WO2005095331A1

WO2005095331A1 - Agent for binding sugar chain to peptide

Info

Publication number: WO2005095331A1
Application number: PCT/JP2005/005810
Authority: WO
Inventors: Shinichiro Nishimura; Akio Takimoto
Original assignee: Shionogi & Co., Ltd.
Priority date: 2004-03-31
Filing date: 2005-03-29
Publication date: 2005-10-13
Also published as: JPWO2005095331A1

Abstract

An agent for binding a sugar chain to a peptide which comprises a compound represented by the general formula (I): X-Y-Z (I) wherein X is H2Z- or HS-; Y is -A1-, -A1-O-A2-, -(CH2CH2O)n-A1-, -(CH2CH2O)n-A1-O-A2-, -A1-NH-C(=O)-A2-, -A1-C(=O)-NH-A2-, -A1-(CH2CH2O)n-NH-C(=O)-A2-, -A1-(CH2CH2O)n-C(=O)-NH-A2-, etc.; and Z is -O-NH(R4), -NH-OR4, -C(=O)-NH-NH-R4, or -C(=S)-NH-NH-R4 (wherein R4 is hydrogen, lower alkyl, or aryl). Also provided is a process for producing a sugar peptide with the agent for binding a sugar chain to a peptide.

Description

Sugar chain-peptide binder

Technical field

The present invention relates to a sugar used for easily producing glycopeptides and glycoproteins having a uniform composition (hereinafter, collectively referred to as “glycopeptides” unless otherwise specified). The present invention relates to a chain peptide binding agent and a method for producing a glycopeptide using the sugar chain peptide binding agent.

Background art

[0002] Peptides and proteins have been studied as typical examples of physiologically active substances. Organisms (especially eukaryotes) produce peptides and proteins through mechanisms of gene transcription and translation, and many peptides and proteins are often post-translationally modified. And is regulated (eg, enhanced or reduced). Post-translational modifications include, for example, those at the amino acid level (ie, cleavage of a signal peptide), and modifications with other biomolecules such as sugar chains and lipids. Among these, the modification with a sugar chain is a typical one, and it is said that very many peptides and proteins in eukaryotes have a sugar chain.

In recent years, attempts have been made to produce artificial conjugated peptides whose functions have been regulated by imitating such sugar chain modifications (Patent Documents 1 to 4), and these are also used as biopharmaceuticals.

However, there are limited methods for introducing a specific sugar chain into a specific site of a peptide or protein, particularly, a method of uniformly introducing a plurality of sugar chains to multiple sites of a peptide or protein. . For example, Non-Patent Document 1 describes a method for introducing a modified sugar obtained through several steps of synthesis reaction into insulin, and Non-Patent Document 2 describes a method for synthesizing a sugar chain after synthesizing a non-natural peptide. Is described. However, it is difficult to say that any of them is a suitable production method from the viewpoint of simplicity and mass production, and it is difficult to produce a biopharmaceutical having a constant composition.

Patent Document 1: JP-A-2000-300287

Patent Document 2: Column 2001—278899 9

Non-patent literature l: Angw. Chem, 2004, 43, 1516

Non-Patent Document 2: Am. Chem. Soc, 2003, 125, 1702

Disclosure of the invention

Problems to be solved by the invention

[0003] An object of the present invention is to provide a sugar chain peptide binding agent used for easily producing a glycopeptide having a constant composition, and a method for producing a glycopeptide using the sugar chain peptide binding agent. Is to do.

Means for solving the problem

That is, the present invention provides (1) a compound represented by the general formula (I):

X-Y-Z (I)

[Where X is H N— or the formula:

2

[Chemical 1]

A group represented by;

Y is —A ¹ -A'-OA ² -,-(CH CH O) —A ^1- (CH CH O) A ¹ —

2 2 n 2 2 n

O-A ² -A ¹ -NH-C (= 0)-A ² -A ¹ -C (= 0)-NH-A ² -A ^1- (CH CH O) NH — C (= 0) — A ² A ¹ — (CH CH O) — C (= 0) — NH—

2 2 n 2 2 n

A ² —, formula:

[Formula 2]

A group represented by or a formula

[Formula 3]

(Wherein A ¹ and A ² are each independently an optionally substituted C 15 alkylene, substituted !, or C 2-15 alkylene or substituted C2-15 alkylene; n is an integer of 1 to 5; R ¹ is hydroxy or NH (R ² ) (where R ² is a hydrogen atom, lower alkyl, aryl or aralkyl); R ³ is a hydrogen atom or —C (= 0) —R ⁵ (where R ⁵ is lower alkyl, aryl or aralkyl)); and Z is O—NH (R ⁴ ), NH—OR ⁴ , C (= 0) —NH—NH—R ⁴ , or C (= S) —NH—NH—R ⁴ (where R ⁴ is a hydrogen atom, lower alkyl, or aryl)] Sugar chain peptide binding agent.

Here, the amino group, imino group and the like present in the compound represented by the formula (I) may be protected by a suitable protecting group.

Specifically, the present invention relates to the following (2) to (11).

(2) X is —NH and Z is —O—NH (R ⁴ ) (where R ⁴ is a hydrogen atom, lower alkyl,

2

Or aryl), the sugar chain peptide binder of (1).

(3) X is the formula:

[Formula 4]

The sugar chain peptide binder according to (1), wherein the group represented by and Z is —O—NH (R ⁴ ), wherein R ⁴ is a hydrogen atom, lower alkyl, or aryl.

(4) General formula (Π): HN— A ¹ — O— NH (R ⁴ ) (II)

2

(Wherein, A ¹ is an optionally substituted CI-15 alkylene; R ⁴ is a hydrogen atom, lower alkyl, or aryl), or a salt thereof.

(5) —General formula (III):

[Formula 5]

(In the formula, A ¹ is an optionally substituted C 1-15 alkylene; R ⁴ is a hydrogen atom, lower alkyl, or aryl), or a salt thereof.

(6) The following steps:

1) General formula (IV):

H N-Y-Z (IV)

2

[Where Y is — A ¹ —, — A ¹ — O— A ² —,-(CH CH O) — A ¹ —,-(CH CH O)

2 2 η 2 2: Α ¹ -Ο-Α ² -A ¹ -NH-C (= 0)-A ² -A ¹ -C (= 0)-NH-A ^2- , A ¹ — (CH CH O ) — NH— C (= 0) — A ² A ¹ — (CH CH O) — C (= 0)

2 2 η 2 2 η NH-Α ^2- , formula:

[Formula 6]

Or a group represented by the formula:

[Formula 7]

Wherein A ¹ and A ² are each independently an optionally substituted C ^{1 —} 1

5 alkylene, substituted !, may be, C2-15 alkylene or substituted !, may be C2-15 alkylene; n is an integer of 1 to 5; R ¹ is hydroxy or NH ( R ² ) (where

R ² is hydrogen atom, lower alkyl, aryl or aralkyl); R ³ is hydrogen atom or —

C (= 0) —R ^{5 where} R ⁵ is lower alkyl, aryl or aralkyl)); and

Z is O—NH (R ⁴ ), one NH—OR ⁴ , one C (= 0) —NH—NH—R ⁴ , or C (= S

) - NH- NH- R ⁴ (wherein, R ⁴ is a hydrogen atom, a lower alkyl or Ariru)] represented by the compound,

A peptide having a glutamine residue,

Reacting in the presence of transdaltaminase, and

2) a step of reacting the peptide obtained in step 1) with a sugar chain,

A method for producing a glycopeptide.

In connection with the production method, 1) a step of reacting the compound represented by the general formula (IV) with a sugar chain, and 2) the peptide obtained in the step 1) and a peptide having a glutamine residue are transtranscribed. A method for producing a glycopeptide, which includes a step of reacting in the presence of sglutaminase, can also be performed.

(7) The following steps:

1) General formula (V):

[Formula 8]

2 2 η 2 2 1 Α ¹ -Ο-Α ^2- , 1 A ¹ -NH-C (= 0)-A ^2- , 1 A ¹ -C (= 0)-NH-A ^2- , 1 A ¹ — (CH CH O) — NH— C (= 0) — A ² —, one A ¹ — (CH CH O) — C (= 0)

2 2 η 2 2 η NH-Α ^2- , formula:

[Formula 9]

(Wherein A ¹ and A ² are each independently an optionally substituted C 15 alkylene, substituted !, or C 2-15 alkylene or substituted C2-15 alkylene; n is an integer of 1 to 5; R ¹ is hydroxy or NH (R ² ) (where R ² is a hydrogen atom, lower alkyl, aryl or aralkyl); R ³ is a hydrogen atom or —C (= 0) —R ⁵ (where R ⁵ is lower alkyl, aryl or aralkyl)); and Z is O—NH (R ⁴ ), NH—OR ⁴ , C (= 0) —NH—NH—R ⁴ , or C (= S) —NH—NH—R ⁴ (where R ⁴ is a hydrogen atom, lower alkyl, or aryl);

Reacting a peptide containing a cysteine residue, and

2) including the step of reacting the peptide obtained in step 1) with a sugar chain,

A method for producing a glycopeptide.

In connection with the production method, 1) reacting the compound represented by the general formula (V) with a sugar chain, and 2) reacting the peptide obtained in the step 1) with the peptide having a cysteine residue. And a method for producing a glycopeptide comprising the step of:

( ⁸ ) The production method according to (6) or (7), wherein Z is —O—NH (R ⁴ ), wherein R ⁴ is a hydrogen atom, lower alkyl, or aryl.

( ⁹ ) The production method according to ( ⁶ ) or (7), wherein Y is —A ¹ — (where A ¹ is an optionally substituted CI—I ⁵ alkylene).

(10) A glycopeptide which can be produced by the production method according to any one of (6) to (9). (11) A pharmaceutical composition containing, as an active ingredient, a glycopeptide that can be produced by the production method according to any one of (6) to (9).

The invention's effect

[0009] The present invention provides a sugar chain-peptide binding agent used for easily producing a glycopeptide having a uniform composition by using a natural or non-natural protein and an unprotected sugar chain. The present invention relates to a method for producing a glycopeptide using a sugar chain-peptide binding agent. According to the present invention, it is possible to provide a glycopeptide having a constant composition, so that it is possible to provide a higher-quality Noro medicine.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described. It should be understood that throughout this specification, the use of the singular includes the plural concept unless specifically stated otherwise. It should also be understood that the terms used in the present specification have the meanings normally used in the art unless otherwise specified.

(the term)

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

[0011] As used herein, the term "sugar chain" refers to a conjugate comprising at least one unit sugar (monosaccharide and Z or a derivative thereof). When two or more unit sugars are linked, each unit sugar is linked by dehydration condensation through glycosidic bonds. Examples of such sugar chains include polysaccharides (glucose, galactose, mannose, fucose, xylose, N-acetyldarcosamine, N-acetylgalatatosamine, sialic acid and (Complexes and derivatives thereof), as well as a wide range of sugar chains degraded or derived from complex biomolecules such as degraded polysaccharides, glycoproteins, proteoglycans, glycosaminodalican, and glycolipids. Not limited to them. Therefore, in this specification, a sugar chain can be used interchangeably with "polysaccharide", "carbohydrate", and "carbohydrate". Unless otherwise specified, the term “sugar chain” in this specification includes both sugar chains and sugar chain-containing substances.

As used herein, “monosaccharide” refers to a compound represented by the general formula CHO, which is not hydrolyzed to a simpler molecule. Where n = 2, 3, 4, 5, 6, 7, 8, 9, and 10 Things are called diose, triose, tetroses, pentoses, hexoses, heptose, otatoses, nonose and decos, respectively. Generally, it corresponds to the aldehyde or ketone of a chain polyhydric alcohol. The former is called aldose and the latter is called ketose.

[0012] As used herein, the term "monosaccharide derivative" refers to a substance in which one or more hydroxyl groups on a monosaccharide are substituted with another substituent and the resulting substance is not within the range of the monosaccharide. Say. Examples of such monosaccharide derivatives include sugars having a carboxyl group (eg, aldonic acid in which the C 1 position has been oxidized to form a carboxylic acid (eg, D-dalconic acid obtained by oxidizing D-glucose), (D-gluconic acid in which D-glucose has been oxidized), a sugar having an amino group or an amino group derivative (eg, an acetylated amino group) (eg, N-acetyl-) D-darcosamine, N-acetyl-D-galactosamine, etc.), a saccharide having both an amino group and a carboxyl group (for example, N-acetylneuraminic acid (sialic acid), N-acetylmuramic acid, etc.), and a sugar that has been deoxylated (for example, 2- (Doxy-D-ribose), sulfated sugars containing sulfate groups, phosphorylated sugars containing phosphate groups, etc., but are not limited thereto. To the Miasetaru structure Te formed sugar odor, glycosides also of Asetaru structure by reacting with alcohol, are within the scope of the monosaccharide derivatives.

As used herein, “sugar chain-containing substance” refers to a substance containing a sugar chain and a substance other than the sugar chain. Such sugar chain-containing substances are found abundantly in living organisms.For example, in addition to polysaccharides contained in living organisms, complex substances such as degraded polysaccharides, glycoproteins, proteodalicans, glycosaminoglycans, glycolipids, etc. The biomolecular force also includes, but is not limited to, a wide range of sugar chains decomposed or derived.

