WO2007114307A1

WO2007114307A1 - Mucin-type glycopeptide having polylactosamine backbone

Info

Publication number: WO2007114307A1
Application number: PCT/JP2007/057001
Authority: WO
Inventors: Shinichiro Nishimura; Masataka Fumoto
Original assignee: National Institute Of Advanced Industrial Science And Technology; Shionogi & Co., Ltd.
Priority date: 2006-03-31
Filing date: 2007-03-29
Publication date: 2007-10-11
Also published as: JP2009143809A

Abstract

The object is to provide: a novel compound which is useful as a primer for use in the production of a mucin-type glycopeptide having a polylactosamine backbone which is useful in a wide variety of applications including a material for a biochemical study, a pharmaceutical and a food and has a difficulty in production before this time; and a method for production of a glycopeptide using the primer. Disclosed are: a mucin-type glycopeptide derivative which has a novel polylactosamine backbone having an aldehyde group or a ketone group at the terminus and also having an amino acid residue which is cleavable with a protease (i.e., a compound represented by the formula (I)); a simple production method for a mucin-type glycopeptide having a polylactosamine backbone by using the derivative as a primer; and a method for production of a mucin-type peptide having a polylactosamine backbone by synthesizing a glycopeptide having a specific peptide sequence using a mucin-type glycoamino acid having a polylactosamine backbone which has been synthesized previously.

Description

Specification

Mucin-type glycopeptides having a borilactosamine skeleton

Technical field

The present invention relates to a novel compound useful as a primer for producing a glycopeptide, and a method for producing a glycopeptide using the primer. The present invention also relates to glycopeptides obtained by the production method.

Background art

[0002] A sugar chain is a major component of a living body along with nucleic acids and proteins, and is a well-known power source for living bodies. In recent years, in vivo information transmission, protein quality control, and structural stabilization. It has become clear that it has various higher-order functions such as labels for protein transport. However, compared to nucleic acids and proteins, sugar chains have not been established as a general preparation method, and the functions of sugar chains often function as complex carbohydrates bound to lipids and proteins. For this reason, there are a lot of unexplained studies on functions including structural information. In addition, in the field of protein research, the ability to look at many things that seem to perform their functions together with sugar chains is very difficult to study in detail.

[0003] In order to advance these studies and further utilize them in medicines, it is necessary to prepare a uniform sample in the state of complex carbohydrates rather than sugar chains alone. For glycopeptides in particular, both sugar chains and peptides are extremely diverse, so it is virtually impossible to procure the required structure from natural products each time, and the development of a rapid production method is impossible. Expected. When various structures are created using a common procedure, the work is a technology that is good at chemical synthesis, as represented by combinatorial chemistry developed in recent years. Based on this background, various glycopeptide production methods have been studied so far, but no practical production method has yet been reported. The main reason for this is that the preparation of the sugar amino acid as a raw material is complicated and it is difficult to prepare sugar amino acids having various sugar chain structures, and sugar amino acids having a large sugar chain structure have large steric hindrance. The yield and reaction rate are slow, and it is also important to extend the sugar chain by chemical synthesis after glycopeptide construction. Position · The power of 3D control is difficult. In other words, with the current technology, the reaction yield is low and the time required for preparation is long. Furthermore, since it is difficult to prepare the synthetic raw material itself for glycopeptide synthesis, it is custom-made to rapidly prepare the necessary sugar chain structure. It is extremely difficult to construct a glycopeptide library containing a complex sugar chain structure that is required for production and comprehensive functional analysis of glycopeptides and glycoproteins.

[0004] In general, glycopeptides are synthesized by Fmoc amino acid (amino acid with amino group protected with 9 fluorenylmethyloxycarboxyl group, hereinafter 9 fluorenylmethyloxycarboxyl group is abbreviated as Fmoc). ) And Fmoc glycosylamino acid, the basic peptide part is synthesized on a solid phase carrier by an automatic peptide synthesizer, the peptide part is released from the solid phase carrier, and once purified, it is purified organically or enzymatically. A method of extending sugar chains one by one using a simple synthesis method is used. For this reason, a complicated operation and a long time are required for sugar chain elongation. Therefore, automatic synthesis of not only the peptide portion but also the oligosaccharide chain portion is very useful for rapid synthesis of glycopeptides and library creation. For nucleic acids and proteins, automated synthesis technology has been established, and this is where everyone acknowledges that research in these fields has made significant progress, and for sugar chains! Longed for.

[0005] There have been several reports on studies aimed at synthesizing a library of glycopeptides, and all of them have been synthesized by solid phase chemical synthesis based on the method of R. B. Merrifield. On the other hand, there are two main methods for synthesizing oligosaccharide chains. One is by chemical synthesis, but a method for stereoselectively binding sugar residues to sugar residues has not been well established, and a process for attaching or removing protecting groups. There is a problem that is complicated. The other is based on enzymatic synthesis, which does not require a protecting group and can be linked stereoselectively between sugar residues and sugar residues, which is very advantageous compared to chemical synthesis. A method capable of automatic synthesis in combination with several polymer carriers has been proposed. This is due to the fact that genes for various glycosyltransferases have recently been isolated, and mass production of glycosyltransferases has become possible by gene recombination technology.

[0006] As such an example, U. Zehavi et al. Linked an aminoethyl group or an aminohexyl group. Solid phase synthesis by glycosyltransferase using a combined polyacrylamide gel as a solid support has been reported (see Non-Patent Documents 1 to 4). In this method, a suitable monosaccharide is converted to a 4-carboxy 2-to-benzylglycoside, and then linked to the above-mentioned carrier amino group directly or via a spacer, as a primer, a sugar chain elongation reaction using a glycosyltransferase. Then, the sugar chain extended by photolysis is released. However, the sugar transfer yield is about 50%, which is not sufficient. Also, this method can obtain oligosaccharides, not glycopeptides.

[0007] As another example, C. — H. Wong et al. Used a glycopeptide linked to amino-silica as a primer, extended the sugar chain using glycosyltransferase, and then hydrolyzed oc-chymotrypsin. We have reported a method to cut out elongated sugar chains in the form of glycopeptides using the degradation action. (See Non-Patent Document 5). The peptide chain of the resulting glycopeptide is as short as Asn (asparagine) Gly (glycine) Phe (furalanine). Furthermore, the yield of the sugar chain elongation reaction by glycosyltransferase is 55 to 65%, which is very sufficient. Not a thing.

[0008] In addition, C. —H. Wong et al. Improved a group bonded to aminated silica as a solid phase carrier, extended a sugar chain by glycosyltransferase, and then released the sugar chain by hydrazine decomposition. It has also been reported that the glycosyltransferase reaction by the enzyme has been performed almost quantitatively (see Non-Patent Document 6). However, the sugar chain compound obtained by this method is not a sugar peptide.

[0009] In addition, C. — H. Wong et al. Extended the peptide chain using Fmoc amino acid and Fmoc-Thr (j8 GlcNAc) —OH to the primer of Non-Patent Document 7 using aminated silica as a solid phase support. Next, the protecting group on the peptide chain is removed, and then the sugar chain is extended to the above-mentioned GlcNAc residue using a glycosyltransferase and treated with tetrakistriphenylphosphine palladium on the solid support. A method for releasing a synthesized glycopeptide will be reported (see Non-Patent Document 7). The glycopeptide chain obtained by this method has 8 amino acid residues and has a sufficient length as a peptide chain, but the obtained glycopeptide was first introduced into a solid support. The yield based on amino acids is less than 10%, which is not sufficient. In addition, impurities such as unreacted substances accumulate through peptide synthesis and sugar chain synthesis, so that isolation and purification of the target product becomes difficult if the peptide chain and sugar chain structure are complex. The In addition, automatic peptide synthesis is usually carried out in an organic solvent, and glycosylation by glycosyltransferase is usually carried out in an aqueous solution, and the properties of the carrier required for each reaction are different. Even automatic synthesis is difficult.

[0010] In addition, M. Meldal et al. Used a glycopeptide derivative bound to a polymer of mono- and di-acryloylated diamethylene polyethylene glycol as a primer, and extended the sugar chain using glycosyltransferase. Subsequently, a method for releasing sugar chains with trifluoroacetic acid has been reported (see Non-Patent Document 8). However, the peptide chain of the glycopeptide obtained by this method is Asn (asparagine) -Gly (glycine), which is too short to be called a glycopeptide. In addition, the C-terminal glycine residue is a glycinamide residue, and in some cases, it is necessary to convert the glycinamide residue to a glycine residue.

S. Roth et al. Disclose the following method in Patent Document 1. First, a glycosyltransferase sugar receptor is bound to a solid phase carrier, this is used as an affinity adsorbent, and a tissue extract containing a glycosyltransferase that can bind to this sugar receptor is contacted. The glycosyltransferase is bound to the affinity adsorbent. Subsequently, the affinity adsorbent to which the glycosyltransferase is bound is brought into contact with a solution containing a sugar nucleotide that can be used as a sugar donor by the glycosyltransferase, thereby releasing the glycosyltransferase and the sugar adsorbent. Extends one sugar residue to the receptor. Furthermore, a tissue extract containing a glycosyltransferase capable of binding to a sugar receptor in which one sugar residue is extended is brought into contact, and the same process is repeated to synthesize a desired sugar chain on a solid support. It is. However, there is no specific data showing the usefulness of this method or its application to the synthesis of non-natural glycopeptides, and a method for releasing the resulting sugar chain on the solid phase carrier is also disclosed. It has not been.

[0012] Nishimura et al. Used a protease-cleavable primer that can be used for the synthesis of glycopeptides or neoglycopeptides (non-natural glycopeptides), a method for producing a glycopeptide using the primer, and synthesis of the primer. A useful polymerizable aromatic amino acid derivative is disclosed (see Patent Document 2). However, this method involves complicated operations such as column purification and polymerization after peptide synthesis, since the peptide having a sugar residue is radically polymerized, and thus is weak in radicals and difficult to prepare glycopeptides containing sulfur atoms. The problem is that it takes time to switch to a sugar chain elongation reaction by an enzyme. The title is left.

[0013] In this way, there are no primers for producing glycopeptides quickly and in a high yield with simple equipment and purification, and automatic peptide synthesis by chemical methods and automatic sugar chain synthesis by enzymatic methods. New technologies that can efficiently link these are very important in the era of post-genomics, glycomics responsible for postproteomics, and glycoproteomics, and their development is envied. In fact, there are no examples of synthesizing glycopeptides intended for instrumentation, such as multi-product synthesis that can be called glycopeptide libraries, and synthesis of glycopeptides containing complex natural sugar chains or multiple sugar chains.

[0014] Mucin is the main glycoprotein of mucus that covers the lumen of the gastrointestinal tract such as the trachea, gastrointestinal tract, and gonads. MUC1 is a membrane-bound glycoprotein of epithelial cells and the first mucin that has been studied in detail. MUC1 is a tandem repeat (HGVTSAPDTRPAPGSTA PPA (SEQ ID NO: 41)), a repetitive amino acid sequence containing serine and threonine that can be attached to an O-linked sugar chain. is there. Since sugar chain attachments do not occur in all serines and threonines, the degree of sugar chain extension varies, so there are many glycoproteins with the same amino acid sequence but different functions. Yes.

[0015] It has been reported that the expression level of MUC1 changes with the progression of canceration (Non-patent Document 9: Nakamori, S .; Ota, DM; Karen, R .; Shirotani, K .; Irimura , T. Gastroenterology, 1994, 106, 353—361.) ₀ For example, in colorectal cancer, increased expression of MUC 1 has been observed in primary tumors and metastatic lesions in the advanced stage. Furthermore, there is a report that the degree of glycosylation of MU C1 (where sugar chains are introduced) and the structure of sugar chains differ between those derived from normal epithelium and those derived from cancer cells (Non-patent Document 10: Lloyd, K. Burcneli, J .; Kudryashov, V .; Yin, BWT; Taylor- Papadimitnou, J. J. Biol. Chem., 1996, 271, 33325- 33334 .; Patent Document 11: Hanisch, F. — G. Mueller, S. Glycobiology, 2000, 10, 439—449 .; H Hundreds! /, Ί え ί, even in normal cells, glycosylated peptides, even in cancer cells, In some cases, the exposed peptide moieties become epitopes, which are derived from lung cancer, breast cancer, colon cancer, and knee cancer. It is found in the cell membrane of epithelial cell lines. Specifically, cytotoxic T lymphocytes isolated from breast cancer patients undergo glycosylation of the MUC1 protein and recognize peptides! On the other hand, Tn, which is a cancer-related sugar chain antigen, and a mother nucleus structure such as 及び, and sialyl Τη, sialyl した, and sialyl Lewis Α antigen and sialyl Lewis X antigen combined with sialic acid, are mucin of cancer cell membrane and cancer patient serum. It is found in the mucin inside.

[0016] In recent years, application to drug discovery and diagnostics targeting specific changes in MUC1 accompanying such canceration has attracted attention (Non-patent Document 12: Koganty, RR; Reddish, MR; Longenecker , BM Drug Discov. Today, 1996, 1, 190-198 .;). For example, Biomira-Merck is developing a synthetic MUC 1 peptide vaccine that incorporates a 25-amino acid sequence of MUC1 cancer mucin in a liposomal formulation: “L BLP25”. Phasell targets lung cancer and prostate cancer. A clinical trial is ongoing. In addition, Bio mira—Merck has developed KLH (Keyhole limp et hemocyanin) that stimulates antibody production and T-cell responses to STn (disaccharide) specifically expressed in mucin on cancer cells. ) As a carrier protein: “Theratope” is currently under clinical development for breast cancer and rectal cancer.

[0017] Nishimura et al. Developed a water-soluble polymer primer for synthesizing glycopeptides (Patent Document 3). In Patent Document 3, a glycopeptide derivative having an aldehyde group or a ketone group at the end and containing a photocleavable linker for peptide solid phase synthesis is converted into a polymer primer, which is used for sugar conversion. The production of peptides is described. Nishimura et al. Also developed a method for synthesizing mucin-type peptides and a glycopeptide related to MUC1 (Patent Document 4). Patent Document 4 describes that a glycopeptide derivative having an aldehyde group or a ketone group at the end and containing an amino acid residue that can be cleaved by a protease is converted into a primer, and a glycopeptide is produced using this. RU

Patent Document 1: Japanese Patent Publication No. 5-500905

Patent Document 2: JP 2001-220399 A

Patent Document 3: International Publication No. 2005Z108417 Pamphlet

Patent Document 4: Pamphlet of International Publication No. 2006Z030840

Non-patent literature l: Carbohvdr. Res., 124, 23 (1983) Non-Patent Document 2: Carbohydr. Res., 228, 255 (1992)

Non-Patent Document 3: React. Polym., 22, 171 (1994)

Non-Patent Document 4: Carbohydr. Res., 265, 161 (1994)

Non-Patent Document 5: J. Am. Chem. Soc., 116, 1136 (1994)

Non-Patent Document 6: J. Am. Chem. Soc., 116, 11315 (1994)

Non-Patent Document 7: J. Am. Chem. Soc., 119, 8766 (1997)

Non-Patent Document 8: J. Chem. Soc. Chem. Commun., 1849 (1994)

Non-Patent Document 9 Gastroenterology, 106, 353-361 (1994)

Non-Patent Document 10: Biol. Chem., 271, 33325-33334 (1996)

Non-Patent Document ll: Glycobiology, 10, 439-449 (2000)

Non-Patent Document 12: Drug Discov. Today, 1, 190-198 (1996)

Disclosure of the invention

Problems to be solved by the invention

[0018] An object of the present invention is to provide a novel compound useful as a primer for producing a mucin-type glycopeptide having a borilactosamine skeleton, and a mucin-type glycopeptide having a polyratatosamine skeleton using the primer. The purpose of the present invention is to provide a production method, and to produce mucin-type glycopeptides having a borilactosamine skeleton, which has been difficult to produce so far, which is useful in a wide range of fields such as biochemical research materials, medicines and foods.

[0019] Another subject of the present invention is a method for producing a glycopeptide having a voralactosamine skeleton by synthesizing a glycopeptide having a desired peptide sequence using a previously synthesized saccharide amino acid having a vorolactosamine skeleton. It is to provide.

Means for solving the problem

As a result of intensive studies, the present inventors have found that a novel glycopeptide derivative having an aldehyde group or a ketone group at the end and containing an amino acid residue that can be cleaved by a protease has an aldehyde group or a ketone group. And this bond does not degrade under the hydrolysis conditions with proteases, and therefore functions as a primer suitable for the production of mucin-type glycopeptides having a polylactosamine skeleton, And the use of this primer, The present inventors have found that a mucin-type glycopeptide having a ratatosamine skeleton can be easily purified, and that a mucin-type glycopeptide having a borilactosamine skeleton can be produced quickly and in a high yield.

[0021] The present inventors also protected a sugar amino acid having a pre-synthesized voralactosamine skeleton with a protecting group, and synthesized a sugar peptide having a desired peptide sequence using the protected amino acid. The present inventors have found that a glycopeptide having a borilactosamine skeleton can be produced. The method is independent of proteases or photocleavage. Therefore, this method makes it possible to synthesize glycopeptides containing amino acid residues having properties that are easily cleaved by protease or photocleavage.

The present invention also provides a mucin-type glycopeptide that has been useful in a wide range of fields such as biochemical research materials, pharmaceuticals, and foods by the method for producing a glycopeptide using the above-described primer and has been difficult to produce so far. The present invention has been completed.

[0023] The MUC1 and MUC1 peptide library of the present invention are effective for elucidating the function of MUC1, and the possibility of new drug discovery based on the knowledge obtained therefrom is considered. Studies using glycopeptides include, for example, immobilization of glycopeptide libraries 'chipies', antibody reaction screening, search for specific antibodies, structure-activity relationship investigations in antigen-antibody reactions, specificity's of highly selective monoclonal antibodies Development of antibody drugs and vaccine therapy using glycopeptides is also possible.

[0024] Thus, the present invention provides the following.

(Item 1)

The following formula:

X— C (= 0) — (CH) — A— A— A (I)

2 n 1 2 3

Wherein X is a hydrogen atom, c-c alkyl, c-

6 c represents a reel or chromophore;

1 30 30

n represents an integer of 0 to 20;

A is — (CH 2) — C (= 0) —, — (CH 2 CH 2 O) —, an oligo with a degree of polymerization of 1-10.

1 2 0 ~ 20 2 2 1 ~ 10

Or polyacrylamide, polymerization degree 1 to: oligo or polypeptide of LO, oxygen atom or NH;

A represents an amino acid residue cleavable by a protease; A is a sugar amino acid residue substantially free of a site cleavable by a protease, or

Three

Alternatively, it represents a glycopeptide residue containing any sugar amino acid without a site cleavable by a protease, and the sugar amino acid residue or the glycopeptide residue is represented by the following formula:

[Chemical 14-1]

In which R ¹ and R ⁶ are each independently hydrogen, N-acetylethylneuraminic acid (Neu5 Ac) group or N-acetylethyldarcosamine (GlcNAc) group;

R ² and R ⁷ are galactose (Gal) groups;

R ³ and R ⁸ are GlcNAc groups;

R ⁴ and R ⁹ are Gal groups;

R ⁵ is a GlcNAc group;

R ¹⁰ is an N-acetyl-a-D-galactosamine (GalNAc) group;

Z ² and Z ³ are each independently hydrogen or a fucose group; and

n and m are each independently an integer of 0 or more;

Here, the bond between R ¹ and R ² is a 2, 3 bond when R ¹ is Neu5Ac group, and j81, 3 bond when R ¹ is GlcNAc group;

The bond between R ² and R ³ is a β ΐ, 4 bond;

The bond between R ³ and R ⁴ is a β ΐ, 3 bond;

The bond between R ⁴ and R ⁵ is a β ΐ, 4 bond;

The bond between R ⁶ and R ⁷ is an α 2,3 bond when R ⁶ is a Neu5Ac group, and a ΐ, 3 bond when R ⁶ is a GlcNA c group;

The bond between R ⁷ and R ⁸ is a β ΐ, 4 bond;

The bond between R ⁸ and R ⁹ is a β ΐ, 3 bond;

The bond between R ⁹ and R ^1G is a β ΐ, 3 bond;

The bond between R ⁵ and R ^1G is a β ΐ, 6 bond; The bond between Z ¹ and R ³ is << 1, 3 bond;

The bond between Ζ ² and R ⁵ is << 1, 3 bond; and

The bond between Ζ ³ and R ⁸ is << 1, 3 bond,

A compound having a sugar residue represented by:

(Item 2)

The cocoon is a protea derived from Bacillus Licheniformis

2. The compound according to item 1, wherein the compound is a glutamic acid residue or a cysteine residue that can be cleaved together.

(Item 3)

At least a partial force of A in mucin-type glycoprotein MUC1-derived SEQ ID NOs: 1 to 60

Three

2. A compound according to item 1, having an amino acid sequence selected from the group consisting of the amino acid sequences shown.

(Item 4)

Selected from the group consisting of the compound described in Item 1 and an optionally protected aminooxy group, N alkylaminooxy group, hydrazide group, azide group, thiosemicarbazide group, 1,2-dithiol group, and cysteine residue A compound obtained by reacting with a carrier containing a functional group.

(Item 5)

The carrier is:

a) Protected !, which may be a polymer or copolymer of a butyl monomer having an aminooxy group or a hydrazide group, or a polymer having a protected amino acid group or hydrazide group Ethers;

b) Protected !, which may be a silica support having a aminooxy group or a hydrazide group, a resin support, a magnetic bead or a metal support; and

c) the following formula:

[(NH OCH C (= 0)) -Lys] Lys— NHCH CH C (= 0) — R ³

2 2 2 2 2 2,

[(NH OCH C (= 0)) -Lys] Lys— NHCH (CH SH) C (= 0) — R ³

2 2 2 2 2,

[(NH OCH C (= 0)) -Lys] — Lys— Cys— NHCH CH C (= 0) — R ³ (sequence Number 61),

{[(NH OCH C (= 0)) -Lys]-Lys-NHCH [C (= O)-R ³ ] CH S},

2 2 2 2 2 2

{[(NH OCH C (= 0)) -Lys]-Lys-NHCH [C (= 0) NHCH CH C (= 0

2 2 2 2 2 2

) -R ³ ] CH -S},

twenty two

{[(NH OCH C (= 0)) -Lys] -Lys} Lys— NHCH CH C (= 0) — R ³ (

2 2 2 2 2 2 2

SEQ ID NO: 62),

{[(NH OCH C (= 0)) -Lys] -Lys} Lys— NHCH (CH SH) C (= 0) —

2 2 2 2 2 2

R ³ (SEQ ID NO: 63),

{[(NH OCH C (= 0)) -Lys] -Lys} Lys— Cys— NHCH CH C (= 0)

2 2 2 2 2 2 2

—R ³ (SEQ ID NO: 64),

[[[(NH OCH C (= 0)) -Lys] -Lys] Lys— NHCH [C (= 0) — R ³ ] CH

2 2 2 2 2 2 S] (SEQ ID NO: 65),

2

[[[(NH OCH C (= 0)) -Lys] -Lys]-Lys-NHCH [C (= 0) NHCH C

2 2 2 2 2 2

HC (= 0) -R ³ ] CH -S] (SEQ ID NO: 66),

2 2 2

[Chemical 14-2]

[(NH ₂ OCH ₂ C (= 0)) 2 Ly s] NHCHC (= 0) — R ³

I

[(NH ₂ OCH ₂ C (= 0)) 2 -Ly s] -NH (CH ₂ ) 4

Or

{[(NH ₂ OCH 2 C (= 0)) 2-L ys] 2 -L ys} -NHCHC (= 0) -R ³

I

{[(NH ₂ OCH ₂ C (-O)) a -L ys] ₂ ~ L ys} 匪 (CH ₂ ) ₄

In the formula, R ³ represents a hydroxyl group or an amino group, Lys represents a lysine residue, Cys represents cysteine,

[Chemical 14-3]

Where n is an integer from 1 to 15 and x: y is 1: 0 to 1: 1000,

5. The compound according to item 4, selected from the group consisting of:

(Item 6)

The following formula:

A N = C (— X) — (CH) A -A -A (II)

4 2 n 1 2 3

Where X is a hydrogen atom, c to

1 c alkyl,

30 c ~

6 c represents aryl or chromophore; 30

n represents an integer of 0 to 20;

A is — (CH 2) — C (= 0) —, — (CH 2 CH 2 O) —

1 2 0 ~ 20 2 2 1 ~ 10

A is a protease derived from Bacillus Licheniformis.

2

A cleavable glutamic acid residue or cysteine residue;

A is a sugar amino acid residue substantially free of a site cleavable by a protease, or

Three

Wherein R and R are each independently hydrogen, Neu5Ac group or GlcNAc group;

R ² and R ⁷ are Gal groups;

R ³ and R ⁸ are GlcNAc groups;

R ⁴ and R ⁹ are Gal groups;

R ⁵ is a GlcNAc group;

R ¹⁰ is a GalNAc group;

Z ² and Z ³ are each independently hydrogen or a Fuc group; and n and m are each independently an integer of 0 or more;

The bond between R ² and R ³ is a β ΐ, 4 bond;

The bond between R ³ and R ⁴ is a β ΐ, 3 bond;

The bond between R ⁴ and R ⁵ is a β ΐ, 4 bond;

The bond between R ⁷ and R ⁸ is a β ΐ, 4 bond;

The bond between R ⁸ and R ⁹ is a β ΐ, 3 bond;

The bond between R ⁹ and R ^1G is a β ΐ, 3 bond;

The bond between R ⁵ and R ^1G is a β ΐ, 6 bond;

The bond between Ζ ¹ and R ³ is << ¹ , 3 bond;

The bond between Ζ ² and R ⁵ is << 1, 3 bond; and

The bond between Ζ ³ and R ⁸ is << 1, 3 bond,

Having a sugar residue represented by: A is the following formula:

Four

[Chemical 14-5]

In the formula, s is an integer of 1 to 15, and x: y is a group represented by 1: 0 to 1: 1000).

(Item 7)

At least a partial force of A in mucin-type glycoprotein MUC1-derived SEQ ID NOs: 1-60

Three

7. A compound according to item 6, having an amino acid sequence selected from the group consisting of the amino acid sequences shown.

(Item 8)

The following steps:

(A) The compound according to any one of items 1 to 3, and an optionally protected aminooxy group, N-alkylaminooxy, which can specifically react with a ketone residue or an aldehyde residue of the compound And a carrier containing a functional group selected from the group consisting of a group, a hydrazide group, an azide group, a thiosemicarbazide group, a 1,2-dithiol group, and a cysteine residue. Process;

(B) Step (G): A glycosyltransferase is allowed to act on the compound obtained in step (A) in the presence of a sugar nucleotide, thereby transferring a sugar residue from the sugar nucleotide to the compound and extending the sugar chain. A step of obtaining a compound comprising a sugar residue selected from the group consisting of the sugar nucleotide force Gal group, GlcNAc group, Fuc group and Neu5Ac group force;

(C) removing unreacted sugar nucleotides and by-product nucleotides as necessary; and

(D) A method for producing a glycopeptide, comprising a step of allowing a protease to act on a compound in which a sugar residue is transferred and a sugar chain is extended.

(Item 9)

The following steps:

(A) By allowing a sugar transferase to act on the compound according to any one of items 4 to 7 in the presence of a sugar nucleotide, the sugar residue is transferred from the sugar nucleotide to the compound, and the sugar chain is Obtaining an elongated compound, the sugar nucleotide force having a sugar residue selected from the group consisting of a Gal group, a GlcNAc group, a Fuc group and a Neu5Ac group;

(B) removing unreacted sugar nucleotides and by-product nucleotides as necessary; and

(C) A method for producing a glycopeptide, comprising a step of allowing a protease to act on a compound having a sugar residue transferred and a sugar chain extended.

(Item 10)

The following steps:

(B) Step (A) is repeated once or twice or more to extend the sugar chain;

(C) removing unreacted sugar nucleotides and by-product nucleotides as necessary; and (D) A method for producing a glycopeptide, comprising the step of allowing a protease to act on a compound in which a plurality of sugar residues are transferred and the sugar chain is elongated.

