ ID NO: 31: GT T ₃ PSPVPTTST T ₁₃ SAP (synthetic MUC5AC peptide in which α-GalNAc is added to [positions 3 and 13]; FIGS. 8 to 10)
(; 8-10 [3, 5, 13 of Synthesis MUC5AC peptide added alpha-GalNAc _{to) GT T 3 P S 5 PVPTTST} T 13 SAP: SEQ ID NO: 32
SEQ ID NO: 33: GT T ₃ PSPVP T ₉ TSTTSAP (synthetic MUC5AC peptide with β-Gal added at [position 9] and α-GalNAc added at [position 3]; FIG. 9)
SEQ ID NO: 34: GT T ₃ PSPVP T ₉ TST T ₁₃ SAP (synthetic MUC5AC peptide in which β-Gal is added to [position 9] and α-GalNAc is added to [positions 3, 13]; FIG. 9)
SEQ ID NO _{35: GT T 3 P S 5} PVP T 9 TST T 13 SAP ([9 -position] to beta-Gal, [3, 5, 13 of Synthesis was added alpha-GalNAc in MUC5AC peptide; Figure 9 )
SEQ ID NO: 36: GT T ₃ PSPVP T ₉ TSTTSAP (synthetic MUC5AC peptide in which α-Fuc is added to [position 9] and α-GalNAc is added to position 3; FIG. 10)
SEQ ID NO: 37: GT T ₃ PSPVP T ₉ TST T ₁₃ SAP (synthetic MUC5AC peptide with α-Fuc added at [position 9] and α-GalNAc added at positions 3 and 13; FIG. 10)
SEQ ID NO _{38: GT T 3 P S 5} PVP T 9 TST T 13 SAP ([9 -position] to alpha-Fuc, [3, 5, 13 of Synthesis was added alpha-GalNAc in MUC5AC peptide; Figure 10 )
SEQ ID NO: 39: PTTDST T ₇ PAPTTK (synthetic EA2 peptide with α-GalNAc added to [position 7]; FIG. 11)
SEQ ID NO: 40: PTTDST T ₇ PAP T ₁₁ TK (synthetic EA2 peptide with α-GalNAc added to [position 7, position 11]; FIG. 11)
SEQ ID NO: 41: P T ₂ TDST T ₇ PAP T ₁₁ TK (synthetic EA2 peptide with α-GalNAc added to [positions 2, 7, 11]; FIG. 11)
SEQ ID NO: 42: P T ₂ T ₃ DST T ₇ PAP T ₁₁ TK (synthetic EA2 peptide in which α-GalNAc is added to [positions 2, 3, 7, 11]; FIG. 11)
SEQ ID NO: 43: PTTDST T ₇ PAP T ₁₁ TK (synthetic EA2 peptide added with α-Man at [position 7] and β-GalNAc at [position 11]; FIG. 12)
SEQ ID NO: 44: P T ₂ TDST T ₇ PAP T ₁₁ TK (synthetic EA2 peptide added with α-Man at [position 7], α-GalNAc at [position 2], and β-GalNAc at [position 11]; )
SEQ ID NO: 45: P T ₂ TDSTTPAP T ₁₁ TK (synthetic EA2 peptide with α-GalNAc added to [position 2] and β-GalNAc added to [position 11]; FIG. 12)
SEQ ID NO: 46: P T ₂ TDSTTPAPTTK (synthetic EA2 peptide with α-GalNAc added to [position 2]; FIG. 12)
SEQ ID NO: 47: PTTDST T ₇ PAP T ₁₁ TK (synthetic EA2 peptide with α-Man added at [position 7] and α-Fuc added at [position 11]; FIG. 16)
SEQ ID NO: 48: P T ₂ TDST T ₇ PAP T ₁₁ TK (synthetic EA2 peptide with α-GalNAc added at [position 2], α-Man added at [position 7], and α-Fuc added at [position 11]; )
SEQ ID NO: 49: P T ₂ TDSTTPAP T ₁₁ TK (synthetic EA2 peptide with α-GalNAc added at [position 2] and α-Fuc added at [position 11]; FIG. 16)
SEQ ID NO: 50: P T ₂ TDST T ₇ PAP T ₁₁ TK (synthetic EA2 peptide with α-GalNAc added at [position 2, 7] and α-Fuc added at [position 11]; FIG. 16)
SEQ ID NO: 51: P T ₂ TDST T ₇ PAPTTK (synthetic EA2 peptide with α-GalNAc added to [position 2 and position 7]; FIG. 16)
SEQ ID NO: 52: AHGVTSAPDTRAHGV T ₁₆ SAPD (synthetic MUC1 peptide with α-Man added to [position 16]; FIG. 17)
SEQ ID NO: 53: AHGVTSAPD T ₁₀ RAHGV T ₁₆ SAPD (synthetic MUC1 peptide with α-Man added at [position 16] and α-GalNAc added at [position 10]; FIG. 17)
SEQ ID NO: 54: AHGVTSAPD T ₁₀ RAHGV T ₁₆ SAPD (synthetic MUC1 peptide with α-Man added at [position 16] and NeuAcα2-6GalNAcα added at [position 10]; FIG. 17)
SEQ ID NO: 55: AHGVTSAPD T ₁₀ RAHGVTSAPD (synthetic MUC1 peptide with NeuAcα2-6GalNAcα added to [position 10]; FIG. 17)
SEQ ID NO: 56: AHGVTSAPD T ₁₀ RAHGV T ₁₆ SAPD (synthetic MUC1 peptide added with α-GalNAc at [position 16] and NeuAcα2-6GalNAcα at [position 10]; FIG. 17)

Claims (17)

The following steps:
(A) a step of obtaining a protected peptide containing two or more amino acid residues having sugar binding ability, wherein at least one of the amino acid residues having sugar binding ability is protected by a protecting group, At least one other amino acid residue having binding ability is not protected;
(B) a step of attaching a modifying group to at least one amino acid residue having a sugar-binding ability that is not protected of the protected peptide obtained in step (A); and (C) obtained in step (B). Deprotecting a protecting group of a protected peptide having a modified group.