[0013] 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 acids may be naturally occurring or non-naturally occurring or modified amino acids. The term may also include those assembled into a complex of multiple polypeptide chains. The term also includes natural or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, Dali Cosylation, 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 an amino acid (eg, including unnatural amino acids, etc.), peptidomimetic compounds (eg, peptoids), and those known in the art! Other modifications are included. The gene product of the present invention usually takes the form of a polypeptide. The gene product of the present invention in such a polypeptide form is useful as a composition for diagnosis, prevention, treatment or prognosis of the present invention. Particularly, a peptide having a physiological activity is preferable. For example, insulin, erythropoietin, thrombopoetin, interleukin 1, interleukin 2, interleukin 3, interleukin 4, interleukin 5, interleukin 6, interleukin 7, interleukin 8, interleukin 9, interleukin 10, interleukin 11, Interleukin 12, Interleukin 13, Interleukin-14, Interleukin-15, Interferon-a, Interferon-13, Interferon-γ, G-CSF, GM-CSF, M-CSF, GLP-1, Stem Senoractor 1, mpl-ligand, somatostatin, vasoplethsin, gnore gon, growth honoremon, leptin, cholecystokine, substance P, adrenaline, ADF, corticosteroid stimulating hormone, j8 endorphin, neoendor Peptides obtained by modifying the base petit de and their working physiologically in the human body such as fin, animal-derived peptides, microbial origin peptides, such as peptides derived from plants.

As used herein, “glycoprotein” includes, but is not limited to, enzymes, hormones, cytokins, antibodies, vaccines, receptors, serum proteins, and the like.

In the present specification, the “sugar chain-peptide binder” refers to a group that acts as a “linker” interposed between the sugar chain and the peptide when forming a glycopeptide. For example, the general formula (I)

X-Y-Z (I)

[Where X is H N— or the formula:

2

[Formula 11]

A group represented by;

Y is —A ¹ —, -A '-OA ² -,-(CH CH O) —A ¹ —,-(CH CH O) A ¹ —

2 2 n 2 2 n

O-A ^2- , one A ¹ -NH-C (= 0)-A ^2- , one A ¹ -C (= 0)-NH-A ^2- , one A ^1- (

CH CH O) NH— C (= 0) — A ² , one A ¹ — (CH CH O) one C (= 0) — NH—

twenty two

A ² —, formula:

[Formula 12]

⁰丫

, Α ¹

A group represented by Η or a formula:

[Formula 13]

(Wherein Α ¹ and Α ² are each independently an optionally substituted C 1-15 alkylene, substituted !, or C 2-15 alkenyl- or substituted C2-15 alkylene; n is an integer of 1 to 5; R ¹ is hydroxy or NH (R ² ) (where R ² is a hydrogen atom, lower alkyl, aryl or aralkyl); R ³ is a hydrogen atom or —C (= 0) —R ⁵ (where R ⁵ is lower alkyl, aryl or aralkyl)); and Z is one O—NH (R ⁴ ), one NH—OR ⁴ , A compound represented by one C (= 0) —NH—NH—R ⁴ or one C (= S) —NH—NH—R ⁴ (where R ⁴ is a hydrogen atom, lower alkyl, or aryl) And a sugar chain peptide-binding agent comprising: In particular,

1) Formula: HN—A ¹ — O—NH (R ⁴ ) (where A ¹ may be substituted CI—15 alkylene

2

R ⁴ is hydrogen atom or lower alkyl),

2) Formula: H NA ¹ -NH-C (= 0) —A ² —O—NH (R ⁴ ) (where A ¹ and A ² are

2

Each separate or C1- 15 alkylene optionally substituted by; is R ⁴ a hydrogen atom or a lower § Norekinore)

^{3): HN- A 1 - C (=} 0) - NH- A 2 - O- NH (R 4) OLE LINK3 ( wherein, A ¹ Oyo

2

And A ² are each independently optionally substituted C1-15 alkylene; R ⁴ is hydrogen atom or lower alkyl) OLE 丄 INK3

4) Formula: HN— (CH CH O) — A ¹ — O— NH (R ⁴ ) (where A ¹ may be substituted

2 2 2 η

C1- 15 alkylene; R ⁴ is a hydrogen atom or a lower alkyl),

5) Formula: HN— (CH CH Ο) — A ¹ — O— A ² — O— NH (R ⁴ ) (where A ¹ and A ^{2 are}

2 2 2 η

Respectively independently optionally substituted C1- 15 alkylene; R ⁴ is a hydrogen atom or a lower alkyl),

6) Formula: HN— (CH CH O) —A ¹ —NH—C (= 0) —A ² — O—NH (R ⁴ ) (where A ¹

2 2 2 η

And Α ² are each independently substituted !, or! /, C 1-15 alkylene; R ⁴ is a hydrogen atom or lower alkyl),

7) Formula: HN— (CH CH O) -A ¹ -C (= 0) —NH—A ² —O—NH (R ⁴ ) (where A ¹

2 2 2 η

8) Formula:

[Formula 14]

(Where A ¹ is C 1-5 alkylene; R ⁴ is a hydrogen atom, lower alkyl, or aryl); and R ¹ is —NH (R ² ) (where R ² is a hydrogen atom, lower alkyl, aryl Or aralkyl))), and 9) General formula (ΠΙ):

[Formula 15]

(In the formula, A ¹ is an optionally substituted CI-15 alkylene; R ⁴ is a hydrogen atom, lower alkyl, or aryl). In the above 1) to 7) and 9), A ¹ is preferably C3-8 alkylene. In the above 1) to 9), R ⁴ is preferably a hydrogen atom or lower alkyl.

[0016] In the present specification, "fluid" can be any fluid as long as it can provide an environment in which the sugar chain peptide binding agent of the present invention interacts with a sugar chain. . Preferably, such fluids are substantially free of substances containing keto groups. This is because, when a substance containing a keto group is significantly contained, the reaction between the aldehyde group in the fluid and the substance of the present invention does not proceed sufficiently. Therefore, the V-form that does not contain a substance containing a keto group is not essential, but is preferably an embodiment.

Accordingly, it is preferred that the fluid used herein is such that it brings the sugar into equilibrium between the cyclic hemiacetal form and the acyclic aldehyde form. Such fluids include, but are not limited to, for example, aqueous solutions, organic solvents, and mixtures thereof. Preferably, the fluid is an aqueous solution.

[0017] In the present specification, "transglutaminase" (sometimes abbreviated as TGase) refers to a γ-carboxyamide group in a glutamine residue (Gin) in a protein or peptide chain and an ε in a lysine residue (Lys) in a lysine residue (Lys). An enzyme that catalyzes an acyl transfer reaction between an amino group or an alkylamine. Transdaltaminase is widely distributed in animals (various tissues, blood cells and plasma) and microorganisms. Thus, transdaltaminase has various molecular forms, but the molecular forms are diverse. Transdaltaminase catalyzes a cross-linking reaction due to _ε (γ daltamyl) lysine isopeptide linkage between or within peptide chains in vivo. Transdaltaminase is a substrate for Gin residues Since the specificity is extremely high, it is possible to modify only a specific Gin residue in a protein by using a molecule such as alkylamine. Transdaltaminase is said to be involved in fibrin molecular cross-linking, keratinization of epidermal cells, semen coagulation, and wound tissue healing.

By reacting the sugar chain-peptide binder with the glutamine-containing peptide in the presence of transdaltaminase, the sugar chain peptide binder is easily introduced into the glutamine residue of the peptide.

The sugar chain-peptide binding agent is allowed to act on the cysteine-containing peptide in the presence of transdaltaminase, whereby the sugar chain peptide binding agent is easily introduced into the cysteine residue of the peptide.

As used herein, “transferase” refers to an enzyme that catalyzes a group transfer reaction. Representative transferases used herein include glycosyltransferases, which catalyze the transfer of sugars. Exemplary glycosyltransferases include, for example, β 1,4-galatatosyltransferase, α-1,3-galatatosyltransferase, | 81,4-galatatosyltransferase, β ,, 3 Galactosyltransferase, β ΐ, 6-galactosyltransferase, 2,2,6 sialyltransferase, 1,1,4 galactosyltransferase, ceramide galatatosyltransferase, 1,1,2 fucosyltransferase, αΐ, 3 fucose Transferase, << 1, 4 fucose transferase, << 1, 6-fucose transferase, —1,3- ァ acetylgalatatosamine transferase, 1,1,6-—Νcetylgalatatosamine transferase, j81, 4—N-acetylgalatasosyltransferase, polypeptide N-acetylacetylgalatasamine-transferase, βΐ, 4Νacetyldarcosamine-transferase, βΐ, 2-diacetyldarcosamine-transferase, βΐ, 3—Psetyldarcosamine transferase, β ΐ, 6 Pasetyldarcosamine transferase, αΐ, 4—Pasetyldarcosamine transferase, βΐ, 4 mannose transferase, << 1,2 mannose transferase, << 1,3-mannose transferase, αΐ, 4-mannose transferase , Αΐ, 6-mannose transferase, αΐ, 2 glucose transferase, αΐ, 3 glucose transferase, α2,3 sialyltransferase, α2,8 sialyltransferase, << 1,6-dalcosamine transferase, << 1,6-xylose transferase, / 3 xylose transferase (proteodalican core structure synthase), j81, 3 glucuronyl transferase and hyaluronic acid synthase It is not limited to these.

[0019] In the present specification, "amino acid" may be natural or non-natural as long as the object of the present invention is satisfied. "Derivative amino acids" or "amino acid analogs" are those that differ from naturally occurring amino acids but have a similar function as the original amino acid. Such derived amino acids and amino acid analogs are well-known in the art. The term “natural amino acid” refers to the L-isomer of a natural amino acid. Natural amino acids include glycine, alanine, norin, leucine, isoleucine, serine, methionine, threonine, pheninolealanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asnolagin, glutamic acid, glutamine, and carboxyglutamic acid. , Arginine, ordinine, and lysine. Unless otherwise indicated, all amino acids referred to herein are L-forms, but forms using D-form amino acids are also within the scope of the present invention. The term “unnatural amino acid” refers to an amino acid not normally found in nature in proteins. Examples of non-natural amino acids include D- or L-forms of norleucine, para-trophe-l-alanine, homophen-l-alanine, para-fluorophen-l-alanine, 3-amino-2-benzylpropionic acid, and homoarginine, and D-fe-l-alanine Is mentioned. "Amino acid analog" refers to a molecule that is not an amino acid, but that is similar to the physical properties and Z or function of the amino acid. Amino acid analogs include, for example, etyonin, canavanine, 2-methyldaltamine and the like. Amino acid mimetics refers to compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. IUPAC-may be referred to herein by any of the single letter symbols recommended by the IUB Biochemica 1 Nomenclature Commission.

[0020] In the present specification, "biological activity" and "bioactivity" are used interchangeably in the present specification, and a certain factor (eg, nucleotide, polynucleotide, protein, etc.) is contained in a living body. It refers to the activity obtained, and includes activities that exert various functions (eg, transcription promoting activity). For example, if two factors interact (eg, insulin acts on the insulin receptor in the liver, etc.), its biological activity is The binding between the two molecules and the resulting biological changes, such as when one molecule is precipitated with an antibody and the other co-precipitates, the two molecules bind It is thought that there is. Therefore, looking at such co-precipitation is one of the judgment methods. In addition, it is possible to associate that a molecule is functionally related to another molecule using neurite extension as an index. Specifically, it includes checking the blood glucose lowering effect of insulin. For example, if a factor is an enzyme, its biological activity includes the enzymatic activity. In another example, where an agent is a ligand, the ligand involves binding to the corresponding receptor. Such a biological activity can be measured by techniques well known in the art.

As used herein, “bioactive substance” refers to any substance having the biological activity as described above. Such substances include, but are not limited to, for example, enzymes, antibodies, vaccines, serum proteins, hormones, cytokins, growth factors, cell adhesion molecules, receptors, signaling factors, and the like.

As used herein, "activity" is the ability to show or reveal binding (either direct or indirect); affect the response (i.e., measure in response to some exposure or stimulus). Refers to various measurable indices, eg, the affinity of a compound that binds directly to a polypeptide or polynucleotide of the invention, or, eg, upstream or downstream after some stimulation or event. A measure of the amount of other proteins or other similar functions can be mentioned. Such activity can be measured by assays such as competitive inhibition.

As used herein, the term “binding” refers to a physical or chemical interaction between two proteins or compounds or related proteins or compounds, or a combination thereof. . Bonds include ionic bonds, non-ionic bonds, hydrogen bonds, van der Waals bonds, hydrophobic interactions, and the like. The physical interaction (binding) can be direct or indirect, the indirect being a force or cause through the effect of another protein or compound. Direct binding refers to interactions that do not occur through or due to the effects of another protein or compound, and are not associated with other substantial chemical intermediates. [0022] In the present specification, unless otherwise specified, "substitution" refers to replacing one or more hydrogen atoms in an organic compound or a substituent with another atom or atomic group. It is also possible to remove one hydrogen atom and substitute a monovalent substituent, and it is also possible to remove two hydrogen atoms and substitute a divalent substituent.

As used herein, the term “alkyl” refers to a monovalent group generated by the loss of one hydrogen atom in an aliphatic hydrocarbon (alkane) such as methane, ethane, and propane.

n 2n + l is represented by one (where n is a positive integer). Alkyl can be straight or branched. As used herein, the term "substituted alkyl" refers to an alkyl in which H of the alkyl is substituted by a substituent 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-C12 alkyl or C1-C12 alkyl, C1-C2-substituted alkyl, C1-C3-substituted alkyl, C1-C4-substituted alkyl, C1-C5-substituted alkyl, C1-C6-substituted Alkyl, C1-C7 substituted alkyl, C1-C8 substituted alkyl, C1-C9 substituted alkyl, C1-C10 substituted alkyl, C1-C11 substituted alkyl or C1-C12 substituted alkyl. Or 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—), iso

3 2 5 3 2 2 Propyl ((CH) CH-), n-butyl (CH CH CH CH-), n-pentyl (CH C

3 2 3 2 2 2 3

H CH CH CH-I), n-Hexyl (CH CH CH CH CH CH-I), n-Heptyl

2 2 2 2 3 2 2 2 2 2

(CH CH CH CH CH CH CH-), n-octyl (CH CH CH CH CH CH CH C

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

H CH—), n—nonyl (CH CH CH CH CH CH CH CH CH—), n—decyl

2 2 3 2 2 2 2 2 2 2 2

(CH CH CH CH CH CH CH CH CH CH-I), C (CH) CH CH CH (C

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

H), —CH CH (CH) and the like. Also, for example, C1-C10 substituted

3 2 2 3 2

Alkyl is C1-C10 alkyl in which one or more hydrogen atoms have been replaced by substituents.

