JP2014156409A - Production method of n-type glycoprotein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a simple and precise production method of N-type glycoprotein useful as a test drug in life science study or as a medicine.SOLUTION: N-type glycoprotein (P+A) can be produced in that a ligand compound (L) is modified with a tertiary amine, mixed with a protein (P) and interacted with a modified ligand compound (L') to obtain a protein (L'+P), with which a 1,3,5-triazine compound (T) is reacted to form a complex (P+L'+T), followed by further reaction with a sugar amine (A), or in that a ligand compound (L) is modified with a tertiary amine to prepare a modified ligand compound (L'), and a 1,3,5-triazine compound (T) is reacted therewith to synthesize a dehydration condensation agent (L'+T), which is interacted with a protein (P) to form a complex (P+L'+T), followed by further reaction with a sugar amine (A).

Description

  The present invention relates to a method for producing an N-type glycoprotein, in which a uniform arbitrary N-type sugar chain is regioselectively introduced into a protein.

  Proteins derived from eukaryotes (enzymes, antibodies, hormones, etc.) are widely used in life science research and pharmaceuticals. Many of these proteins express their functions by adding sugar chains.

As sugar chains that bind to amino acid residues of proteins, there are two types of sugar chains, O-type sugar chains (serine / threonine-linked type) and N-type sugar chains (asparagine-linked type), depending on the binding mode. These sugar chains play various roles in a wide range of life phenomena such as quality control of the protein, immunity / receptor recognition, inflammation, and cancer metastasis by binding to the protein.
In particular, asparagine (N) -linked sugar chains have a complex structure consisting of polysaccharides, and there are many variations thereof, so a technique for efficiently synthesizing uniform N-type glycoproteins having specific sugar chains is required. It has become.

In general, culture of genetically modified E. coli is widely used for large-scale protein preparation. However, since E. coli, a prokaryote, does not have a mechanism for introducing a sugar chain into a protein, glycoproteins cannot be synthesized by this method.
Therefore, so far, it has been produced by eukaryotic cell culture (for example, incorporating a gene for a protein serving as a drug such as human cytokine or antibody into a Chinese hamster ovary (CHO) cell). However, the production method using eukaryotic cell culture is expensive, can only obtain glycoproteins with a heterogeneous sugar chain structure and slightly different from that of human origin, and often has antigenicity in the human body. However, there is a problem that it does not show a sufficient pharmacological effect, and a technique for introducing a uniform sugar chain having an arbitrary function is desired.

  Recently, a method for producing a glycoprotein having a completely uniform sugar chain structure using an organic synthetic chemistry method has been reported. In this method, a short partial peptide having a human-type sugar chain introduced at an arbitrary position on the N-terminal side of a protein is chemically synthesized, and thionyl ester addition is performed, while the remaining C-terminal is set so that the terminal is a cysteine residue. The peptide on the side is expressed in recombinant E. coli, and both peptide chains are linked by native chemical ligation (Non-patent Document 1). However, this method has a problem that it can be applied only to a protein having a sugar chain in a relatively short peptide chain on the N-terminal side of the cysteine residue.

  On the other hand, a method for producing an N-type glycoprotein having a completely uniform sugar chain structure by adding an N-type sugar chain based on an N-type glycoprotein introduced with N-acetylglucosamine (GlcNAc) is known. ing. In this method, the reducing end of the sugar chain to be imparted is converted to GlcNAc-oxazoline, and a mutant glycosyltransferase of endoglucosidase M (Endo-M) derived from the filamentous fungus Mucor hiemalis called Endo-M-N175Q is used. In other words, a sugar chain is added to the 4-position of GlcNAc of the N-type glycoprotein into which GlcNAc has been introduced (Non-patent Document 2). However, the N-type glycoprotein introduced with GlcNAc as a raw material can be obtained by digesting naturally-occurring N-type glycoprotein with endoglucosidase H (Endo-H), or aspartic acid in which the side chain carboxy group is amide-linked with GlcNAc amine. N-type sugar chains could not be directly introduced into proteins having no sugar chain.

In addition, many methods have been reported in which sugar chains are selectively introduced into the side chain thiol group of a cysteine residue or the amino group of a lysine residue of a protein (Non-patent Document 3). However, since its binding mode is very different from that of natural ones, it does not always exert its intended function, and such a non-natural structure is considered to be an antigen when administered to a living body. There are problems with its application to pharmaceuticals.
From such a background, a technique capable of directly introducing a natural N-type sugar chain into a protein is strongly desired.

  On the other hand, the present inventors recently labeled a target polymer by mixing a tertiary amine having a ligand, a 1,3,5-triazine compound, and a labeled compound with a target polymer (protein etc.) and reacting them. An affinity labeling method for introducing a compound was found (Patent Document 1). The affinity labeling method uses an amine compound containing a labeling site in the molecular structure as a labeling compound, and introduces the labeling compound by forming an amide bond with a carboxy group near the ligand binding site of the target polymer. It is. However, the amine compounds that can be used in the affinity labeling method are limited to specific amines having a labeling functional group, such as amines having no labeling site and polyfunctional sugar amines (having amino groups). There was no disclosure of sugar chains and the like.