A method for producing a modified peptide, wherein an amino acid residue having at least one sugar-binding ability is modified with the modifying group. The production method according to claim 1, wherein the amino acid residue having sugar binding ability is at least one selected from asparagine, serine, threonine, hydroxyproline, hydroxylysine and tyrosine. The method according to claim 1, wherein the protecting group is at least one substituent selected from the group consisting of a sugar chain, a phosphate group, and a sulfate group. The protecting group is selected from the group consisting of α-glucose, β-glucose, β-galactose, α-mannose, β-GlcNAc, α-Fuc and β-GalNAc, and disaccharides and modified sugars thereof. Item 2. The method according to Item 1. The method according to claim 1, wherein the modifying group is a sugar chain, and the step (B) is performed using a glycosyltransferase. The protecting group is a sugar chain, the step (A) is carried out by a chemical synthesis method, a modifying group is bound using a glycosyltransferase in the step (B), and a sugar hydrolase in the step (C) The method according to claim 1, wherein the deprotection reaction is carried out using. The sugar chain is α-glucose, β-glucose, β-galactose, α-mannose, β-acetylglucosamine, α-fucose, β-acetylgalactosamine or galactosyl-β1,3-N-acetylgalactosamine, The sugar hydrolase is α-glucosidase, β-glucosidase, β-galactosidase, α-mannosidase, β-hexosaminidase, α-fucosidase, β-hexosaminidase or O-glucosidase, respectively. The method described in 1. The glycosyltransferases are β1,4-galactosyltransferase, α-1,3-galactosetransferase, β1,4-galactosetransferase, β1,3-galactosetransferase, β1,6-galactosetransferase, α2, 6-sialyltransferase, α1,4-galactose transferase, ceramide galactose transferase, α1,2-fucose transferase, α1,3-fucose transferase, α1,4-fucose transferase, α1,6-fucose transferase Enzyme, α1,3-N-acetylgalactosamine transferase, α1,6-N-acetylgalactosamine transferase, β1,4-N-acetylgalactosamine transferase, polypeptide N-acetylgalactosamine transferase, β1,4-Nacetyl Glucosamine transferase, β1,2-N acetylglucosamine transferase, β1,3-N acetylglucose Saminyltransferase, β1,6-N acetylglucosaminyltransferase, α1,4-N acetylglucosaminyltransferase, β1,4-mannosetransferase, α1,2-mannosetransferase, α1,3-mannosetransferase, α1, 4-mannose transferase, α1,6-mannose transferase, α1,2-glucose transferase, α1,3-glucose transferase, α2,3-sialyltransferase, α2,8-sialyltransferase, α1, 6-glucosamine transferase, α1,6-xylose transferase, β-xylose transferase (proteoglycan core structure synthase), β1,3-glucuronic acid transferase, hyaluronic acid synthase; UDP-N-acetylglucosamine: polypeptide N -Acetylglucosaminyltransferase, protein O-fucose transferase, protein O-mannose transfer A group consisting of an enzyme, protein O-glucosyltransferase; O-fucosyl peptide 3-β-acetylglucosaminyltransferase; and UDP-N-acetylglucosamine protein O-mannose β1,2-N-acetylglucosaminyltransferase The method of claim 6, wherein the method is more selected. The step (B) is performed by a chemical method, and when the protecting group and the modifying group have a hydroxyl group, the hydroxyl group is protected, and after the step (B) is finished, as the step (B2), The method of Claim 1 which performs the deprotection process of this hydroxyl-protecting group. The following steps:
(D) a step of obtaining a protected peptide comprising 3 or more amino acid residues having sugar binding ability, wherein at least two of the amino acid residues having sugar binding ability are protected by a protecting group, At least one other amino acid residue having binding ability is not protected;
(E) a step of attaching a modifying group to at least one amino acid residue having a sugar-binding ability that is not protected of the protected peptide obtained in the step (D);
(F) a step of deprotecting one of the protecting groups of the protected peptide having a modifying group obtained in the step (E);
(G) Modification of the protected peptide having a modifying group obtained in the step (F) to at least one amino acid residue having sugar-binding ability selected from unprotected amino acid residues having sugar-binding ability A step of bonding a group; and (H) a step of deprotecting another type of the protected peptide having a modifying group obtained in (G), which is different from that in the step (F), of the protected group.
Including
A method for producing a modified peptide, wherein at least two amino acid residues having sugar-binding ability are modified with a modifying group. The manufacturing method according to claim 10, wherein the modifying group in the step (E) is different from the modifying group in the step (G). The step (E) and the step (G) are carried out by a chemical method. When the protecting group and the modifying group have a hydroxyl group, the hydroxyl group is protected, and the step (E) and the step (G), respectively. The method according to claim 10, wherein after the step is completed, a deprotecting step of the protecting group of the hydroxyl group is performed as the step (E2) and the step (G2), respectively. The manufacturing method of Claim 10 which has at least 1 characteristic of Claims 2-9. The method according to claim 10, further comprising a step of attaching a modifying group to at least one amino acid residue having sugar-binding ability that is not protected. 11. A method for producing a library of peptides to which a modifying group is attached, comprising the step of performing the method according to claim 1 or 10. A peptide to which a modifying group produced by the method according to claim 1 is bound. The library of the peptide which the modification group produced by the method of Claim 15 couple | bonded.