[0023] In the present specification, "lower alkyl" is preferably C1 to C6 alkyl, and more preferably C1 to C3 alkyl. In the present specification, “alkylene” is a divalent group derived from “alkyl”, for example, methylene, ethylene, trimethylene, tetramethylene, pentamethylene, hexamethylene, heptamethylene, octamethylene, nonane Methylene, decanmethylene, pendecamethylene, dodecamethylene and the like can be mentioned.

As used herein, the term "alkenyl" refers to a monovalent group such as ethylene or propylene, which is generated by the loss of one hydrogen atom of an aliphatic hydrocarbon having one double bond in the molecule, Generally represented by CH (where n is a positive integer greater than or equal to 2). "Replaced

n 2n_ l

The term "modified alkyl" refers to an alkyl in which H of the alkyl is substituted by a substituent defined below. Specific examples include C2-C3 alkenyl, C2-C4 alkenyl, C2-C5 alkenyl, C2-C6 alkenyl, C2-C7 alkenyl, C2-C8 alkenyl, C2-C9 alkenyl. C2 to C10, K2 to C11, K2 to C12, C2 to C3 substituted C, C2 to C4 substituted C, C2 to C5 Substituted alkyl, C2-C6 substituted alkyl, C2-C7 substituted alkyl, C2-C8 substituted alkyl, C2-C9 substituted alkyl, C2-C It may be a 10-substituted, a 2- to 11-substituted or a 2- to 12-substituted. Here, for example, C2-C10 alkyl means a straight-chain or branched alkylene having 2 to L0 carbon atoms, such as butyl (CH = CH—), aryl

2

(CH = CHCH—), CH CH = CH—, and the like. Also, for example, C2-C

2 2 3

A 10-substituted alkylene is a C2-C10 alkylene, in which one or more hydrogen atoms are substituted with a substituent.

In the present specification, “alkene” is a divalent group derived from “alkenyl”, and examples include biylene, probenylene, and butylene.

As used herein, the term “alkynyl” refers to a monovalent group such as acetylene formed by the loss of one hydrogen atom from an aliphatic hydrocarbon having one triple bond in the molecule, and is generally CH 2 (Where n is a positive integer greater than or equal to 2). "Substituted al n 2n_3

“Kyl” refers to an alkyl in which H of the alkyl is substituted by a substituent defined below. Specific examples include C2-C3 alkyl, C2-C4 alkyl, C2-C5 alkyl, C2-C6 alkyl, C2-C7 alkyl, C2-C8 alkyl, C2- C9 Alkyl, C2-C10 Alkyl, C2-C11 Alkyl, C2-C12 Alkynyl, C2-C3-Substituted Alkyl, C2-C4-Substituted Alkyl, C2-C5-Substituted Alkyl -Alkyl, C2-C6-substituted alkyl, C2-C7-substituted alkyl, C2-C8-substituted alkyl, C2-C9-substituted alkyl, C2-C10-substituted It can be an alkyl, a C2-C11 substituted alkyl or a C2-C12 substituted alkyl. Here, for example, C2-C10 alkyl means, for example, a linear or branched alkyl containing 2 to 10 carbon atoms, such as ethur (CH≡C—), 1 propynyl ( CH C≡C). Also, for example, C2-C10 substituted alkyl

Three

Nyl refers to C2-C10 alkyl in which one or more hydrogen atoms are substituted with a substituent.

In the present specification, “alkylene” is a divalent group derived from “alkyl”, and includes, for example, propylene, butylene and the like.

In the present specification, "optionally substituted C1-15 alkylene", "optionally substituted C2-15 alkylene" or "substituted! As the substituent, is substituted or substituted by a substituent selected from Substituent Group A, aryl, heteroaryl which may be substituted by a substituent selected from Substituent Group A, A non-aromatic carbocyclic group optionally substituted by a substituent selected from Group A, a non-aromatic heterocyclic group optionally substituted by a substituent selected from Substituent Group A , Hydroxy, alkyloxy, mercapto, alkylthio, halogen, nitro, shear-input ruboxy, alkyloxycarbol, haloalkyl, haloalkyloxy, optionally substituted aminocarboxy which may be substituted Phenyl, acryl, acryloxy, aryloxy (for example, phenyloxy), aralkyloxy (for example, benzyloxy), lower alkylsulfonyl, guadino, azo, etc. These are one in every possible position. The above can be substituted.

As used herein, “substituted or substituted by a substituent selected from substituent group A, V” means that one or more substituent groups A described below are substituted. Well, that means.

Substituent group A: alkyl, hydroxy, alkyloxy, mercapto, alkylthio, halo Gen, nitro, carboxy, alkyloxycarboyl, haloalkyl, haloalkyloxy, haloalkyloxy, optionally substituted aminocarbyl which may be substituted, acyl, acyloxy, aryloxy (for example, phenyloxy) Aralkyloxy (for example, benzyloxy), lower alkylsulfonyl, guadino, azo group, substituent group B. The substituent may be substituted with a selected substituent, or may be substituted with an aryl or substituent group B. A heteroaryl, substituted with a substituent selected from a substituent group B, a non-aromatic carbocyclic group, and a substituent selected from a substituent group B A non-aromatic heterocyclic group which may be substituted by a group;

In the present specification, the term "substituent group B may be substituted by a selected substituent" means that the substituent group B may be substituted with one or more substituent groups below. You.

Substituent group B: alkyl, hydroxy, alkyloxy, mercapto, alkylthio, halogen, nitro, carboxy, carboxy, alkyloxycarbo, haloalkyl, haloalkyloxy, optionally substituted, optionally substituted Aminocarbyl, acetyl, acryloxy, aryloxy (eg, phenyloxy), aralkyloxy (eg, benzyloxy), lower alkylsulfonyl, guadino, azo group, aryl, heteroaryl, non-aromatic carbocyclic group, and Aromatic heterocyclic group.

As used herein, “aryl” refers to a group formed by the removal of one hydrogen atom bonded to a ring of an aromatic hydrocarbon, and is included in the “carbocyclic group” in this specification. Examples thereof include phenyl, a-naphthyl, 13-naphthyl, anthyl, indul, fenanthryl and the like. “Substituted aryl” refers to aryl substituted with a substituent selected below.

In the present specification, when the “protecting group” is a protecting group for an amino group and an imino group, examples thereof include benzyl, benzoyl, acetyl, t-butoxycarbonyl, and benzyloxycarbonyl. Acetyl is preferred. The protection reaction and the deprotection reaction can be performed according to the method described in ci ci, for example, Protective uroups in Organic Synthesis, Theodora W ureen (John Wiley & Sons).

In the present specification, Cl, C2,... Cn represent the number of carbon atoms. Therefore, C1 has 1 carbon Used to represent a substituent of

[0027] In this specification, unless otherwise specified, the term "substitution" refers to replacing one or more hydrogen atoms in an organic compound or a substituent with another atom or atomic group. It is also possible to remove one hydrogen atom and substitute with a monovalent substituent, and it is also possible to remove two hydrogen atoms and substitute with a divalent substituent.

Examples of the substituent include alkyl, cycloalkyl, alkaryl, cycloalkyl, alkyl, alkoxy, carbocyclic group, heterocyclic group, halogen, hydroxy, thiol, cyano, nitro, amino, carboxy, carbamoyl, and acyl. , Asilamino, thiocarboxy, amide, carbol, thiocarbol, sulfol or sulfiel, but are not limited thereto. When the substituent is substituted, alkyl, cycloalkyl, alkenyl, cycloalkyl, alkyl, alkoxy, alkenyl, alkoxy, alkoxyalkyl, halogenoalkyl, halogen, nitro, succinyl , Acyloxy, hydroxy, mercapto, carboxy, thiocarboxy, alkoxycarbyl, aryloxycarbol, carbamoyl, optionally substituted amino. The “optionally substituted amino” means an amino, which may be substituted at one or two places with alkyl, aryl, aryl or arylalkyl.

[0028] (Organic chemistry)

Regarding organic chemistry, for example, Morrison Boyd Organic Chemistry (upper) (middle) (lower) 5th edition (published by Tokyo Chemical Doujinshi (1989)), March, Advanced Organic Chemstry 4th edition (Wiley Intersience, JOHN WILEY & SONS, 1992) and the like, the relevant portions of which are incorporated herein by reference.

As used herein, the term "protection reaction" refers to Boc (t-butoxycarbol), acetyl, benzyl, Cbz (benzyloxycarbol), Fmoc (9-fluorenylmethoxycarbonyl), and the like. A reaction in which a protecting group is added to a functional group for which protection is desired. By protecting a functional group with a protecting group, the reaction of a functional group having higher reactivity can be suppressed, and only the functional group having lower reactivity can be reacted.

As used herein, the term "deprotection reaction" refers to a reaction for removing a protecting group as listed above. As for the deprotection reaction, trifluorene is used for Boc (t-butoxycarbol). Reaction with acetic acid (TF A) and H, PdZC for benzyl ether

2

Reactions such as the original reaction can be mentioned. The `` protection reaction '' and `` deprotection reaction '' are described in detail, for example, in TW Greene and PGM Wuts, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, 3rd edition (JOHN WILE Y & SONS, Inc. (1999)). The contents are incorporated herein by reference.

(General techniques used in this specification)

The techniques used herein are within the skill of the art, unless otherwise indicated, in analytical chemistry, organic chemistry, biochemistry, genetic engineering, molecular biology, microbiology, unless otherwise indicated. Uses conventional techniques well known in the genetics and related fields. Such techniques are explained fully, for example, in the literature listed below and in the references cited elsewhere herein.

The molecular biological, biochemical, and microbiological techniques used herein are well known and commonly used in the art, and are described, for example, in Maniatis, T. et al. (, 1989). . 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, 101 58 (2000); Innis, MA (1990) .PCR Protocols: A Guide to Methods and Applications, Academic Press; Innis, MA et al. (1995) .PCR St rategies, Academic Press; Sninsky, JJ et al. "999) .PCR Applications s: Protocols for Functional Genomics, Academic Press; Gait Oligonucleotide Synthesis: A Practical Approach, IRL Press; Git, MJ (1990) .Oliigonucleotide Synthesis: A Practical Approach, IR L Press; Eckstein, F. (1991) .Oliigonucleotides and Analogues: A Prac tical Approac, IRL Press; Adams, RL et al. (1992) .The Biochemis try of the Nucleic Acids, Chapman &Hall; Shabarova, Z. et al. (1994) .Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn , GM et al. (1996) .Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, GT (1996) .Bioconjugate Te. chniques, Academic Press; Method in Enzymology 230, 242, 247, Academic Press, 1994; Separate experimental medicine `` Experimental Method for Gene Transfer and Expression Analysis '' Yodosha, 1997; Hatanaka, Nishimura et al., Carbohydrate Science and Engineering , Kodansha Scientifiq, 1997; Design and Physiological Function of Glycochain Molecules, edited by The Chemical Society of Japan, Academic Publishing Center, 2001, etc., and these are relevant portions (which may be all) in this specification. Incorporated as reference (modification of genes, protein molecules, etc.)

In certain protein molecules (eg, insulin), certain amino acids in the sequence may be used in other protein structures, such as cationic regions or substrate molecule binding sites, without appreciable loss or loss of interaction binding capacity. Amino acid. It is the interaction capacity and properties of a protein that define the biological function of a protein. Thus, certain amino acid substitutions may be made in the amino acid sequence, or at the level of its DNA coding sequence, resulting in a protein that retains its original properties after the substitution. Accordingly, various modifications may be made to the peptide disclosed herein or the corresponding DNA encoding this peptide without appreciable loss of biological utility.

In designing such modifications, the hydrophobicity index of amino acids can be considered. The importance of the hydrophobic amino acid index in conferring interactive biological functions on proteins is generally recognized in the art (Kyte. J and Doolittle, RFJ Mol. Biol. 157 (1): 105-132, 1982). The hydrophobic nature of amino acids contributes to the secondary structure of the resulting protein, which in turn defines the interaction of that protein with other molecules (eg, enzymes, substrates, receptors, DNA, antibodies, antigens, etc.). Each amino acid is assigned a hydrophobicity index based on its hydrophobicity and charge properties. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); hueralanine (+2.8); cysteine Z cystine (+2.5); methionine (+ 1.9); alanine (+1.8); glycine (0.4); threonine (0.7); serine (0.8); tryptophan (0.9); tyrosine (1.1). .3); Proline (-1.6); Histidine (1.3.2); Gnoletamic acid (13.5); Gnoletamine (13.5); Aspartic acid (13.5); Asparagine (1. 3.5); lysine (1-3.9); and arginine (1-4.5) ).

It is well known in the art that one amino acid can be replaced by another amino acid having a similar hydrophobicity index and still yield a protein having a similar biological function (e.g., an equivalent protein in enzymatic activity). It is well known. In such amino acid substitutions, the hydrophobicity index is preferably within ± 2, more preferably within ± 1, and even more preferably within ± 0.5. It is understood in the art that such substitution of amino acids based on hydrophobicity is efficient. As described in US Pat. No. 4,554,101, the following hydrophilicity indices have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); Aspartic acid (+ 3.0 ± 1); Glutamic acid (+ 3.0 ± 1); Serine (+0.3); Asparagine (+0.2); Glutamine (+0.2); Glycine (0); Threonine (10.4); Proline (10.5 ± 1); Alanine (10.5); Histidine (10.5); Cystine (11.0); Methionine (11.3); Valine (1-1.5); Leucine (1-1.8); Isoloisin (1-1.8); Tyrosine (1-2.3); Hue-alanan (1-2.5); and Tryptophan (3.4) . It is understood that an amino acid can be substituted for another that has a similar hydrophilicity index and still provide a bioisostere. In such amino acid substitutions, the hydrophilicity index is preferably within ± 2, more preferably within ± 1, and even more preferably within ± 0.5.