(Item 11)

The following steps:

(A) A step of obtaining a compound according to any one of items 1 to 3 by performing peptide solid-phase synthesis using amino acids, sugar amino acids, and keto acids or aldehyde acids that can be cleaved by a protease as raw materials;

(B) The compound obtained in step (A) and an optionally protected aminooxy group, N-alkylaminooxy group, hydrazide capable of reacting specifically with the ketone residue or aldehyde residue of the compound Reacting with a carrier comprising a functional group selected from the group consisting of a group, an azide group, a thiosemicarbazide group, a 1,2-dithiol group and a cysteine residue;

(C) Step (B) By reacting the compound obtained in step (B) with a glycosyltransferase in the presence of a sugar nucleotide, a sugar residue is transferred from the sugar nucleotide to the compound, and the sugar chain is elongated. A step of obtaining a compound comprising a sugar residue selected from the group consisting of the sugar nucleotide force Gal group, GlcNAc group, Fuc group and Neu5Ac group force;

(D) removing unreacted sugar nucleotides and by-product nucleotides as necessary; and

(E) A method for producing a glycopeptide, comprising a step of allowing a protease to act on a compound in which a sugar residue is transferred and a sugar chain is elongated.

(Item 12)

The following steps:

(B) The compound obtained in step (A) and an optionally protected aminooxy group, N-alkylaminooxy group, hydrazide group capable of reacting specifically with the ketone residue or aldehyde residue of the compound And a carrier containing a functional group selected from the group consisting of an azide group, a thiosemicarbazide group, a 1,2-dithiol group and a cysteine residue, and at the same time, the step (A) Removing unreacted material in the step;

(C) A sugar transfer enzyme is allowed to act on the compound bound to the carrier obtained in step (B) in the presence of a sugar nucleotide to transfer a sugar residue from the sugar nucleotide to the compound, A step of obtaining a compound in which a sugar chain is extended, the sugar nucleotide having a sugar residue selected from the group consisting of a Gal group, a GlcNAc group, a Fuc group and a Neu5Ac group;

(D) Step (C) is repeated once or twice or more to extend the sugar chain;

(E) removing unreacted sugar nucleotides and by-product nucleotides as necessary; and

(F) A method for producing a glycopeptide, comprising the step of causing a protease to act on a compound in which a plurality of sugar residues are transferred and the sugar chain is elongated.

(Item 13)

Keto acid or aldehyde acid power in the step (A)

X-C (= 0)-(CH) -A -COOH (III)

2 n 1

In which X is a hydrogen atom, c1-c alkyl,

30 c represents 6 to c aryl or chromophore;

30

n represents an integer of 0 to 20;

A represents a linker having a length of 1 to 20 methylene chains,

13. The method according to item 11 or 12, which is a compound represented by:

(Item 14)

The following steps:

(A) By allowing a sugar transferase to act on the compound according to any one of items 1 to 3 in the presence of a sugar nucleotide, the sugar residue is transferred from the sugar nucleotide to the compound, and the sugar chain is Obtaining an elongated compound, the sugar nucleotide force having a sugar residue selected from the group consisting of a Gal group, a GlcNAc group, a Fuc group and a Neu5Ac group;

(B) Step (A) is repeated one or more times as necessary to extend the sugar chain; (C) a compound in which the sugar residue is transferred and the sugar chain is extended, and the ketone residue of the compound Protected, which can react specifically with a group or aldehyde residue, may be an aminooxy group, N-alkylaminooxy group, hydrazide group, azide group, thiosemicarbazide group, 1,2-dithiol group and cysteine. Reacting with a carrier containing a functional group selected from the group consisting of residues ;and

(D) removing unreacted sugar nucleotides and by-product nucleotides as necessary;

A process for producing a glycopeptide comprising

(Item 15)

The following steps:

(B) Step (A) is repeated one or more times as necessary to extend the sugar chain as necessary;

(C) a compound in which a sugar residue is transferred and a sugar chain is extended, and a protected compound capable of reacting specifically with a ketone residue or an aldehyde residue of the compound, and may be an aminooxy group, N-alkyl Reacting a carrier containing a functional group selected from the group consisting of an aminooxy group, a hydrazide group, an azide group, a thiosemicarbazide group, a 1,2-dithiol group and a cysteine residue;

(Item 16)

Said glycopeptide has the following formula:

[Chemical 14-6]

X ¹ X ¹ X ¹

Y '-His- Gly-Va 卜 Thr-Ser- Ala-Pro- Asp- Thr-Arg-Y ²

(SEQ ID NO: 2)

[Chemical 14-7]

X ¹ X ¹ X ¹

Y ¹ -Ala-His- Gly- Va 卜 Thr-Se `` AI a- Pro-Asp-Thr-Arg- Y ² (SEQ ID NO: 21)

Or

[Chemical 14-8]

X ¹ X ¹ X ¹ X ¹ X ¹

Y'-His-Gly-Val-Thr-Ser-Ala-Pro-Asp-Thr-Arg-Pro-Ala-Pro-Gly-Ser-Thr-Ala-Pro-Pro-Ala-Y ²

(SEQ ID NO: 41)

In the formula, each X ¹ independently represents a hydrogen atom or the following formula:

[Chemical 14-9]

In which R ¹ and R ⁶ are each independently hydrogen, Neu5Ac group or GlcNAc group;

R ² and R ⁷ are Gal groups;

R ³ and R ⁸ are GlcNAc groups;

R ⁴ and R ⁹ are Gal groups;

R ⁵ is a GlcNAc group;

R ¹⁰ is a GalNAc group;

The bond between R ² and R ³ is a β ΐ, 4 bond;

The bond between R ³ and R ⁴ is a β ΐ, 3 bond;

The bond between R ⁴ and R ⁵ is a β ΐ, 4 bond;

The bond between R ⁷ and R ⁸ is a β ΐ, 4 bond;

The bond between R ⁸ and R ⁹ is a β ΐ, 3 bond; The bond between R ⁹ and R ^1G is a β,, 3 bond;

The bond between R ⁵ and R ^1G is a β,, 6 bond;

The bond between Ζ ¹ and R ³ is << ¹ , 3 bond;

The bond between Ζ ² and R ⁵ is << 1, 3 bond; and

The bond between Ζ ³ and R ⁸ is << 1, 3 bond,

Represents a group represented by the formula, except when all of X ¹ are hydrogen atoms;

Υ ¹ represents a hydrogen atom, acetyl, acyl, alkyl or aryl;

Υ ² represents a hydroxyl group, ΝΗ, alkyl or aryl.

2

16. The method according to any one of items 8 to 15, which is a glycopeptide represented by:

(Item 17)

The following formula:

[Chemical 14-10]

X ¹ X ¹ X ¹

Y ¹ -His- Gly-Va 卜 Thr-Ser- Ala-Pro- Asp- Thr-Arg-Y ²

(SEQ ID NO: 2)

[Chem. 14-11]

X ¹ X ¹ X ¹

Y '-Ala-His- Gly- Va 卜 Thr-Se `` AI a- Pro-Asp- Thr-Arg- Y ²

(SEQ ID NO: 21)

Or

[Chemical 14-14]

X ¹ X ¹ X ¹ X ¹ X ¹

Y '-His-Gly-Val-Thr-Ser-Ala-Pro-Asp-Thr-Arg-Pro-Ala-Pro-Gly-Ser-Thr-Ala-Pro -Pro-Ala-Y ²

(SEQ ID NO: 41)

[Chemical 14-14]

Wherein R ¹ and R ⁶ are each independently hydrogen, Neu5 Ac group or GlcNAc group. Yes;

R ² and R ⁷ are Gal groups;

R ³ and R ⁸ are GlcNAc groups;

R ⁴ and R ⁹ are Gal groups;

R ⁵ is a GlcNAc group;

R ¹⁰ is a GalNAc group;

The bond between R ² and R ³ is a β ΐ, 4 bond;

The bond between R ³ and R ⁴ is a β ΐ, 3 bond;

The bond between R ⁴ and R ⁵ is a β ΐ, 4 bond;

The bond between R ⁷ and R ⁸ is a β ΐ, 4 bond;

The bond between R ⁸ and R ⁹ is a β ΐ, 3 bond;

The bond between R ⁹ and R ¹⁰ is a β ΐ, 3 bond;

The bond between R ⁵ and R ^1G is a β ΐ, 6 bond;

The bond between Ζ ¹ and R ³ is << ¹ , 3 bond;

The bond between Ζ ² and R ⁵ is << 1, 3 bond; and

The bond between Ζ ³ and R ⁸ is << 1, 3 bond,

Υ ¹ represents a hydrogen atom, acetyl, acyl, alkyl or aryl;

Υ ² represents a hydroxyl group, ΝΗ, alkyl or aryl.

2

A glycopeptide represented by

(Item 18)

A method for producing a desired glycopeptide comprising the following steps:

R ² and R ⁷ are Gal groups;

R ³ and R ⁸ are GlcNAc groups;

R ⁴ and R ⁹ are Gal groups;

R ⁵ is a GlcNAc group;

R ¹⁰ is a GalNAc group;

R ¹¹ is a hydrogen atom or a methyl group;

Here, the bond between R ¹ and R ² is a 2, 3 bond when R ¹ is Neu5Ac group, and j8 1, 3 bond when R ¹ is GlcNAc group;

The bond between R ² and R ³ is a β,, 4 bond;

The bond between R ³ and R ⁴ is a β,, 3 bond;

The bond between R ⁴ and R ⁵ is a β,, 4 bond;

The bond between R ⁶ and R ⁷ is an α 2,3 bond when R ⁶ is a Neu5Ac group, and a,, 3 bond when R ⁶ is a GlcNA c group; The bond between R ⁷ and R ⁸ is a β,, 4 bond;

Bond between R ⁸ and R ^9, β ΐ, is 3 bonds;

The bond between R ⁹ and R ^1G is a β,, 3 bond;

The bond between R ⁵ and R ^1G is a β,, 6 bond;

The bond between Ζ ¹ and R ³ is << ¹ , 3 bond;

The bond between Ζ ² and R ⁵ is << 1, 3 bond;

The bond between Ζ ³ and R ⁸ is << 1, 3 bond; and

Ρ is a 9-fluormethylcarboxyl group or a tert-butoxycarbol group,

A sugar amino acid having a sugar chain protected by protecting the sugar chain of the sugar amino acid represented by the following: a acetyl group, a benzoyl group, a methyl group, a methoxymethyl group, A step selected from the group consisting of trimethylsilyl group, t-butyldimethylsilyl group, dimethylphenol group and triisopropylpropyl group, benzyl group, benzylidene group, isopropylidene group, di-tert-butylsilylidene group power;

(B) Using the sugar amino acid in which the sugar chain obtained in step (A) is protected and an amino acid N-protected with a 9-fluorenylmethyloxyl group or a tert-butoxycarbol group, the desired peptide sequence is prepared. Synthesizing a glycopeptide having a protected sugar chain; and

(C) a step of deprotecting the glycopeptide protected in sugar chain obtained in step (B) to produce the desired glycopeptide;

Including the method.

The invention's effect

In the present invention, a sugar chain amino acid containing about 1 to 3 sugars, which is relatively easy to prepare, is used for synthesizing a mucin-type glycopeptide having a borilactosamine skeleton, and the sugar chain is elongated after the peptide synthesis. It is possible to synthesize a glycopeptide having a chain and to prepare a library of each sugar chain structure that is an intermediate of a sugar chain elongation reaction. Furthermore, since the sugar chain elongation reaction is carried out by carrying a glycopeptide on a water-soluble polymer, the acceleration effect of the reaction and the simplification of the molecular operation become possible, and the sugar chain elongation reaction can be automated. This It is possible to prepare a library of glycopeptides having a simple structure of a sugar chain, which is extremely difficult with conventional techniques, and having a complex sugar chain structure. For example, the present invention makes it possible to synthesize mucin-type glycopeptides that are useful in a wide range of fields such as biochemical research materials, pharmaceuticals, and foods, and that have been difficult to produce.

[0026] The present invention also protects a sugar amino acid having a pre-synthesized voralactosamine skeleton with a protecting group, and synthesizes a glycopeptide having a desired peptide sequence using the protected amino acid, thereby It enables the production of glycopeptides having The method is independent of proteases or photocleavage. As a result, it is possible to synthesize a glycopeptide containing an amino acid residue having the property of being easily cleaved by protease or photocleavage.

[0027] The obtained glycopeptide library can be used as a standard sample for structural analysis and biochemical tests. In addition, this glycopeptide library can be placed on a chip for comprehensive detection of glycopeptide recognition proteins, pathological diagnosis, cell adhesion sequence search, sequence analysis related to cell growth and apoptosis, etc. become.

[0028] These and other advantages of the present invention will be apparent to those of ordinary skill in the art upon reading and understanding the following detailed description with reference to the accompanying drawings.

Brief Description of Drawings

[0029] FIG. 1 shows the procedure up to the production of the glycopeptide of the present invention. The indicated numbers indicate the compound numbers in the examples.

Sequence listing free text

[0030] (Description of array)

SEQ ID NO: 1 to 20: Partial amino acid sequence of mucin type glycoprotein MUC1 10 residues SEQ ID NO: 21 to 40: Partial amino acid sequence of mucin type glycoprotein MUC1 SEQ ID NO: 41 to 60: Mucin type glycoprotein MUC 1 A partial amino acid sequence of 20 residues of SEQ ID NOs: 61 to 66: Examples of amino acid sequences contained in a carrier contained in a compound BEST MODE FOR CARRYING OUT THE INVENTION

[0031] The present invention will be described below with reference to the best mode. Throughout this specification, it should be understood that the singular forms also include the plural concept unless otherwise stated. It is. Thus, it should be understood that singular articles (eg, “a”, “an”, “the”, etc. in English) also include the plural concept unless otherwise stated. In addition, it should be understood that the terms used in the present specification are used in the meaning normally used in the above field 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.

[0032] It is understood that the embodiments provided below are provided for a better understanding of the present invention, and the scope of the present invention should not be limited to the following description. Therefore, it is obvious that those skilled in the art can make appropriate modifications within the scope of the present invention in consideration of the description in the present specification.

[0033] (Terminology)

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

In the present specification, the term “sugar amino acid” means a combination of a sugar residue and an amino acid residue, and is used interchangeably with “sugar amino acid residue”.

In the present specification, the term “sugar amino acid residue substantially not including a site cleavable by a protease” means that a compound represented by the above item (4) is treated with a protease. Even so, it refers to a sugar amino acid residue in which the sugar amino acid moiety is not cleaved by more than 50% by protease, preferably a sucrose amino acid residue not cleaved by more than 20%.

In the present specification, “glycopeptide residue” means a peptide residue containing at least one sugar amino acid, and is used interchangeably with “glycopeptide”.

[0037] The sugar residue constituting the sugar amino acid contained in the glycopeptide residue is not particularly limited, but monosaccharide to monosaccharide or monosaccharide to trisaccharide derivatives are preferred. Inductive materials are more preferably used.

[0038] As used herein, the term "sugar chain" refers to a compound comprising one or more unit sugars (monosaccharide and Z or a derivative thereof). When two or more unit sugars are connected, each unit sugar is linked by dehydration condensation using a glycosidic bond. Examples of such sugar chains include polysaccharides contained in the living body (glucose, galactose, mannose, fucose, etc. , Xylose, N-acetyl dalcosamine, N-acetyl galatatosamine, sialic acid and their complexes and derivatives), as well as degraded polysaccharides, glycoproteins, proteoglycans, glycosaminodarlicans, glycolipids Examples include, but are not limited to, sugar chains that are decomposed or derived from complex biomolecules. Therefore, in the present specification, the sugar chain can be used interchangeably with “polysaccharide”, “sugar”, and “carbohydrate”. Further, unless otherwise specified, in this specification, “sugar chain” may include both sugar chains and sugar chain-containing substances.

[0039] As used herein, "monosaccharide" refers to a polyhydroxy aldehyde or polyhydroxy ketone and a hydrolyzate thereof that is not hydrolyzed into a simpler molecule and contains at least one hydroxyl group and at least one aldehyde group or ketone group. Refers to a derivative. Usually, monosaccharides are represented by the general formula C H O, but are not limited to them: fucose (deoxyhexose), N n n 2n n

Also included are cetinoregenorecosamine. Here, the above formula [koo !, n = 2, 3, 4, 5, 6, 7, 8, 9 and 10 is converted into diose, triose, tetrose, pentose, hexose, heptose, respectively. , Otatos, nonose and decourse. It is generally equivalent to an aldehyde or ketone of a chain polyhydric alcohol. The former is called aldose and the latter is called ketose.

[0040] The nomenclature and abbreviations used to describe sugars in the present invention follow conventional nomenclature. For example, β D galactose

[0041] [Chemical 15]

f}--Galac ose

Is Gal;

N acetyleno β D darcosamine

[0042] [Chemical 16] M-Acetyl-pD-glucosam ine

GlcNAc;

: L course

[0043] [Chemical 17] α-L-Fucose

Fuc;

a—N acetylneuraminic acid

[Chemical 18]

α-Ν-Acetyl neuraminic acid

Is Neu5Ac;

N Acetinore a D Galactosamine [0045] [Chem. 18-1] N-Acety [-cr-D-galactosamine

Is represented by GalNAc. The two cyclic anomers are represented by α and j8. It may be expressed as a or b for display reasons. Therefore, in the present specification, α, a, and b are used interchangeably with respect to the anomeric notation.

[0046] In the present specification, galactose refers to any isomer, but is typically j8-D galactose, and is used to refer to j8-D-galactose unless otherwise specified.

As used herein, acetylyldarcosamine refers to any isomer, but is typically N-acetyleno β D darcosamine, and unless otherwise specified, ァ acetylthio β-D— Used to refer to darcosamine.

[0048] In the present specification, fucose refers to any isomer, but is typically a L-fucose, and is used to refer to α L-fucose unless otherwise specified.

In the present specification, sialic acid is a general term for sialic acid and derivatives of neuraminic acid. As sialic acid, N-acyl (N-acetyl or N-glycolyl) neuraminic acid and N-acyl-0-acetylethylneuraminic acid are naturally known. Although sialic acid is rarely present in the free state, it is mostly an acid-labile bond (α-ketoside bond) in a monosaccharide, polysaccharide, glycoprotein, glycopeptide, or glycosphingolipid molecule. Exists. The sialic acid used herein is preferably acetylethylneuraminic acid.

[0050] In the present specification, acetylylgalatatosamine refers to any isomer, but is typically Νacetylyl-a-D galactosamine, and unless otherwise specified, N-acetylyl-a-D galactosamine. Used as a pointer. [0051] In the present specification, it should be noted that sugar symbols, designations, abbreviations (Glc and the like) and the like are different when representing a monosaccharide and when used in a sugar chain. In the sugar chain, the unit sugar has a dehydration condensation with another unit sugar to which it is bonded, so that the mutual force also exists in a form excluding hydrogen or hydroxyl group. Therefore, when these abbreviations for sugars are used to represent monosaccharides, all hydroxyl groups are present, but when used in a sugar chain, the hydroxyl groups are bound to the hydroxyl groups of the sugars to which they are attached. It is understood that only oxygen remains after dehydration condensation. Thus, as used herein, a “sugar residue” for a sugar refers to those with 1 to n hydrogens, usually because sugars are linked to other chemical moieties via hydroxyl groups. A sugar residue is a hydroxyl group of a sugar that has a hydrogen power of ^ to n (in this case, n is the number of hydroxyl groups present in the sugar), but is not limited to this. If there is, it can indicate that the hydrogen has been removed. Therefore, the term “sugar residue” may be a monovalent, divalent, or trivalent to nvalent residue depending on the situation in which it is used. In the present specification, specific names are expressed in the form of “sugar name” + “group”, for example, galactose group, Gal group, and the like.

[0052] Monosaccharides are generally joined by glycosidic bonds to form disaccharides and polysaccharides. The direction of the bond with respect to the plane of the ring is indicated by α and j8. Also described are specific carbon atoms that form a bond between two carbons.

[0053] In the present specification, the sugar chain is

[0054] [Chemical 19]

Is represented by Thus, for example, a sugar chain in which C 6 of N-acetyldylcosamine and C-1 of N-acetyldylcosamine are linked by a 13 glycoside bond is represented by GlcNAc6-1 βGlcNAc.

[0055] Branches of sugar chains are represented by parentheses, and are arranged and placed immediately to the right of the unit sugar to be bound. For example,

[0056] [Chemical 20]

And in parentheses are

[0057] [Chemical 21] Monosaccharide

It is written. Thus, for example, C-6 of N-acetylyldarcosamine is bonded to C-1 of N-acetylyldarcosamine with a j8 glycoside, and C-3 of N-acetylyldarcosamine is further linked to C-1 and 13 glycosides of galactose. When bound, it is expressed as GlcNAc (3-1 β Gal) 6-1 ι8 GlcNAc. Monosaccharides are represented on the basis of numbering as small as possible (latent) carbonyl groups. According to the general standard of organic chemical nomenclature, even when an atomic group superior to a (latent) carbon group is introduced into a molecule, it is usually represented by the above numbering.

[0058] [Chemical 22]

[0059] [Chemical 23]

As specifically referred to herein, a “monosaccharide derivative” refers to a substance that results from the substitution of one or more hydroxyl groups on an unsubstituted monosaccharide with another substituent. That Examples of such monosaccharide derivatives include saccharides having a carboxyl group (for example, aldonic acids in which the C-1 position is oxidized to form carboxylic acids (for example, D-dalconic acid in which D-glucose is oxidized), terminal C Uronic acid (D-glucose oxidized D-glucuronic acid) with carboxylic acid atom, sugar having amino group or amino group derivative (eg acetylated amino group) (eg N-acetyl- D-darcosamine, N-acetyl-D-galatatosamine, etc., sugars that have both amino and carboxyl groups (eg, Neu5Ac (sialic acid), N-acetylethylmuramic acid, etc.), deoxylated sugars (eg, 2 -Deoxycy D-ribose), sulfate-containing sucrose containing sulfate groups, phosphate-containing sucrose containing phosphate groups, etc., but not limited to them. If say, also encompasses the derivatives. Alternatively, in the forming the Miasetaru structure sugars, glycosides also of Aseta Lumpur structure by reacting with alcohol, are within the scope of monosaccharide.

[0060] The “amino acid residue” constituting the glycopeptide residue of the present invention is not particularly limited as long as it has an amino group and a carboxy group in the molecule. The Gly (glycine) residue and the Ala (alanine) residue are not particularly limited. Group, Val (parin) residue, Leu (leucine) residue, lie (isoleucine) residue, Tyr (tyrosine) residue, Trp (tributophane) residue, Glu (glutamic acid) residue, Asp (aspartic acid) residue Group, Lys (lysine) residue, Arg (arginine) residue, His (histidine) residue, Cys (cystine) residue, Met (methionine) residue, Ser (serine) residue, Thr (threonine) residue And a-amino acid residues such as Asn (asnolagin) residue, Gin (glutamine) residue or Pro (proline) residue, or | 8-amino acid residue such as j8-Ala residue, etc. The The amino acid residue may be either D-form or L-form, but the L-form is preferred. As the glycopeptide residue, the above-mentioned amino acid residues or 2-30 glycopeptide residues are preferred. More preferred are glycopeptide residues having 4 to 20 forces. In this specification, in the structural formulas, Gly or the like may be omitted when it is clear that a residue is indicated. For example,

[0061] [Chemical 23-1] Craft

2 ZH

2

Q H- One H

2_iCH 0NHNHaHcCHc C-II-GHCNI CH CN-II-II-I H H H H

100 oo o o O o

○ 1 o— o

II II

o

T00 .S0 / Z, 00Zdf / X3d 38 LO £ ni / LOOZ OAV Is

[0062] [Chemical 23-2]

His— Gly—Va 卜 Thr—Ser— Ala-Pro— Asp— Thr—Arg—NH ₂

(SEQ ID NO: 2).

[0063] [Chemical 23-23]

Is

[0064] [Chemical 23-4]

Ala-His-Gly-Val -Thr-Ser-AI a- Pro-Asp-Thr-Arg- NH ₂

(SEQ ID NO: 21).

[0065] [Chemical 23-23]

\ „

Jp. S c? ¾c4 one *, ..

lOOLSO / LOOZdi / lDd L0 £ m / L00Z OAV Is

[0066] [Chemical 23-23]

His-Gly-Val-Thr-Ser-Ala-Pro-Asp-Thr-Arg-Pro-Ala-Pro-Gly-Ser-Thr- Ala-Pro -Pro-Ala-NH2

(SEQ ID NO: 41).

[0067] Amino acids have the general formula:

[0068] [Chemical 23-23]

H

H ₂ NC-C00H

R

(Wherein R represents a side chain).

[0069] Proline is:

[0070] [Chemical 23-23]

It is expressed.

[0071] When the amino acid is a peptide bond, the structure is as follows:

[0072] [Chemical 23-23]

H 0 R

H ₂ NCC- NC-C00H

R H H

(Wherein R represents a side chain). For example, if both R are H,

[0073] [Chemical 23-23]

H 0 H

H ₂ NCC- NC-C00H

Expressed as HHH. The above structure is also expressed as Gly—Gly. Here, “Gly-” or “-Gly” located at the end of the peptide chain represents a monovalent glycine residue. For example, the peptide chain When the constituent amino acid residue includes a proline residue and is expressed as Gly—Pro, the structure is as follows:

[0074] [Chemical 23-23]

H 0 H

H ₂ NCCNC-COOH

I / \

H CH ₂ CH ₂

ノ

Expressed as CH ₂ .

[0075] When referring to a peptide in which three amino acids are bound, the following:

[0076] [Chemical 23-23]

(Wherein R ^{A to} R ^e represent side chains). For example, R ^A is H, R ^B force SCH

Three

, ^Re is CH (CH) CH, the peptide chain is also represented as Gly—Ala—Val. here

3 3

“Gly—” represents a monovalent glycine residue, “—Ala—” represents a divalent alanine residue, and “—Val” represents a monovalent palin residue.

[0077] When the peptide chain includes a serine residue or threonine residue as an amino acid residue, when the serine residue or threonine residue is located at the end of the peptide chain, the serine residue is "Ser-" or " It is expressed as “Ser”, and the threonine residue is expressed as “Thr-” or “-Thr” and is a monovalent amino acid residue. Sugar chains may be bound to these amino acid residues. When sugar chains are attached, these residues are divalent, and serine and threonine residues are as follows:

[0078] [Chemical 23-23]

¾er— Also expressed as Ser; Thr- or One Thr.

[0079] When a serine residue or a threonine residue is present at a position other than the end of the peptide chain, These residues are divalent and are represented by “-Ser-” and “-Thr-”, respectively. Sugar chains may be bound to these amino acid residues. When sugar chains are attached, these residues are trivalent, and serine and threonine residues are as follows:

[0080] [Chemical 23-23]

— Ser— ； One Thr—

It is expressed.