As used herein, the term "conservative substitution" refers to an amino acid substitution in which the hydrophilicity index or Z and hydrophobicity index of the amino acid to be replaced with the original amino acid are similar to those described above. Replacement. Examples of conservative substitution include, for example, those having a hydrophilicity index or a hydrophobicity index within ± 2, preferably within ± 1, more preferably within ± 0.5. But not limited to them. Thus, examples of conservative substitutions are well known to those skilled in the art and include, for example, substitutions within each of the following groups: arginine and lysine; glutamic and aspartic acid; serine and threonine; glutamine and asparagine; Leucine, and isoleucine, but are not limited thereto.

As used herein, the term "variant" refers to a substance in which a substance such as an original peptide or polypeptide is partially changed. Such variants include substitutional variants , Addition variants, deletion variants, truncated variants, allelic variants and the like. Such variants include one or more substitutions, additions and Z or deletions, or one or more substitutions, additions and Z or deletions with respect to a reference nucleic acid molecule or peptide. But not limited to them. Alleles refer to genetic variants that belong to the same locus and are distinct from each other. Therefore, “allelic variant” refers to a variant that has an allelic relationship to a certain gene. Such allelic variants usually have sequences that are identical or very similar to their corresponding alleles, usually have nearly the same biological activity, but rarely have different biological activities. May have activity. "Species homolog or homolog" refers to homology (preferably 60% or more homology, more preferably 80% or more) of a certain gene at the amino acid level or nucleotide level with a certain gene. , 85% or more, 90% or more, 95% or more homology). Homology can be calculated using BLAST software. A method for obtaining such a species homolog is apparent from the description of the present specification. The term "ortholog" is also called an orthologous gene, and refers to a gene derived from a common ancestral species, Tseidai, which has two genes. For example, taking the hemoglobin gene family with a multigene structure as an example, human and mouse α-hemoglobin genes are orthologs.Human α-hemoglobin gene and j8 hemoglobin gene are paralogs (genes generated by gene duplication). is there. Orthologs are useful for estimating molecular phylogenetic trees. Orthologs can usually perform the same function as the original species in another species, so the orthologs of the present invention can also be useful in the present invention.

As used herein, the term "conservative (modified) variant" applies to both amino acid sequences and nucleic acid sequences. With respect to a particular nucleic acid sequence, a conservatively modified variant refers to a nucleic acid that encodes the same or essentially the same amino acid sequence, and essentially the same if the nucleic acid does not encode an amino acid sequence. Sequence. Due to the degeneracy of the genetic code, many functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, that codon is The codons can be changed to any of the corresponding codons described without altering the polypeptide. Such nucleic acid variation is a "silent modification (mutation)," which is one type of conservatively modified mutation. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. In the art, each codon in the nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) 1S Produces functionally identical molecules It will be understood that this can be modified. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence. Preferably, such modifications may be made to avoid substitution of the amino acid cysteine, which greatly affects the conformation of the polypeptide. Examples of such base sequence modification methods include cleavage with a restriction enzyme or the like, ligation treatment with a DNA polymerase, Klenow fragment, DNA ligase, or the like, site-specific base substitution using a synthetic oligonucleotide, or the like. (Site-directed mutagenesis; Mark Zoller and Michael Smith, Methods in Enzymology, 100, 468-500 (1983)) You can do it too.

As used herein, in addition to amino acid substitution, amino acid addition, deletion, or modification can also be performed to produce a functionally equivalent polypeptide. Substitution of an amino acid refers to substitution of the original peptide with one or more, for example, 1 to: L0, preferably 1 to 5, more preferably 1 to 3 amino acids. The addition of amino acids refers to adding one or more, for example, 1 to 10, preferably 1 to 5, and more preferably 1 to 3 amino acids to the original peptide chain. Amino acid deletion refers to deletion of one or more, for example, 1 to 10, preferably 1 to 5, and more preferably 1 to 3, amino acids from the original peptide. Amino acid modifications include amidation, carboxylation, sulfation, halogenation, shortening, lipidation, phosphorylation, alkylation, glycosylation, phosphorylation, hydroxylation, hydroxylation, acylation (eg, acetyl ) And other forces. The amino acid to be substituted or added may be a natural amino acid or a non-natural amino acid or an amino acid analog. Natural amino acids are preferred.

As used herein, the term “peptide derivative” refers to a compound that differs from a peptide. But at least one chemical or biological function equivalent to that of the peptide. Thus, peptide analogs include those in which one or more amino acid analogs or amino acid derivatives have been added or substituted with the original peptide.

. Peptide analogs have the same functional capabilities as the original peptide (e.g., similar _P Ka values, similar functional groups, similar binding modes to other molecules, Such additions or substitutions have been made to be substantially similar to those of similar gender. Such peptide analogs can be made using techniques well known in the art. Thus, a peptide analog can be a polymer that includes an amino acid analog.

As used herein, a nucleic acid molecule may have a portion of the nucleic acid sequence deleted or other as described above, as long as the expressed peptide has substantially the same activity as the native peptide. It may be replaced by a base or another nucleic acid sequence may be partially inserted. Alternatively, another nucleic acid may be bound to the 5 'end and the Z or 3' end. Alternatively, a nucleic acid molecule which encodes a peptide-encoding gene under stringent conditions and encodes a peptide having substantially the same function as the peptide may be used. Such genes are known in the art and can be used in the present invention.

Such a nucleic acid can be obtained by a well-known PCR method, and can also be chemically synthesized. These methods may be combined with, for example, a site-specific displacement induction method, a hybridization method, or the like.

As used herein, the term "substitution, addition or deletion" of a peptide or polypeptide refers to the replacement, addition or removal of an amino acid or a substitute thereof with respect to the original peptide or polypeptide, respectively. That means. Techniques for such substitution, addition or deletion are well known in the art, and examples of such techniques include site-directed mutagenesis techniques. The number of substitutions, additions or deletions may be any number as long as the number is one or more.Such numbers may vary depending on the variant having the substitution, addition or deletion, and the intended function (for example, hormone As long as the information transfer function of the site power-in is maintained. For example, such a number can be one or several, and And preferably less than 20%, less than 10%, or less than 100, less than 50, less than 25, etc. of the total length.

(Genetic engineering)

The physiologically active peptide and the like, and fragments and variants thereof used in the present invention can be produced using genetic engineering techniques.

As used herein, when referring to a gene, the term "vector" or "recombinant vector" refers to a vector capable of transferring a target polynucleotide sequence to a target cell. . Such vectors are capable of autonomous replication in host cells such as prokaryotic cells, yeast, animal cells, plant cells, insect cells, animal individuals and plant individuals, or capable of incorporation into chromosomes, Those containing a promoter at a position suitable for transcription of the polynucleotide of the present invention are exemplified. Of the vectors, those suitable for cloning are referred to as “cloning vectors”. Such a closing vector usually contains a multiple closing site containing a plurality of restriction enzyme sites. Such restriction enzyme sites and multiple closing sites are well known in the art, and those skilled in the art can appropriately select and use them according to the purpose. Such techniques are described in the literature described herein (eg, Sambrook et al., Supra). Preferred vectors include plasmids, phages, cosmids, episodes, viral particles or viruses, and DNA fragments that can be integrated (ie, fragments that can be integrated into the host genome by homologous recombination). It is not limited to. Preferred viral particles include, but are not limited to, adenovirus, baculovirus, parvovirus, herpes virus, box virus, adeno-associated virus, semliki forest virus, vaccinia virus and retrovirus.

One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors (eg, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) are capable of autonomous replication in a host cell into which they are introduced. Other vectors (e.g., non-episomal mammalian vectors) are capable of introducing host cell genes into their host cells upon introduction. It is integrated into the nom and is thereby replicated with the host genome. In addition, certain vectors may direct the expression of genes to which they are operably linked. One such vector is referred to herein as an “expression vector”.

Therefore, as used herein, the term "expression vector" refers to a nucleic acid sequence in which various regulatory elements are operably linked in a host cell in combination with a structural gene and a promoter that regulates its expression. Say. The regulatory elements may preferably include a terminator, a selectable marker such as a drug resistance gene, and an enhancer. It is well known to those skilled in the art that the type of expression vector of an organism (eg, an animal) and the type of regulatory element used may vary depending on the host cell.

Examples of the "recombinant vector" for prokaryotic cells that can be used in the present invention include pc DNA3 (+), pBluescript-SK (+ Z-I), pGEM-T, pEF-BOS, pEGFP, pHAT, pUC18, pFT-DESTTM42GATEWAY ( Examples of the “recombinant vector” for animal cells that can be used in the present invention include pcDNAI / Amp, pcDNAI, pCDM8 (all sold from Funakoshi), pAGE107 [Japanese Unexamined Patent Publication No. 3-229 (Invitrogen). ), PAGE103 Q [. Biochem., 101, 1307 (1987)], ρΑΜo, pAMoAQ [. Biol. Chem., 268, 22782--22787 (1993)], mouse stem cell virus (Murine Stem Cell Virus) (MSCV ), PEF-BOS, pEGFP and the like.

As used herein, the term "terminator 1" is a sequence located downstream of a region encoding a gene protein and involved in terminating transcription when DNA is transcribed into mRNA and adding a poly A sequence. Terminators are known to be involved in mRNA stability and affect gene expression levels.

As used herein, the term "promoter" refers to a region on DNA that determines the transcription start site of a gene and directly regulates the frequency of transcription, and is a base sequence to which RNA polymerase normally starts transcription by binding. . Therefore, in the present specification, a portion of a gene having a promoter function is referred to as a “promoter portion”. The promoter region is usually within about 2 kbp upstream of the first exon of the putative protein coding region. Therefore, the promoter region can be estimated by predicting the protein coding region in the genomic base sequence using DNA analysis software. The putative promoter region varies for each structural gene, but is usually, but not limited to, upstream of the structural gene, and may be downstream of the structural gene. Preferably, the putative promoter region is also within about 2 kbp upstream of the first exon translation initiation force.

As used herein, the “origin of replication” refers to a specific region on a chromosome where DNA replication starts. The origin of replication can either be provided by constructing the vector to include an endogenous origin, or can be provided by the chromosomal replication machinery of the host cell. If the vector integrates into the host cell chromosome, the latter may be sufficient. Alternatively, rather than using a vector containing a viral origin of replication, one of skill in the art can transform mammalian cells by a method of co-transforming a selectable marker with the DNA of the present invention. Examples of suitable selectable markers are dihydrofolate reductase (DHFR) or thymidine kinase (see US Pat. No. 4,399,216).

For example, by expressing nucleic acids using tissue-specific regulatory elements, recombinant mammalian expression vectors can preferentially direct expression of the nucleic acid in certain cell types. Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include developmentally regulated promoters (eg, the mouse hox promoter (Kessel and Gruss (1990) Science 249, 374-379) and α-fe Topoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3, 537-546)), albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1, 268-277), lymph-specific promoter ( Calame and Eaton (1988) Adv. Immunol. 43, 235—275), especially T cell receptors (Winoto and Baltimore (1989) EMBO J. 8, 729—733) and immunoglobulins (Banerji et al. (1983) Cell

33, 729—740; Queen and Baltimore (1983) Cell 33, 741-748), neuron-specific promoters (eg, nerve fiber promoters; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86) , 5473-5477), a spleen-specific promoter (Edlund et al. (1985) Science 230, 912-916), and a mammary gland-specific promoter (eg, whey promoter; US Pat. No. 4,873,316 and European Application Publication Nos. 264, 166).

[0038] In the present specification, the term "ennosense" refers to a sequence used to increase the expression efficiency of a target gene. Such enos are well known in the art. A plurality of enhancers can be used, but one may be used! /, And no need to use! /.

As used herein, the term “operably linked” refers to a transcription / translation control sequence (eg, promoter, henno, saccharin, etc.) or translation control that has expression (operation) of a desired sequence. It means to be placed under the control of the sequence. In order for a promoter to be operably linked to a gene, the promoter is usually, but not necessarily, positioned immediately upstream of the gene.

In the present specification, any technique for introducing a nucleic acid molecule into a cell may be used. Examples thereof include transformation, transduction, and transfection. Techniques for introducing such nucleic acid molecules are well known and commonly used in the art, and are described in, for example, Ausubel FA et al. (1988), Current Protocols in Molecular Biotechnology, Wiley, New York, NY ; Sambrook J et al. (1987) Molecular Cloning: A Laboratory Manual, 2nd Ed. And its third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Tsuchiya, 1997. Introduction of the gene can be confirmed using the methods described herein, such as Northern plot, Western plot analysis, or other well-known conventional techniques.

As a method for introducing a vector, any of the above-described methods for introducing DNA into cells can be used, and examples thereof include transfection, transduction, and transformation (for example, calcium phosphate method, Ribosome method, DEAE dextran method, electroporation method, method using particle gun (gene gun), etc.).

[0039] As used herein, the term "transformant" refers to all or a part of an organism such as a cell produced by transformation. Examples of the transformant include prokaryotic cells, yeast, animal cells, plant cells, insect cells, and the like. A transformant is also called a transformed cell, a transformed tissue, a transformed host, or the like, depending on the target. The cells used in the present invention may be transformants. In the present invention! For genetic manipulation, etc.! When a prokaryotic cell is used, the prokaryotic itoda cell may be a prokaryotic cell belonging to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Panyu, Microbacterium, Pseudomonas, or the like, for example, Escherichia coli XL1-Blue II Escherichia coli XL2-Blue Escherichia coli DH1, BL-21 and JM109 are exemplified.

As used herein, animal cells include mouse's myeloma cells, rats, CHO cells, BHK cells, African green monkeys, which are myeloma cells, mouse'ino, ibridoma cells, Chinese'no, and Muster cells. Examples include kidney cells, human leukemia cells, HBT5637 (JP-A-63-299), and human colon cancer cell lines. African green monkeys such as ps20 and NSO for mouse 'myeloma cells, YB2Z0 for rat' myeloma cells, HEK293 (ATCC: CRL-1573) for human fetal kidney cells, and BALL-1 for human leukemia cells Kidney cells include COS-1 and COS-7, human colon cancer cell lines include HCT-15, human neuroblastoma SK—N—SH, SK—N—SH—5Y, mouse neuroblastoma Neuro2A, etc. Is exemplified.

As used herein, any method for introducing a recombinant vector can be used as long as it is a method for introducing DNA, for example, a calcium chloride method, an etatroporation method (Methods.Enzymol. , 194, 182 (1990)], lipofection method, spheroplast method [Proc. Natl. Acad. Sci. USA, 84, 1929 (1978)], lithium acetate method Q [. Bacteriol., 153, 163 (1983) Natl. Acad. Sci. USA, 75, 1929 (1978).

As used herein, a method for infecting a retrovirus is well known in the art, for example, as described in Current Protocols in Molecular Biology, supra (particularly, Units 9.9 to 9.14). Sing 幹胚【sing 後後こここここ、、、、、、、、、、、、、、後、、、後後、、、、、、、. By co-culturing for 1 to 2 hours, a sufficient amount of infected cells can be obtained. The transient expression of the Cre enzyme, DNA mapping on the chromosome, etc., used in the method for removing a genome or a locus used in the present specification, may be performed by cell type. It is well known in this field, as described in the separate study protocol series “FISH Experiment Protocol Human: Genome Analysis Power Chromosomes and Genetic Diagnosis” by Kenichi Matsubara and Hiroshi Yoshikawa, supervised by Shujunsha (Tokyo).

As used herein, “detection” or “quantification” of gene expression (eg, mRNA expression, polypeptide expression) can be achieved using an appropriate method including, for example, mRNA measurement and immunological measurement methods. Examples of the molecular biological measurement method include a Northern blot method, a dot plot method, and a PCR method. Examples of the immunological measurement method include, for example, an ELISA method using a microtiter plate, an RIA method, a fluorescent antibody method, a Western blot method, and an immunohistochemical staining method. Examples of the quantification method include an ELISA method and an RIA method. Genetic analysis using an array (for example, a DNA array or a protein array) can also be performed. The details of DNA arrays are widely reviewed in Shujunsha, edited by Cell Engineering, “DNA Microarrays and Latest PCR Methods”. Protein arrays are described in detail in Nat Genet. 2002 Dec; 32 Suppl: 526-32. Methods for analyzing gene expression include, but are not limited to, RT-PCR, RACE, SSCP, immunoprecipitation, two-hybrid systems, and in vitro translation, as described above. Such a further analysis method is described in, for example, Genome Analysis Experimental MethodYusuke Nakamura Lab 'Mayuaru, Editing' Yusuke Nakamura Yodosha (2002), etc. All are incorporated by reference. As used herein, “expression level” refers to the level of expression of a polypeptide or mRNA in a target cell or the like. Such expression levels are evaluated using the antibody of the present invention by any appropriate method including immunological measurement methods such as ELISA, RIA, fluorescent antibody, western blotting, and immunohistological staining. The expression level of the polypeptide of the present invention at the protein level or the polypeptide of the present invention evaluated by any appropriate method including molecular biological measurement methods such as Northern blotting, dot blotting, and PCR. The expression level of the peptide at the mRNA level can be mentioned. The term “change in expression level” refers to the expression level of the polypeptide of the present invention at the protein level or mRNA level, which is evaluated by any appropriate method including the above-described immunological measurement method or molecular biological measurement method. Means increase or decrease. As used herein, the term “upstream” refers to a position at the 5 ′ end of a particular reference point force polynucleotide.

As used herein, the term “downstream” also indicates a position at which a specific reference point force is also located at the 3 ′ end of the polynucleotide.

[0042] As used herein, the expressions "base paired" and "Watson & Crick base paired" are used synonymously in the present specification, and similar to those found in double-stranded DNA, adenine residues are used. The nucleotides that are capable of hydrogen bonding to each other based on the identity of the sequence where the group is bonded to a thymine or peracyl residue by two hydrogen bonds and the cytosine and guanine residues are bonded by three hydrogen bonds. (See Stryer, L., Biochemistry, 4th edition, 1995).

As used herein, the term "complementary" or "complement" is used herein to refer to a polynucleotide that is capable of forming a Watson & Crick base pair with another specific polynucleotide in its entirety. Shows the sequence. For the purposes of the present invention, a first polynucleotide is considered to be complementary to a second polynucleotide if each base of the first polynucleotide is paired with its complementary base. The complementary bases are generally A and T (or A and U) or C and G. As used herein, the term "complementary" is used as a synonym for "complementary polynucleotide," "complementary nucleic acid," and "complementary nucleotide sequence." These terms apply to a pair of polynucleotides based solely on their sequence, and do not apply to a particular set of two polynucleotides in effect that are in association.

(Method for producing peptide)

A transformant derived from a microorganism, animal cell, or the like having a recombinant vector into which DNA encoding the peptide (for example, insulin or a variant or fragment thereof) used in the present invention has been incorporated, can be subjected to ordinary culture. The peptide of the present invention can be produced by culturing according to the method to produce and accumulate the polypeptide of the present invention, and collecting the peptide of the present invention from the culture of the present invention.

The method of culturing the transformant of the present invention in a medium can be performed according to a usual method used for culturing a host. A prokaryote such as Escherichia coli, or a medium for culturing a transformant obtained by using a eukaryote such as yeast as a host may be assimilated by the organism of the present invention. As long as the medium contains a carbon source, a nitrogen source, inorganic salts, and the like, and can efficiently culture the transformant, a natural medium or a synthetic medium may be used.

[0044] As the carbon source, glucose, flatatose, sucrose, glycerol, carbohydrates such as molasses, starch or starch hydrolysate, acetic acid, and propion containing these can be used as long as each microorganism can assimilate. Organic acids such as acids and alcohols such as ethanol and propanol can be used.

Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate, and other inorganic or organic acid ammonium salts, and other nitrogen-containing substances. In addition, peptone, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digested products thereof can be used.

As the inorganic salt, potassium (II) phosphate, potassium (II) phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, and the like can be used. . Culture is performed under aerobic conditions such as shaking culture or deep aeration stirring culture.

[0045] The culture temperature is 15 to 40 ° C, and the culture time is usually 5 hours to 7 days. During the cultivation, the pH is maintained at 3.0 to 9.0. The pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like. If necessary, an antibiotic such as ampicillin or tetracycline may be added to the medium during the culture.

When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium, if necessary. For example, when culturing a microorganism transformed with an expression vector using the lac promoter, culture a microorganism transformed with an expression vector using the trp promoter such as isopropyl-1-β-D-thiogalatatopyranoside. In addition, indole acrylic acid or the like may be added to the medium. Cells or organs into which the gene has been introduced can be cultured in large quantities using a jar armmenter.

For example, when animal cells are used, the medium for culturing the cells of the present invention may be a commonly used RPMI 1640 medium (The Journal of the American Medical Association). , 199, 519 (1967)), Eagle's MEM medium (Science, 122, 501 (1952)), DMEM medium (Virology, 8, 396 (1959)), 199 medium (Proceedings of the Society for the Biological Medicine) 73, 1 (1950)), or a medium obtained by adding fetal calf serum or the like to such a medium.

Cultivation is usually carried out for 1 to 7 days under conditions such as pH 6 to 8, 25 to 40 ° C, and the presence of 5% C02. If necessary, antibiotics such as kanamycin, penicillin, and streptomycin may be added to the medium during the culturing.

In order to isolate or purify the peptide used in the present invention from a culture of a transformant transformed with the nucleic acid sequence encoding the peptide used in the present invention, a conventional enzyme commonly used in the art is used. Isolation or purification techniques can be used. For example, when the peptide used in the present invention secretes the peptide used in the present invention extracellularly from the transformant for producing the peptide of the present invention, the culture is treated by a method such as centrifugation. , To obtain a soluble fraction. From the soluble fraction, use a solvent extraction method, salting out method using ammonium sulfate, etc., a precipitation method using an organic solvent, and a resin such as getylaminoethyl (DEAE)-Sepharose, DIAION HPA-75 (Mitsubishi Dirigaku). Anion exchange chromatography, cation exchange chromatography using a resin such as S-Sepharose FF (Pharmacia), and hydrophobic chromatography using a resin such as butyl sepharose and phenyl sepharose. A purified sample can be obtained by a method such as gel filtration using molecular sieve, affinity chromatography, chromatofocusing, and electrophoresis such as isoelectric focusing.

When the peptide (for example, insulin) used in the present invention accumulates in a dissolved state in the cells of the transformant for producing the peptide used in the present invention, the culture is centrifuged to remove the peptide from the culture. After collecting the cells and washing the cells, the cells are crushed with an ultrasonic crusher, a French press, a Mantongaulin homogenizer, a Dynomill or the like to obtain a cell-free extract. The supernatant obtained by centrifuging the cell-free extract is subjected to solvent extraction, salting out using ammonium sulfate, desalting, precipitation with an organic solvent, getylaminoethyl (DEAE)-Sepharose, DIAION HPA-75 Anion exchange chromatography using resin (Mitsubishi Chemical), using resin such as S-Sepharose FF (Pharmacia) Cation exchange chromatography, hydrophobic chromatography using resins such as butyl sepharose, phenyl sepharose, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, isoelectrics, etc. A purified sample can be obtained by using an electrophoresis method such as point electrophoresis.

[0047] When the peptide used in the present invention is expressed by forming an insoluble substance in the cells, cells are similarly collected, crushed, and centrifuged to obtain a precipitate fraction obtained by a conventional method. After recovering the polypeptide of the present invention, the insoluble form of the polypeptide is solubilized with a polypeptide denaturing agent. This soluble solution is diluted or dialyzed into a solution that does not contain a polypeptide denaturing agent or has a concentration of the polypeptide denaturing agent that is not so large as to denature the peptide, or is dialyzed to obtain a peptide used in the present invention. After having a normal tertiary structure, a purified sample can be obtained by the same isolation purification method as described above.

Further, the protein can be purified according to the usual protein purification method Q [Evan. Sadler et al .: Methods in Enzymology, 83, 458]. Further, the peptide used in the present invention can be produced as a fusion protein with another protein, and a substance having affinity for the fused protein can be purified by using affinity chromatography. [Yamakawa, Akio, Experimental Medicine, 13, 469–474 (1995)]. For example, according to the method described by Lowe et al. [Proc. Natl. Acad. Sci., USA, 86, 8227-8231 (1989), Genes Devlop., 4, 1288 (1990)] Can be produced as a fusion protein with peptide A and purified by affinity chromatography using immunoglobulin G.

[0048] In addition, the peptide used in the present invention can be produced as a fusion protein with a FLAG peptide and purified by affinity chromatography using an anti-FLAG antibody [Proc. Natl. Acad. Sci., USA , 86, 8227 (1989), Genes Develop., 4, 1288 (1990)]. In such a fusion protein, in the expression vector, the proteolytic cleavage site is combined with the fusion moiety and the recombinant protein in order to allow the separation of the recombinant protein from the fusion moiety following purification of the fusion protein. And is introduced at the junction. Such enzymes and their cognate recognition sequences include Factor Xa, thrombin, and enterokinase. Representative fusion expression vectors include PGEX (Pharmacia Biotech; Smith and Johnson (1988) Gene 67, 31-40), pMAL (New England), which fuses on-S transferase (GST), maltose E binding protein, or protein A to the target recombinant protein Biolabs, Beverly, Mass.) And pRIT5 (Pharmacia, Piscataway, NJ). Furthermore, the antibody against the peptide itself used in the present invention can be purified by affinity chromatography. The peptide used in the present invention can be transcribed in vitro according to a known method I. Biomolecular NMR, 6, 129-134, Science, 242, 1162-1164, Biochem., 110, 166-168 (1991)]. · It can be produced using a translation system.

The peptide used in the present invention can also be produced by a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbol method) or the tBoc method (t-butyloxycarbol method) based on the amino acid information. Chemical synthesis using a peptide synthesizer such as Advanced ChemTech, Applied Biosystems, Pharmacia Biotech, Protein Technology Instrument ^ Synthecell-Vega, PerSeptive, Shimadzu Corporation.

Structural analysis of the purified peptide used in the present invention can be performed by a method commonly used in protein tradition, for example, the method described in Protein Structural Analysis for Gene Cloying (Hisashi Hirano, Tokyo Kagaku Dojin, 1993). Can be implemented. The physiological activity of the peptide used in the present invention can be measured according to a known measuring method.

The production of soluble peptides useful in the present invention can also be achieved by various methods known in the art. For example, peptides can also induce intact transmembrane P75 polypeptide molecular strength by proteolysis by using specific endopeptidases in combination with exopeptidase, Edman degradation, or both. This intact P75 peptide molecule can be purified from its natural source using conventional methods. Alternatively, the intact p75 peptide can be produced by recombinant DNA technology utilizing well-known techniques for cDNA, expression vectors and recombinant gene expression.Preferably, the soluble peptides useful in the present invention are produced directly. And therefore Eliminates the need for the entire p75 peptide as starting material. This can be achieved by the power that can be achieved by conventional chemical synthesis techniques, or by well-known recombinant DNA techniques, where only the DNA sequence encoding the desired peptide is expressed in the transformed host. Can be done. For example, a gene encoding the desired soluble p75 peptide can be synthesized by chemical means using an oligonucleotide synthesizer. Such oligonucleotides are designed based on the amino acid sequence of the desired soluble p75 peptide. The specific DNA sequence encoding the desired peptide can also be derived from the full length DNA sequence by isolation of a specific restriction endonuclease fragment or by PCR synthesis of a specific region from cDNA.