[0081] The sugar amino acid of the present invention defined above is not particularly limited as long as the amino acid residues and sugar residues listed above can theoretically be combined, but preferably, the combination is not limited. ,

Ser-1 a GalNAc (3 ~ 1 β Gal) 6— 1 β GlcNAc4— 1 β Gal [3— 1 β GlcNAc4 -1β Gal] n

• Ser- 1 a GalNAc (3 — 1 j8 Gal) 6— 1 β GlcNAc4— 1 β Gal [3— 1 β GlcNAc4 —113 Gal] n3 ~ 1β GlcNAc

Ser-1 a GalNAc (3 ~ 1β Gal) 6— 1 β GlcNAc4— 1 β Gal [3— 1 β GlcNAc4

Ser-la GalNAc (3—1 j8 Gal [3 ~ 1β GlcNAc4 ~ 1β Gal] m) 6 ~ 1β GlcNAc 4-1 β Gal [3 -1β GlcNAc4 ~ 1β Gal] n

Ser-la GalNAc (3—1 j8 Gal [3 ~ 1β GlcNAc4 ~ 1β Gal] m3 ~ 1β GlcNAc)

6-1 β GlcNAc4 ~ 1β Gal [3 ~ 1β GlcNAc4 ~ 1β Gal] n

Ser-la GalNAc (3—1 j8 Gal [3 ~ 1β GlcNAc4 ~ 1β Gal] m3 -2a Neu5Ac

) 6-1 β GlcNAc4 ~ 1β Gal [3 ~ 1β GlcNAc4 ~ 1β Gal] n

Ser-la GalNAc (3 — 1 j8 Gal [3 ~ 1β GlcNAc4 ~ 1β Gal] m) 6 ~ 1β GlcNAc

4-1 β Gal [3 -1β GlcNAc4 ~ 1β Gal] n3 ~ 1β GlcNAc

Ser-la GalNAc (3—1 j8 Gal [3 ~ 1β GlcNAc4 ~ 1β Gal] m3 ~ 1β GlcNAc)

6-1 β GlcNAc4 ~ 1β Gal [3 ~ 1β GlcNAc4 ~ 1β Gal] n3 ~ 1β GlcNAc

Ser-la GalNAc (3—1β Gal [3-1—GlcNAc4—1β Gal] m3-2α Neu5Ac β β β () ua Ser 1 GalNAc31 Gal3 GlcNAc Galm 61 GlcNAc • 111

GlcNAc

β β β β) () ua 61 GlcNAc31 Fuc 41 Gal3 GlcNAc Galn3111

β β (u aα Ser 1 GalNAc31 Gal3 GlcNAc Galm32 Neu5Ac • 111

GlcNAc

β β β β β () uα 61 GlcNAc31 Fuc 41 Gal3 GlcNAc Galn3111

β β β () u a Ser 1 GalNAc31 Gal3 GlcNAc Galm3 GlcNAc • 11

β β β β () ua31 Fuc 41 Gal3 GlcNAc Galn3 GlcNAc11

β β () u a Ser 1 GalNAc31 Gal3 GlcNAc Galm 61 GlcNAc • 111

β β β) () Ua 61 GlcNAc31 Fuc 41 Gal3 GlcNAc Gan 111

β β (u aα Ser 1 GalNAc31 Gal3 GlcNAc Galm32 Neu5Ac • 111

β β β β () Uα 61 GlcNAc31 Fuc 41 Gal3 GlcNAc Gan111

β β β () u a Ser 1 GalNAc31 Gal3 GlcNAc Galm3 GlcNAc • 11

β β β () Ua31 Fuc 41 Gal3 GlcNAc Gan11

β β () u a Ser 1 GalNAc31 Gal3 GlcNAc Galm 61 GlcNAc • 111

β βuα 1 GlcNAc Galn32 Neu5Ac 1

β () () s aa Ser 1 GalNAc31 Gal 61 GlcNAc31 Fuc 41 Ga • 11111 I

β β βu 1 GlcNAc 1 Galn31 GlcNAc 1l

β () () s aa Ser 1 GalNAc31 Gal 61 GlcNAc31 Fuc 41 Ga • 11111 I

! lUss l GlcNAcl Gan I

β () () s aa Ser 1 GalNAc31 Gal 61 GlcNAc31 Fuc 41 Ga • 11111 I

β β β) uα 61 GlcNAc Gal3 GlcNAc Galn32 Neu5Ac 11

β β (u αα Ser 1 GalNAc31 Gal3 GlcNAc Galm32 Neu5Ac • 111

β β β βuα 61 GlcNAc Gal3 GlcNAc Galn32 Neu5Ac11

β β β () u a Ser 1 GalNAc31 Gal3 GlcNAc Galm3 GlcNAc • 11

β βuα 1 Gal3 GlcNAc Galn32 Neu5Ac 11

β β () ua Ser 1 GalNAc31 Gal3 GlcNAc Galm 61 GlcNAc • 111 β β β β) u 61 GlcNAc Gal3 GlcNAc Galn3 GlcNAc 1 () uaa cNAc31 Fuc 1 Galn31 GlcNAc31 Fuc 1111

β β β β) (M aa G1CNAC31 Fuc 61 GlcNAc31 FUC 1 Gal31 Gl111Il

β β (() u aa Ser 1 GalNAc31 Gal3 GlcNAc31 Fuc 41 Galm3 • 1111

βU a an3 GlcNAc 31 Fuc 1

β β β β (M (Maa GlcNAc31 FUC 1 Gal31 GlcNAc31 FUC 1 G1Il1I

β β (()) u aa Ser 1 GalNAc31 Gal3 GlcNAc31 Fuc 41 Galm 6 • 1111! β β) () (asa2 Neu5 Ac 61 GlcNAc31 Fuc 41 Gal3 GlcNAc3 11111

β β (() u aa Ser 1 GalNAc31 Gal3 GlcNAc31 Fuc 41 Galm3 • 1111

β β () Ua 1 GlcNAc31 Fuc 41 Gan11

β () () s aa Ser 1 GalNAc31 Gal 61 GlcNAc31 Fuc 41 Ga • 11111 I β β β) () uaα 61 GlcNAc31 Fuc 41 Gal3 GlcNAc Galn321111

β β (u aα Ser 1 GalNAc31 Gal3 GlcNAc Galm32 Neu5Ac • 111 β β β β () uαα 61 GlcNAc31 Fuc 41 Gal3 GlcNAc Galn321111

β β β () ua Ser 1 GalNAc31 Gal3 GlcNAc Galm3 G1CNAC • 11 β β β () uαα31 Fuc 41 Gal3 GlcNAc Galn32 Neu5 Ac111 β β β β) (u 61 GlcNAcI Gal3 GlcNAc Galn3 GlcNAc 1 I

β β β ((u aThr 1 GalNAc31 Gal31 GlcNAc 1 Galm31 GlcNAc l1l 1l

β β β [u c 1 Gal3 GlcNAc Galn3 GlcNAc 1

β β β () (u aThr 1 GalNAc31 Gal31 GlcNAc 1 Galm 61 G1CNA

β β β) (U 61 GlcNAc Gal3 GlcNAc Gan 1

β β ([u ssaThr 1 GalNAc31 Gal3 GlcNAc 1 Galm32 Neu Ac • l 111

β β β) (U 61 GlcNAc Gal3 GlcNAc Gan 1

β β β ((u aThr 1 GalNAc31 Gal31 GlcNAc 1 Galm31 GlcNAc l1l 1l

β β [U c 1 Gal31 GlcNAc 1 Gan 1l 1

β β β () (u aThr 1 GalNAc31 Gal31 GlcNAc 1 Galm 61 G1CNA • l1l 11 β β β () (aThr 1 GalNAc31 Gal 61 GlcNAc 1 Gal31 GlcNAc

β βu Galn3 GlcNAc

β β β () (aThr 1 GalNAc31 Gal 61 GlcNAc 1 Gal31 GlcNAc • l111l β β β () (aThr 1 GalNAc31 Gal 61 GlcNAc 1 Gal31 GlcNAc • l111l

β) u aa 1 Fuc 41 Galn32 Neu5Ac11

β β) () ((asa2 Neu5 Ac 61 GlcNAc31 Fuc 41 Gal3 GlcNAc3 11111

β β (() (u aa Ser 1 GalNAc31 Gal3 GlcNAc31 Fuc 41 Galm3 • 1111

I () u aS a cNAc31 FucIGaln32 Neu Ac1 Il

β β β β) (M (aa G1CNAC31 Fuc 61 GlcNAc31 FUC 1 Gal31 Gl111Il

β β (() (u aa Ser 1 GalNAc31 Gal3 GlcNAc31 Fuc 41 Galm3 • 1111

ua aln32 Neu Ac 1

β β β β (M (M (aa GlcNAc31 FUC 1 Gal31 GlcNAc31 FUC 1 G1Il1I

β β (()) (u aa Ser 1 GalNAc31 Gal3 GlcNAc31 Fuc 41 Galm 6 • 1111

) u a a 1 Fuc 1 Galn31 GlcNAc31 Fuc 111

β β) () ((asa2 Neu5 Ac 61 GlcNAc31 Fuc 41 Gal3 GlcNAc3 11111 β β (() (u aa Ser 1 GalNAc31 Gal3 GlcNAc31 Fuc 41 Galm3 • 1111 GlcNAc

β β β β) () (ua 61 GlcNAc31 Fuc 41 Gal3 GlcNAc Galn3111

β β ([u ssaThr 1 GalNAc31 Gal3 GlcNAc 1 Galm32 Neu Ac • l 111

GlcNAc

β β β β) () (ua 61 GlcNAc31 Fuc 41 Gal3 GlcNAc Galn3111

β β β ((u aThr 1 GalNAc31 Gal31 GlcNAc 1 Galm31 GlcNAc l1l 1l

β β β β () (u a c31 Fuc 41 Gal3 GlcNAc Galn3 GlcNAc 11

β β β () (u aThr 1 GalNAc31 Gal31 GlcNAc 1 Galm 61 G1CNA

β β β) () (Ua 61 GlcNAc31 Fuc 41 Gal3 GlcNAc Gan 111

β β ([u ssaThr 1 GalNAc31 Gal3 GlcNAc 1 Galm32 Neu Ac • l 111

β β β) () (Ua 61 GlcNAc31 Fuc 41 Gal3 GlcNAc Gan 111

β β β ((u aThr 1 GalNAc31 Gal31 GlcNAc 1 Galm31 GlcNAc l1l 1l

β β β () (U a c31 Fuc 41 Gal3 GlcNAc Gan 11

β β β () (u aThr 1 GalNAc31 Gal31 GlcNAc 1 Galm 61 G1CNA

β βuα 1 GlcNAc Galn32 Neu5Ac 1

β () (M [aThr 1 GalNAc31 Gal 61 GlcNAc31 FUC 1 Gal3 • 1111I 1

β β βu 1 GlcNAc 1 Galn31 GlcNAc 1l

β () (M [aThr 1 GalNAc31 Gal 61 GlcNAc31 FUC 1 Gal3 • 1111I 1

! lUss l GlcNAcl Gan I

β () (M [aThr 1 GalNAc31 Gal 61 GlcNAc31 FUC 1 Gal3 • 1111I 1

β β β) (uα 61 GlcNAc Gal3 GlcNAc Galn32 Neu5Ac 11

β β ([u ssaThr 1 GalNAc31 Gal3 GlcNAc 1 Galm32 Neu Ac • l 111

β β β) (uα 61 GlcNAc Gal3 GlcNAc Galn32 Neu5Ac 11

β β β ((u aThr 1 GalNAc31 Gal31 GlcNAc 1 Galm31 GlcNAc l1l 1l

β β [uα c 1 Gal3 GlcNAc Galn32 Neu5Ac 11

β β β () (u aThr 1 GalNAc31 Gal31 GlcNAc 1 Galm 61 G1CNA

β β β β) (u 61 GlcNAc Gal3 GlcNAc Galn3 GlcNAc 1! β β ((u ssaThr 1 GalNAc31 Gal3 GlcNAc 1 Galm32 Neu Ac • 1111 β β β β) (M sa G1CNAC31 Fuc 61 GlcNAc31 FUC 1 Gal31 Gl111Il

β β ((Mu aThr 1 GalNAc31 Gal3 GlcNAc31 FUC 1 Galm3 • l 11I

βU a an3 GlcNAc 31 Fuc 1

β β β β (M (Maa GlcNAc31 FUC 1 Gal31 GlcNAc31 FUC 1 G1Il1I

β β ((M) u aThr 1 GalNAc31 Gal3 GlcNAc31 FUC 1 Galm 6 • l 11I! β β) () (asa2 Neu5 Ac 61 GlcNAc31 Fuc 41 Gal3 GlcNAc3 11111

β β ((Mu aThr 1 GalNAc31 Gal3 GlcNAc31 FUC 1 Galm3 • l 11I

β () (MaThr 1 GalNAc31 Gal 61 GlcNAc31 FUC 1 Gal3 • l 111I 1 β β β) () uaα 61 GlcNAc31 Fuc 41 Gal3 GlcNAc Galn321111

β β (u ssaThr 1 GalNAc31 Gal3 GlcNAc 1 Galm32 Neu Ac • l 111 β β β) () uaα 61 GlcNAc31 Fuc 41 Gal3 GlcNAc Galn321111

β β β (u aThr 1 GalNAc31 Gal31 GlcNAc 1 Galm31 GlcNAc l1l 1l

β β β () u aα c31 Fuc 41 Gal3 GlcNAc Galn32 Neu5 Ac 111 β β β () u aThr 1 GalNAc31 Gal31 GlcNAc 1 Galm 61 G1CNA • l1l 11 cNAc (3— 1 a Fuc) 4— 1 j8 Gal] n3— 1 j8 GlcNAc3— 1 a Fuc

Thr-ΐα GalNAc (3— 1 j8 Gal [3 ~ 1β GlcNAc (3— 1 a Fuc) 4— 1 β Gal] m3 -2a Neu5Ac) 6— 1 j8 GlcNAc (3— 1 a Fuc) 4— 1 j8 Gal [3 ~ 1β GlcNAc (3 — 1 a Fuc) 4— 1 j8 Gal] n3 — 1 j8 GlcNAc3— 1 a Fuc

Thr-la GalNAc (3— 1 j8 Gal [3 ~ 1β GlcNAc (3 — 1 a Fuc) 4— 1 β Gal] m) 6 —113 GlcNAc (3-la Fuc) 4-1 β Gal [3— 1 β GlcNAc (3-1 a Fuc) 4-1 β G al] n3— 2a Neu5Ac

Thr-la GalNAc (3— 1 j8 Gal [3 ~ 1β GlcNAc (3-la Fuc) 4— 1 β Gal] m3 —113 GlcNAc3— 1 a Fuc) 6— 1 β GlcNAc (3-la Fuc) 4- 1 β Gal [3— 1 β Gl cNAc (3— 1 a Fuc) 4— 1 j8 Gal] n3— 2 a Neu5Ac

Thr-la GalNAc (3— 1 j8 Gal [3 to 1β GlcNAc (3-la Fuc) 4— 1 β Gal] m3 -2a Neu5Ac) 6— 1 j8 GlcNAc (3 — 1 a Fuc) 4 to 1β Gal [ 3 ~ 1β GlcNAc (3 — 1 a Fuc) 4-1 β Gal] n3 -2a Neu5Ac

Is mentioned. Here, n represents an integer of 1 to 10, Gal represents galactose, Glc represents gnoleose, Man represents mannose, Xyl represents xylose, GlcNAc represents N-acetyl D darcosamine, GalNAc represents N-acetyl D represents galactosamine.

In the present specification, the term “N-terminal” means an amino group that may be substituted at the end of the peptide main chain.

[0083] As used herein, the term "C-terminus" refers to a substitution located at the end of the peptide backbone! And, it means a carboxyl group.

In the present specification, the “side chain” means a functional group extending from the peptide main chain in a direction orthogonal to the direction in which the peptide main chain extends or a portion containing the functional group.

In the present specification, the “primer” means a substance having an action that triggers the initiation of a reaction in an enzyme reaction.

In the present specification, the term “transferase” is a general term for enzymes that catalyze a group transfer reaction. In the present specification, “transferase” may be used interchangeably with “transferase”. The group transfer reaction is represented by the following formula (1): X-Y + ZH XH + ZY (1)

As shown, the group Y is transferred from one compound (donor) to another compound (acceptor).

As used herein, “glycosyltransferase” refers to a sugar (corresponding to group Y in formula (1) above; unit sugar or sugar chain) separated from a certain place (corresponding to compound X—Y in formula (1) above). The enzyme has an action of catalyzing the transfer to the site (corresponding to the compound Z—H of the above formula (1)). Examples of the glycosyltransferase include galactose transferase, glucose transferase, sialic acid transferase, mannose transferase, fucose transferase, xylose transferase, N-acetylylcosamine transferase, and N-acetyl galatatosamine transferase. However, it is not limited to them. Some glycosyltransferases are specific for a particular binding mode. For example, j81,3-N-acetylylcosamine transferase is a glycosyltransferase that binds the 3rd position of a sugar such as galactose and the 1st position of N-acetylethyldarcosamine. The glycosyltransferase used in the present invention is only required to be able to use sugar nucleotides as a sugar donor. For example, β ΐ, 4 galactose transferase, α-1,3 galactose transferase, β 1,4 galactose Transferase, β 1,3 galactose transferase, β 1,6-galatose transferase, α 2,6 sialyltransferase, << 1, 4 galactose transferase, ceramid galatose transferase, αΐ, 2 fucose transferase, 1,3 fucose transferase, αΐ, 4-fucose transferase, αΐ, 6 fucose transferase, α 1, 3—Ν acetylceta ratatosamine transferase, αΐ, 6—Ν acetyl galatatosamine transferase, β ΐ, 4 -Ν- Acetylgalatatosamine transferase, polypeptide ァ Acetylgalatatosamine transferase, β 1, 4—Ν Acetyl darcosamine transferase, 131, 2—Ν Acetyl darcosamine transferase Containing, β ΐ, ₃ -Ν § cetyl Darco Sa Min transferase, β ΐ, 6-Ν- Asechirudaru Kosamin transferase, α 1, _4-Ν § Cetyl Darco Sa Min transferase, β ΐ, 4 mannose transferase, Ai , 2 Mannose transferase, << 1, 3 Mannose transferase, << 1, 4 Mannose transferase, << 1, 6 Mannose transferase, << 1, 2 Glucosetransferase, αΐ, 3 Glucosetransferase, α2, 3 Sial Acid transferase, α 2,8 sialyl transferase, αΐ, 6-darcosamine transferase, << 1, 6 xylose transferase, β xylose transferase (proteodalycan core structure synthase), j81, 3-glucuron Acid transfer fermentation Saccharide, hyaluronic acid synthase, glycosyltransferase using other sugar nucleotides as sugar donors, and glycosyltransferase using dolcor phosphate type sugar donors.

In the present invention, “sugar chain elongation reaction” refers to a reaction in which the chain length of a sugar chain is elongated in the presence of a glycosyltransferase as defined above.

[0089] As used herein, the term "biomolecule" refers to a molecule related to a living body.

Samples containing such biomolecules are sometimes referred to herein as biological samples. As used herein, “living body” refers to a biological organism, including but not limited to animals, plants, fungi, viruses, and the like. Therefore, a biomolecule includes a molecule extracted from a living body, but is not limited thereto, and any molecule that can affect a living body falls within the definition of a biomolecule. Such biomolecules include proteins, polypeptides, oligopeptides, peptides, glycopeptides, polynucleotides, oligonucleotides, nucleotides, sugar nucleotides, nucleic acids (eg, DNA such as cDNA, genomic DNA, mRNA Such as RNA), polysaccharides, oligosaccharides, lipids, small molecules (eg, hormones, ligands, signaling substances, small organic molecules, etc.), complex molecules thereof, and the like. In the present specification, the biomolecule may preferably be a sugar chain or a complex molecule containing a sugar chain (eg, glycoprotein, glycolipid, etc.).

[0090] The source of such biomolecules may be any animal, plant, bacteria, or virus, as long as it is a material to which a biological sugar chain is bound or attached. More preferably, an animal-derived biological sample is used. Preferable examples include whole blood, plasma, serum, sweat, saliva, urine, knee fluid, amniotic fluid, and cerebrospinal fluid, and more preferable examples include plasma, serum, and urine. Biological samples include biological samples that have not been previously separated from individuals. For example, it includes mucosal tissue that can be contacted with a test solution from the outside, or glandular tissue, preferably ductal epithelium attached to breast, prostate, and spleen.

[0091] 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. . The polymer may be linear or branched or cyclic. The amino acid may be a modified amino acid, which may be natural or non-natural. The term also includes assembly into a complex of multiple polypeptide chains. May be included. The term also encompasses natural or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, daricosylation, 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 non-natural amino acids, etc.), peptidomimetic compounds (eg, peptoids), and the art! Other modifications are included

In the present specification, the term “sugar nucleotide” means a nucleotide to which a sugar residue as defined above is bound, and the sugar nucleotide used in the present invention can be used by the above enzyme. If it is, it will not specifically limit. For example, uridine 5, monodiphosphate galactose, uridine 5, monodiphosphate-N acetyl dalcosamine, uridine 5, mono diphosphate N acetyl galactosamine, uridine-5, monodiphosphate glucuronic acid, uridine 5, 1 Examples include diphosphate xylose, guanosine 5, monodiphosphate fucose, guanosine 5, monodiphosphate mannose, cytidine-5, monomonophosphate 1N acetylneuraminic acid, and sodium salts thereof.

[0093] (Organic Chemistry)

The organic chemistry is described in, for example, Organic Chemistry, R. T. Morrison, R. N. Boyd 5th ed. (1987), and these are incorporated by reference in the relevant part in this specification.

In this specification, unless otherwise specified, “substitution” refers to replacement of one or more hydrogen atoms in an organic compound or substituent with another atom or atomic group. One hydrogen atom can be removed and substituted with a monovalent substituent, and two hydrogen atoms can be removed and substituted with a divalent substituent.

As used herein, “alkyl” refers to a monovalent group formed by loss of one hydrogen atom in an aliphatic hydrocarbon (alkane) force such as methane, ethane, or propane. CH

n 2n + l is represented by one (where n is a positive integer). Alkyl can be linear or branched. As used herein, “substituted alkyl” refers to an alkyl in which one or more hydrogen atoms are each independently substituted with a substituent as defined below. Examples of these are: C1-C2 alkyl, C1-C3 alkyl, C1-C4 alkyl, C1-C5 alkyl, C1-C6 alkyl, C1-C7 alkyl, C1-C8 alkyl, C1-C9 alkyl, C1-C10 alkyl, C1-C11 alkyl C1-C12 alkyl, C1-C15 alkyl, Cl-C20 alkyl, C1-C25 alkyl or C1-C30 alkyl. Here, for example, C 1 -C 10 alkyl means a linear or branched alkyl having 1 to 10 carbon atoms, such as methyl (CH—), ethyl (CH 1), n-propyl. (CH CH CH one)

3 2 5 3 2 2

, Isopropyl ((CH) CH—), n-butyl (CH CH CH CH —), n-pentyl (

3 2 3 2 2 2

CH CH CH CH CH 1), n-hexyl (CH CH CH CH CH CH 1), n-

3 2 2 2 2 3 2 2 2 2 2 butyl (CH CH CH CH CH CH CH CH—), n-octyl (CH CH CH CH CH

3 2 2 2 2 2 2 3 2 2 2

CH CH CH ―), n-nonyl (CH CH CH CH CH CH CH CH CH ―), n-

2 2 2 2 3 2 2 2 2 2 2 2 2 Decyl (CH CH CH CH CH CH CH CH CH CH —), C (CH) CH CH

3 2 2 2 2 2 2 2 2 2 3 2 2 2

CH (CH), one CH CH (CH), etc. are exemplified.

3 2 2 3 2

[0096] As used herein, "aryl" refers to a monovalent aromatic of 6 to 30 carbon atoms derived by removing one hydrogen atom from one carbon atom of the parent aromatic ring system. This refers to the hydrocarbon radical. Representative aryl groups include, but are not limited to, benzene, naphthalene, anthracene, biphenyl and the like.

As used herein, the term “chromophore” refers to a functional group having an absorption band in the ultraviolet light or visible light region, or emitted light in the visible light region excited by electromagnetic waves in the ultraviolet light or visible light region. A functional group that emits. For example, nitro group, benzyl group, thiophenol group, paranitrophenol group, 2,4 di-trifluoro group, dansyl group, 2-aminobenzyl group, fluorescein isothiocyanate (FITC) group, 4-methoxy group Examples thereof include, but are not limited to, β-naphthylamide group.

In the present specification, “keto acid” is a general term for compounds having a carboxyl group and a carbocycle group of a ketone.

In the present specification, “aldehyde acid” is a general term for compounds having a carboxyl group and a carboxylic group of an aldehyde.

[0100] Such keto acids or aldehyde acids are, for example, X—C (= 0)-(CH) —A—CO

2 n 1

OH (III) (wherein X represents a hydrogen atom, C to C alkyl, C to C aryl or chromophore)

1 30 6 30 N represents an integer of 0 to 20; A represents a linker having a length of 1 to 20 methylene chains).

[0101] As used herein, the term "protection reaction" refers to a reaction in which a protective group such as Boc (tert-butoxycarbonyl group) is added to a functional group desired to be protected. By protecting the functional group with a protecting group, the reaction of the functional group with higher reactivity can be suppressed, and only the functional group with lower reactivity can be reacted.

[0102] As used herein, "deprotection reaction" refers to a reaction for removing a protecting group such as Boc. Examples of deprotection reactions include reactions with trifluoroacetic acid (TFA) and reduction reactions with PdZC.

In the present specification, examples of the “protecting group” include, for example, a fluorenylmethoxycarbol (F moc) group, a acetyl group, a benzyl group, a benzoyl group, a tert-butoxycarbol group, t-Butyldimethyl group, silyl group, trimethylsilylethyl group, N-phthalimidyl group, trimethylsilylethyloxycarbonyl group, 2-trow 4,5 dimethoxybenzyl group, 2-trow 4,5-dimethoxybenzyloxy Carbon group, strong rubamate group, methyl group, methoxymethyl group, trimethylsilyl group, t-butyldimethylsilyl group, dimethylphenyl group, triisopropylpropylsilyl group, benzylidene group, isopropylidene group, ditert-butylsilylidene group, etc. Are listed as representative protecting groups. The protecting group can be used, for example, to protect a reactive functional group such as an amino group or a force lpoxyl group. Depending on the reaction conditions and purpose, various protecting groups can be used properly. As the protecting group for the aminooxy group and the N-alkylaminooxy group, a trimethylsilylethyloxycarboxyl group, a 2-trow 4,5 dimethoxybenzyloxycarboxyl group or a derivative thereof is preferable.

[0104] In each method of the present invention, the target product is a contaminant (unreacted weight loss, by-product, solvent, etc.) from the reaction solution, and a method commonly used in the art (for example, extraction, distillation, After removal by washing, concentration, precipitation, filtration, drying, etc., followed by treatment by a combination of post-treatment methods commonly used in the art (eg adsorption, elution, distillation, precipitation, precipitation, chromatography, etc.) obtain.

[0105] (General technique used in this specification) The techniques used herein are organic chemistry, biochemistry, genetic engineering, molecular biology, microbiology, genetics and within the skill of the art, unless specifically indicated otherwise. Well-known and conventional techniques in the relevant fields are used. Such techniques can be found, for example, in the documents listed below and in references cited elsewhere in this specification! Explained enough! Speak.

[0106] The organic chemical methods used in this specification are well known and commonly used in the art, and are described in, for example, Organic Chemistry, RT Morrison, RN Boyd 5th ed. (1987). Which are incorporated herein by reference in their entirety (which may be all).

[0107] Molecular biological techniques, biochemical techniques, and microbiological techniques used in this specification are well known and commonly used in the art. For example, Maniatis, T. et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, FM, et al. Eds, Current Protocols in Molecular Biology, John Wiley & Sons Inc., NY, 1015 8 ( 2000); Innis, MA (1990) .PCR Protocols: A Guide to Methods and Applications, Academic Press; Innis, MA et al. (1995) .PCR Str ategies, Academic Press; Sninsky, JJ et al. (1999) PCR Applications s: Protocols for Functional Genomics, Academic Press; Gait, MJ (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; G ait, MJ (1990). Oligonucleotide Synthesis: A Practical Approach, IR L Press; Eckstein , F. (1991). Oligonucleotides and Analogues: A Prac tical Approac, IRL Press; Adams, RL et al. (1992). The Biochemis try of the Nuclei c Acids, Chapman &Hall; Shabarova, Z. et al. (19 94). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blac kburn, GM et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, GT (1996). Bioconjugate Technics, Academic Press; Method in Enzymology 230, 242, 247, Ac ademic Press, 1994; 997; Hatanaka, Nishimura et al., Glycoscience and Engineering, Kodansha Scientific, 1997; Design and Physiology of Glycomolecules, The Chemical Society of Japan, Society Publishing Center, 2001, etc. Relevant parts in the description (may be all) are incorporated by reference

(Description of Preferred Embodiment)

Hereinafter, preferred embodiments of the present invention will be described. The embodiments provided below are provided for a better understanding of the present invention, and it is understood that the scope of the present invention should not be limited to the following description. Therefore, it is obvious that those skilled in the art can make modifications within the scope of the present invention with reference to the description of the present specification.

[0109] In one aspect, the present invention provides the following formula:

X— C (= 0) — (CH) — A— A— A (I)

2 n 1 2 3

(Wherein X represents a hydrogen atom, C-C alkyl, C-C aryl or chromophore; n

1 30 6 30

Represents an integer from 0 to 20; A is — (CH) — C (= 0) —,-(CH CH O) —,

1 2 0 ~ 20 2 2 1 ~ 10 Degree of polymerization 1 ~: LO oligo or polyacrylamide, degree of polymerization 1 ~: LO oligo or polypeptide, oxygen atom or NH; A is cleavable by protease Amino acid residue

2

A represents a sugar amino acid substantially free of a site cleavable by a protease.

Three

And a glycopeptide residue containing any sugar amino acid without a site cleavable by a protease). By using this as a primer, purification of a glycopeptide, which conventionally required multi-step purification, can be simplified, and the glycopeptide can be produced quickly and with high yield. Since the compound of the above formula (I) of the present invention always has an aldehyde group or a ketone group at the terminal, it may be protected with an aminooxy group, an N alkylaminooxy group, a hydrazide group, an azide group, a thiosemicarbazide group. , 1,2-dithiol group and a carrier containing a functional group selected from the group consisting of cysteine residues, by reacting with a carrier containing a functional group selected from the group consisting of the above-mentioned formula (I) and using it as a polymer primer can do. The bond obtained by this reaction is a strong bond that does not decompose under the subsequent hydrolysis with a protease (pH condition, etc.), so that there is an advantage that the purification of hydrolysis is very simple.