(Method for producing variant peptide)

Deletion, substitution or addition (including fusion) of amino acids of the peptide (for example, insulin or the like) used in the present invention can be carried out by a site-directed mutagenesis method which is a well-known technique. One or several amino acids are deleted, substituted or added.Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor

Laboratory Press (1989), Current Protocols in Molecular Biology, Supplement 1-38, John Wiley & Sons (1987-1997), Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci., USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. Acad. Sci USA, 82, 488 (1985), Proc. Natl. Acad. Sci., USA, 81, 5662 (1984), Science, 224, 1431 (1984), PCT WO85 / 00817 (1985), Nature, 316, 601 (1985), etc. it can.

(Pharmaceutical composition)

In another aspect, the present invention relates to medicaments (for example, medicaments such as vaccines, health foods, medicaments whose protein or lipid has reduced antigenicity) and cosmetics. The medicament and cosmetics may further include a pharmaceutically acceptable carrier and the like. The pharmaceutically acceptable carrier contained in the medicament of the present invention includes any substance known in the art.

Such suitable formulation materials or pharmaceutically acceptable carriers include antioxidants , Preservatives, colorants, flavors, and diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, diluents, excipients and / or pharmaceutical adjuvants. But not limited to them. Typically, the medicament of the invention will be administered in the form of a composition comprising the compound, or a variant or derivative thereof, together with one or more physiologically acceptable carriers, excipients or diluents. . For example, suitable vehicles may be water for injection, physiological solutions, or artificial cerebrospinal fluid, which may be supplemented with other materials common in compositions for parenteral delivery. is there.

As used herein, an acceptable carrier, excipient, or stabilizer is non-toxic to recipients, and is preferably inert at the dosages and concentrations employed, and Phosphate, citrate, or other organic acid; ascorbic acid, a tocopherol; low molecular weight polypeptide; protein (eg, serum albumin, gelatin or immunoglobulin); hydrophilic polymer (eg, Amino acids (eg, glycine, glutamine, asparagine, arginine or lysine); monosaccharides, disaccharides and other carbohydrates (including glucose, mannose, or dextrin); chelating agents (eg, EDTA); Sugar alcohol (eg, mantole or sorbitol); salt form Counterions (such as sodium); and Z or non-ionic surface-active agents (e.g., Tween, pull port nick (pluronic) or polyethylene glycol (PEG)).

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

Hereinafter, a general method for preparing the pharmaceutical composition of the present invention will be described. In addition, animal drug compositions, quasi-drugs, marine drug compositions, food compositions, ligne cosmetic compositions, and the like can also be produced by known preparation methods.

The peptide into which the sugar chain of the present invention has been introduced is compounded with a pharmaceutically acceptable carrier. Orally or parenterally as solid preparations such as tablets, capsules, granules, powders, powders, suppositories, etc., or liquid preparations such as syrups, injections, suspensions, solutions, sprays, etc. be able to. Pharmaceutically acceptable carriers include, as described above, excipients, lubricants, binders, disintegrants, disintegration inhibitors, absorption promoters, adsorbents, humectants, and dissolution aids in solid preparations. , Stabilizers, solvents in liquid preparations, solubilizers, suspending agents, tonicity agents, buffers, soothing agents and the like. If necessary, pharmaceutical additives such as preservatives, antioxidants, coloring agents and sweeteners can be used. Further, the composition of the present invention can also contain substances other than the polynucleotide, polypeptide and the like of the present invention. Parenteral routes of administration include, but are not limited to, intravenous injection, intramuscular injection, nasal, rectal, vaginal and transdermal.

Examples of excipients in solid preparations include glucose, ratatose, sucrose, D-mantol, crystalline cellulose, starch, calcium carbonate, light anhydrous silicic acid, sodium chloride, kaolin and urea.

Examples of the lubricant in the solid preparation include, but are not limited to, magnesium stearate, calcium stearate, powdered boric acid, colloidal citric acid, talc, polyethylene glycol and the like.

Examples of the binder in the solid preparation include water, ethanol, propanol, sucrose, D-mantol, crystalline cellulose, dextrin, methylcellulose, hydroxypropinoresenolerose, hydroxypropinolemethinoresenorelose, canoleboxy methinoresenorelose, Examples include starch solutions, gelatin solutions, polyvinylpyrrolidone, calcium phosphate, potassium phosphate, and shellac.

Disintegrants in solid preparations include, for example, starch, carboxymethylcellulose, carboxymethylcellulose calcium, agar powder, laminaran powder, croscarmellose sodium, sodium carboxymethyl starch, sodium alginate, sodium hydrogen carbonate, calcium carbonate, polyoxyethylene Examples include, but are not limited to, sorbitan fatty acid esters, sodium lauryl sulfate, starch, monoglyceride stearate, ratatose, and calcium cellulose glycolate.

Preferred examples of the disintegration inhibitor in a solid preparation include hydrogenated oil, sucrose, stearin , Cocoa butter and hardened oil, but are not limited thereto.

Examples of the absorption enhancer in the solid preparation include, but are not limited to, quaternary ammonium bases and sodium lauryl sulfate.

Examples of the adsorbent in the solid preparation include, but are not limited to, starch, ratatose, kaolin, bentonite, and colloidal keic acid.

Examples of the humectant in the solid preparation include, but are not limited to, glycerin and starch.

Examples of the solubilizer in the solid preparation include, but are not limited to, arginine, glutamic acid, and aspartic acid.

Examples of the stabilizer in the solid preparation include, but are not limited to, human serum albumin, ratatose, and the like.

When preparing tablets, pills and the like as solid preparations, they may be coated with a film of a gastric or enteric substance (sucrose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate, etc.) if necessary. Tablets include tablets coated with usual coatings as necessary, such as sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets or double tablets, and multilayer tablets. Capsules include hard capsules and soft capsules. When molding into a suppository form, for example, higher alcohols, higher alcohol esters, semi-synthetic glycerides, etc. can be added in addition to the additives listed above, but the invention is not limited thereto.

Preferred examples of the solvent in the liquid preparation include water for injection, alcohol, propylene glycol, macrogol, sesame oil, corn oil and the like.

Preferred examples of solubilizers in liquid formulations include polyethylene glycol, propylene glycol, D-mantol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate and sodium citrate. And the like, but are not limited thereto.

Preferred examples of the suspending agent in the liquid preparation include stearyltriethanolamine, sodium radium sulfate, laurylaminopropionic acid, lecithin, benzalco-pum chloride, Surfactants such as salted benzene, glyceryl monostearate, etc., for example, polybutyl alcohol, polybutylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethinoresenorelose, hydroxyethinoresenorelose, hydroxyethinorescellulose And hydrophilic polymers such as hydroxypropylcellulose, but are not limited thereto.

Preferable examples of the tonicity agent in the liquid preparation include, but are not limited to, sodium salt salt, glycerin, D-mantol and the like.

Suitable examples of the buffer in the liquid preparation include, but are not limited to, buffers such as phosphate, acetate, carbonate, and citrate.

Preferable examples of the soothing agent in the liquid preparation include, but are not limited to, benzyl alcohol, benzalco-pum chloride and proforce hydrochloride.

Preferred examples of the preservative in the liquid preparation include, but are not limited to, noraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, 2-phenylethyl alcohol, dehydroacetic acid, and sorbic acid.

Suitable examples of antioxidants in liquid formulations include, but are not limited to, sulfites, ascorbic acid, a-tocopherol and cysteine.

When prepared as an injection, the solutions and suspensions are preferably sterilized and isotonic with blood and blood. Usually, these are sterilized by filtration using a nocteria retention filter or the like, blending of a bactericide or irradiation. Further, after these treatments, the mixture is solidified by freeze-drying or the like, and sterile water or a sterile injectable diluent (lidocaine hydrochloride solution, physiological saline) is used immediately before use.

Water, an aqueous glucose solution, ethanol or a mixed solution thereof). In addition, if desired, the pharmaceutical compositions may contain coloring agents, preservatives, flavors, flavors, sweeteners and the like, as well as other agents.

The medicament of the present invention can be administered orally or parenterally. Alternatively, the medicament of the present invention can be administered intravenously or subcutaneously. When administered systemically, the medicament used in the present invention may be in the form of a pyrogen-free, pharmaceutically acceptable aqueous solution. The preparation of such pharmaceutically acceptable compositions takes into account pH, isotonicity, stability, etc. By doing so, those skilled in the art can easily carry out. In the present specification, the administration method includes oral administration, parenteral administration (for example, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, mucosal administration, rectal administration, vaginal administration, local administration to the affected area, Skin administration). Formulations for such administration may be provided in any formulation. Such preparations include, for example, solutions, injections, and sustained-release preparations.

The medicament of the present invention may contain a physiologically acceptable carrier, excipient or stabilizing agent as necessary (Japanese Pharmacopoeia 14th edition or its latest edition, Remington's Pharmaceutical sciences, 18th Edition, AR Gennaro, ed., MacK Publishing Company, 1990) and a glycan composition having the desired degree of purity, which can be prepared and stored in the form of a lyophilized cake or aqueous solution .

Various delivery systems are known and can be used to administer the compounds of the invention (eg, ribosomes, microparticles, microcapsules, etc.). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compound or composition can be administered by any convenient route, such as by injection or bolus injection, by absorption through epithelial or mucosal linings such as oral, rectal and intestinal mucosa, and It can be administered together with other biologically active agents. Administration can be systemic or local. In addition, the pharmaceutical compounds or compositions of the present invention can be administered by any suitable route, including intraventricular injection and intrathecal injection; intraventricular injection can involve, for example, a brain attached to a reservoir, such as an Ommaya reservoir. It may be desirable to introduce it into the central nervous system by means of an intravenous catheter). Pulmonary administration may also be used, for example, with the use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

In certain embodiments, it may be desirable to administer the polypeptide, polynucleotide or composition of the invention locally to the area in need of treatment (eg, central nervous system, brain, etc.); Although not intended, for example, local injection during surgery, topical application (eg, in combination with a wound dressing after surgery), injection, by catheter, by suppository, or implant (this implant may be (Including porous, non-porous, or glue-like materials, including membranes or fibers such as sialastic membranes) Can be Preferably, when administering a protein of the present invention, including antibodies, no protein must be absorbed! Care must be taken to use the material! / ヽ.

In another embodiment, the compound or composition can be delivered encapsulated in vesicles, especially ribosomes (Langer, Science 249: 1527-1533 (1990); Treat et al., Liposomes m The Therapy of Infectious). Disease and Cancer, Lo pez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lo pez-Berestein, ibid., Pp. 317-327; see broadly ibid).

In yet another embodiment, the compound or composition can be delivered in a controlled release system. In one embodiment, a pump may be used (Langer, supra; Seft on, CRC Crit. Ref. Biomed. Eng. 14: 201 (1987); Buchwald et al., Surgery 88: 507 (1980); Saudek et al. , N. Engl. J. Med. 321: 574 (1989)). In another embodiment, polymeric materials may be used (Medical Applications of

Controlled

Release, Langer and Wise (eds.), CRC Pres., Boca

Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds), Wiley, New York (19).

84); Ranger and Peppas, J., MacromoL Sci. Rev. MacromoL Chem. 23:

61 (1983); Levy et al., Science 228: 190 (1985); During et al., Ann.

Neurol. 25: 351 (1989); see also Howard ^, J. Neurosurg. 71: 105 (1989)).

In yet another embodiment, a controlled sustained release system can be placed near the therapeutic target, ie, the brain, and thus requires only a portion of the systemic dose (eg, Goodson, Medical

Applications of Controlled Release, supra, Vol. 2, pp. 115-138 (1984)).

Other controlled release systems are discussed in a review by Langer (Science 249: 1527-1533 (1990)).

The amount of the composition used in the treatment method of the present invention depends on the purpose of use, the target disease (type, severity, etc.), the patient's age, weight, sex, medical history, cell morphology or type, etc. With this in mind, those skilled in the art can easily determine. The frequency of applying the treatment method of the present invention to a subject (or patient) also depends on the purpose of use, target disease (type, severity, etc.), age, weight, sex, medical history, and course of treatment of the patient. It can be easily determined by a person skilled in the art in consideration of the above. The frequency includes, for example, administration once every few months (eg, once a week, once a month). It is preferable to administer once a week to one month while observing the course.

The dose of the peptide into which the sugar chain of the present invention is introduced varies depending on the age, body weight, symptoms, administration method and the like of the subject, and is not particularly limited. Olmg ~: LOg, preferably 0.1 mg ~: Lg, lmg ~: L00 mg, 0.1 mg ~: LOmg, etc. In the case of parenteral administration, it can be 0.01 mg to 100 mg, preferably 0.01 mg to 100 mg, 0.1 mg to 100 mg, 1 mg to 100 mg, 0.1 mg to 10 mg, and the like.

As used herein, the term "administer" refers to a cell or tissue of an organism, alone or in combination with a pharmaceutical composition containing the peptide into which the sugar chain of the present invention has been introduced or a pharmaceutical composition containing the same. Means that the compound is incorporated into the compound. The combination may be administered, for example, either simultaneously as a mixture, separately but simultaneously or concurrently; or sequentially. This includes presentations where the combined agents are administered together as a therapeutic mixture, and also procedures where the combined agents are administered separately but simultaneously (e.g., through separate intravenous lines to the same individual). Also includes. "Combination" administration further includes separately administering one of the compounds or agents given first, followed by the second.