[0110] The protease used in the hydrolysis in the present invention and the protease The combination with the cleavable amino acid residue (A ² ) is a bond produced by the reaction of at least the terminal aldehyde or ketone group of the compound of formula (I) with the carrier in a pH range where hydrolysis by a protease can occur. Any combination is acceptable as long as it does not decompose. Amino acid residues force of some or all and A ² of the polypeptide of A ¹ also Narupepu tide recognizing protease may also be used. Examples of such combinations include a combination of a protease (glutamidase) derived from Bacillus Licheniformis and a glutamic acid residue or cysteine residue that can be cleaved by this protease; Combination of peptidase and Asn (recognition site (A ² )) (cleaves the C-terminus of asparagine (Asn)); Combination of arginyl endopeptidase and Arg (recognition site (A ² )) (arginine ( Cleaves C-terminal of Arg)); Combination of Achromopacter protease I and lysine (Lys) (recognition site (A ² )) (cleaves C-terminal of lysine (Lys)); , Arginine (Arg) or lysine (Lys) (recognition site (A ² )) combination (when Arg is recognized, C-terminal of arginine (Arg) is cleaved and lysine (Lys) is recognized) Cleave the C-terminus of (Lys)); a combination of chymotrypsin and Phe, Tyr or Trp (recognition site (A ² )) (if Phe is recognized, cleave the C-terminus of ferulanine (Phe) When Tyr is recognized, the C-terminal end of tyrosine (Tyr) is cleaved. When Trp is recognized, the C-terminal end of tributophan (Trp) is cleaved.); V8 protease and Glu (recognition site (A ² )) (Cleaves the C-terminus of glutamic acid (Glu)); Factor Xa (factor Xa) and —lie—Glu—Gly—Arg— (recognition site, according to the definition in this document, the recognition site ( A ² ) is arginine (Arg) and —lie—Glu—G1 y— is the end of A ¹ ; this cleaves the C terminus of arginine (Arg).) Enterokinase and Asp— Asp— Asp— Asp— Lys— (recognition site, according to the definition of this specification, the recognition site (A ² ) is lysine (Ly a s), -Asp- Asp- A sp- Asp- is the terminus of A ^1;. This cleaves the C-terminal lysine (Lys)) ₀ as such a combination, Bacillus RIQUET - Horumisu ( Cleaved by this protease with a protease derived from Bacillus Licheniformis (glutamidase (eg, protease specific to glutamic acid residues derived from Bacillus Lichenif ormis) (BLase: manufactured by Shionogi & Co., Ltd.)) Possible combinations with glutamic acid residues or cysteine residues Combination is preferred. BLase can be produced by the method described in JP-A-4-166085 (Patent No. 3046344). BLase is produced by Bacillus spp., In particular by Bacillus lichen niformis ATCC 14580. This strain can be obtained from the American Type Culture Collection (ATCC). If necessary, the genomic DNA of Bacillus liquor-formis ATCC 14580 strain can be prepared from cultured cells of the strain according to known methods (M. Stahl et al., Journal of Bacteriology, 154, 406-412 (1983)). In a preferred embodiment of the present invention, at least A contained in the compound of the above formula (I)

3 is also partly the following SEQ ID NO: 1-60:

AHGVTSAPDT (SEQ ID NO: 1),

HGVTSAPDTR (SEQ ID NO: 2),

GVTSAPDTRP (SEQ ID NO: 3),

VTSAPDTRPA (SEQ ID NO: 4),

TSAPDTRPAP (SEQ ID NO: 5),

SAPDTRPAPG (sequence number 6),

APDTRPAPGS (SEQ ID NO: 7),

PDTRPAPGST (SEQ ID NO: 8),

DTRPAPGSTA (SEQ ID NO: 9),

TRPAPGSTAP (SEQ ID NO: 10),

RPAPGSTAPP (SEQ ID NO: 11),

PAPGSTAPPA (SEQ ID NO: 12),

APGSTAPPAH (SEQ ID NO: 13),

PGSTAPPAHG (SEQ ID NO: 14),

GSTAPPAHGV (SEQ ID NO: 15),

STAPPAHGVT (SEQ ID NO: 16),

TAPPAHGVTS (SEQ ID NO: 17),

APPAHGVTSA (SEQ ID NO: 18),

PPAHGVTSAP (SEQ ID NO: 19), PAHGVTSAPD (SEQ ID NO: 20),

AHGVTSAPDTR (SEQ ID NO: 21),

HGVTSAPDTRP (SEQ ID NO: 22),

GVTSAPDTRPA (SEQ ID NO: 23),

VTSAPDTRPAP (SEQ ID NO: 24),

TSAPDTRPAPG (SEQ ID NO: 25),

SAPDTRPAPGS (sequence number 26),

APDTRPAPGST (SEQ ID NO: 27),

PDTRPAPGSTA (SEQ ID NO: 28),

DTRPAPGSTAP (SEQ ID NO: 29),

TRPAPGSTAPP (SEQ ID NO: 30),

RPAPGSTAPPA (SEQ ID NO: 31),

PAPGSTAPPAH (SEQ ID NO: 32),

APGSTAPPAHG (SEQ ID NO: 33),

PGSTAPPAHGV (SEQ ID NO: 34),

03? ? Eight 110 yen (sequence number 35),

STAPPAHGVTS (SEQ ID NO: 36),

Dingpachi? ? 8110 ¥ 3-8 (sequence number 37),

APPAHGVTSAP (SEQ ID NO: 38),

PPAHGVTSAPD (SEQ ID NO: 39),

PAHGVTSAPDT (SEQ ID NO: 40),

HGVTSAPDTRPAPGSTAPPA (SEQ ID NO: 41), GVTSAPDTRPAPGSTAPPAH (SEQ ID NO: 42), VTSAPDTRPAPGSTAPPAHG (SEQ ID NO: 43), TSAPDTRPAPGSTAPPAHGV (SEQ ID NO: 44), SAPDTRPAPGSTAPPAHGVT (SEQ ID NO: 45), APDTRPAPGSTAPPAHGVTS (SEQ ID NO: 46), PDPA, TRPA No. DTRPAPGSTAPPAHGVTSAP (SEQ ID NO: 48),

TRPAPGSTAPPAHGVTSAPD (SEQ ID NO: 49),

RPAPGSTAPPAHGVTS APDT (SEQ ID NO: 50),

PAPGSTAPPAHGVTSAPDTR (SEQ ID NO: 51),

APGSTAPPAHGVTSAPDTRP (SEQ ID NO: 52),

PGSTAPPAHGVTSAPDTRPA (SEQ ID NO: 53),

GSTAPPAHGVTSAPDTRPAP (SEQ ID NO: 54),

STAPPAHGVTSAPDTRPAPG (SEQ ID NO: 55),

TAPPAHGVTSAPDTRPAPGS (SEQ ID NO: 56),

APPAHGVTSAPDTRPAPGST (SEQ ID NO: 57),

PPAHGVTSAPDTRPAPGSTA (SEQ ID NO: 58),

PAHGVTSAPDTRPAPGSTAP (SEQ ID NO: 59) and

AHGVTSAPDTRPAPGSTAPP (SEQ ID NO: 60),

The amino acid sequence derived from the mucin-type glycoprotein MUC1 selected from the group power consisting of the amino acid sequence shown in FIG. Furthermore, it is an amino acid sequence derived from a mucin type protein including a sequence in which any one of SEQ ID NOs: 41 to 60 is repeated twice or three times.

[0112] The polymer carrier that can be used in the present invention can bind the group represented by the formula (I), and after the coupling, the compound of formula (I) can act by the action of glycosyltransferase as described below. There is no particular limitation as long as it can transfer a further sugar residue to the sugar residue of the represented group.For example, a vinyl monomer having an aminooxy group or a hydrazide group may be protected. Polymers or copolymers of the above (the above-mentioned butyl monomers include acrylamides, methacrylate amides, acrylic acids, methacrylic acids, styrenes, fatty acid butyl esters, etc.) or may be protected. , Polyethers which may have an aminooxy group or hydrazide group; silica carriers, rosin carriers, magnetic beads or amino beads having an aminooxy group or hydrazide group which may be protected Metallic substrate (e.g., the following expression:

[0113] [Chemical 24]

Silica carrier, rosin carrier and magnetic beads, metal carrier [wherein, ◯ represents silica, rosin, magnetic beads, metal carrier]; and Maps used for peptide synthesis (Multiple carrier similar to the antigen peptide systems method; for example:

[(NH OCH C (= 0)) -Lys] Lys— NHCH CH C (= 0) — R ³ ,

2 2 2 2 2 2

[(NH OCH C (= 0)) -Lys] Lys— NHCH (CH SH) C (= 0) — R ³ ,

2 2 2 2 2

[(NH OCH C (= 0)) -Lys] — Lys— Cys— NHCH CH C (= 0) — R ³ (sequence

twenty two

Number 61),

{[(NH OCH C (:: 0)) -Lys]-Lys-NHCH [C (= O)-R] CH S},

2 2 2 2 2

{[(NH OCH C (:: 0)) -Lys]-Lys-NHCH [C (= O) NHCH CH C (= 0

twenty two

) -R ³ ] CH -S},

twenty two

{[(NH OCH C (:: 0)) -Lys] -Lys} -Lys -NHCH CH C (= 0) — R (

twenty two

SEQ ID NO: 62),

{[(NH OCH C (= 0)) -Lys} -Lys- NHCH (CH SH) C (= 0)

2 2 2

R ³ (SEQ ID NO: 63),

{[(NH OCH C (= 0)) -Lys} —Lys— Cys— NHCH CH C (= 0)

2 2 2

—R ³ (SEQ ID NO: 64),

[[[(NH OCH C (= 0)) -Lys]-Lys-NHCH [C (= 0) -R] CH

twenty two

S] (SEQ ID NO: 65),

[[[(NH OCH C (= 0)) -Lys] -Lys]-Lys-NHCH [C (= 0) NHCH C

2 2 2 2 2 2

HC (= 0) R ³ ] CH—S] (SEQ ID NO: 66),

2 2 2

[Chemical 25]

[(NH ₂ OCH ₂ C (= 0)) 2 -Ly s] -NHCHC (= 0) R ³

[(NH ₂ OCH 2 C (= 0)) 2 -L ys] -NH (CH ₂ ) 4

Or

{[(NH ₂ OCH ₂ C (= 0)) 2-L ys] ₂ — L ys} -NHCHC (= 0) R ³ {[(NH ₂ OCH ₂ C (= 0)) 2 -L ys] ₂ — Ly s} — NH (CH ₂ ) 4 (Wherein R ³ represents a hydroxyl group or an amino group, Lys represents a lysine residue, and Cys represents a cysteine residue)

The compound etc. which are represented by these are mentioned.

[0115] The above-mentioned polymer or copolymer of a bull monomer having an aminooxy group or a hydrazide group which may be protected is at least one of a polymer or copolymer of an unsubstituted vinyl monomer. A method in which a part is protected with an aminooxy group or a hydrazide group, or a method in which a vinyl monomer having an aminooxy group or a hydrazide group is protected or polymerized or copolymerized It is prepared by.

[0116] Examples of the acrylamides include N-alkyl acrylamides such as acrylamide, N-ethyl acrylamide, N-isopropyl acrylamide, etc., which may have protected amino group or hydrazide group. Illustrated.

[0117] Examples of the methacrylamides include methacrylamide, N-methyl methacrylamide N-ethyl methacrylamide, N-isopropyl methacrylamide, etc., which may have an aminooxy group or a hydrazide group which may be protected. N-alkyl methacrylamide and the like are exemplified.

[0118] Examples of the acrylic acid include acrylic acid such as acrylic acid, methyl acrylate, ethyl acrylate, hydroxyethyl acrylate, and dimethylaminoethyl acrylate, which may have an aminooxy group or hydrazide group which may be protected. Examples include acid esters.

[0119] The above methacrylic acids are protected and may have an aminooxy group or a hydrazide group. Examples include methacrylic acid esters.

[0120] Examples of the styrenes include styrene, p-hydroxystyrene, p-hydroxymethylstyrene and the like, which may be protected but may have an aminooxy group or a hydrazide group.

[0121] Examples of the fatty acid vinyl ester include vinyl acetate and vinyl butyrate which may be protected and have an aminooxy group or a hydrazide group. In addition, the polymer or copolymer of fatty acid bule ester in the present invention includes an alkali after polymerization reaction, etc. In this case, all or part of the ester bond is hydrolyzed.

[0122] The above polyethers may be protected! Polyethylene glycol which may have an aminooxy group or a hydrazide group, or may be protected, and may have an aminooxy group or a hydrazide group. Examples thereof include polyethylene and polyethylene glycol substituted with aryl groups.

[0123] The polymer carrier here may be either water-insoluble or water-soluble, but water-soluble is preferred. The general molecular weight is about 10,000 to about 5000000, preferably 20000 to 2000000, more preferably <50,000 to 1000000. In the case of a water-insoluble carrier, the form includes a bead shape, a fiber shape, a film shape, and a film shape, but is not particularly limited.

[0124] As a more preferred carrier, the following formula:

[0125]

The polymeric carrier represented by these is mentioned. Here, n is an integer of 1 to 15, preferably 1 to 10, and more preferably 1 to 5. The ratio of x: y is 1: 0 to 1: 1000, preferably 1: 0 to 1: 100. The molecular weight of the polymer carrier is about 10,000 to about 5000000, preferably <is 20000 to 2000000, more preferably <is 50000 to 1000000. [0126] In a preferred embodiment, the invention provides a compound of the following formula:

X— C (= 0) — (CH) — A — A — A (I)

2 n 1 2 3

In which X represents a hydrogen atom, c-c alkyl, c-c aryl or chromophore;

1 30 6 30

n represents an integer of 0 to 20;

A is — (CH 2) — C (= 0) —, — (CH 2 CH 2 O) —

1 2 0 ~ 20 2 2 1 ~ 10

A represents an amino acid residue cleavable by a protease;

2

Three

[0127] [Chemical 26— 1]

Z1 Z2 Z2

R1 ー R2_ _R 3 _R 4 _ _R 5 R5

R ^6- (R ⁷ -R ⁸ " ⁹ -R ^{10 or} R9—R ¹⁰

Z3

R ² and R ⁷ are Gal groups;

R ³ and R ⁸ are GlcNAc groups;

R ⁴ and R ⁹ are Gal groups;

R ⁵ is a GlcNAc group;

R ¹⁰ is a GalNAc group;

ZZ ² and Z ³ are each independently hydrogen or a Fuc group; and n and m are each independently an integer greater than or equal to 0;

The bond between R ² and R ³ is a β ΐ, 4 bond;

The bond between R ³ and R ⁴ is a β ΐ, 3 bond; The bond between R ⁴ and R ⁵ is a β ΐ, 4 bond;

The bond between R ⁶ and R ⁷ is an α 2,3 bond when R ⁶ is a Neu5Ac group, and a β ΐ, 3 bond when R ⁶ is a GlcNA c group;

The bond between R ⁷ and R ⁸ is a β ΐ, 4 bond;

The bond between R ⁸ and R ⁹ is a β ΐ, 3 bond;

The bond between R ⁹ and R ^1G is a β ΐ, 3 bond;

The bond between R ⁵ and R ^1G is a β ΐ, 6 bond;

The bond between Ζ ¹ and R ³ is << ¹ , 3 bond;

The bond between Ζ ² and R ⁵ is << 1, 3 bond; and

The bond between Ζ ³ and R ⁸ is << 1, 3 bond,

The compound which has the sugar residue represented by this is provided.

[0128] In another preferred embodiment, the invention provides a compound of the following formula:

X— C (= 0) — (CH) — Α— Α— A (I)

2 n 1 2 3

Wherein X is a hydrogen atom, c to

1 c alkyl, ~

30 c 6 c represents a reel or chromophore;

30

n represents an integer of 0 to 20;

A is — (CH 2) — C (= 0) —, — (CH 2 CH 2 O) —

1 2 0 ~ 20 2 2 1 ~ 10

A represents an amino acid residue cleavable by a protease;

2

Three

[0129] [Chemical 26-2] R —

In which R ¹ is hydrogen, a sialic acid group or a GlcNAc group;

R ² is a Gal group; R ³ is a GlcNAc group;

R ⁴ is a Gal group;

R ⁵ is a GlcNAc group;

R ¹⁰ is a GalNAc group;

R ⁸ is hydrogen or a sialic acid group;

R ⁹ is a Gal group;

Z ¹ and Z ² are each independently hydrogen or a Fuc group; and

n is an integer greater than or equal to 0;

Where the bond between R ¹ and R ² is an a 2, 3 bond when R ¹ is a sialic acid group, and a j8 1, 3 bond when R ¹ is a GlcNAc group;

The bond between R ² and R ³ is a β,, 4 bond;

The bond between R ³ and R ⁴ is a β,, 3 bond;

The bond between R ⁴ and R ⁵ is a β,, 4 bond;

The bond between R ⁸ and R ⁹ is << 2, 3 bond;

The bond between R ⁹ and R ¹⁰ is a β,, 3 bond;

The bond between R ⁵ and R ^1G is a β,, 6 bond;

The bond between Ζ ¹ and R ³ is << ¹ , 3 bond; and

The bond between Ζ ² and R ⁵ is << 1, 3 bond,

The compound which has the sugar residue represented by this is provided.

In another preferred embodiment, the present invention provides the following formula:

Α N = C (— X) — (CH) A -A -A (II)

4 2 n 1 2 3

[Where X is a hydrogen atom, c to ally

1 c alkyl,

30 c ~

6 c represents a chromophore or chromophore; 30

n represents an integer of 0 to 20;

A is — (CH 2) — C (= 0) —, — (CH 2 CH 2 O) —

1 2 0 ~ 20 2 2 1 ~ 10

A is a protease derived from Bacillus Licheniformis.

2

A cleavable glutamic acid residue or cysteine residue; A is a sugar amino acid residue substantially free of a site cleavable by a protease, or

Three

[Chem 26-3]

R ² and R ⁷ are Gal groups;

R ³ and R ⁸ are GlcNAc groups;

R ⁴ and R ⁹ are Gal groups;

R ⁵ is a GlcNAc group;

R ¹⁰ is a GalNAc group;

The bond between R ² and R ³ is a β ΐ, 4 bond;

The bond between R ³ and R ⁴ is a β ΐ, 3 bond;

The bond between R ⁴ and R ⁵ is a β ΐ, 4 bond;

The bond between R ⁷ and R ⁸ is a β ΐ, 4 bond;

The bond between R ⁸ and R ⁹ is a β ΐ, 3 bond;

The bond between R ⁹ and R ^1G is a β ΐ, 3 bond;

The bond between R ⁵ and R ^1G is a β ΐ, 6 bond;

The bond between Ζ ¹ and R ³ is << ¹ , 3 bond; The bond between Z ² and R ⁵ is << 1, 3 bond; and

The bond between Ζ ³ and R ⁸ is << 1, 3 bond,

Having a sugar residue represented by:

Α is the following formula:

Four

[Chemical 26-4]

(Wherein, s is an integer of 1 to 15, and: is 1: 0 to 1: 1000)] is provided.

A N = C (— X) — (CH) A -A -A (II)

4 2 n 1 2 3

[Wherein X represents a hydrogen atom, c-c alkyl, c-c aryl or chromophore;

1 30 6 30

n represents an integer of 0 to 20;

A is — (CH 2) — C (= 0) —, — (CH 2 CH 2 O) —

1 2 0 ~ 20 2 2 1 ~ 10

A is a protease derived from Bacillus Licheniformis. A cleavable glutamic acid residue or cysteine residue;

Three

[Chemical 27] R ¹

In which R ¹ is hydrogen, a sialic acid group or a GlcNAc group;

R ² is a Gal group;

R ³ is a GlcNAc group;

R ⁴ is a Gal group;

R ⁵ is a GlcNAc group;

R ¹⁰ is a GalNAc group;

R ⁸ is hydrogen or a sialic acid group;

R ⁹ is a Gal group;

Z ¹ and Z ² are each independently hydrogen or a Fuc group; and

n is an integer greater than or equal to 0;

Where the bond between R ¹ and R ² is an a 2, 3 bond when R ¹ is a sialic acid group, and a β ΐ, 3 bond when R ¹ is a GlcNAc group;

The bond between R ² and R ³ is a j8 1, 4 bond;

The bond between R ³ and R ⁴ is a 1, 3 bond;

The bond between R ⁴ and R ⁵ is] 3 1, 4 bond;

The bond between R ⁸ and R ⁹ is a 2, 3 bond;

The bond between R ⁹ and R ^1G is a j8 1,3 bond;

The bond between R ⁵ and R ^1G is a j8 1,6 bond;

The bond between Z ¹ and R ³ is an α ΐ, 3 bond; and

The bond between Ζ ² and R ⁵ is al, 3 bond, Having a sugar residue represented by:

A is the following formula:

Four

[Chemical 27-1]

(Wherein, s is an integer of 1 to 15, and: is 1: 0 to 1: 1000)] is provided.

In the present invention, the mucin-type glycopeptide having a voralactosamine skeleton provided in the present invention, in the formula (I) or (または), in A, n is an integer of 0 or more, and preferably, n is 0 to Five

Three

More preferably, n is an integer of 4 or less. Further, in the above formula, in A, Z ¹

Three

˜Z ³ may or may not be present, but preferably m is 0 to 5, and when n is 1, Z ¹ and Z ² are sialic acid groups. More preferably, the sugar residue of the glycopeptide is [Chemical 27-2]

It can be a sugar residue or a derivative of a sugar residue selected from the group consisting of

[0137] In another aspect, the present invention provides a composition for a primer for producing a glycoamino acid or glycopeptide comprising the compound described in the above formula (I) or (IV).

[0138] The synthesis and purification of the compounds of the present invention useful as primers for producing glycopeptides involves the following procedures:

1) Protected amino acids (including amino acid residues cleavable by proteases), sugar amino acids with pre-synthesized protecting groups, and keto acids or aldehyde acids as raw materials for peptide solid-phase synthesis, with ketone residues at the ends Or has an aldehyde residue and is cleaved by a protease Synthesize glycopeptides (glycans / amino acid protectors) containing cleavable amino acid residues (if necessary, inactivate the cabbing reaction after each step of the amino acid coupling reaction, that is, unreacted amino acid coupling product) Reaction);

2) By acid treatment, the glycopeptide containing an amino acid residue that has a ketone residue or an aldehyde residue at the end and can be cleaved by protease is released to the solid phase carrier force, and at the same time, the amino acid side chain protecting group is removed Protect (if the amino acid side chain protecting group is not removed by acid treatment, the protecting group may be separately deprotected by a deprotection reaction);

3) The reaction mixture or the mixture obtained by the ether precipitation method is purified by HPLC, and a glycopeptide (sugar chain protector) containing an amino acid residue that has a ketone residue or an aldehyde residue at the end and can be cleaved by a protease is obtained. Isolate;

4) Deprotect the sugar chain protecting group;

5) Purification by HPLC to isolate a glycopeptide containing a ketone residue or an aldehyde residue at the end and containing an amino acid residue that can be cleaved by a protease

It is done in the procedure. This method can also be applied to the synthesis of peptides that do not contain sugar amino acids, in which case step 4) is omitted.

[0139] Synthesis and purification of a polymeric primer from a glycopeptide containing an amino acid residue having a ketone residue or an aldehyde residue at the terminal and thus cleavable by a protease are as follows:

6) reacting the glycopeptide obtained above with a polymer carrier;

7) Purify by gel filtration column chromatography to obtain a polymer primer.

[0140] Another general synthesis and purification of the compounds of the invention is as follows:

1) Protected amino acids (including amino acid residues cleavable by proteases), sugar amino acids with pre-synthesized protecting groups, and keto acids or aldehyde acids as raw materials for peptide solid-phase synthesis, with ketone residues at the ends Alternatively, synthesize a glycopeptide (sugar chain / amino acid protected form) containing an aldehyde residue and an amino acid residue that can be cleaved by a protease (if necessary, a cubbing reaction after each step of the amino acid coupling reaction, that is, Performing a reaction to inactivate unreacted amino acid coupling); 2) By acid treatment, the glycopeptide containing an amino acid residue that has a ketone residue or an aldehyde residue at the end and is cleavable by protease is released to the solid phase carrier force, and at the same time, the protecting group of the amino acid side chain is removed. Protect (if the amino acid side chain protecting group is not removed by acid treatment, the protecting group may be separately deprotected by a deprotection reaction);

3) Deprotect the sugar chain protecting group;

4) A polymer carrier is introduced into the reaction solution containing the glycopeptide of 3) and selectively reacted with the glycopeptide;

5) The glycopeptide bound to the carrier is purified by gel filtration or dialysis, ultrafiltration, etc .;

6) Hydrolyze the glycopeptide bound to the carrier with a protease to release the glycopeptide, remove the carrier, and isolate the desired glycopeptide.

It is done in the procedure.

According to this procedure, it is possible to guide the polymer primer in one pot without isolating each step. Synthesis and purification of a polymeric primer from a glycopeptide containing an amino acid residue having a ketone residue or an aldehyde residue at the terminal and thus cleavable by a protease is as follows:

1) Protected amino acids (including amino acid residues that can be cleaved by proteases), sugar amino acids with pre-synthesized protecting groups, and keto acids or aldehyde acids as raw materials for peptide solid-phase synthesis, and ketone residues at the ends Alternatively, a glycopeptide (sugar chain / amino acid protected form) having an aldehyde residue and containing an amino acid residue that can be cleaved by a protease is synthesized (if necessary, a cubbing reaction after each step of the amino acid coupling reaction, that is, Performing a reaction to inactivate unreacted amino acid coupling);

2) By acid treatment, the glycopeptide containing an amino acid residue having a ketone residue or an aldehyde residue at the end and cleavable by a protease is released to the solid phase carrier force, and at the same time, the protecting group of the amino acid side chain is removed. Protect (if the amino acid side chain protecting group is not removed by acid treatment, the protecting group may be separately deprotected by a deprotection reaction);

3) Deprotect the sugar chain protecting group;

4) A polymer carrier is introduced into the reaction solution containing the glycopeptide of 3) to selectively react with the glycopeptide. React;

5) Purify the glycopeptide bound to the carrier by gel filtration or dialysis, ultrafiltration, etc. to obtain a primer.

It is done in the procedure.

[0142] Preferably, according to the embodiment, the keto acid or aldehyde acid used in the above step 1) has the following formula:

X-C (= 0)-(CH) -A -COOH (III)

2 n 1

Wherein X is a hydrogen atom, c-c alkyl,

1 30 c represents 6-c aryl or chromophore;

30

n represents an integer of 0 to 20;

(A represents a linker having a length of 1 to 20 methylene chains).

[0143] In one preferred embodiment, the method for producing a glycopeptide of the present invention comprises the following steps:

(A) the compound according to any one of the above items (1) to (3), an aminooxy group which may be specifically protected and which can specifically react with a ketone residue or an aldehyde residue of the compound; Reacting a carrier containing a functional group selected from the group consisting of N alkylaminoxy group, hydrazide group, azide group, thiosemicarbazide group, 1,2-dithiol group and cysteine residue;

(B) By reacting the compound obtained in step (A) with a glycosyltransferase in the presence of a sugar nucleotide, the sugar residue is transferred from the sugar nucleotide to the compound, and the sugar chain is elongated. A step of obtaining a compound comprising a sugar residue selected from the group consisting of the sugar nucleotide force Gal group, GlcNAc group, Fuc group and sialic acid group;

(D) a step of causing a protease to act on a compound in which sugar residues are transferred and sugar chains are elongated.

[0144] The glycosyltransferase used in the present invention is preferably 13 1, 4-galactose transfer. Enzyme, j8 1,3-Galactosyltransferase, 1,3-Fucose transferase, / 3 1,3-N-Acetyldarcosaminetransferase, 13 1,6-N-Acetyldarcosaminetransferase, α 2,3-sialyltransferase, << 2,6-sialyltransferase.

[0145] In another preferred embodiment, the method for producing a glycopeptide of the present invention comprises the following steps:

(Ii) A sugar residue is transferred from the sugar nucleotide to the compound by allowing a glycosyltransferase to act on the compound according to any one of items (4) to (7) in the presence of the sugar nucleotide. , A step of obtaining a compound in which a sugar chain is elongated, wherein the sugar nucleotide has a sugar residue selected from the group consisting of a Gal group, a GlcNAc group, a Fuc group and a sialic acid group. ,

(Ii) removing unreacted sugar nucleotides and by-product nucleotides as necessary; and

(C) a step of allowing a protease to act on a compound in which sugar residues are transferred and sugar chains are elongated. Furthermore, the process of isolating glycopeptide may be included. In this production method, by-products other than the glycopeptide containing the target glycopeptide and the carrier can be easily separated.

[0146] In still another preferred embodiment, the method for producing a glycopeptide of the present invention comprises the following steps:

(Ii) Step (ii) Repeating step (1) one or more times to extend the sugar chain;

(D) including a step of allowing a protease to act on a compound in which a plurality of sugar residues are transferred and sugar chains are elongated. [0147] In still another preferred embodiment, the method for producing a glycopeptide of the present invention comprises the following steps:

(A) Peptide solid phase synthesis is performed using amino acids, sugar amino acids, and keto acids or aldehyde acids that can be cleaved by proteases, and the compound according to any one of items (1) to (3) is obtained. Obtaining step;

(B) The compound obtained in step (A) and an optionally protected aminooxy group, N-alkylaminooxy group, hydrazide group capable of specifically reacting with the ketone residue or aldehyde residue of the compound Reacting with a carrier comprising a functional group selected from the group consisting of: azide group, thiosemicarbazide group, 1,2-dithiol group and cysteine residue;

(C) By allowing a glycosyltransferase to act on the compound obtained in step (B) in the presence of a sugar nucleotide, the sugar residue is transferred from the sugar nucleotide to the compound, and the sugar chain is elongated. A step of obtaining a compound comprising a sugar residue selected from the group consisting of the sugar nucleotide force Gal group, GlcNAc group, Fuc group and sialic acid group;

(E) a step of allowing a protease to act on a compound in which sugar residues are transferred and sugar chains are elongated.

[0148] In another preferred embodiment, the method for producing a glycopeptide of the present invention comprises the following steps:

(B) The compound obtained in step (A) and an optionally protected aminooxy group, N-alkylaminooxy group, hydrazide group capable of reacting specifically with the ketone residue or aldehyde residue of the compound Reacting with a carrier comprising a functional group selected from the group consisting of: azide group, thiosemicarbazide group, 1,2-dithiol group and cysteine residue;

(D) Step (C) is repeated once or twice or more to extend the sugar chain;

(F) a step of allowing a protease to act on a compound in which a plurality of sugar residues are transferred and sugar chains are elongated.

[0149] In still another preferred embodiment, the method for producing a glycopeptide of the present invention comprises the following steps:

(B) The compound obtained in step (A) and an optionally protected aminooxy group, N-alkylaminooxy group, hydrazide group capable of reacting specifically with the ketone residue or aldehyde residue of the compound , A reaction with a carrier containing a functional group selected from the group consisting of an azide group, a thiosemicarbazide group, a 1,2-dithiol group and a cysteine residue, and simultaneously removing unreacted substances in step (A);

(C) The sugar residue is transferred from the sugar nucleotide to the compound by allowing a sugar transferase to act on the compound bound to the carrier obtained in step (B) in the presence of the sugar nucleotide. Obtaining a compound having an extended chain, wherein the sugar nucleotide has a sugar residue selected from the group consisting of a Gal group, a GlcN Ac group, a Fuc group and a sialic acid group; and And

(D) A step of allowing protease to act on the compound obtained by extending the sugar chain obtained in step (C) is included.

[0150] In still another preferred embodiment, the method for producing a glycopeptide of the present invention comprises the following steps:

(A) A peptide solid-phase synthesis is carried out using amino acids, sugar amino acids, and keto acids or aldehyde acids that can be cleaved by a protease, as described in any one of items (1) to (3) Obtaining a compound;

(B) The compound obtained in step (A) and an optionally protected aminooxy group, N-alkylaminooxy group, hydrazide group capable of reacting specifically with the ketone residue or aldehyde residue of the compound , A reaction with a carrier containing a functional group selected from the group consisting of azide group, thiosemicarbazide group, 1,2-dithiol group and cysteine residue, and at the same time, removing unreacted substances in step (A) ;

(C) The sugar residue is transferred from the sugar nucleotide to the compound by allowing a sugar transferase to act on the compound bound to the carrier obtained in step (B) in the presence of the sugar nucleotide. Obtaining a compound having an extended chain, wherein the sugar nucleotide has a sugar residue selected from the group consisting of a Gal group, a GlcN Ac group, a Fuc group and a sialic acid group;

(D) Step (C) is repeated once or twice or more to extend the sugar chain;

In another preferred embodiment, the method for producing the glycopeptide of the present invention comprises the following steps:

(A) A sugar residue is transferred from the sugar nucleotide to the compound by allowing a glycosyltransferase to act on the compound according to any one of items (1) to (3) in the presence of the sugar nucleotide. A step of obtaining a compound in which a sugar chain is elongated, wherein the sugar nucleotide has a sugar residue selected from the group consisting of a Gal group, a GlcNAc group, a Fuc group and a sialic acid group

(C) a compound in which a sugar residue is transferred and a sugar chain is extended, and a protected compound capable of reacting specifically with a ketone residue or an aldehyde residue of the compound, and may be an aminooxy group, N-alkyl Reacting with a carrier comprising a functional group selected from the group consisting of an aminooxy group, a hydrazide group, an azide group, a thiosemicarbazide group, a 1,2-dithiol group and a cysteine residue; and (D) removing unreacted sugar nucleotides and by-product nucleotides as necessary,

including.

[0152] In still another preferred embodiment, the method for producing a glycopeptide of the present invention comprises the following steps:

(E) a step of causing a protease to act on a compound in which a sugar residue is transferred and a sugar chain is elongated.

[0153] In still another preferred embodiment, the method for producing a glycopeptide of the present invention comprises the following steps:

(B) The compound obtained in step (A) and an optionally protected aminooxy group, N-alkylaminooxy group, hydrazide group capable of reacting specifically with the ketone residue or aldehyde residue of the compound , Azide group, thiosemicarbazide group, 1,2-dithiol group and cysteine Reacting with a soluble carrier containing a functional group selected from the group consisting of residues and removing unreacted substances in step (A) by reprecipitation, gel filtration, or ultrafiltration;

(C) By allowing a glycosyltransferase to act on the compound solublely bound to the carrier obtained in step (B) in the presence of a sugar nucleotide, the sugar residue is transferred from the sugar nucleotide to the compound, A step of obtaining a compound in which a sugar chain is elongated, wherein the sugar nucleotide has a sugar residue selected from the group consisting of a Gal group, a GlcNAc group, a Fuc group and a sialic acid group

(D) Step (C) is repeated once or twice or more to extend the sugar chain;

(E) removing unreacted sugar nucleotides and by-product nucleotides as necessary;

(F) reacting the compound in which the sugar residue is transferred and the sugar chain is extended with an insoluble carrier having keto acid or aldehyde acid bound to the surface, and immobilizing on the surface; and

(G) a step of removing the reagents and enzymes used in the sugar chain elongation reaction as necessary.

In still another preferred embodiment, the method for producing a glycopeptide of the present invention comprises the following steps:

(B) The compound obtained in step (A) and an optionally protected aminooxy group, N-alkylaminooxy group, hydrazide group capable of reacting specifically with the ketone residue or aldehyde residue of the compound , An azide group, a thiosemicarbazide group, a 1,2-dithiol group, and a soluble carrier containing a functional group selected from the group consisting of cysteine residues, and then reacting with a soluble carrier, followed by reprecipitation, gel filtration, or ultrafiltration. Removing unreacted substances in (A);

(C) By allowing a glycosyltransferase to act on the compound solublely bound to the carrier obtained in step (B) in the presence of a sugar nucleotide, the sugar residue is transferred from the sugar nucleotide to the compound, A step of obtaining a compound having an extended sugar chain, wherein the sugar nucleotide has a sugar residue selected from the group consisting of a Gal group, a GlcNAc group, a Fuc group and a sialic acid group @;

(D) Step (C) is repeated once or twice or more to extend the sugar chain;

(F) reacting the compound in which the sugar residue is transferred and the sugar chain is extended with an insoluble carrier having keto acid or aldehyde acid bound to the surface, and immobilizing it on the surface;

(G) removing the reagents and enzymes used in the sugar chain elongation reaction as necessary; and

(H) a step of allowing a protease to act on the compound in which the sugar chain immobilized in step (F) is elongated.

[0155] The glycopeptide used in the method for producing a glycopeptide of the present invention is preferably

And the following formula:

[0156] [Chemical 28-1]

(SEQ ID NO: 2)

[0157] [Chemical 28-2]

(SEQ ID NO: 21)

Or

[0158] [Chemical 28-3]

X ¹ X ¹ X ¹ X ¹ X ¹

(SEQ ID NO: 41)

[0159] [Chemical 28- 4]

R ² and R ⁷ are Gal groups;

R ³ and R ⁸ are GlcNAc groups;

R ⁴ and R ⁹ are Gal groups;

R ⁵ is a GlcNAc group;

R ¹⁰ is a GalNAc group;

The bond between R ² and R ³ is a β ΐ, 4 bond;

The bond between R ³ and R ⁴ is a β ΐ, 3 bond;

The bond between R ⁴ and R ⁵ is a β ΐ, 4 bond;

The bond between R ⁷ and R ⁸ is a β ΐ, 4 bond;

The bond between R ⁸ and R ⁹ is a β ΐ, 3 bond;

The bond between R ⁹ and R ¹⁰ is a β ΐ, 3 bond;

The bond between R ⁵ and R ^1G is a β ΐ, 6 bond;

The bond between Ζ ¹ and R ³ is << ¹ , 3 bond;

The bond between Ζ ² and R ⁵ is << 1, 3 bond; and

The bond between Ζ ³ and R ⁸ is << 1, 3 bond,

Υ ¹ represents a hydrogen atom, acetyl, acyl, alkyl or aryl;

Υ ² represents a hydroxyl group, ΝΗ, alkyl or aryl.

2

It is a glycopeptide represented by these.

Further preferably, in the above formula, each X ¹ independently represents a hydrogen atom or the following formula: [Chemical 28— 5]

In which R ¹ is hydrogen, a sialic acid group or a GlcNAc group;

R ² is a Gal group;

R ³ is a GlcNAc group;

R ⁴ is a Gal group;

R ⁵ is a GlcNAc group;

R ¹⁰ is a GalNAc group;

R ⁸ is hydrogen or a sialic acid group;

R ⁹ is a Gal group;

Z ¹ and Z ² are each independently hydrogen or a Fuc group; and

n is an integer greater than or equal to 0;

The bond between R ² and R ³ is a β,, 4 bond;

The bond between R ³ and R ⁴ is a β,, 3 bond;

The bond between R ⁴ and R ⁵ is a β,, 4 bond;

The bond between R ⁸ and R ⁹ is a << 2, 3 bond;

The bond between R ⁹ and R ^1G is a β,, 3 bond;

The bond between R ⁵ and R ^1G is a β,, 6 bond;

The bond between Ζ ¹ and R ³ is << ¹ , 3 bond; and

The bond between Ζ ² and R ⁵ is << 1, 3 bond,

It is a glycopeptide represented by these.

In the mucin-type glycopeptide having a voralactosamine skeleton provided in the present invention, n is an integer of 0 or more, preferably n is 0 to 5, more preferably , N is an integer of 4 or less. Further, in A above, ¹ to ³ may or may not be present.

2

Force Preferably, m is 0-5, preferably when n is 1, Z ¹ and Z ² are sialic acid groups. More preferably, the sugar residue of the glycopeptide is

[Chemical 28-6]

[0163] In an even more preferred embodiment, the method for producing a glycopeptide of the present invention comprises the following steps:

(A)

The following formula:

[0164] [Chemical 28—8]

Where R ¹ and R. Each independently is hydrogen, Neu5Ac group or GlcNAc group;

R ² and R ⁷ are Gal groups;

R ³ and R ⁸ are GlcNAc groups;

R ⁴ and R ⁹ are Gal groups;

R ⁵ is a GlcNAc group;

R ¹⁰ is a GalNAc group;

R ¹¹ is a hydrogen atom or a methyl group;

The bond between R ² and R ³ is a β,, 4 bond;

The bond between R ³ and R ⁴ is a β,, 3 bond; The bond between R ⁴ and R ⁵ is a β,, 4 bond;

The bond between R ⁶ and R ⁷ is an α 2,3 bond when R ⁶ is a Neu5Ac group, and a β,, 3 bond when R ⁶ is a GlcNA c group;

The bond between R ⁷ and R ⁸ is a β,, 4 bond;

Bond between R ⁸ and R ^9, β ΐ, is 3 bonds;

The bond between R ⁹ and R ^1G is a β,, 3 bond;

The bond between R ⁵ and R ^1G is a β,, 6 bond;

The bond between Ζ ¹ and R ³ is << ¹ , 3 bond;

The bond between Ζ ² and R ⁵ is << 1, 3 bond;

The bond between Ζ ³ and R ⁸ is << 1, 3 bond; and

Ρ is an amino acid protecting group (for example, 9 fluorenylmethyloxycarbonyl group, ter rtbutoxycarbol group, etc.),

Wherein the sugar chain of the sugar amino acid is protected with a protecting group to produce a sugar amino acid in which the sugar chain is protected, wherein the protecting group is a protecting group of a sugar alcohol group (for example, acetyl group, benzoyl group). Group, methyl group, methoxymethyl group, trimethylsilyl group, t-butyldimethylsilyl group, dimethylphenol group, triisopropylpropylsilyl group, benzyl group, benzylidene group, isopropylidene group, ditert-butylsilylidene group, etc. Yeah

Including the method.

In a still further preferred embodiment, the method for producing a glycopeptide of the present invention comprises the following steps:

(A) The following formula:

[Chemical 29]

R "

P, person ^{0 H}

^H & wherein R ¹ is hydrogen, a sialic acid group or a GlcNAc group;

R ² is a Gal group;

R ³ is a GlcNAc group;

R ⁴ is a Gal group;

R ⁵ is a GlcNAc group;

R ¹⁰ is a GalNAc group;

R ⁸ is hydrogen or a sialic acid group;

R ⁹ is a Gal group;

R ¹¹ is a hydrogen atom or a methyl group;

Z ¹ and Z ² are each independently hydrogen or a Fuc group; and

n is an integer greater than or equal to 0;

The bond between R ² and R ³ is a β,, 4 bond;

The bond between R ³ and R ⁴ is a β,, 3 bond;

The bond between R ⁴ and R ⁵ is a β,, 4 bond;

The bond between R ⁸ and R ⁹ is << 2, 3 bond;

The bond between R ⁹ and R ^1G is a β,, 3 bond; The bond between R ⁵ and R ^1G is a / 3 ^1,6 bond;

The bond between Z ¹ and R ³ is << 1, 3 bond;

The bond between Z ² and R ⁵ is << 1, 3 bond; and

P is a protecting group for an amino acid group (for example, a 9-fluorenylmethyloxycarbonyl group, a tert-butoxycarboxyl group, etc.),

Including the method.

[0167] In the method for producing a glycopeptide of the present invention, the series of reactions using the glycosyltransferase described above is carried out by using a dispensing device (dispensing device) or the like that can control the temperature of the reaction part as necessary. It can be done automatically by using.

[0168] (Mucin-type glycopeptide)

The present invention is useful in a wide range of fields such as biochemical research materials, pharmaceuticals, and foods by the novel primer described above and the method for producing glycopeptides using the same, and mucin type, which has been difficult to produce so far. Glycopeptides can be synthesized. Examples of mucin-type glycopeptides include the following formula:

[0169] [Chemical 29-1]

X ¹ X ¹ X ¹

Y '-His- Gly-Va 卜 Thr-Ser- Ala-Pro- Asp- Thr-Arg-Y ² (SEQ ID NO: 2)

[0170] [Chemical 29-2]

X ¹ X ¹ X ¹

Y ¹ -Ala-His- Gly-Val-Thr-Ser-AI a- Pro-Asp- Thr-Arg- γ2

(SEQ ID NO: 21)

Or

[0171] [Chemical 29- 3]

X ¹ X ¹ X ¹ X ¹ X ¹

(SEQ ID NO: 41)

[0172] [Chemical 29-4]

Wherein R ¹ and R ⁶ are each independently hydrogen, Neu5 Ac group or GlcNAc group;

R ² and R ⁷ are Gal groups;

R ³ and R ⁸ are GlcNAc groups;

R ⁴ and R ⁹ are Gal groups;

R ⁵ is a GlcNAc group;

R ¹⁰ is a GalNAc group;

The bond between R ² and R ³ is a β,, 4 bond;

The bond between R ³ and R ⁴ is a β,, 3 bond;

The bond between R ⁴ and R ⁵ is a β,, 4 bond; The bond between R ⁶ and R ⁷ is an α 2,3 bond when R ⁶ is a Neu5Ac group, and a j8 1,3 bond when R ⁶ is a GlcNA c group;

The bond between R ⁷ and R ⁸ is a β,, 4 bond;

Bond between R ⁸ and R ^9, β ΐ, is 3 bonds;

The bond between R ⁹ and R ^1G is a β,, 3 bond;

The bond between R ⁵ and R ^1G is a β,, 6 bond;

The bond between Ζ ¹ and R ³ is << ¹ , 3 bond;

The bond between Ζ ² and R ⁵ is << 1, 3 bond; and

The bond between Ζ ³ and R ⁸ is << 1, 3 bond,

Υ ¹ represents a hydrogen atom, acetyl, acyl, alkyl or aryl;

Υ ² represents a hydroxyl group, ΝΗ, alkyl or aryl.

2

The glycopeptide represented by these is mentioned.

[0173] The mucin-type glycopeptide of the present invention preferably has the following formula:

[0174] [Chemical 30]

(SEQ ID NO: 2)

[0175] [Chemical 31]

(SEQ ID NO: 21)

Or

[0176] [Chemical 32]

X ¹ X ¹ X ¹ X ¹ X ¹

(SEQ ID NO: 41)

[0177] [Chemical 33]

In which R ¹ is hydrogen, a sialic acid group or a GlcNAc group;

R ² is a Gal group;

R ³ is a GlcNAc group;

R ⁴ is a Gal group;

R ⁵ is a GlcNAc group;

R ¹⁰ is a GalNAc group;

R ⁸ is hydrogen or a sialic acid group;

R ⁹ is a Gal group;

Z ¹ and Z ² are each independently hydrogen or a Fuc group; and

n is an integer greater than or equal to 0;

The bond between R ² and R ³ is a β,, 4 bond;

The bond between R ³ and R ⁴ is a β,, 3 bond;

The bond between R ⁴ and R ⁵ is a β,, 4 bond;

The bond between R ⁸ and R ⁹ is << 2, 3 bond;

The bond between R ⁹ and R ¹⁰ is a β,, 3 bond;

The bond between R ⁵ and R ^1G is a β,, 6 bond;

The bond between Ζ ¹ and R ³ is << ¹ , 3 bond; and

The bond between Ζ ² and R ⁵ is << 1, 3 bond,

Υ ¹ represents a hydrogen atom, acetyl, acyl, alkyl or aryl;

Υ ² represents a hydroxyl group, ΝΗ, alkyl or aryl.

2

The glycopeptide represented by these is mentioned.

The mucin-type glycopeptide having a borilactosamine skeleton provided in the present invention is In serial X ^1, n is an integer of 0 or more, preferably, n is 0-5, more preferably, n is an integer of 4 or less. Further, in X ¹ , ¹ to ³ may or may not be present. Preferably, m is 0 to 5, and preferably, when n is 1, Z ¹ and Z ² are sialic acid groups. is there. More preferably, the sugar residue of the glycopeptide is

[Chemical 33-1]

[0179] (Mucin-type sugar amino acid)

According to the present invention, a mucin-type sugar amino acid can be provided. Examples of mucin-type sugar amino acids include the following formula (M ¹ ):

[0180] [Chemical 34]

R ² and R ⁷ are Gal groups;

R ³ and R ⁸ are GlcNAc groups;

R ⁴ and R ⁹ are Gal groups;

R ⁵ is a GlcNAc group;

R ¹⁰ is a GalNAc group;

R ¹¹ is a hydrogen atom or a methyl group;

The bond between R ² and R ³ is a β,, 4 bond;

The bond between R ³ and R ⁴ is a β,, 3 bond;

The bond between R ⁴ and R ⁵ is a β,, 4 bond; The bond between R ⁶ and R ⁷ is a 1,3 bond when R ⁶ is a Neu5Ac group, and a j81, 3 bond when R ⁶ is a GlcNA c group;

The bond between R ⁷ and R ⁸ is a j81, 4 bond;

The bond between R ⁸ and R ⁹ is a / 31,3 bond;

The bond between R ⁹ and R ^1G is a 1, 3 bond;

The bond between R ⁵ and R ^1G is a βΐ, 6 bond;

The bond between Ζ ¹ and R ³ is αΐ, 3 bond;

The bond between Ζ ² and R ⁵ is αΐ, 3 bond;

The bond between Ζ ³ and R ⁸ is an αΐ, 3 bond; and

Ρ is an amino acid protecting group (for example, 9 fluorenylmethyloxycarboxyl group, ter rt butoxycarbonyl group, etc.)

The sugar amino acid represented by these is mentioned.

[0181] The mucin-type sugar amino acid of the present invention preferably has the following formula (M ² ):

[0182] [Chemical 35]

Z1 Z2

2— R3 _R 4— _R 5

_R 8_ _R 9_ _R 10

In which R ¹ is hydrogen, a sialic acid group or a GlcNAc group;

R ² is a Gal group;

R ³ is a GlcNAc group;

R ⁴ is a Gal group;

R ⁵ is a GlcNAc group;

R ¹⁰ is a GalNAc group; R ⁸ is hydrogen or a sialic acid group;

R ⁹ is a Gal group;

R ¹¹ is a hydrogen atom or a methyl group;

Z ¹ and Z ² are each independently hydrogen or a Fuc group; and

n is an integer greater than or equal to 0;

The bond between R ² and R ³ is a β,, 4 bond;

The bond between R ³ and R ⁴ is a β,, 3 bond;

The bond between R ⁴ and R ⁵ is a β,, 4 bond;

The bond between R ⁸ and R ⁹ is << 2, 3 bond;

The bond between R ⁹ and R ¹⁰ is a β,, 3 bond;

The bond between R ⁵ and R ^1G is a β,, 6 bond;

The bond between Ζ ¹ and R ³ is << ¹ , 3 bond;

The bond between Ζ ² and R ⁵ is << 1, 3 bond; and

The sugar amino acid represented by these is mentioned.

In the present invention, the mucin-type sugar amino acid having a voralactosamine skeleton provided in the present invention is the above formula (M ¹ ) (M ² ), wherein n is an integer of 0 or more, preferably n is 0-5. Yes, and more preferably, n is an integer of 4 or less. Furthermore, in the above formula (M ¹ ) (M ² ), Τ˜τ ³ may or may not be present, but preferably m is 0-5, and preferably, when n is 1, Z ¹ and Z ² are sialic acid groups. Even more preferably, the above formulas (M ¹ ) and (M ² ) are

It may have a sugar or sugar derivative selected from the group consisting of

(Medicine and its treatment, prevention, etc.)

In another aspect, the present invention relates to a medicament (for example, a pharmaceutical product such as salmon cutin, a health food, a residual protein) containing a glycopeptide obtained by the production method of the present invention (for example, a mucin-type glycopeptide having a borilactosamine skeleton). Or lipids that have reduced antigenicity) Related. The medicament 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.

[0185] In the present specification, "borilactosamine" refers to a sugar chain containing a repeating structure of ratatosamine (eg, — 3Gal | 8 1-4GlcNA _{C j} 8 1-) unit as an oligosaccharide component. Polylactosamine is thought to be involved in congenital erythropoietic anemia (HEMPAS), neuronal differentiation, and reduction of autoimmune responses to immunocompromised carbohydrate antigens in fetal erythrocytes. In addition, borilactosamine forms part of specific glycoproteins such as laminin and lysosomal membrane protein (LAMP), and regulates cell-cell adhesion and signal transduction through the interaction of these molecules with lectins. There is a possibility that it is.

[0186] In this description! The “mucin-type glycopeptide having a borilactosamine skeleton” refers to a glycopeptide containing voractactamine as a skeleton. `` Mucin-type glycopeptides with a borilactosamine skeleton '' are related to congenital erythropoiesis anemia (HEMPAS), neuronal differentiation, reduction of autoimmune responses to immunocompromised carbohydrate antigens in fetal erythrocytes, etc.

[0187] Therefore, "mucin-type glycopeptide having a polylactosamine skeleton" is useful as a pharmaceutical related to the control of congenital erythropoiesis anemia (HEMPAS), cancer, neuronal differentiation, and the like.

[0188] Suitable formulation materials or pharmaceutically acceptable carriers for the medicament of the present invention include antioxidants, preservatives, colorants, flavors, and diluents, emulsifiers, suspending agents, solvents, fillers. Including, but not limited to, bulking agents, buffering agents, delivery vehicles, diluents, excipients and / or pharmaceutical adjuvants. Typically, the medicament of the present invention comprises a composition comprising an isolated pluripotent stem cell, 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 common substances in compositions for parenteral delivery. is there.

[0189] Acceptable carriers, excipients or stabilizers as used herein are Non-toxic and preferably inert at the dosages and concentrations used, and include: phosphates, citrates, or other organic acids; ascorbic acid A tocopherol; low molecular weight polypeptide; protein (eg, serum albumin, gelatin or immunoglobulin); hydrophilic polymer (eg, polybulurpyrrolidone); amino acid (eg, glycine, glutamine, asparagine, arginine or lysine); Monosaccharides, disaccharides and other carbohydrates (including glucose, mannose, or dextrin); chelating agents (eg, EDTA); sugar alcohols (eg, mantol or sorbitol); salt-forming counterions (eg, sodium) As well as Z or non-ionic surfactants ( For example, Tween, pulluric or polyethylene glycol (PEG)).

[0190] 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 Tris buffer at pH 7.0—8.5 or acetate buffer at pH 4.0—5.5, which are sardine, sorbitol or suitable substitutes thereof. Can be included.

[0191] The medicament of the present invention may 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 (e.g., intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, mucosal administration, rectal 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 preparation form include liquids, injections, and sustained release agents.

[0192] The medicament of the present invention may be a physiologically acceptable carrier, excipient, or stabilizer as necessary (Japanese Pharmacopoeia 14th edition or its latest edition, Remington's Pharmaceutical sciences, 18th Edition, AR Gennaro, ed., MacK Publishing Compan y, 1990, etc.) and a composition obtained by the production method of the present invention and containing a glycopeptide having a desired degree of purity (for example, a mucin-type glycopeptide) is frozen. It can be prepared and stored in the form of a dried cake or an aqueous solution.

[0193] The amount of the composition containing a glycopeptide (for example, a mucin-type glycopeptide having a borilactosamine skeleton) used in the treatment method of the present invention depends on the purpose of use, target disease (type, severity, etc.), patient's A person skilled in the art can easily determine the age, weight, sex, medical history, cell morphology or type, and the like. 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, gender, 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 once a few months every day (for example, once a week-once a month). It is preferable to administer once a week – once a month, while monitoring the course.

[0194] As described above, the power of the present invention exemplified by using the preferred embodiment of the present invention. The present invention should not be understood to be limited to this embodiment. It is understood that the scope of the present invention should be construed only by the claims. Those skilled in the art will understand that the description of specific preferred embodiments of the present invention can be implemented within the equivalent scope based on the description of the present invention and common general technical knowledge. The patents, patent applications, and documents cited herein are to be incorporated by reference in their entirety, as if the contents themselves were specifically described herein.

Example

[0195] Abbreviations used herein have the following meanings.

[0196] This study will be described in more detail with reference to the following examples, but this study is not limited thereto. An outline of the example described below is shown in FIG.

[0197] Abbreviations used in this example have the following meanings.