An abnormal condition can also be prevented or treated by administering the compound to a group of cells having an abnormality in a signaling pathway to the organism. The effect of administering the compound on the biological function can then be monitored. The organism is preferably a mouse, a rat, a heron, a guinea pig, or a goat, more preferably a monkey (monkey or ape), and most preferably a human.

In the present specification, the “instruction” refers to a physician, a patient, or the like who administers a method of administering or diagnosing the medicament or the like of the present invention, or a person diagnosing (possibly a patient). It is described. This instruction describes a word indicating a procedure for administering the diagnostic agent, the medicine and the like of the present invention. This instruction is prepared in accordance with the format prescribed by the competent authority of the country where the present invention is implemented (for example, the Ministry of Health, Labor and Welfare in Japan and the Food and Drug Administration (FDA) in the United States) and is issued by the competent authority. The approval is clearly stated. Instructions are so-called package inserts, which are usually provided on paper media, but are not limited to, for example, electronic media (eg, homepages (websites) provided on the Internet, emails, etc.). ) Can also be provided.

Judgment of termination of treatment according to the method of the present invention may be based on standard laboratory results from commercially available atsays or instrumentation or clinical features characteristic of disorders such as insulin (eg, neurological disorders). It can be supported by the disappearance of symptoms. Treatment can be resumed with a recurrence of a disease associated with insulin or the like (eg, a neurological disease). The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more components of the pharmaceutical composition of the present invention. Manufacture of pharmaceuticals or biological products,

: p I

A notice in the form of an agency that regulates use or sale may optionally accompany such containers, and this notice represents the agency's approval of manufacture, use or sale for administration to humans.

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

Hereinafter, a preferred embodiment of the present invention will be described.

The sugar chain peptide binding agent of the present invention can be obtained by the following method.

[0059] (Method 1)

[Formula 16]

_A 1 A ¹ CHal ₄ / Ph ₃ P _A 1

H ₂ N OH HN OH H Hal

Pro

(Where A ¹ has the same meaning as (1), Pro is a protecting group, Hal is halogen, Ph P is (Fin and Alk are alkyl)

(Method 2)

[Formula 17]

(In the formula, A ¹ has the same meaning as (1), Pro is a protecting group, and DEAD is getyl azodicarboxylate.)

(Method 3)

[Formula 18]

(Where Pro is a protecting group)

(Method 4)

[Formula 19]

(Wherein, A ¹ and R ⁴ have the same meanings as (1), and Pro is a protecting group) (Reaction of Peptide with the Sugar Chain Peptide Binding Agent)

In one aspect, the present invention provides a glycopeptide in which glutamine is modified with a sugar chain. This glycopeptide is characterized in that at least one amino acid residue has glutamine in the amino acid sequence of a peptide having a biological activity, and the glutamine is modified with a sugar chain. Here, modification means direct or indirect binding. The glutamine modified with a sugar chain is preferably at least one dartamine contained in the peptide, and more preferably a plurality (for example, two or three), or all of the glutamine present are modified with a sugar chain. May be. The bond is usually, but not limited to, a covalent bond, as long as the carbohydrate is capable of constituting the same entity as the peptide to which it is attached, other interactions (eg, hydrophobic bonds, hydrogen bonds, etc.). Bonding or the like).

[0064] In one embodiment, the biologically active form of the peptide used is preferably water-soluble under at least one condition under which the enzymatic activity of transdaltaminase is exerted. Such conditions vary depending on the transdaltaminase and the substrate used, and those skilled in the art can appropriately set such conditions. Exemplary conditions include, but are not limited to, for example, pH 4 to pH 9, salt concentration OmM to: LM, and a temperature of 0 ° C to 42 ° C. Any buffer salt may be used. As such a buffer salt, any kind of buffer solution can be used as long as the buffer solution is pH4 to pH9.For example, Hepes buffer solution, Tris phosphate buffer solution (PH 8.0), phosphate Buffers (pH 6.4 to 7.4) and the like. In addition, since animal-derived transdaltaminase is often a calcium-dependent protein, it is necessary to add calcium salts such as calcium chloride and calcium carbonate to the reaction system to express the enzyme activity. The preferred amount of addition is usually 0.1 to: LOO millimolar concentration is preferred. Since transdaltaminase derived from microorganisms is not dependent on calcium, the addition of potassium salt with potassium salt is not necessary. Examples of transdaltaminase include those produced by microorganisms such as Streptoverticillium sp. (Eg, Streptoverticillium sp. Strain s-8112), those derived from animals such as guinea pigs and humans, and those derived from fish. Can also be used. In addition, it is also commercially available as an adhesive for general-purpose foods, and in the present invention, such a commercially available transdaltaminase is also used. More preferably, such conditions may be “mild conditions”. Such mild conditions include, but are not limited to, conditions under which the intramolecular disulfide bond is not broken and conditions under which the steric structure is not changed. Conditions that determine such mild conditions include, for example, peptide or protein concentration, substrate concentration, calcium ion concentration, PH, temperature, type and concentration of coexisting salt, the presence or absence of GSH, and the presence or absence of a reducing agent. But not limited to them. If necessary, a plurality of these factors may be considered. Specific examples of such conditions include, for example, insulin (0.9 lmM), Lac. Amino Hex (0.9 mM) ゝ CaC12 (5 mM) ゝ Tris-HCl (20 mM, pH 7.5), transglutaminase (3.5 U / ml); Examples include, but are not limited to, reaction at 37 ° C and 30 minutes. The addition of GSH promotes the reaction of transdaltaminase with wild-type insulin, but such a supplemented koji is not preferred because it has an adverse effect on the biological activity of the peptide. Specific examples of other mild conditions include, for example, setting the concentration of a peptide or protein (eg, a protein containing a lysine residue (eg, insulin)) to 0.2 mM or less (preferably, 0. ImM or less). The calcium concentration must be 2 mM or more (preferably 5 mM or more, more preferably 10 mM), and the reaction at 37 ° C when GSH is removed is shortened (for example, 3 hours or less, preferably 1 hour or less). In the case where a commercially available transdaltaminase is used, the use of a reduced amount of the reducing agent by exchanging a small amount or a buffer without affecting the reducing agent may be mentioned, but is not limited thereto.

The transdaltaminase used in the method of the present invention may be of any origin, including those derived from animals (derived from various tissues and plasma components), those derived from microorganisms, and those derived from microorganisms. It may be made in

When a sugar chain is introduced using transdaltaminase, the reaction product is subjected to centrifugation, chromatography (eg, reversed-phase HPLC, affinity chromatography with a lectin column, gel filtration, etc.) in the relevant field. Can be isolated and purified from the reaction mixture by a known method. Alternatively, the obtained glycan-linked insulin can be isolated as a mixture of a plurality of types. In addition, it may include those modified with a plurality of different sugar chains or other substituents.

Analysis of reaction products includes high performance liquid chromatography, nuclear magnetic resonance spectra, and mass spectrometry. (Such as MALDI TOF mass spectrum analysis).

Preferably, in the embodiment, the amino acid residue substituted with glutamine in the peptide having a biological activity in the present invention may be present on the surface of the peptide. preferable. By converting amino acid residues on the surface to glutamine and attaching sugar chains to them, it is possible to protect the peptide molecule against external aggression that could not be achieved conventionally. Identification of amino acid residues on such a surface can be performed by crystal structure analysis well known in the art. Methods for determining the spatial conformation of a peptide are known in the art and include, for example, X-ray crystallography, and two-dimensional nuclear magnetic resonance spectroscopy. Methods for identifying parts present on a surface include methods for identifying epitopes. Identification of such epitopes in a given protein is readily accomplished using techniques well known in the art. See, eg, Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81: 3998 (a general method for rapidly synthesizing peptides to determine the location of immunogenic epitopes on a given antigen); No. 4,708,871 (Procedure for identifying and chemically synthesizing antigenic epitopes); and Geysen et al. (1986) Molecular Immunology 23: 709 (Peptides with high affinity for a given antibody) Techniques for identifying).

In another embodiment, glutamine may be present in the wild-type form of the biologically active peptide or, if not present in the wild-type, may replace or add glutamine. Alternatively, the production of such additional or substituted peptides can be performed synthetically or genetically. Such production methods can be performed using techniques well known to those skilled in the art, as described herein above and illustrated in the Examples. The position at which glutamine is substituted or added is preferably at a site susceptible to protease digestion. Easily subject to protease digestion! / The ability to convert to glutamine and “mask” it with sugar chains to allow it to undergo protease digestion. Whether the site is susceptible to such protease digestion can be determined by incubating the peptide with any protease and determining the sequence of the digested partial peptide. The number of glutamine residues into which a sugar chain is introduced is at least one. Even if sugar chains are introduced into two or more, three or more glutamine residues, it does not matter. When sugar chains are introduced into a plurality of glutamine residues, the sugar chains to be introduced may be the same or different. Alternatively, even when a sugar chain is introduced into one glutamine residue, the introduced sugar chain may be uniform or a mixture. I prefer something that is even! /.

The reaction occurs only by mixing a peptide having a free cysteine residue (thiol group) and a sugar chain-peptide binder containing a compound represented by the general formula (V) in a buffer solution. A derivative into which the chain peptide binding agent has been introduced is obtained.

(Method for producing peptide)

In the glycopeptide of the present invention, the peptide moiety can be produced synthetically or genetically using techniques well known in the art, and such synthetic and genetic engineering methods are described elsewhere herein. The place is detailed. Further, the production of a peptide having glutamine at at least one amino acid residue can also be produced using synthesis and Z or genetic engineering techniques well known in the art. In such a case, if the wild-type peptide does not have glutamine at at least one amino acid residue, the amino acid sequence can be modified to have glutamine within the range. Such modification methods are also well known in the art and are described herein.

V, it is listed elsewhere. Glutamine can be introduced in any position! / ,. In another embodiment, the glycopeptide of the invention has at least one selected from the group consisting of substitution, addition and Z or deletion of one or several amino acid residues, and Z or sugar chain and lipid group. It may be advantageous to further include a biomolecule. Such further modifications of the glycopeptide can be made when it is desired to impart or eliminate or regulate the desired function. Making such modifications can be performed by one of ordinary skill in the art.

[0068] In another aspect, the present invention provides a method for preparing a functional glycopeptide via a sugar chain-peptide binding agent. Such a method includes the steps of: A) providing a target peptide containing at least one dartamine at an amino acid residue; and B) introducing a sugar chain-peptide binding agent into the glutamine. Here, the peptide of interest has at least one amino acid residue other than glutamine in the wild-type form of the peptide. The peptide may be provided by substituting with a min, or a wild-type peptide having glutamine at such a position may be directly provided. Such a glutamine residue may be present within 12 amino acids from the C-terminal or N-terminal of the peptide, but is not limited thereto.

In a preferred embodiment, the method of the present invention further comprises the step of identifying a residue in the amino acid sequence of the peptide of interest that does not lose the biological activity of the peptide of interest even when converted to glutamine. . By having such a step, in the present invention, a functional glycopeptide can be steadily produced. Here, at least one amino acid residue in which glutamine is replaced may be a residue identified by such a method, and a residue other than such a residue may be modified. Such an identification step may include, for example, identifying amino acid residues on the surface of the glycopeptide. The target peptide is provided by identifying a site susceptible to protease digestion in the wild-type form of the target peptide and, if glutamine is not present at that site, substituting the site susceptible to protease digestion with glutamine. obtain.

The peptide to be modified may be a wild-type peptide or a synthetic artificially modified peptide.

The introduction of a sugar chain into glutamine usually utilizes transdaltaminase, and the sugar chain contains, but is not limited to, an amino group. Since transdaltaminase has high specificity for glutamine, it is advantageously used to introduce a sugar chain into only glutamine residues. The amino group and the sugar chain may be directly bonded, or may be bonded via a spacer.

(Sugar chains)

Any sugar chains can be used in the present invention. Preferably, the sugar chains used advantageously comprise ratatose or sialic acid. More preferably, the sugar chain used further advantageously comprises sialic acid.

Accordingly, examples of the sugar chain used in the present invention include, for example, Lac, Sia a2, 3 — Lac, Sia a2, o— Lac, uc α 丄, 2— Lac, Sia a2, 8— a 2, 3— Lac, ¾ia 2, 8— ¾ia 2, 6— Lac, Sia 2, 8— Sia a 2, 3— (Fuc 1, 2—) Lac, ¾ia 2 , 8-Sia- a ~ 2, 6— (Fuc—a— 1, 2—) Lac and other trisaccharides, tetrasaccharides, pentasaccharides, hexasaccharides, or n-saccharides (where n is 7 or more) , And the like are not limited thereto. These exemplified sugar chains may be linear, branched, or 榭 -shaped. When the sugar chain is branched, the sugar chain is preferably in the form of a double chain, a triple chain, or a quadruple chain.

The size of the sugar chain to be introduced may also be any. Preferably, the sugar chains used are at least disaccharide in size, more preferably at least trisaccharide in size. Such sugar chains may be linear, branched or cyclized.

In a preferred embodiment, the method for producing a glycopeptide via a sugar chain-peptide binding agent of the present invention may further include a step of further elongating the sugar chain. By having an elongation step, in principle, sugar chains can be added indefinitely. In addition, when transglutaminase is used, the limit of the size of the sugar chain to be introduced, which is a limit, is effectively eliminated.