[0198] DMF = N, N dimethylformamide,

DCM = dichloromethane,

HOBT = N hydroxybenzotriazolone,

HBTU = 1-(Bis (dimethylamino) methylene) monobenzotriazolium 3 oxide Hexafluorophosphate,

DIEA = diisopropylethylamine,

Boc group = tert butoxycarbol group,

Fmoc group = 9 Fluorenylmethoxycarbo

Pbf group = 2, 2, 4, 6, 7- -5-sulfol group TFA = trifluoroacetic acid,

Fmoc— Ala— OH = N— a Fmoc— L-Afnin,

Fmoc-Gly-OH = N— — Fmoc— L glycine,

Fmoc— Pro— OH = N— a— Fmoc— L Proline,

Fmoc— Arg (Pbf) — OH = Ν- α -Fmoc-Νγ (2, 2, 4, 6, 7 penta-snorehoninole) L anoreginine,

Fmoc -Asp (OtBu) — OH = Ν— a—Fmoc— L Asanolagic acid β—t— butyl ester,

Fmoc-Gin (OtBu) OH = N— α— Fmoc—: L-gnoretamic acid β-t-butenoestestole,

Fmoc— Phe— OH = N-a Fmoc— L

Fmoc— Val— OH = N— a Fmoc— L—valine,

Fmoc— His (Trt) — OH = N— a— Fmoc— N— im Trityl mono L histidine,

Fmoc— Thr— OH = N— — Fmoc— L

Fmoc— Ser— OH = N— a Fmoc— L-serine,

Fmoc -Thr (Ac7core2)-OH = N— α— Fmoc— O— {O— (2, 3, 3, 4, 6, 6, tetra-tetra-O acetyleno β-D-galatatopyranosyl)-(1 , → 3) -0- [2 "—acetamide 1” 3 ”, 4”, 6, 1 tri 1 O acetyl 1 2, 1 doxy 1 13—D—Darkovyran (1 ”→ 6)] — 2 Acetamido 2 Deoxy a— D Galactopyranosyl} — L

Fmoc- Ser (Ac7core2)-OH = N— α— Fmoc— O— {O— (2, 3, 3, 4, 6, 6, tetra-tetra-O acetyleno β-D-galatatopyranosyl)-(1 , → 3) -0- [2 "—case Toamide 1 3 ", 4", 6, 1 Tri 1 O Acetyl 1 2, 1 Deoxy 1 β-D-Darcobyranosyl (1 "→ 6)] — 2 Acetamido 2 Deoxy a- D Galactopyranosyl} — L-serine,

(Example 1: Synthesis of MUC1-related glycopeptide derivatives (1) to (8) having a ketone derivative at the heel end)

(1.1 Synthesis of compounds (1) to (2))

[Chemical 36]

2y_g heGIUHisGlvalThrserAlaproAS> ThrArNH -----------

ho

T00.S0 / .00Zdf / X3d s LOZnilLOOZ OAV Tentagel S RAM (registered trademark) (Hipep Laboratories, 0.25 mmol / g) Using 0.24 g (0.06 mmol) as a carrier, the following N-protected amino acids and keto acids are sequentially condensed by the FmocZHBTU ZHOBt method. A glycopeptide derivative was synthesized. Fmoc— Arg (Pbf) — OH, Fmoc -Thr (Ac7core2) — OH, Fmoc— Asp (OtBu) — OH, Fmoc— Pro -OH, Fmoc— Ala— OH, Fmoc— Ser (tBu) — OH, Fmoc— Thr (tBu) -OH, Fmoc-Val-OH, Fmoc—Gly—OH, Fmoc—His (Trt) —OH, Fmoc—Glu (OtBu) —OH, Fmoc-Phe-OH, 5-ketohexanoic acid. After the peptide extension reaction, the protecting group on the peptide residue was removed by reacting in 95% TFA aqueous solution at room temperature for 2 hours, and the compound (1) was released from the solid phase carrier. The resin was filtered off and TFA was distilled off, and t-butyl methyl ether was added to precipitate the product. The resulting slurry was centrifuged, the supernatant was removed, and t-butyl methyl ether was added again to wash the precipitate. Centrifugation was performed again to remove the supernatant, and the resulting precipitate was dissolved in 3.0 ml of methanol. A 1N sodium hydroxide aqueous solution was added to this solution to adjust the pH to 12 to 12.5, and the mixture was stirred at room temperature for 1.5 hours to carry out a DeAc protection reaction. After the reaction, neutralize by adding 1N acetic acid, and then remove the solvent to remove the residue. Reverse phase HPLC (Inertsil (registered trademark) OD S-3 20 X 250 mm column (GL Sciences Inc., Tokyo), transfer Phase A: Purified with 0.1% TFA aqueous solution B: 0.1% TFA-containing acetonitrile with 2% to 50% gradient to give 27 mg of compound (2) (23% yield). MALDI-TOF / MS: [M (average) + H] ⁺ = 1997.0 (theoretical value: [M (average) + H] + = 1996.0).

[0200] (1.2 Synthesis of Compounds (3) to (4))

[0201] [Chemical 37]

yP2gG PheGluHis slValTh "erAlaproASThrArNH ---------- l-

l00Z.S0 / Z.00Zdf / X3d III Z.0CMI / Z.00Z OAV // D / O looz-soz-ooidTId / -οεΗΪζ-οοίAV 8_

V

^S HN-6 ”VJ 丄 -dsv-0Jd-eiV- S-JLU- | BA-A | £) -s! Hn | 〇-9Ljd

ΗΟΘΙΛΙ I HO ^ N

Tentagel S RAM (registered trademark) (Hipep Laboratories, 0.25 mmol / g) 0.67 g (0.045 mmol) as a carrier N-protected amino acid and keto acid shown below were sequentially condensed by Fmoc / HBT UZHOBt method, The desired glycopeptide derivative was synthesized. Fmoc— Arg (P bf) — OH, Fmoc— Thr (tBu) — OH, Fmoc— Asp (OtBu) — OH, Fmoc -Pro -OH, Fmoc— Ala— OH, Fmoc— Ser (tBu) — OH, Fmoc — Thr (Ac7core2) — OH, Fmoc-Val-OH, Fmoc— Gly—OH, Fmoc -His (Trt) —OH, Fmoc -Glu (OtBu) -OH, Fmoc— Phe— OH, 5-ketohexanoic acid . After the peptide elongation reaction, the protecting group on the peptide residue was removed by reacting in a 90% TFA aqueous solution at room temperature for 2 hours, and the compound (3) was released from the solid support. The resin was filtered off and TFA was removed by evaporation, and t-butyl methyl ether was added to precipitate the product. The resulting slurry was centrifuged, the supernatant was removed, and t-butyl methyl ether was added again to wash the precipitate. Centrifugation was performed again to remove the supernatant, and the resulting precipitate was dissolved in 3.Oml of methanol. A 1N aqueous sodium hydroxide solution was added to this solution to adjust the pH to 12 to 12.5, and the mixture was stirred at room temperature for 1.5 hours to carry out a DeAc protection reaction. After the reaction, 1N acetic acid was added for neutralization, the solvent was distilled off, and the residue was subjected to reverse phase HPLC (Inertsil (registered trademark) ODS 3 20 X 250 mm column (GL Science Inc., Tokyo), mobile phase A: 0. Purified by B: 1% TFA aqueous solution: 0.1% TFA-containing acetonitrile (2% to 50% dargent) to obtain 32 mg of Compound (4) (yield 36%). MALDI-TOF / MS: [M (average) + H] ⁺ = 1997.2, (theoretical value: [M (average) + H] + = 1996.0).

[0202] (1.3 Synthesis of Compounds (5) to (6))

[0203] [Chemical 38]

T00.S0 / .00Zdf / X3d 1-31.OCMT / .OOZ OAV Tentagel S RAM (registered trademark) (Hipep Laboratories, 0.25 mmol / g) 0.1 g (0.025 mmol) as a carrier, the following N-protected amino acid and keto acid are sequentially condensed by the Fmoc / HBT UZHOBt method, The desired glycopeptide derivative was synthesized. Fmoc-Ala-O H, Fmoc— Pro— OH, Fmoc— Pro— OH, Fmoc— Ala— OH, Fmoc— Thr (Ac 7core2) — OH, Fmoc— Ser (tBu) — OH, Fmoc— Gly— OH, Fmoc— Pro— OH, Fmoc— Ala— OH, Fmoc— Pro— OH, Fmoc— Arg (Pbf) — OH, Fmoc— T hr (tBu) — OH, Fmoc-Asp (OtBu) — OH, Fmoc— Pro— OH, Fmoc -Ala-OH, Fmoc-Ser (tBu) — OH, Fmoc -Thr (tBu) — OH, Fmoc— Val— OH, F moc— Gly— OH, Fmoc -His (Trt) — OH, Fmoc— Glu (OtBu) —OH, Fmoc-Phe-OH, 5 Ketohexanoic acid. After the peptide elongation reaction, the protecting group on the peptide residue was removed by reacting in 90% TFA aqueous solution at room temperature for 2 hours, and the compound (5) was released from the solid phase carrier. The coconut resin was filtered off and TFA was evaporated off, and t-butyl methyl ether was added to precipitate the product. The obtained slurry was centrifuged, the supernatant was removed, and t-butyl methyl ether was added again to wash the precipitate. Centrifugation was performed again to remove the supernatant, and the resulting precipitate was dissolved in 1.5 ml of methanol. A 1N sodium hydroxide aqueous solution was added to this solution to adjust the pH to 12 to 12.5, and the mixture was stirred at room temperature for 1.5 hours to carry out a DeAc protection reaction. After the reaction, the reaction mixture was neutralized by adding 1N acetic acid, the solvent was distilled off, and the residue was purified by reversed-phase HPLC (Inertsil (registered trademark) ODS 3 20 X 250 mm column (GL Science Inc., Tokyo), mobile phase A: 0 Purification with B to 1% TFA aqueous solution (2% to 50% gradient of 0.1% TFA-containing acetonitrile) yielded 0.5 mg of compound (6) (yield 7%). MALDI-TOF / MS: [M (average) + H] + = 2844.3 (theoretical value: [M (average) + H] + = 2843.0).

[0204] (1.4 Synthesis of Compounds (7) to (8))

[0205] [Chemical 39]

// u / O soz-soz-osdrld / -οεΗΪζ-οοίAV

Tentagel S RAM (registered trademark) (Hipep Laboratories, 0.25 mmol / g) 0.10 g (0. 0025 mmol) as a carrier, the following N-protected amino acids and keto acids are sequentially condensed by the FmocZHB TUZHOBt method, A glycopeptide derivative was synthesized. Fmoc-Ala-OH, Fmoc— Pro— OH, Fmoc— Pro— uH, Fmoc— Ala uH, Fmoc— Thr (Ac7core2) — OH, Fmoc— Ser (tBu) — OH, Fmoc— Gly— OH, Fmoc -Pro -OH, Fmoc— Ala— OH, Fmoc— Pro— OH, Fmoc— Arg (Pbf) — OH, Fmoc— Thr (Ac7core2) — OH, Fmoc— Asp (OtBu) — OH, Fmoc— Pro— OH, Fmoc — Ala— OH ゝ Fmoc -Ser (tBu) — OH, Fmoc— Thr (tBu) — OH, Fmoc— Val — OH, Fmoc— Gly— OH, Fmoc -His (Trt) — OH, Fmoc— Glu (OtBu) — OH, Fmoc-Phe-OH, 5 ketohexanoic acid. After the peptide elongation reaction, the protecting group on the peptide residue was removed by reacting in a 90% TFA aqueous solution at room temperature for 2 hours, and the compound (7) was released from the solid phase carrier. The coconut resin was filtered off and TFA was evaporated off, and t-butyl methyl ether was added to precipitate the product. After the resulting slurry was centrifuged, the supernatant was removed, and t-butyl methyl ether was added again to wash the precipitate. Centrifugation was performed again to remove the supernatant, and the resulting precipitate was dissolved in 1.5 ml of methanol. A 1N sodium hydroxide aqueous solution was added to this solution to adjust the pH to 12 to 12.5, and the mixture was stirred for 1.5 hours at room temperature to carry out a DeAc protection reaction. After the reaction, 1N acetic acid was added to neutralize, the solvent was distilled off, and the residue was subjected to reverse phase HPLC (Inertsil (registered trademark) ODS 3 20 X 250 mm force ram (GL Sciences Inc., Tokyo), mobile phase A: Purification by B: 0.1% TFA aqueous solution (2% to 50% gradient of 0.1% TFA-containing acetonitrile) gave 0.6 mg of compound (8) (yield 6%). MALDI—TOFZMS: [M (average) + H] + = 3 412.0 (theoretical value: [M (average) + H] + = 3411.5).

(Example 2: Synthesis of polymer primer for glycopeptide synthesis)

(2.1 Synthesis of compounds (10) to (11))

[0207] [Chemical 40] yP2GGsheIUHislvalThre "AlaproASThrAr? NH -----------

Ye

Yes

WOLSO / LOOZdT / lJd 931-OC I / OOZ OAV

"

A reaction solution of 1.5 ml of 10 mM glycopeptide derivative (2), 15 mM (converted to oxiamine residue) water-soluble polymer (9) and 50 mM sodium acetate buffer (pH 5.5) was stirred at room temperature for 24 hours. After completion of the reaction, the reaction solution was centrifuged and concentrated with an ultrafiltration filter 10K Apollo (registered trademark) 20 ml (Orbital Biosciences, LLC (MA, USA)), and 25 mM HEP ES buffer (pH 7.0) was added thereto. Washing was carried out by concentrating again, and 10 mM (theoretical content of glycopeptide) polymer (10) was obtained by adding water to a final volume of 1.5 ml. To identify the polymer (10), a part of the polymer (10) is treated with BLase, and the product (11) Done by getting.

MALDI-TOF / MS of compound (11): [M (average) + H] + = 1608.5 (theoretical value: [

M (average) + H] ⁺ = 1607. 6).

[0208] (2.2 Synthesis of Compounds (12) to (13))

[0209] [Chemical 41]

// u soz-soz-osdrld / -οεΗΪ-οοί

N-6JV-JLU-dsv-o "de | V- S- Up- A-person l〇-s! Hn | 〇-ei] d

9HNV! Ldlvul> I! Hv6JJdsOJeJ①Jles_A ------ ----

10 ml of a reaction solution of 10 mM glycopeptide derivative (4), 20 mM (converted to oxiamine residue) water-soluble polymer (9), 50 mM sodium acetate buffer (pH 5.5) at room temperature

Stir for hours. After completion of the reaction, the reaction solution was centrifuged and concentrated with an ultrafiltration filter 10K Apollo (registered trademark) 20 ml (Orbital Biosciences, LLC (MA, USA)), and 25 mM HEPES buffer (pH 7.0) was added thereto, and again. Washing was carried out by concentrating, and finally 10 ml (theoretical content of glycopeptide) of polymer (12) was obtained by adding water so that the volume became 1. Oml. To identify the polymer (12), a part of the polymer (12) is treated with BLase, This was done by obtaining the product (13).

MALDI—TOFZMS of compound (13): [M (average) + H] + = 1609.6 (theoretical value: [M (average) + H] ⁺ = 1608. 6).

[0210] (Example 3: Glycoelongation reaction of glycopeptide and polymer primer for glycopeptide synthesis)

(3.1 Synthesis of Compounds (14) to (15))

[0211] [Chemical 42]

UDP-galax

β1,4-galactosyltransferase

Phe-Glu-His-Gly-Val-Thr-Ser-Ala-Pro-Asp-Thr-Ara-NH ₂

Yes

s 9 ¾Νν MlvdlvMll>! ß '' sOJeJeJesi- -------

50 mM HEPES buffer (pH 7.6), 0.2 U / ml human-derived j8 1,4-galactose transferase (manufactured by Toyobo Co., Ltd.), 10 mM salty manganese, 9 mM uridine-1,5, monophosphate galactose ( UDP-Gal) and lml reaction solution containing 3.3 mM glycopeptide derivative (10) were stirred at 25 ° C for 90 minutes. After completion of the reaction, the reaction solution is centrifuged and concentrated by ultrafiltration filter ULTRAFRE- MC 10, OOONMWL Filter Unit (Millipore), and then 50mM HEPES buffer (pH 7.0) is added thereto and concentrated again. Finally, water was added so that the volume became 0.33 ml to obtain 10 mM (glycopeptide theoretical content) polymer (14). To identify the polymer (14), a part of the transglycosylation reaction solution is taken, B Lase (manufactured by Shionogi & Co., Ltd.) is added thereto, and the reaction solution is subjected to reverse phase HPLC (Inertsil (registered trademark) ODS-3 4.6 X Compound (15) was purified by purification using a 250 mm column, mobile phase A: B: 2% to 20% gradient of acetonitrile, to 25 mM ammonium acetate buffer (pH 6.5). Done by getting. MALDI—TOFZMS of compound (15): [M (average) + H]

= 1770. 3 [M (average) + H] ⁺ = 1769. 8).

[0212] (3.2 Synthesis of Compounds (16) to (17))

[0213] [Chemical 43]

Ye

v J "--one-, i

VHN KU HN O.

Ee oH〇,

HNV-lsvdlvl〇ll! __ lld6JJ _l ds0 "e" 3J _l _esl _l nl "----------

25 mM HEPES buffer ¾¾ (pH 7.5), 0.02 mU / ml derived from j8 1, 3— N-acetyl dalcosamine transferase (manufactured by Toyo Denshi Co., Ltd.), 20 mM sodium chloride, 40 mM uridine — 5, 2-dilin 1 ml of the reaction solution containing acid N-acetylyldarcosamine (UDP-GlcNAc) and 2.5 mM glycopeptide derivative (14) was stirred at 37 ° C for 24 hours. After completion of the reaction, the reaction solution is centrifuged and concentrated with an ultrafiltration filter ULTRAFRE- MC 10, OOONMWL Filter Unit (Millipore), and 50 mM HEPES buffer (pH 7.0) is added to the solution and concentrated again. The polymer was washed and water was added so that the final volume was 0.25 ml. Thus, a 10 mM (glycopeptide theoretical content) polymer (16) was obtained. Part of the polymer (16) was identified by treating with BLase (manufactured by Shionogi & Co., Ltd.), and the reaction mixture was reversed-phase HPLC (Inertsil (registered trademark) ODS— 3 4.6 X 250 mm column, mobile phase A: 25 mM Purification by B: acetonitrile gradient 2% to 20% of ammonium acetate buffer (pH 6.5) was carried out to obtain compound (17). Compound (17) MALDI— TOFZMS: [M (av

18

[] 0214

TOg poultry. ) U ([()) 瞄 erae + H 1974.0: Maverae + Η 1973.0 = = + S

50 mM HEPES buffer (pH 7.6), 0.4 U / ml baboon-derived j8 1,4-galactose transferase (manufactured by Toyobo Co., Ltd.), 10 mM salt-manganese, 10 mM uridine-5, dilin An lml reaction solution containing acid galactose (UDP-Gal) and 2 mM glycopeptide derivative (16) was stirred at 25 ° C for 60 minutes. After completion of the reaction, the reaction solution is centrifuged and concentrated by ultrafiltration filter ULTRAFRE- MC 10, OOONMWL Filter Unit (Millipore), and then 50 mM HEPES buffer (pH 7.0) is added and concentrated again. Finally, water was added so that the volume became 0.2 ml to obtain 10 mM (theoretical content of glycopeptide) polymer (18). Part of the polymer (18) was identified by treating with BLase (manufactured by Shionogi & Co., Ltd.), and the reaction mixture was reversed-phase HPLC (Inertsil (registered trademark) ODS— 3 4.6 X 250 mm column, mobile phase A: B: 2% of acetonitrile for 25 mM ammonium acetate buffer (pH 6.5) It was carried out by obtaining the compound (19) by purifying with a force of 20% gradient. MALDI—TOFZMS of compound (19): [M (average) + H] + = 21136. 1 (theoretical value: [M (ave)) + H] ⁺ = 21135. 1).

[0216] (3.4 Synthesis of Compounds (20) to (21))

[0217] [Chemical 45]

18

U DP- N-Acetylglucosamine

β1,3-Ν-acetylglucosamine transferase

-NH ₂

20

25 mM HEPES buffer ¾¾ (pH 7.5), 0.2 mU / ml derived from J8 1, 3— N-acetyldarcosamine transferase (manufactured by Toyo Denshi Co., Ltd.), 20 mM manganese salt, 40 mM uridine-5, diphosphoric acid A 1 ml reaction solution containing N-acetylyldarcosamine (UDP-GlcNAc) and 2.5 mM glycopeptide derivative (18) was stirred at 37 ° C. for 24 hours. After completion of the reaction, the reaction solution was filtered with an ultrafiltration filter ULTRAFRE— MC 10, OOONMWL Filter Unit (Millipo Reconcentrate by centrifugation, add 50 mM HEPES buffer (pH 7.0) to it, wash again by concentrating, and finally add water to a volume of 0.25 ml. To 10 mM (glycopeptide theoretical content) polymer (20). Part of the polymer (20) was identified by treating with BLase (manufactured by Shionogi & Co., Ltd.), and the reaction mixture was reversed-phase HPLC (Inertsil (registered trademark) ODS- 3 4.6 X 250 mm column, mobile phase A: 25 mM Purification by B: acetonitrile gradient from ammonium acetate buffer (pH 6.5) to give compound (21). MALDI-TOFZMS of compound (21): [M (average) + H] ⁺ = 23339. 0 (theoretical value: [M (average) + H] + = 23338.3).

[0218] (3.5 Synthesis of Compounds (22) to (23))

[0219] [Chem 46]

U DP-galactose

β1, 4-galactosyltransferase

Three

50mM HEPES buffer (pH 7.0), 0.4U / ml baboon-derived j8 1, 4-galactose transferase (manufactured by Toyobo), lOmM salt 匕 manganese, lOmM uridine-5, 2-dilin Acid gala An lml reaction solution containing kutose (UDP-Gal) and 2.3 mM glycopeptide derivative (20) was stirred at 25 ° C for 45 minutes. After completion of the reaction, the reaction solution is concentrated by centrifugation using an ultrafiltration filter ULTRAFRE—MC 10,000 NMWL Filter Unit (Millipore), and then added with 50 mM HEPES buffer (pH 7.0) and concentrated again. Finally, 10 mM (theoretical content of glycopeptide) polymer (22) was obtained by adding water to a volume of 0.23 ml. A part of the polymer (22) was identified by BLase (manufactured by Shionogi & Co., Ltd.), and the reaction mixture was reversed-phase HPLC (Inertsil (registered trademark) ODS— 3 4.6 X 250 mm column, mobile phase A: Purification by B: acetonitrile gradient 2% to 20% of 25 mM ammonium acetate buffer (pH 6.5) gave compound (23). MALDI—TOFZMS of compound (23): [M (average) + H] + = 2501.6 (theoretical value: [M (a verage) + H] ⁺ = 2500.4).

[0220] (3.6 Synthesis of compounds (24) to (25))

[0221] [Chemical 47]

UDP-N-acetyl glucosamine

β1,3-Ν-acetylglucosamine transferase

H '

s 3 ¾Νν lvdlv41l>! e β ”dsOJeJ①Jes ---------

50 mM HEPES buffer (pH 7.5), 0.2 U / ml porcine j8 1, 3-— N-acetyl darcosamine transferase (manufactured by Toyobo Co., Ltd.), 20 mM manganese salt, 40 mM uridine— -A 1 ml reaction solution containing N-acetyldarcosamine diphosphate (UDP-GlcNAc) and 2. OmM glycopeptide derivative (22) was stirred at 37 ° C for 18 hours. After completion of the reaction, the reaction solution is concentrated by centrifugation using an ultrafiltration filter ULTRAFRE— MC 10, OOONMWL Filter Unit (Millipore), and then concentrated again by adding 50 mM HEPES buffer (pH 7.0). The polymer was washed and water was added so that the final volume was 0.2 ml to obtain 10 mM (glycopeptide theoretical content) polymer (24). Part of the polymer (24) was identified with BLase (manufactured by Shionogi & Co., Ltd.), and the reaction mixture was reversed-phase HPLC (Inertsil (registered trademark) ODS- 3 4.6 X 250 mm column, mobile phase A: 25 mM) Purification by B: acetonitrile gradient from 2% to 20% of ammonium acetate buffer (pH 6.5) gave compound (25). MALDI-TOFZMS of compound (25): [M (average) + H] ⁺ = 2704.0 (theoretical value: [M (average) + H] + = 2703. 6).

[0222] (3.7 Synthesis of Compounds (26) to (27))

[0223] [Chemical 48]

U DP-Garac

β1 galactose transferase

26

¾NVMlvdlvsill> lo! H6JJdsOJe ”9e> s ---------

1 ml of a reaction solution containing kutose (UDP-Gal) and 1.8 mM glycopeptide derivative (24) was stirred at 25 ° C. for 45 minutes. After completion of the reaction, the reaction solution is centrifuged and concentrated by ultrafiltration filter ULTRAFRE- MC 10, OOONMWL Filter Unit (Millipore), and then 50mM HEPES buffer (pH 7.0) is added thereto and concentrated again. Finally, 10 mM (theoretical content of glycopeptide) polymer (26) was prepared by adding water so that the volume was 0.18 ml. Part of the polymer (26) was identified by BLase (manufactured by Shionogi & Co., Ltd.), and the reaction mixture was reversed-phase HPLC (Inertsil (registered trademark) ODS— 3 4.6 X 250 mm column, mobile phase A: This was carried out by purifying with B: acetonitrile 2% to 20% gradient against 25 mM ammonium acetate buffer (pH 6.5) to obtain compound (27). MALDI—TOFZMS of the compound (27): [M (average) + H] + = 28867.0 (theoretical value: [M (a verage) + H] ⁺ = 28865.8).

[0224] (3.8 Synthesis of Compounds (28) to (29))

[0225] [Chemical 49]

26

UDP-N-acetyl glucosamine

β1,3-Ν-acetylglucosamine transferase

OAV

50 mM HEPES buffer (pH 7.5), 0.2 U / ml pig-derived j8 1,3— N-acetyl darcosamine transferase (manufactured by Toyobo), 20 mM manganese salt, 40 mM uridine-5, —diphosphate N -Acetyldarcosamine (UDP- GlcNAc), 1.5 ml of a reaction solution containing 1.5 mM glycopeptide derivative (26) was stirred at 25 ° C for 45 minutes. After completion of the reaction, the reaction solution is concentrated by centrifugation using an ultrafiltration filter ULTRAFRE— MC 10, OOONMWL Filter Unit (Millipore), and then concentrated again by adding 50 mM HEPES buffer (pH 7.0). The polymer was washed and water was added so that the final volume was 0.15 ml to obtain a 10 mM (glycopeptide theoretical content) polymer (28). Part of the polymer (28) was identified with BLase (manufactured by Shionogi & Co., Ltd.), and the reaction mixture was reversed-phase HPLC (Inertsil (registered trademark) ODS- 3 4.6 X 250 mm column, mobile phase A: 25 mM. Purification by B: acetonitrile gradient from ammonium acetate buffer (pH 6.5) to give compound (29). MALDI-TOFZMS of compound (29): [M (average) + H] ⁺ = 3070.0 (theoretical value: [M (average) + H] + = 3069. 0).

[0226] (3.9 Synthesis of Compounds (30) to (31))

[0227] [Chemical 50]

28

30

/ DId / -οεΗΪ-οοί

50 mM HEPES buffer (pH 7.0), 0.2 U / ml human-derived j8 1,4-galactose transferase (manufactured by Toyobo Co., Ltd.), 10 mM salty manganese, 10 mM uridine-5, galactose diphosphate ( UDP-Gal) and lM reaction solution containing ImM glycopeptide derivative (28) were stirred at 25 ° C for 90 minutes. After completion of the reaction, the reaction solution is centrifuged and concentrated by ultrafiltration filter ULTRAFRE- MC 10, OOONMWL Filter Unit (Millipore), and then 50 mM HEPES buffer (pH 7.0) is added and concentrated again. Finally, water was added so that the volume became 0.1 ml, so that a 10 mM (theoretical content of glycopeptide) polymer (30) was obtained. Part of the polymer (30) was identified by treating with BLase (manufactured by Shionogi & Co., Ltd.), and the reaction solution was reversed-phase HPLC (Inertsil (registered trademark) ODS— 3 4.6 X 250 mm column, mobile phase A: This was carried out by purifying with 25 mM ammonium acetate buffer (pH 6.5) using B: acetonitrile at 2% strength and 20% gradient to obtain compound (31). MALDI—TOF / MS of compound (31): [M (average) + H] + = 23232.4 (theoretical value: [M (av erage) + H] ⁺ = 3231.1).

[0228] (3.10 Synthesis of Compounds (32) to (33))

[0229] [Chemical 51]

12

UDP-galactose

β1,4-galactose transferase

-Ser-Ala-Pro-Asp-Thr-Arg-NH ₂

50 mM HEPES buffer ¾¾ (pH 7.0), 0.05 U / ml Human-derived j8 1,4-galactose transferase (manufactured by Toyobo Co., Ltd.), lOmM salt 匕 manganese, ImM uridine-5, diphosphate galactose (UDP) — Gal), 0.1% BSA, containing 5 mM glycopeptide derivative (12) 1.2 ml of the reaction solution was stirred at 25 ° C for 60 minutes. After completion of the reaction, the reaction solution was concentrated by centrifugation using an ultrafiltration filter ULTRAFRE-MC 10, OOONMWL Filter Unit (Millipore), and 50 mM HEPES buffer (pH 7.0) was added to it and concentrated again. LOmM (sugar) by washing and adding water to a final volume of 0.6 ml. Theoretical peptide content) polymer (32). Part of the polymer (32) was identified by treatment with BLase (Yoshio Shiono Pharmaceutical), and the reaction mixture was reversed-phase HPLC (Inertsil (registered trademark) ODS— 3 4.6 X 250 mm column, mobile phase A: 10 mM B: 10% ammonium acetate buffer (pH 6.5) B: 10% 10 mM ammonium acetate buffer (pH 6.5) containing acetonitrile containing 2% strength and 40% gradient) ). MA LDI-TOF / MS of compound (33): [M (average) + H] ⁺ = 1770.8 (theoretical value: [M (average) + H] + = 1770.8).

[0230] (3.1 Synthesis of Compounds (34) to (35))

[0231] [Chemical 52]

UDP-N-Acetyldarcosamine

β1,3-Ν-acetylglucosamine transferase

¾NvsMlvdlovUp> l! H6 ”Jdso” eJL> s ----------

50 mM HEPES buffer (pH 7.5), 0.019 mU / ml derived from j8 1, 3—N-acetyl laccosamine transferase (manufactured by Toyobo Co., Ltd.), 20 mM salty manganese, 40 mM uridine— 2. Oml reaction solution containing N-acetylethylcolcamine phosphate (UDP-GlcNAc), 0.5% BSA, and 2.5 mM glycopeptide derivative (32) was stirred at 37 ° C for 24 hours. Anti After completion of the reaction, the reaction solution is centrifuged and concentrated with an ultrafiltration filter ULTRAFRE- MC 10, OOONMWL Filter Unit (Millipore), and 50 mM HEPES buffer solution (PH7.0) is added to it and concentrated again. After washing, water was added to a final volume of 0.5 ml to obtain a 10 mM (glycopeptide theoretical content) polymer (34). For identification of the polymer (34), a part of it was treated with BLase (manufactured by Shionogi & Co., Ltd.), and the reaction solution was reversed-phase HPLC (Inertsil (registered trademark) ODS— 3 4.6 X 250 mm column, mobile phase A: 10mM acetate Ann mode - © beam buffer (ρΗ ⁶ 5.) for B: 10% 10mM acetate ammonium - © beam buffer (. pH ⁶ 5) to give gradient) from 2% to 40% of the content Asetonitoriru This was carried out by obtaining compound (35). MALDI-TOFZMS of compound (35): [M (average) + H] "= 1974.3 (theoretical value: [M (avemge) + H] + = 1973. 0).

[0232] (3.12 Synthesis of Compounds (36) to (37))

[0233] [Chemical 53]

U DP-galactose

β1,4-galactosyltransferase

S ¾Nv_lvdlvlll> I! ”LdsOJeJe JleAs ----------

卜

50 mM HEPES buffer (pH 7.0), 0.05 U / ml Human-derived j8 1, 4-galactose transferase (manufactured by Toyobo Co., Ltd.), 10 mM sodium chloride, 20 mM uridine-5, diphosphate Contains galactose (UDP-Gal), 0.1% BSA, 5 mM glycopeptide derivative (34) 1. The Oml reaction was stirred at 25 ° C for 45 minutes. After completion of the reaction, the reaction solution is centrifuged and concentrated with an ultrafiltration filter, ULTRAFRE-MC 10,000 NMWL Filter Unit (Millipore), and 50 mM HEPES buffer (pH 7.0) is added thereto and concentrated again. After washing, water was added so that the final volume became 0.5 ml to obtain a 10 mM (glycopeptide theoretical content) polymer (36). Part of the polymer (36) was identified by treating with BLase (Yoshio Shiono Pharmaceutical), and the reaction mixture was reversed-phase HPLC (Inertsil (registered trademark) ODS— 3 4.6 X 250 mm column, mobile phase A: 10 mM Compound (37) purified by B: 10% 10 mM ammonium acetate buffer (pH 6.5) containing 2% strength of 40% gradient of ammonium acetate buffer (pH 6.5) against ammonium acetate buffer (pH 6.5) Was done by MA LDI-TOF / MS of compound (37): [M (average) + H] + = 213.61 (theoretical value: [M (average) + H] + = 21135. 1).

[0234] (3.13 Synthesis of Compounds (38) to (39))

[0235] [Chemical 54]

UDP-N-acetyl crucosamine

β1,3-Ν-acetylglucosamine transferase

50 mM HEPES buffer (pH 7.5), 0.019 mU / ml Pig-derived j8 1, 3— N-Acetyldarcosamine transferase (manufactured by Toyobo Co., Ltd.), 20 mM salted manganese salt, 40 mM uridine — 5, 2 2. Oml reaction solution containing N-acetylyldarcosamine phosphate (UDP-GlcNAc), 0.5% BSA, 2.5 mM glycopeptide derivative (36) was stirred at 37 ° C for 24 hours. After completion of the reaction, the reaction mixture is centrifuged and concentrated with an ultrafiltration filter ULTRAFRE—MC 10,000 NMWL Filter Unit (Millipore), and 50 mM HEPES buffer (pH 7.0) is added to it and concentrated again. After washing with water, 10 mM (theoretical content of glycopeptide) polymer (38) was obtained by adding water to a final volume of 0.5 ml. For identification of the polymer (38), a part was treated with BLase (manufactured by Shionogi Pharmaceutical Co., Ltd.), and the reaction solution was reversed-phase HPLC (Inertsil (registered trademark) ODS— 3 4.6 X 250 mm column, mobile phase A: 10mM acetate Ann mode - © beam buffer (ρΗ ⁶ 5.) for B: 10% 10mM acetate ammonium - © beam buffer (. pH ⁶ 5) to give gradient) from 2% to 40% of the content Asetonitoriru This was carried out by obtaining compound (39). MALDI—TOFZMS of compound (39): [M (average) + Na] ⁺ = 236.0.0 (theoretical value: [M (average) + H] + = 23361.3).

[0236] (3.14 Synthesis of Compounds (40) to (41))

[0237] [Chemical 55]

U DP-galactose

β1,4-Gala exogenous monotransferase

BLase

T00.S0 / .00Zdf / X3d OilOC I / OOZ OAV 50 mM HEPES buffer (pH 7.0), 0.05 U / ml Human-derived j8 1, 4-galactose transferase (manufactured by Toyobo Co., Ltd.), 10 mM sodium chloride, 20 mM uridine-5, diphosphate galactose (UDP) Gal), 0.1% BSA, 5 mM glycopeptide derivative (38) containing 0.8 ml of the reaction solution was stirred at 25 ° C. for 105 minutes. After completion of the reaction, the reaction solution is concentrated by centrifugation using an ultrafiltration filter, ULTRAFRE- MC 10, OOONMWL Filter Unit (Millipore), and 50 mM HEPES buffer (pH 7.0) is added thereto and concentrated again. After washing, water was added so that the final volume became 0.4 ml to obtain a 10 mM (glycopeptide theoretical content) polymer (40). Part of the polymer (40) was identified using BLase (manufactured by Shionogi & Co., Ltd.), and the reaction mixture was reversed-phase HPLC (Inertsil (registered trademark) ODS— 3 4.6 X 250 mm column, mobile phase A: 10 mM B against Ammonium acetate buffer (pH 6.5): Compound purified by purification with B: 10% 10 mM ammonium acetate buffer (pH 6.5) 2% strength of acetonitrile containing 40% gradient (41) Was done by MA LDI-TOF / MS of the compound (41): [M (average) + H] + = 24999.5 (theoretical value: [M (average) + H] + = 2500.4).

[0238] (3. 15 Synthesis of Compounds (42) to (43))

[0239] [Chem 56]

UDP-N-acetyl glucosamine

β1,3-Ν-acetylglucosamine transferase

42

50 mM HEPES buffer (pH 7.5), 0.019 mU / ml Pig-derived j8 1, 3— N-Acetyldarcosamine transferase (manufactured by Toyobo Co., Ltd.), 20 mM salted manganese salt, 40 mM uridine — 5, 2 1.6 ml of a reaction solution containing N-acetyldarcosamine phosphate (UDP-GlcNAc), 0.5% BSA, and 2.5 mM glycopeptide derivative (40) was stirred at 37 ° C. for 24 hours. After completion of the reaction, the reaction mixture is centrifuged and concentrated with an ultrafiltration filter ULTRAFRE—MC 10,000 NMWL Filter Unit (Millipore), and 50 mM HEPES buffer (pH 7.0) is added to it and concentrated again. After washing with water, 10 mM (theoretical content of glycopeptide) polymer (42) was obtained by adding water to a final volume of 0.4 ml. For identification of the polymer (42), a part of it was treated with BLase (manufactured by Shionogi & Co., Ltd.), and the reaction solution was reversed-phase HPLC (Inertsil (registered trademark) ODS— 3 4.6 X 250 mm column, mobile phase A: 10mM acetate Ann mode - © beam buffer (ρΗ ⁶ 5.) for B: 10% 10mM acetate ammonium - © beam buffer (. pH ⁶ 5) to give gradient) from 2% to 40% of the content Asetonitoriru This was done by obtaining compound (43). MALDI-TOFZMS of the compound (43): [M (average) + H] "= 27033.2 (¾ | ^ fg: [M (average) + H] ⁺ = 2703. 6).

[0240] (3. Synthesis of compounds (44) to (45))

[0241] [Chemical 57]

42

UDP-galactose

β1,4-Gala exogenous monotransferase

His-Gly-Va ^ r-Ser-Ala-Pro-Asf Thr-Arg-NH ₂

50 mM HEPES buffer (pH 7.0), 0.05 U / ml Human-derived j8 1, 4-galactose transferase (manufactured by Toyobo Co., Ltd.), 10 mM sodium chloride, 20 mM uridine-5, diphosphate galactose (UDP) (Gal), 0.1% BSA, 5 mM glycopeptide derivative (42) containing 0.6 ml of the reaction solution was stirred at 25 ° C. for 60 minutes. After completion of the reaction, the reaction solution is concentrated by centrifugation using an ultrafiltration filter, ULTRAFRE- MC 10, OOONMWL Filter Unit (Millipore), and 50 mM HEPES buffer (pH 7.0) is added thereto and concentrated again. After washing, water was added so that the final volume was 0.3 ml to obtain 10 mM (theoretical content of glycopeptide) polymer (44). Part of the polymer (44) was identified using BLase (manufactured by Shionogi & Co., Ltd.), and the reaction mixture was reversed-phase HPLC (Inertsil (registered trademark) ODS— 3 4.6 X 250 mm column, mobile phase A: 10 mM Compound B (45) against ammonium acetate buffer (pH 6.5): purified by B: 10% 10 mM ammonium acetate buffer (pH 6.5) with 2% strength of acetonitrile containing 40% gradient Was done by MA LDI-TOF / MS of compound (45): [M (average) + H] + = 28655.5 (theoretical value: [M (average) + H] + = 2865. 8).

[0242] (3. 17 Synthesis of compounds (46) to (47))

[0243] [Chemical 58]

UDP good acetyl glucosamine

β1,3-N-acetyl glucosamine transferase

// D / O looz-soz-ooidTId / -οεΗΪζ-οοίAV 6Ζ_ ·

50 mM HEPES buffer (pH 7.5), 0.019 mU / ml Pig-derived j8 1, 3— N-Acetyldarcosamine transferase (manufactured by Toyobo Co., Ltd.), 20 mM salted manganese salt, 40 mM uridine — 5, 2 0.8 ml of a reaction solution containing N-acetyldarcosamine phosphate (UDP-GlcNAc), 0.5% BSA, and 2.5 mM glycopeptide derivative (44) was stirred at 37 ° C for 24 hours. After completion of the reaction, the reaction mixture is centrifuged and concentrated with an ultrafiltration filter ULTRAFRE—MC 10,000 NMWL Filter Unit (Millipore), and 50 mM HEPES buffer (pH 7.0) is added to it and concentrated again. After washing with water, 10 mM (theoretical content of glycopeptide) polymer (46) was obtained by adding water to a final volume of 0.2 ml. For identification of the polymer (46), a part of it was treated with BLase (manufactured by Shionogi & Co., Ltd.), and the reaction solution was reversed-phase HPLC (Inertsil (registered trademark) ODS— 3 4.6 X 250 mm column, mobile phase A: 10mM acetate Ann mode - © beam buffer (ρΗ ⁶ 5.) for B: 10% 10mM acetate ammonium - © beam buffer (. pH ⁶ 5) to give gradient) from 2% to 40% of the content Asetonitoriru This was done by obtaining compound (47). MALDI—TOFZMS of the compound (47): [M (average) + H] ”= 3067.1 (theoretical value: [M (average) + H] + = 3069.0).

[0244] (3. 18 Synthesis of Compounds (48) to (49))

[0245] [Chemical 59]

UDP-outside—

β1,4-galactosyltransferase

48

T00.S0 / .00Zdf / X3d 381 50 mM HEPES buffer (pH 7.0), 0.05 U / ml Human-derived j8 1, 4-galactose transferase (manufactured by Toyobo Co., Ltd.), 10 mM sodium chloride, 20 mM uridine-5, diphosphate galactose (UDP) Gal), 0.1% BSA, 5 mM glycopeptide derivative (46) containing 0.2 ml reaction solution was stirred at 25 ° C. for 60 minutes. After completion of the reaction, the reaction solution is concentrated by centrifugation using an ultrafiltration filter, ULTRAFRE- MC 10, OOONMWL Filter Unit (Millipore), and 50 mM HEPES buffer (pH 7.0) is added thereto and concentrated again. After washing, water was added so that the final volume was 0.1 ml to obtain a 10 mM (glycopeptide theoretical content) polymer (48). Part of the polymer (48) was identified with BLase (manufactured by Shionogi & Co., Ltd.), and the reaction mixture was reversed-phase HPLC (Inertsil (registered trademark) ODS— 3 4.6 X 250 mm column, mobile phase A: 10 mM B against Ammonium acetate buffer (pH 6.5): Compound purified by purification with B: 10% ammonium acetate buffer (pH 6.5) containing 2% strength of 40% gradient (49) Was done by MA LDI-TOF / MS of the compound (49): [M (average) + H] + = 32333.4 (theoretical value: [M (average) + H] + = 3231.1).

[0246] (3. 19 Synthesis of Compound (50))

[0247] [Chemical 60]

25 mM HEPES buffer (pH 7. 6), 0.05 U / ml Human-derived j8 1, 4-galactose transferase (manufactured by Toyobo), lOmM salt 匕 manganese, 2.4 mM uridine-5, diphosphate galactose ( UDP-Gal) containing 0.5% BSA and 0.088 mM glycopeptide derivative (6) 2. The Oml reaction solution was stirred at 25 ° C for 180 minutes. After completion of the reaction, the reaction solution was purified by reverse phase HPLC to obtain compound (50). MALDI—TOFZMS of compound (50): [M (ave rage) + H] ⁺ = 3006.0 (¾ | ^ fg: [M (average) + H] ⁺ = 3005.1).

[0248] (3. 20 Synthesis of Compound (51))

[0249] [Chemical 61]

50 mM HEPES buffer (pH 7.5), 4. 55 mU / l Pig-derived j8 1,3— N-acetyldarcosamine transferase (manufactured by Toyo Denshi Co., Ltd.), 20 mM manganese salt, 40 mM uridine-5, diphosphate Reaction solution of 20 0 / z 1 containing N-acetylcylcosamine (UDP—GlcNAc), 0. ImM glycopeptide derivative (50) (estimated from theoretical yield from compound (6) to compound (50)) The mixture was stirred at 37 ° C for 24 hours. After completion of the reaction, the reaction solution was purified by reverse phase HPLC to obtain compound (51). MALDI—TOFZMS of the compound (51): [M (average) + H] ⁺ = 3209.9 (M m: [M (average) + H] ⁺ = 32088.3).

[0250] (3.2 Synthesis of Compound (53))

[0251] [Chemical 62]

50 mM HEPES buffer (pH 7.0), 0.1 uU / ml human-derived j8 1,4-galactose transferase (manufactured by Toyobo), 0.0185 mUZml rat recombinant a 2, 3- (N) -sialic acid transfer Enzyme (Calbiochem), 0.0175mU / ml rat recombinant α 2, 3— (Ο) —sialyltransferase (Calbiochem) 10mM manganese chloride, 5mM uridine 5, galactose monophosphate (UDP—) Gal), 5 mM cytidine-5, -phosphosialic acid (CMP—N ANA), 0.1% BSA, 0.02 mM glycopeptide derivative (51) (from compound (6) to compound (51) 1. The Oml reaction solution was stirred at 25 ° C for 24 hours. After completion of the reaction, the reaction solution was purified by reverse phase HPLC to obtain Compound (53). MALDI—TOF / MS of compound (53): [M (average) + ΗΓ = 3954.1 (theoretical value: [M (average) + H] + = 3953. 0).

[0252] (3.2 Synthesis of Compound (54))

[0253] [Chemical 63]

mM HEPES buffer (pH 7.0), lOmM salt and manganese, 0.1% BSA, 0.1 U Zml human recombinant α ΐ, 3-Fucose transferase V (Calbiochem), 2 mM guanosine-5, monodiphosphate fucose (GDP-Fuc), 0.08 mM glycopeptide derivative (53) (compound (6) To the compound (53) (estimated from the theoretical yield) to 200 μl of the reaction solution was stirred at 25 ° C. for 24 hours. After completion of the reaction, the reaction solution was purified by reverse phase HPLC to obtain a compound (54). MALDI—TOFZMS of the compound (54): [M (average) + H] + = 424 5.4 (¾ | ^ fg: [M (average) + H] ⁺ = 42245.2).

[0254] (3.2 Synthesis of Compound (55))

[0255] [Chemical 64]

25 mM HEPES buffer (pH 7. 6), 0.05 U / ml Human-derived j8 1, 4--galactose transferase (manufactured by Toyobo Co., Ltd.), lOmM salt and manganese, 2.4 mM uridine-5, diphosphate Contains galactose (UDP-Gal) and 0.08 mM glycopeptide derivative (8) 2. The Oml reaction was stirred at 25 ° C for 180 min. After completion of the reaction, the reaction solution was purified by reverse phase HPLC to obtain a compound (55). MALDI of compound (55) —TOFZMS: [M (average) + H] + = 3 736.5 (¾ | ^ fg: [M (average) + H] ⁺ = 3735. 8).

[0256] (Synthesis of 3.24 Compound (56))

[0257] [Chemical 65]

50 mM HEPES buffer (pH 7.5), 3. 85 mU / l pig-derived j8 1, 3— N-acetyldarcosamine transferase (manufactured by Toyo Corporation), 20 mM manganese salt, 40 mM uridine— 500-μl reaction solution containing 5, -diphosphate N-acetyldarcosamine (UDP-GlcNAc), 0.5% BSA, 0. ImM glycopeptide derivative (55) was stirred at 37 ° C for 60 hours . After completion of the reaction, the reaction solution was purified by reverse phase HPLC to obtain compound (50). M of compound (56)

ALDI-TOF / MS: [M (average) + H] + = 414.3 (Theoretical value: [M (average) +

H] ⁺ = 4142. 2).

[0258] (3. 25 Synthesis of Compound (58))

[0259] [Chemical 66]

idan! —- 翻 Nio¾ ΛΞ ヽ 1ii: f ---

50 mM HEPES buffer (pH 7.0), 0.1 uU / ml human-derived j8 1,4-galactose transferase (manufactured by Toyobo), 0.0185 mUZml rat recombinant a 2, 3- (N) -sialic acid transfer Enzyme (Calbiochem), 0.0169mU / ml Rat recombinant α 2, 3— (Ο) —Sialyltransferase (Calbiochem) 10mM manganese chloride, 5mM uridine 5, monophosphate galatose (UDP—) Gal), 5 mM cytidine-5, -phosphosialic acid (CMP— N ANA), 0.1% BSA, 0.05 mM glycopeptide derivative (56) (from compound (8) to compound (56) 1. The Oml reaction solution was stirred at 25 ° C for 24 hours. After completion of the reaction, the reaction solution was purified by reverse phase HPLC to obtain compound (58). MALDI—TOFZMS of compound (5 8): [M (average) + ΗΓ = 5631.5 (theoretical value: [M (average) + H] + = 5631.9).

[0260] (3. 26 Synthesis of Compound (59))

[0261] [Chemical 67]

0 mM HEPES buffer ¾¾ (pH 7.0), lOmM salt-manganese, 0.1% BSA, 0.1 U ml human recombinant a 1,3-fucose transferase V (Calbiochem), 2 mM guano A reaction solution of 625 1 containing Shin-5, monodiphosphate fucose (GDP-Fuc) and 0.08 mM glycopeptide derivative (58) was stirred at 25 ° C for 24 hours. After completion of the reaction, the reaction solution was purified by reverse phase HPL C to obtain compound (59). MALDI of compound (59) —TOFZMS: [M (av erage) + H] ⁺ = 62216.8 (theoretical value: [M (average) + H] + = 6216. 0).

[0262] (3. 27 Synthesis of Compounds (60) to (61))

[0263] [Chemical 68]

T00.S0 / .00Zdf / X3d 003 .0CMT / .00Z OAV

10mM Glycopeptide Derivatives (6) 0. lml, lOmM (converted to oxiamine residue) High water solubility A total of 0.4 ml of molecule (9) 0.2 ml, 50 mM sodium acetate buffer (pH 5.5) 0.1 ml was stirred at room temperature for 21 hours. After completion of the reaction, the reaction solution is concentrated by centrifugation using an ultrafiltration filter 10K Apollo (registered trademark) 20 ml (Orbital Biosciences, LLC (MA, USA)), and 25 mM HEPES buffer (pH 7.0) is added thereto and concentrated again. After washing, water was added so that the volume finally reached 961 to obtain a 10 mM (glycopeptide theoretical content) polymer (60). The polymer (60) was identified by treating a part of the polymer (60) with BLase to obtain the product (61).

MALDI—TOFZMS of compound (61): [M (average) + H] + = 2456. 1 (theoretical value: [

M (average) + H] + = 2454. 6).

[0264] (3. 28 Synthesis of Compounds (62) to (63))

[0265] [Chemical 69]

// D O soz-soz-ooidTId / -οεΗΪ-οοίAV εοδ

峯 ^

// u O soz-soz-ooidrld / -οεΗΪ-οοίAV

N-eiV-o ”d—oJd-e | V-J

Transfer enzyme (manufactured by Toyobo Co., Ltd.), 10 mM salt-manganese, ImM uridine-1,5 monodiphosphate galactose (UDP—Gal), 0.1% BSA, 0. ImM glycopeptide derivative (60) 50 μ The reaction solution of 1 was stirred at 25 ° C for 45 minutes. After completion of the reaction, the reaction solution was concentrated by centrifugation using an ultrafiltration filter ULTRAFRE-MC 10, OOONMWL Filter Unit (Millipore), and 50 mM HEPES buffer (pH 7.0) was added to it and concentrated again. Then, it was washed and water was added so that the volume finally reached 71. Thus, a polymer of 0.71 mM (theoretical content of glycopeptide) was obtained (62). A part of the polymer (62) was identified by treating with BLase (Yoshio Shiono Pharmaceutical Co., Ltd.), and the reaction mixture was reversed-phase HPLC (Inertsil (registered trademark) ODS— 3 4.6 X 250 mm column, mobile phase A: 0 Purified by B: 1% TFA aqueous solution B: 0.1% TFA-containing acetonitrile, 2% to 50% gradient) to obtain compound (63). MALDI—TOFZMS of compound (63): [M (average) + H] + = 2617.6 (theoretical value: [M (average) + H] + = 2616.7).

[0266] (3. 29 Synthesis of Compounds (64) to (65))

[0267] [Chemical 70]

// D / O soz-soz-ooidTId / -οεΗΪζ-οοίAV 90S

39

BLase

His-Gly-Va 卜 Thr-Ser-Ala-Pro-Asp-Thr-Arg-Pro-Ala-P `` o-Gly-Ser-Thr-Ala-Pro-Pro-Ala-NH2

50 mM HEPES buffer (pH 7.5), 3. 85 mU / l Pig-derived j8 1,3-—N-acetyl darcosamine transferase (Toyobo), 20 mM Manganese chloride, 10 mM uridine-5, —diphosphate N —40 μl of a reaction solution containing acetylyldarcosamine (UDP—GlcNAc), 0.5% BSA, and 0.13 mM glycopeptide derivative (62) was stirred at 37 ° C. for 43 hours. After completion of the reaction, the reaction solution is concentrated by centrifugation using an ultrafiltration filter ULTRAFRE— MC 10, OOONMWL Filter Unit (Millipore), and then concentrated by adding 50 mM HEPES buffer (pH 7.0) and concentrating it again. Then, water was added so that the volume finally reached 18 1 to obtain 0.28 mM (glycopeptide theoretical content) polymer (64). Part of the polymer (6 4) was identified by treating with BLase (manufactured by Shionogi & Co., Ltd.), and the reaction mixture was reversed-phase HPLC (Inert sil (registered trademark) ODS— 3 4.6 X 250 mm column, mobile phase A : 0.1% TFA aqueous solution B: 0.1% TFA-containing acetonitrile: 2% to 50% gradient). MALDI-TOFZMS of the compound (65): [M (average) + H] "= 2820.7 (Mm: [M (average) + H] ⁺ = 2819.9).

[0268] (3. 30 Synthesis of Compounds (66) to (67))

[0269] [Chemical 71]

// D / O soz-soz-ooidTId / -οεΗΪζ-οοίAV 60S

OAV 9

50 mM HEPES buffer (pH 7.0), 0.05 U / ml Human-derived j8 1, 4--galactose transferase (manufactured by Toyobo Co., Ltd.), 10 mM sodium chloride manganese, 1. OmM uridine-5, diphosphate gallium A 90 1 reaction solution containing ratatoose (UDP-Gal) and 0.06 mM glycopeptide derivative (64) was stirred at 25 ° C. for 90 minutes. After completion of the reaction, the reaction solution is centrifuged and concentrated with an ultrafiltration filter ULTRAFR E-MC 10, OOONMWL Filter Unit (Millipore), and 50 mM HEPES buffer (pH 7.0) is added to the solution and concentrated again. After washing with water, water was added so that the final volume was 22 1 to obtain 0.23 mM (glycopeptide theoretical content) polymer (66). Part of the polymer (66) was identified with BLase (manufactured by Shionogi & Co., Ltd.), and the reaction mixture was reversed-phase HPLC (Inertsil (registered trademark) ODS— 3 4.6 X 250 mm force ram, mobile phase A: 0.1% TFA aqueous solution B: 0.1% TFA-containing acetonitrile was purified by 2% strength (50% gradient). MALDI—TOF / MS of compound (67): [M (average) + H] + = 2982.0 (theoretical value: [M (average) + H] + = 29820.0).

[0270] (3. 31 Synthesis of Compounds (68) to (69))

[0271] [Chemical 72]

// D / O looz-soz-ooidTId / -οεΗΪζ-οοίAV

| _ |

// u O soz-soz-oozdrld / -οεΗΪ-οοίAV ε

50 mM HEPES buffer (pH 7.5), 7.7 mU / l Pig-derived j8 1, 3-— N-acetyl darcosamine transferase (manufactured by Toyobo Co., Ltd.), 20 mM manganese salt, 10 mM uridine-5, —diphosphate N —Acetyldarcosamine (UDP—GlcNAc), 0.5% BSA, 0.06 mM glycopeptide derivative (66) containing 80 μ 1 reaction solution was stirred at 37 ° C. for 24 hours. After completion of the reaction, the reaction solution is concentrated by centrifugation using an ultrafiltration filter ULTRAFRE— MC 10, OOONMWL Filter Unit (Millipore), and then concentrated by adding 50 mM HEPES buffer (pH 7.0) and concentrating it again. Then, water was added so that the final volume was 161 to obtain 0.3 ImM (glycopeptide theoretical content) polymer (68). Part of the polymer (6 8) was identified with BLase (manufactured by Shionogi & Co., Ltd.), and the reaction mixture was reversed-phase HPLC (Inert sil (registered trademark) ODS— 3 4. 6 X 250 mm column, mobile phase A : 0.1% TFA aqueous solution B: 0.1% TFA-containing acetonitrile: 2% to 50% gradient). MALDI—TOFZMS of the compound (69): [M (average) + H] ”= 3184.8 (¾ | ^ fg: [M (average) + H] ⁺ = 318.5.2).

[0272] (3.3 Synthesis of Compounds (70) to (71))

[0273] [Chemical 73]

yPy2 heGIUHisGlvalThrserAlaproASTh ArapoAlaproGlserThAlaproproNH ---------------------

TOO.SO/.OOZdf/X3d 91-3 LOZnilLOOZ OAV

mM HEPES buffer (pH 7.0), 0.05 U / ml Human-derived j8 1, 4-galatato Transfer enzyme (manufactured by Toyobo Co., Ltd.), 10 mM salt and manganese, 1. OmM uridine-5, -diphosphate galactose (UDP-Gal), 0.0 1 mM solution containing 80 1 reaction solution (68) The mixture was stirred at 25 ° C for 60 minutes. After completion of the reaction, the reaction solution is centrifuged and concentrated with an ultrafiltration filter ULTRAFR E-MC 10, OOONMWL Filter Unit (Millipore), and 50 mM HEPES buffer (pH 7.0) is added to the solution and concentrated again. After washing with water, water was added so that the final volume was 19.3 1 to obtain 0.26 mM (theoretical content of glycopeptide) polymer (70). Part of the polymer (70) was identified by treatment with BLase (manufactured by Shionogi & Co., Ltd.), and the reaction mixture was reversed-phase HPLC (Inertsil (registered trademark) ODS— 3 4.6 X 250 mm column, mobile phase A: 0.1% TFA aqueous solution B: 2% to 50% gradient of 0.1% TFA-containing acetonitrile. MA LDI-TOF / MS of compound (71): [M (average) + H] + = 3348.8 (theoretical value: [M (average) + H] + = 3347.4).