Such sugar chain elongation is due to β は, 4-galatatotransferase, 1,3-galatatotransferase, βΐ, 4 galactosyltransferase, / 31,3-galactosyltransferase, / 31 , 6—galactosyltransferase, a 2,6 sialyltransferase, 1,4 galactose transferase, ceramide galatatosyltransferase, 1,2 fucose transferase, 1,3 fucose transferase, al, 4 fucose Transferase, al, 6-fucose transferase, a 1,3-N-acetylgalatatosamine transferase, al, 6-N-acetylgalatatosamine transferase, β 4,4Ν-acetylgalatatosamine transferase , Polypeptide ァ acetylgalatasamine transferase, / 31,4Νacetyl darcosamine transferase, 131,2- Νacetyl darcosamine transferase, βΐ, 3 、チルチルチルΐ, 6—Psetyldarcosamine transferase, Shed 1, 4-N § cetyl Darco Sa Min transferase, β ΐ, 4 mannose transferases enzyme, _a l, 2 mannosyltransferase, _a l, 3 mannosyltransferase, a 1, 4 mannose transferase, al, 6 Mannose transferase, al, 2 glucose transferase, al, 3 glucose transferase, << 2,3 sialyltransferase, << 2,8 sialyltransferase, al, 6-darcosamine transferase, al, 6-xylose Transferase, β-xylose An enzyme selected from the group consisting of glucosyltransferase (proteodarican core structure synthase), βΐ, 3-glucuronyltransferase and hyaluronic acid synthase may be used, but is not limited thereto. The enzymes used for sugar chain elongation are preferably used under appropriate conditions.

[0071] The present invention has been described above by showing preferred embodiments for easy understanding. Hereinafter, the present invention will be described based on examples. However, the above description and the following examples are provided for illustrative purposes only, and are not provided for limiting the present invention. Therefore, the scope of the present invention is not limited to the embodiments and examples specifically described in this specification, but is limited only by the appended claims.

Example

Next, the present invention will be described with reference to examples, but the scope of the present invention is not limited to these examples.

In the examples, the following abbreviations are used.

Boc: t—butoxycanoleboninole

Me: methyl

Et: Ethil

Ph: Feel

DMF: dimethylformamide

THF: tetrahydrofuran

TFA: trifluoroacetic acid

TGase: transglutaminase

DTT: Dithiothreitol

Ac: Asetinore

Example 1 Synthesis of Compound (1)

[Formula 20]

Me,, ΟΗ (Boc) ₂₀ e..OH

NH _HC , Et ₃ NI MeOH

Boc

1 [0074] N-methylhydroxylamine hydrochloride (lOg) was dissolved in about 10 times the volume of methanol (100 ml) and cooled in an ice bath. After adding 1 equivalent (16.7 ml) of triethylamine, 1.1 equivalents of di-t-butyl dicarbonate (28.7 g) were added dropwise. After stirring for about 1 hour, the temperature was raised to room temperature and further stirred for 1 hour. The solvent was distilled off under reduced pressure, ethyl acetate and water were added to the residue for extraction, and the organic layer was dried over magnesium sulfate. Purification was performed by silica gel column chromatography to obtain the desired product (yield 63%). 'H-NMR CCDCl), δ 3.16 (s, 3H), 1.49 (s, 9H)

Three

(Example 2) Synthesis of compound (2)

[Formula 21]

[0076] 6 amino-11-hexanol (17.7 g) was added to a 9-fold amount of a 4.4% aqueous sodium hydroxide solution.

(164.4 g), and about 6 times the volume of acetonitrile (100 ml) was added. After cooling in an ice bath, 1.2 equivalents of di-tert-butyl dicarbonate (39.3 g) was added dropwise. After stirring for about 1 hour, 0.2 equivalent of di-t-butyl dicarbonate (6.6 g) was added, and the mixture was further stirred for 1 hour. The reaction solution was heated to room temperature, and acetonitrile was distilled off under reduced pressure, and then ethyl acetate was added. The organic layer was washed with water, extracted and dried over magnesium sulfate, and then ethyl acetate was distilled off under reduced pressure. Purification was performed by silica gel column chromatography to quantitatively obtain the target compound.

— NMR (CDC1), 6.45 (br, 1H), 3.63 (bt, 2H), 3.15, 3.12, 3.10

Three

, 3.07 (dd, 2H), 1.67-1.20 (m, 17H)

Example 3 Synthesis of Compound (3)

[Formula 22]

[0078] Compound (2) (10.9 g) and 1.2 equivalents of carbon tetrabromide (19.9 g) were dissolved in about 40-fold amount of dichloromethane (400 ml). After cooling in an ice bath, 1.2 equivalents of 15.7 g of triphenylphosphine Was added and stirred for about 1 hour. The reaction solution was warmed to room temperature and stirred for another 2 hours. The reaction solution was poured into a saturated aqueous solution of sodium hydrogen carbonate, the organic layer was washed with water, extracted, dried over magnesium sulfate, and the solvent was distilled off under reduced pressure. Purification was performed by silica gel column chromatography to obtain the desired product (yield: 91%).

— NMR (CDC1), 6 4.53 (br, 1H), 3.42, 3.40, 3.37 (t, 2H), 3.14

Three

, 3. 12, 3. 10, 3.07 (dd, 2H), 1.92- 1.80 (m, 2H), 1.60—1.23 (m, 15H)

(Example 4) Synthesis of compound (4)

[Formula 23]

Me,., OH ^NaH

M N

Boc DMF I THF Boc oc H

. · ,, ^ / \ /-\ / ^, „

Me 〜 ~ Boc

Four

[0080] Compound (1) (3. lg) was dissolved in about 32 times the amount of DMF (100 ml), and about 32 times the amount of THF (100 ml) was added. 7272% sodium hydride Oily oil (0.96 g) was stirred. 0.95 equivalent of compound (3) (5.58 g) was added, and the mixture was heated to 50 to 60 ° C and stirred for 1 hour. The reaction solution was poured into 0.1 mol of hydrochloric acid, and ethyl acetate was added. The organic layer was washed with water, extracted, dried over magnesium sulfate, and the solvent was distilled off under reduced pressure. Purification was performed by silica gel column chromatography to obtain the desired product (93% yield). OLE 丄 INK2

'H-NMR CCDCl), δ OLE LINK24.52 (br, 1H), 3.84, 3.82, 3.79 (t, 2

Three

H), 3.20- 3.00 (s, m, 5H), 1.72-1.24 (m, 26H)

Example 5 Synthesis of Compound (5)

[Formula 24]

Four

Compound (4) (1. Og) was dissolved in about 10 volumes of a 90% dichloromethane solution of trifluoroacetic acid (10 ml) and stirred at room temperature for 1.5 hours. Thereafter, the solvent was distilled off under reduced pressure to obtain a target product, trifluoroacetate. This solution was adjusted to pH 68 with an aqueous sodium hydroxide solution to obtain a 200 mM aqueous solution.

— NMR (D O), 64.03, 4.01, 3.99 (t, 2H), 2.93— 2.85 (s, m, 5H),

2

1.65-1.50 (m, 4H), 1.40-1.25 (m, 4H)

(Example 6) Introduction of Compound (5) Derivative into Peptide

[Formula 25]

TGase (manufactured by Wako Pure Chemical Industries, Ltd.) was added to a reaction solution (100 mM Tris-HCl pH 7.5 10 mM CaCl 5 mM DTT) containing ImM mastoparan and 20 mM oxamine linker.

2

U) was added and reacted at 37 ° C for 4 hours. On the way, TGase (5.6U) was added. The reaction was stopped by adding the same amount of 10% TFA as the reaction solution. The main product was isolated by purification by reverse phase HPLC (Inertsil® ODS-3 20X 250 mm column, mobile phase A: 0.1% TF A: mobile phase against aqueous solution B: 10% force of 0.1% TFA in acetonitrile and a gradient of 40%), MALDI-TOF / MS [M (average) + H] + = 1764.3, [M ( average) + Na] ⁺ = 1786.3, [M (average) + K] ⁺ = 1802.5 peak (theoretical [M (average) + H] ⁺ = 1765.2, [M (average) + Na] ⁺ = 1787.2, [M (average) + K] ⁺ = 180 3. 3) to confirm the target.

(Example 7) Introduction of sugar

[Formula 26]

[0086] A reaction solution (50 mM AcOH / Na buffer pH 4.5) containing Imox's oxyamine linker with persimmon mastoparan and lOOmM glucose was reacted at 37 ° C for 24 hours. Reversed phase HPLC of the reaction solution (Inertsil® ODS-34.6 × 250 mm column, moving bed A: against 0.1% TFA in water B: 20% force of 0.1% TFA in acetonitrile 40% Gradient), and the peak of [M (ave rage) + H] ⁺ = 1927.3 (theoretical value [M (average) + H] + = 1927.3) is obtained with OLE 丄 INK1MALDI—TOFZMSOLE 丄 INK1. By confirming, the production of the target product was confirmed.

[0087] Similar to Example 7, N-acetyldarcosamine, ratatose, and maltose were also masts. «The sugar chain was introduced into the orchid, and [M (average) + H] + = 1968.0 (N-acetyldarcosamine) by reversed-phase HPLC (same conditions as in Example 7) and MALDI-T OF / MS, respectively. : Theoretical value [M (avemge) + H] + = 1968. 4), [M (average) + H] + = 2088.3 (Lattose: theoretical value [M (avemge) + H] + = 2088.9) ), [M (average) + H] + = 2089.7 (maltose: theoretical value [M (avemge) + H] + = 2099.5), confirming the production of the target product .

Industrial applicability

The present invention relates to a sugar chain-peptide binding agent used for easily producing a glycopeptide having a uniform composition using a natural or unnatural protein and an unprotected sugar chain, and the sugar chain. The present invention relates to a method for producing a glycopeptide using a peptide binding agent.

According to the present invention, it is possible to provide a glycopeptide having a constant composition, so that it is possible to provide a higher-quality Noro medicine.

Claims

The scope of the claims

[1]-General formula (I):

[Where X is Η Ν—or the formula:

2

[Chemical 1]

A group represented by;

Υ an Alpha ¹ -, one Α ¹ - Ο- Α ² -, one (CH CH Omicron) one Alpha ¹ -, one (CH CH Omicron)

2 2 η 2 2

Ο-Α ^2- , one A ¹ -NH-C (= 0)-A ^2- , one A ¹ -C (= 0)-NH-A ^2- ,

CH CH O) One NH— C (= 0) — A ² —, One A ¹ — (CH CH O) — C (= 0) _

2 2 η 2 2 η

Α ^2- , formula:

[Formula 2]

Or a group represented by the formula:

[Formula 3]

Wherein A ¹ and A ² are each independently an optionally substituted C 1-15 alkylene, substituted !, may! /, C 2-15 alkylene or ! is substituted, also good C2- 15 alkynylene; n is an integer of from 1 to 5; R ¹ is hydroxy or - NH (R ²⁾ (wherein, R ² is hydrogen atom, lower alkyl, aryl or aralkyl); R ³ is hydrogen atom or —C (= 0) —R ⁵ (where R ⁵ is lower alkyl, aryl or aralkyl)); O—NH (R ⁴ ), one NH—OR ⁴ , one C (= 0) —NH—NH—R ⁴ , or one C (= S) —NH—NH—R ⁴ (where R ⁴ is hydrogen Atom, lower alkyl, or aryl))].

[2] X is —NH and Z is —O—NH (R ⁴ ) (where R ⁴ is a hydrogen atom, lower alkyl, or

2

2. The sugar chain peptide binding agent according to claim 1, which is

[3] X is the formula:

[Formula 4]

The sugar chain peptide binding agent according to claim 1, wherein the group represented by and Z is —O—NH (R ⁴ ), wherein R ⁴ is a hydrogen atom, lower alkyl, or aryl.

[4] General formula (II):

HN— A ¹ — O— NH (R ⁴ ) (II)

2

[5] General formula (ΠΙ):

[Formula 5]

(In the formula, A ¹ is an optionally substituted C 1-15 alkylene; R ⁴ is a hydrogen atom, lower alkyl, or aryl), or a salt thereof. The following steps:

1) General formula (IV):

H N-Y-Z (IV)

2

2 2 η 2 2 η NH-Α ^2- , formula:

[Formula 6]

C (= 0) —R ^{5 where} R ⁵ is lower alkyl, aryl or aralkyl)); and

A peptide having a glutamine residue,

Reacting in the presence of transdaltaminase, and

2) a step of reacting the peptide obtained in step 1) with a sugar chain, A method for producing a glycopeptide.

The following steps:

1) General formula (V):

[Formula 8]

Wherein Y is -A ¹ OA ² —, one (CH CH O) one A ^1- (CH CH O)

twenty two

2

A NH— C (= 0) — A 'C (= 0) — NH— A ² —,

-A NH-C (= 0) -A ^2- (CH CH O) C (= 0) A '

[Formula 9]

Or a group represented by the formula:

[Formula 10]

(Wherein A ¹ and A ² are each independently an optionally substituted C 1-15 alkylene, substituted !, or C 2-15 alkylene or substituted And C2-15 alkylene; n is an integer of 1 to 5; R ¹ is hydroxy or NH (R ² ) (where R ² is a hydrogen atom, lower alkyl, aryl or aralkyl); R ³ is a hydrogen atom or —C (= 0) —R ⁵ (where R ⁵ is lower alkyl, aryl or aralkyl)); Z is one O—NH (R ⁴ ), one NH—OR ⁴ , one C (= 0) —NH—NH—R ⁴ , or one C (= S) —NH—NH—R ⁴ (where R ⁴ is a hydrogen atom, lower alkyl, or aryl))],

Reacting a peptide containing a cysteine residue, and

2) a step of reacting the peptide obtained in step 1) with a sugar chain,

A method for producing a glycopeptide.

[8] The production method according to claim 6, wherein Z is —O—NH (R ⁴ ), wherein R ⁴ is a hydrogen atom, lower alkyl, or aryl.

[9] The production method according to claim 6 or 7, wherein Y is —A ¹ — (wherein A ¹ is C1-15 alkylene which may be substituted).

[10] A glycopeptide that can be produced by the production method according to claim 6 or 7.

[11] A pharmaceutical composition comprising, as an active ingredient, a glycopeptide which can be produced by the production method according to claim 6 or 7.