[0274] (3.3 Synthesis of Compounds (72) to (73))

[0275] [Chemical 74]

70

UDP-N-ase-rudarcosamine P N-acetylmethyl] samine ¾transfer enzyme

50 mM HEPES buffer (pH 7.5), 3. 85 mU / l pig-derived j8 1,3— N-acetyl darcosamine transferase (manufactured by Toyobo Co., Ltd.), 20 mM sodium chloride manganese, lOmM uridine-5, —70 μl of a reaction solution containing N-acetyldarcosamine diphosphate (UDP—GlcNAc), 0.5% BSA, 0.07 mM glycopeptide derivative (70) was stirred at 37 ° C. for 24 hours. After completion of the reaction, the reaction solution is concentrated by centrifugation using an ultrafiltration filter ULTRAFRE- MC 10,000 NMWL Filter Unit (Millipore), and then concentrated by adding 50 mM HEPES buffer (pH 7.0) and concentrating it again. Then, water was added so that the final volume was 42.2 1 to obtain 0.12 mM (theoretical content of glycopeptide) polymer (72). Part of the polymer (72) was identified by treatment with BLase (manufactured by Shionogi & Co., Ltd.), and the reaction mixture was reversed-phase HPLC (Inertsil (registered trademark) ODS— 3 4.6 X 250 mm column, mobile phase A: 0.1% TFA aqueous solution B: 0.1% TFA-containing acetonitrile: 2% to 50% gradient). MALDI—TOFZMS of compound (73): [M (average) + H] + = 3552.0 (theoretical value: [M (average) + H] + = 3550. 6).

[0276] (3. Synthesis of Compounds (74) to (75))

[0277] [Chemical 75]

T00.S0 / .00Zdf / X3d YZZ

50 mM HEPES buffer (pH 7.0), 0.05 U / ml Human-derived j8 1, 4--galactose transferase (manufactured by Toyobo), lOmM salt 匕 manganese, 1. OmM uridine-5, diphosphate gallium The 55 1 reaction solution containing ratatoose (UDP-Gal) and 0.09 mM glycopeptide derivative (72) was stirred at 25 ° C for 60 minutes. After completion of the reaction, the reaction solution is centrifuged and concentrated with an ultrafiltration filter ULTRAFR E-MC 10,000 NMWL Filter Unit (Millipore), and 50 mM HEPES buffer (pH 7.0) is added to the solution and concentrated again. After washing with water, water was added so that the final volume was 12.41. Thus, a 0.4 mM (theoretical content of glycopeptide) polymer (74) was obtained. Part of the polymer (74) was identified by treatment with BLase (manufactured by Shionogi & Co., Ltd.), and the reaction mixture was reversed-phase HPLC (Inertsil (registered trademark) ODS— 3 4.6 X 250 mm column, mobile phase A: 0.1% TFA aqueous solution B: 2% to 50% gradient of 0.1% TFA-containing acetonitrile. MA LDI-TOF / MS of compound (75): [M (average) + H] + = 3714.6 (theoretical value: [M (average) + H] + = 3712.7).

[0278] (3. 35 Synthesis of Compounds (76) to (77))

[0279] [Chem 76]

50 mM HEPES buffer (pH 7.5), 3. 85 mU / l pig-derived j8 1,3— N-acetyl darcosamine transferase (manufactured by Toyobo Co., Ltd.), 20 mM sodium chloride manganese, lOmM uridine-5, —45 μl of a reaction solution containing N-acetyldarcosamine diphosphate (UDP—GlcNAc), 0.5% BSA and 0.1 lmM glycopeptide derivative (74) was stirred at 37 ° C. for 24 hours. After completion of the reaction, the reaction solution is concentrated by centrifugation using an ultrafiltration filter ULTRAFRE- MC 10,000 NMWL Filter Unit (Millipore), and then concentrated by adding 50 mM HEPES buffer (pH 7.0) and concentrating it again. Then, water was added so that the final volume was 33.7 1 to obtain 0.15 mM (glycopeptide theoretical content) polymer (76). Part of the polymer (76) was identified by treating with BLase (manufactured by Shionogi & Co., Ltd.), and the reaction mixture was reversed-phase HPLC (Inertsil (registered trademark) ODS— 3 4.6 X 250 mm column, mobile phase A: 0.1% TFA aqueous solution B: 0.1% TFA-containing acetonitrile: 2% to 50% gradient). MALDI—TOFZMS of the compound (77): [M (average) + H] + = 3917. KM mi ^: [M (average) + H] ⁺ = 3915. 9).

[0280] (Example 4: Synthesis of sugar amino acids (79) to (83))

(4.1 Synthesis of Compound (79))

[0281] [Chemical 77]

67.6 mg of compound (78) was dissolved in 14 ml of 10 mM sodium hydroxide in methanol and stirred at room temperature for 1.5 hours. After completion of the reaction, the reaction solution was neutralized with acetic acid, and the solvent was distilled off under reduced pressure. The obtained mixture was dissolved in 2.8 ml of 60% aqueous acetone solution containing 11.8 mg of sodium bicarbonate, and 35.4 mg of 9-fluorenylmethylsuccinimidyl carbonate dissolved in 4.2 ml of acetone was further dissolved. The mixture was further stirred at room temperature for 2 hours. After completion of the reaction, the solvent was distilled off under reduced pressure, and the resulting mixture was purified by reverse phase HPLC to obtain 45. lmg of compound (79) (yield 67%). ESI-HRMS: [M + Na] ⁺ = 988. 3890, (theoretical: [M + Na] + = 988. 3903). [0282] (4.2 Synthesis of Compound (80))

[0283] [Chemical 78]

50 mM HEPES buffer (pH 7.0), β 1,4 galactose transferase (Toyobo) 4.7 U, uridine mono-1, monodiphosphate galactose (UDP—Gal) 57 mg, 10 mM mangan chloride, 0.1 % Sodium azide, 0.1% Bovine serum albumin, 10 mM 2, 6 Di-O-methyl-β-cyclodextrin, compound (79) 45. Img containing 23.4 ml reaction solution at 25 ° C for 15 hours Stir. After completion of the reaction, the reaction solution was purified by reverse phase HPLC to obtain 45.6 mg of Compound (80) (yield 87%). ESI— HRMS: [M + Na] + = 1150. 4440, (theoretical: [M + Na] + = 1150. 4431).

[0284] (4.3 Synthesis of Compound (81))

[0285] [Chemical 79]

50 mM Tris— HC1 buffer (pH 8.0), β 1, 3— Ν Acetyldarcosamine transferase Elemental 606mU, Uridine-5, -Diphosphate-N-Acetyldarcosamine (UDP-GlcNAc) 65.8mg, 10mM manganese chloride, 10mM magnesium chloride, 0.1% sodium azide, 0.1% cattle A 20.2 ml reaction solution containing serum albumin, 0.2% Triton X-100, lOOmUZml alkaline phosphatase, 45.6 mg of compound (80) was stirred at 25 ° C for 48 hours. After completion of the reaction, the reaction solution was purified by reverse phase HPLC to obtain 38.7 mg of Compound (81) (yield 72%). ESI— HRMS: [M + Na] + = 1353. 5199, (theoretical: [M + Na] + = 1353. 5223).

[0286] (4.4 Synthesis of Compound (82))

[0287] [Chemical 80]

38.7 mg of compound (81) was dissolved in 29.1 ml of acetic anhydride Zpyridine Z acetic acid (5Z3Z2, vZvZv) solution and stirred at room temperature for 19 hours. After completion of the reaction, the solvent was distilled off under reduced pressure, and the resulting mixture was purified by reverse phase HPLC to obtain 51.6 mg of Compound (82) (yield 94%). ESI— HRMS: [M + Na] ⁺ = 1899. 6631, (theoretical: [M + Na] + = 1899. 6598).

[0288] (4.5 Synthesis of Compound (83))

[0289] [Chemical 81]

51.6 mg of Compound (82) was dissolved in 10 ml of 95% TFA aqueous solution and stirred at room temperature for 1 hour. After completion of the reaction, the solvent was evaporated and the resulting mixture was purified by reverse phase HPLC to obtain 39.2 mg of Compound (83) (yield 78%). ESI— HRMS: [M + Na] + = 1843. 5978, (Theoretical: [M + Na] + = 1843. 5972).

[0290] (Example 5: Synthesis of glycopeptides (84) to (85))

(5.1 Synthesis of Compounds (84) to (85))

[0291] [Chemical 82]

Glycopeptide solid phase synthesis

Rink Amide PEGA resin (0.O55mmol / g) 25.9mg (13.8mol) as a carrier, the following N-protected amino acids were condensed sequentially by FmocZHBTUZHOBt method, The desired glycopeptide was synthesized. Fmoc— Arg (Pbf) — OH, Fmoc-Thr (Ac7core2) — OH, Fmoc-Asp (OtBu) — OH, Fmoc— Pro— OH, Fmoc— Ala— OH, F moc— Ser (tBu) — OH, compound (83), Fmoc—Val—OH, Fmoc—Gly—OH, F moc-His (Trt) —OH, Fmoc—Ala—OH. After the peptide elongation reaction, the protecting group on the peptide residue is removed by reacting in TFAZ triisopropyl silane Z water (95Z2.5 / 5 / 2.5, vZvZv) at room temperature for 2 hours. Compound (84) was released from the carrier. The coconut resin was filtered off and TFA was evaporated off, and t-butyl methyl ether was added to precipitate the product. The obtained slurry was centrifuged, and the precipitate was purified by reverse phase HPLC to obtain 1.28 mg of a crude product of compound (84). The obtained crude product was dissolved in 10 ml of a methanol solution of 10 mM sodium hydroxide and stirred at room temperature for 3 hours. After completion of the reaction, the reaction solution was neutralized with acetic acid, and the solvent was evaporated. The obtained mixture was purified by reverse phase HPLC to obtain 1. Omg of compound (85) (yield 2.8%). ₀ ESI-HRMS: [MH] "= 2610. 1057 (theoretical value: [M — H] _ = 2610. 1053).

Industrial applicability

[0292] According to the present invention, it is possible to prepare a library of glycopeptides having a comprehensive sugar chain structure and a complex sugar chain structure, which has been extremely difficult with the prior art. For example, the present invention makes it possible to synthesize mucin-type glycopeptides having a borilactosamine skeleton that have been useful in a wide range of fields such as biochemical research materials, pharmaceuticals, and foods, and have been difficult to produce.

[0293] The obtained mucin-type glycopeptide library having a borilactosamine skeleton can be used as a standard sample for structural analysis and biochemical tests. In addition, a mucin-type glycopeptide library having this voralactosamine skeleton is placed on the chip for detection of glycopeptide recognition proteins, pathological diagnosis, search for cell adhesion sequences, sequence analysis related to cell growth and apoptosis, etc. It becomes possible to do it comprehensively.

Claims

The scope of the claims

The following formula:

X— C (= 0) — (CH) — A— A — A (I)

2 n 1 2 3

Wherein X represents a hydrogen atom, c-c alkyl, c-c aryl or chromophore;

1 30 6 30

n represents an integer of 0 to 20;

A is — (CH 2) — C (= 0) —, — (CH 2 CH 2 O) —

1 2 0 ~ 20 2 2 1 ~ 10

A represents an amino acid residue cleavable by a protease;

2

Three

[Chemical 1]

R ² and R ⁷ are galactose (Gal) groups;

R ³ and R ⁸ are GlcNAc groups;

R ⁴ and R ⁹ are Gal groups;

R ⁵ is a GlcNAc group;

R ¹⁰ is an N-acetyl-a-D-galactosamine (GalNAc) group;

Z ² and Z ³ are each independently hydrogen or a fucose group; and

n and m are each independently an integer of 0 or more;

Here, the bond between R ¹ and R ² is a 2, 3 bond when R ¹ is Neu5Ac group, and β ΐ, 3 bond when R ¹ is GlcNAc group; The bond between R ² and R ³ is a β,, 4 bond;

The bond between R ³ and R ⁴ is a β,, 3 bond;

The bond between R ⁴ and R ⁵ is a β,, 4 bond;

The bond between R ⁶ and R ⁷ is an α 2,3 bond when R ⁶ is a Neu5Ac group, and a,, 3 bond when R ⁶ is a GlcNA c group;

The bond between R ⁷ and R ⁸ is a β,, 4 bond;

Bond between R ⁸ and R ^9, β ΐ, is 3 bonds;

The bond between R ⁹ and R ¹⁰ is a β,, 3 bond;

The bond between R ⁵ and R ^1G is a β,, 6 bond;

The bond between Ζ ¹ and R ³ is << ¹ , 3 bond;

The bond between Ζ ² and R ⁵ is << 1, 3 bond; and

The bond between Ζ ³ and R ⁸ is << 1, 3 bond,

A compound having a sugar residue represented by:

[2] The cocoon is a protea derived from Bacillus Licheniformis.

2. The compound according to claim 1, which is a glutamic acid residue or a cysteine residue cleavable with one enzyme.

[3] At least a partial force of the above A to Mucin-type glycoprotein MUC1 derived SEQ ID NOs: 1 to 60

Three

2. The compound of claim 1 having an amino acid sequence selected from the group consisting of the amino acid sequences shown.

[4] From the compound according to claim 1 and an optionally protected aminooxy group, N-alkylaminooxy group, hydrazide group, azide group, thiosemicarbazide group, 1,2-dithiol group, and cysteine residue A compound obtained by reacting with a carrier containing a functional group selected from the group consisting of:

[5] The carrier includes the following:

b) Protected !, but silica carriers having aminooxy groups or hydrazide groups, resin A carrier, magnetic beads or metal carrier; and

C) The following formula:

[(NH OCH C (= 0)) -Lys] Lys— NHCH CH C (= 0) — R ³ ,

2 2 2 2 2 2

[(NH OCH C (= 0)) -Lys] Lys— NHCH (CH SH) C (= 0) — R ³ ,

2 2 2 2 2

[(NH OCH C (= 0)) -Lys] — Lys— Cys— NHCH CH C (= 0) — R ³ (sequence

2 2 2 ""

Number 61),

{[(NH OCH C (= 0)) -Lys] Lys— NHCH [C (= 0) — R ³ ] CH— S},

2 2 2 2 2 2

{[(NH OCH C (= 0)) -Lys]-Lvs-NHCH (C (= 0) NHCH CH C (= 0

2 2 2 2 2 2

) -R ³ ] CH -S},

twenty two

{[(NH OCH C (= 0)) -Lys] -Lys} Lys— NHCH CH C (= 0) — R ³ (

2 2 2 2 2 2 2

SEQ ID NO: 62),

{[(NH OCH C (= 0)) -Lys] -Lys} -Lys- NHCH (CH SH) C (= 0) —

2 2 2 2 2 2

R ³ (SEQ ID NO: 63),

{[(NH OCH C (= 0)) -Lys] -Lys} Lys— Cys— NHCH CH C (= 0)

2 2 2 2 2 2 2

—R ³ (SEQ ID NO: 64),

[[[(NH OCH C (= 0)) -Lys] -Lys] Lys— NHCH [C (= 0) — R ³ ] CH

twenty two

S] (SEQ ID NO: 65),

2

[[[(NH OCH C (= 0)) -Lys] -Lys]-Lys-NHCH [C (= 0) NHCH C

2 2 2 2 2 2

HC (= 0) -R ³ ] CH -S] (SEQ ID NO: 66),

2 2 2

[Chemical 2]

[(NH ₂ OCH ₂ C (= 0)) 2 Ly s] NHCHC (= 0) — R ³

I

[(Title ₂ OCH ₂ C (= 0)) 2 -Ly s] -NH (CH ₂ ) 4

Or

{[(NH ₂ OCH2 C (= 0)) 2 Ly s] 2 Lys} NHCHC (= 0) — R ³

I

{[(NH ₂ OCH ₂ C (= 0)) ≥ -L ys] ₂ Ly s} NH (CH ₂ )

[Chemical 3]

Where n is an integer from 1 to 15 and x: y is 1: 0 to 1: 1000,

The compound according to claim 4, wherein the compound represented by

[6] The following formula:

A N = C (— X) — (CH) A -A -A (II)

4 2 n 1 2 3

Wherein X is a hydrogen atom, c-c alkyl,

30 c ~

6 c represents a reel or chromophore;

1 30

n represents an integer of 0 to 20;

A is — (CH 2) — C (= 0) —, — (CH 2 CH 2 O) —

1 2 0 ~ 20 2 2 1 ~ 10

A is a protease derived from Bacillus Licheniformis.

2

A cleavable glutamic acid residue or cysteine residue;

Three

[Chemical 4]

In which R ¹ and R are each independently hydrogen, Neu5Ac group or GlcNAc group;

R ² and R ⁷ are Gal groups;

R ³ and R ⁸ are GlcNAc groups;

R ⁴ and R ⁹ are Gal groups;

R ⁵ is a GlcNAc group;

R ¹⁰ is a GalNAc group;

The bond between R ² and R ³ is a β ΐ, 4 bond;

The bond between R ³ and R ⁴ is a β ΐ, 3 bond;

The bond between R ⁴ and R ⁵ is a β ΐ, 4 bond;

The bond between R ⁷ and R ⁸ is a β ΐ, 4 bond;

The bond between R ⁸ and R ⁹ is a β ΐ, 3 bond;

The bond between R ⁹ and R ^1G is a β ΐ, 3 bond;

The bond between R ⁵ and R ^1G is a β ΐ, 6 bond;

The bond between Ζ ¹ and R ³ is << ¹ , 3 bond;

The bond between Ζ ² and R ⁵ is << 1, 3 bond; and

The bond between Ζ ³ and R ⁸ is << 1, 3 bond,

Having a sugar residue represented by:

Α is the following formula: [Chemical 5]

[7] At least a partial force of the above A to Mucin-type glycoprotein MUC1

Three

7. A compound according to claim 6 having an amino acid sequence selected from the group consisting of the amino acid sequences shown.

[8] The following steps:

(A) The compound according to any one of claims 1 to 3, and an optionally protected aminooxy group, N-al which can specifically react with the ketone residue or aldehyde residue of the compound Reacting with a carrier containing a functional group selected from the group consisting of a kilaminoxy group, a hydrazide group, an azide group, a thiosemicarbazide group, a 1,2-dithiol group and a cysteine residue;

(B) By reacting the compound obtained in step (A) with a glycosyltransferase in the presence of a sugar nucleotide, the sugar residue is transferred from the sugar nucleotide to the compound, and the sugar chain is elongated. A step of obtaining a compound comprising a sugar residue selected from the group consisting of the sugar nucleotide force Gal group, GlcNAc group, Fuc group and Neu5Ac group force;

[9] The following steps:

(A) A sugar residue is transferred from the sugar nucleotide to the compound by allowing a sugar transferase to act on the compound according to any one of claims 4 to 7 in the presence of the sugar nucleotide. A step of obtaining a chain-extended compound having a sugar residue selected from the group consisting of the sugar nucleotide force Gal group, GlcNA c group, Fuc group and Neu5Ac group;

[10] The following steps:

(B) Step (A) is repeated once or twice or more to extend the sugar chain;

(D) A method for producing a glycopeptide, comprising the step of allowing a protease to act on a compound in which a plurality of sugar residues are transferred and the sugar chain is elongated.

[11] The following steps:

The compound according to any one of claims 1 to 3, wherein (A) peptide solid phase synthesis is performed using amino acids, sugar amino acids, and keto acid or aldehyde acid that can be cleaved by a protease as raw materials. Obtaining a product;

The following steps:

(A) a step of obtaining a compound according to any one of claims 1 to 3 by performing solid phase peptide synthesis using amino acids, sugar amino acids, and keto acids or aldehyde acids that can be cleaved by a protease as raw materials;

(B) The compound obtained in step (A) and an optionally protected aminooxy group, N-alkylaminooxy group, hydrazide group capable of reacting specifically with the ketone residue or aldehyde residue of the compound , An azide group, a thiosemicarbazide group, a 1,2-dithiol group, and a carrier containing a functional group selected from the group consisting of cysteine residues, and at the same time, removing unreacted substances in step (A) ;

(D) Step (C) is repeated once or twice or more to extend the sugar chain;

(E) Remove unreacted sugar nucleotides and by-product nucleotides as necessary And

[13] Keto acid or aldehyde acid power in the step (A) The following formula:

X-C (= 0)-(CH) -A -COOH (III)

2 n 1

In which X is a hydrogen atom, c1-c alkyl,

30 c represents 6 to c aryl or chromophore;

30

n represents an integer of 0 to 20;

A represents a linker having a length of 1 to 20 methylene chains,

The method of Claim 11 or 12 which is a compound represented by these.

[14] The following steps:

(A) A sugar residue is transferred from the sugar nucleotide to the compound by allowing a sugar transferase to act on the compound according to any one of claims 1 to 3 in the presence of the sugar nucleotide. A step of obtaining a chain-extended compound having a sugar residue selected from the group consisting of the sugar nucleotide force Gal group, GlcNA c group, Fuc group and Neu5Ac group;

(B) Step (A) is repeated one or more times as necessary to extend the sugar chain; (C) a compound in which the sugar residue is transferred and the sugar chain is extended, and the ketone residue of the compound Protected, which can react specifically with a group or aldehyde residue, may be an aminooxy group, N-alkylaminooxy group, hydrazide group, azide group, thiosemicarbazide group, 1,2-dithiol group and cysteine. Reacting with a carrier containing a functional group selected from the group consisting of residues

;and

A process for producing a glycopeptide comprising

[15] The following steps:

(A) A sugar residue is transferred from the sugar nucleotide to the compound by allowing a sugar transferase to act on the compound according to any one of claims 1 to 3 in the presence of the sugar nucleotide, A step of obtaining a chain-extended compound comprising a sugar residue selected from the group consisting of the sugar nucleotide force Gal group, GlcNA c group, Fuc group and Neu5Ac group; (B) Step (A) is repeated one or more times as necessary to extend the sugar chain as necessary;

Said glycopeptide has the following formula:

[Chemical 6]

X ¹ X ¹ X ¹

Y ¹ -His- Gly-Va 卜 Thr-Ser- Ala-Pro- Asp- Thr-Arg-Y ²

(SEQ ID NO: 2)

[Chemical 7]

X ¹ X ¹ X ¹

Y '-Ala-His- Gly- Va 卜 Thr-Se `` AI a- Pro-Asp- Thr-Arg- Y ²

(SEQ ID NO: 21)

Or

[Chemical 8]

X ¹ X ¹ X ¹ X ¹ X ¹

(SEQ ID NO: 41)

[Chemical 9]

Wherein R ¹ and R ⁶ are each independently hydrogen, Neu5Ac group or GlcNAc group. Yes;

R ² and R ⁷ are Gal groups;

R ³ and R ⁸ are GlcNAc groups;

R ⁴ and R ⁹ are Gal groups;

R ⁵ is a GlcNAc group;

R ¹⁰ is a GalNAc group;

Here, the bond between R ¹ and R ² is a 2, 3 bond when R ¹ is a Neu5Ac group, and a β ΐ, 3 bond when R ¹ is a GlcNAc group;

The bond between R ² and R ³ is a β ΐ, 4 bond;

The bond between R ³ and R ⁴ is a β ΐ, 3 bond;

The bond between R ⁴ and R ⁵ is a β ΐ, 4 bond;

The bond between R ⁷ and R ⁸ is a β ΐ, 4 bond;

The bond between R ⁸ and R ⁹ is a β ΐ, 3 bond;

The bond between R ⁹ and R ¹⁰ is a β ΐ, 3 bond;

The bond between R ⁵ and R ^1G is a β ΐ, 6 bond;

The bond between Ζ ¹ and R ³ is al, 3 bond;

The bond between Ζ ² and R ⁵ is an αΐ, 3 bond; and

The bond between Ζ ³ and R ⁸ is αΐ, 3 bond,

Υ ¹ represents a hydrogen atom, acetyl, acyl, alkyl or aryl;

Υ ² represents a hydroxyl group, ΝΗ, alkyl or aryl.

2

The method according to any one of claims 8 to 15, which is a glycopeptide represented by the formula:

The following formula:

[Chemical 10] X ¹ X ¹ X ¹

Y ¹ -His- Gly-Va 卜 Thr-Ser- Ala-Pro- Asp- Thr-Arg-Y ²

(SEQ ID NO: 2)

[Chemical 11]

X ¹ X ¹ X ¹

Y ¹ -Ala-His- Gly-Val-Thr-Ser-AI a- Pro-Asp- Thr-Arg- γ2

(SEQ ID NO: 21)

Or

[Chemical 12]

X ¹ X ¹ X ¹ X ¹ X ¹

Y ¹ -His— Gly—Va 卜 Thr—Ser— Ala-Pro— Asp— Thr—Arg— Pro-Ala— Pro-Gly-Ser-Thr-Ala— Pro-Pro— Ala— Υ ²

(SEQ ID NO: 41)

[Chemical 13]

R ² and R ⁷ are Gal groups;

R ³ and R ⁸ are GlcNAc groups;

R ⁴ and R ⁹ are Gal groups;

R ⁵ is a GlcNAc group;

R ¹⁰ is a GalNAc group;

The bond between R ² and R ³ is a β,, 4 bond;

The bond between R ³ and R ⁴ is a β,, 3 bond; The bond between R ⁴ and R ⁵ is a / 31, 4 bond;

The bond between R ⁷ and R ⁸ is a β ΐ, 4 bond;

The bond between R ⁸ and R ⁹ is a β ΐ, 3 bond;

The bond between R ⁹ and R ^1G is a β ΐ, 3 bond;

The bond between R ⁵ and R ^1G is a β ΐ, 6 bond;

The bond between Ζ ¹ and R ³ is << ¹ , 3 bond;

The bond between Ζ ² and R ⁵ is << 1, 3 bond; and

The bond between Ζ ³ and R ⁸ is << 1, 3 bond,

Υ ¹ represents a hydrogen atom, acetyl, acyl, alkyl or aryl;

Υ ² represents a hydroxyl group, ΝΗ, alkyl or aryl.

2

A glycopeptide represented by

A method for producing a desired glycopeptide comprising the following steps:

(Α)

The following formula:

[Chemical 14]

Where R ¹ and R. Are each independently hydrogen, Neu5 Ac group or GlcNAc group; R ² and R ⁷ are Gal groups;

R ³ and R ⁸ are GlcNAc groups;

R ⁴ and R ⁹ are Gal groups;

R ⁵ is a GlcNAc group;

R ¹⁰ is a GalNAc group;

R ¹¹ is a hydrogen atom or a methyl group;

The bond between R ² and R ³ is a β ΐ, 4 bond;

The bond between R ³ and R ⁴ is a β ΐ, 3 bond;

The bond between R ⁴ and R ⁵ is a β ΐ, 4 bond;

The bond between R ⁷ and R ⁸ is a β ΐ, 4 bond;

The bond between R ⁸ and R ⁹ is a β ΐ, 3 bond;

The bond between R ⁹ and R ¹⁰ is a β ΐ, 3 bond;

The bond between R ⁵ and R ^1G is a β ΐ, 6 bond;

The bond between Ζ ¹ and R ³ is << ¹ , 3 bond;

The bond between Ζ ² and R ⁵ is << 1, 3 bond;

The bond between Ζ ³ and R ⁸ is << 1, 3 bond; and

Ρ is a 9-fluormethylcarboxyl group or a tert-butoxycarbol group,

A sugar amino acid having a sugar chain protected by protecting the sugar chain of the sugar amino acid represented by the following: a acetyl group, a benzoyl group, a methyl group, a methoxymethyl group, Trimethylsilyl group, t-butyldimethylsilyl group, dimethylphenol group and triisopropylpropylsilyl group, benzyl group, benzylidene group, isopropylidene group, di-tert- The

A process selected from the group consisting of:

(B) Using the sugar amino acid in which the sugar chain obtained in step (A) is protected and an N-protected amino acid with a 9-fluorenylmethylcarboxyl group or a tert-butoxycarbol group, the desired peptide Synthesizing a glycopeptide in which the sugar chain having the sequence is protected; and

Including the method.