JP 2009-175139 A

Hirano, K. et al. et al. , Angew. Chem. Int. Ed. 48, 9557 (2009). M.M. Umekawa et al. Biochimica et Biophysica Acta, 2010, 1800, 1203 Gamblin, D.M. P. et al. , Chem. Rev. 109, 131-163 (2009)

  The present invention inexpensively produces an N-type glycoprotein in which any uniform N-type sugar chain structure, which has been difficult to obtain by a conventional production method using eukaryotic cell culture, is introduced into a protein in a position-selective manner. It is an object to provide a method.

  As a result of intensive studies in order to solve the above problems, the present inventors have found that the side chain carboxy group of the sugar amine compound (A) and the protein (P) is amide bond-forming reaction via the ligand compound (L). It was found that the N-type glycoprotein (P + A) bound by can be synthesized, and the present invention was completed.

The present invention includes the following.
[1] A method for producing an N-type glycoprotein comprising the following steps:
1) a step of modifying a ligand compound (L) with a tertiary amine to prepare a modified ligand compound (L ′);
2) A step of mixing the modified ligand compound (L ′) and the protein (P);
3) A step of reacting the protein (L ′ + P) interacting with the modified ligand compound (L ′) with a 1,3,5-triazine compound (T) to synthesize a complex (P + L ′ + T);
4) A step of further reacting the sugar amine compound (A) to synthesize an N-type glycoprotein (P + A).
Here, protein (P) is a compound having a carboxy group in the side chain and / or C-terminus,
The ligand compound (L) is a compound that can interact with the protein (P).
[2] A method for producing an N-type glycoprotein comprising the following steps:
1) a step of modifying a ligand compound (L) with a tertiary amine to prepare a modified ligand compound (L ′);
2 ′) reacting the modified ligand compound (L ′) with the 1,3,5-triazine compound (T) to synthesize a dehydrating condensing agent (L ′ + T);
3 ′) the step of mixing the dehydrating condensing agent (L ′ + T) and the protein (P) of the above 2 ′) to synthesize a complex (P + L ′ + T);
4) A step of further reacting the sugar amine compound (A) to synthesize an N-type glycoprotein (P + A).
Here, protein (P) is a compound having a carboxy group in the side chain and / or C-terminus,
The ligand compound (L) is a compound that can interact with the protein (P).
[3] The 1,3,5-triazine compound (T) is represented by the following formula (I):

[Wherein, R ¹ and R ² are each independently a hydrogen atom, an alkyl group, a hydroxyalkyl group, a sulfo group, a phospho group, —R ³ —R ⁴ (where R ³ represents — (C _m H _2m Q) _n , Q is O or NR ¹³ , R ⁴ is a hydrogen atom, an alkyl group, a sulfo group, a phospho group, or an alkyl group having an amino group, an ammonio group, a sulfo group, or a phospho group R ¹³ is an alkyl group or an alkyl group having an amino group, an ammonio group, a sulfo group, or a phospho group, m is an integer of 1 to 30, and n is an integer of 1 to 120.) And a substituent selected from the group consisting of an amino group, an ammonio group, a sulfo group, or an alkyl group having a phospho group, and X has a chlorine atom, a fluorine atom, a bromine atom, an iodine atom, and a substituent. Also There alkylsulfonyloxy group, an optionally substituted aryl sulfonyloxy group, or an N- methyl morpholinium group. ] The method of the said [1] or [2] description which is a compound represented by this.
[4] The method according to [1] or [2] above, wherein the 1,3,5-triazine compound (T) is 2-chloro-4,6-dimethoxy-1,3,5-triazine.
[5] The ligand compound (L) is biotin, warfarin, phenytoin, phenylbutazone, bucolome, acetylsalicylic acid, ketoprofen, flurbiprofen, N-acetyl-L-tryptophan, naproxen, fenoprofen, diclofenac sodium, diazepam , Digitoxin, indomethacin, ibuprofen, hexamethonium, decamethonium, and a structure selected from any one of the above [1] to [4], comprising a structure selected from Phos-tag (registered trademark).
[6] The tertiary amine is represented by the following formula (II):

[Wherein, one or two of R ⁵ , R ⁶ and R ⁷ are substituents containing a functional group capable of forming a bond to the ligand compound (L), and the remaining R ⁵ , R ⁶ And R ⁷ are each independently an alkyl group, an alkenyl group, an alkynyl group, an aryl group, an arylalkyl group, —CH ₂ COOR ⁸ , or CH ₂ CONR ⁹ R ¹⁰ (where R ⁸ , R ⁹ , and R ¹⁰ is each independently a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, an arylalkyl group, or — (C _y H _2y O) _z R ¹⁴ , and R ¹⁴ is a hydrogen atom, an alkyl group , An alkenyl group, an alkynyl group, an aryl group, or an arylalkyl group, y is an integer of 1 to 30, and z is an integer of 1 to 120], or the following formula (III):

[Wherein R ¹¹ is a substituent containing a functional group capable of forming a bond to the ligand compound (L), and R ¹⁵ is an alkyl group, an alkenyl group, an alkynyl group, an aryl group, or an arylalkyl group. is there. ] The method as described in any one of said [1]-[5] which is a compound represented by.
[7] The sugar amine compound (A) is
N-acetylglucosamineamine, N-acetylgalactosamineamine, N-acetylmannosamineamine, alloseamine, taloseamine, growthamine, glucoseamine, altroseamine, mannoseamine, galactoseamine, glucuronic acidamine, galacturonic acidamine, deoxy Riboseamine, fucoseamine, rhamnoseamine, idoseamine, riboseamine, lyxoseamine, xyloseamine, arabinoseamine, erythroseamine, threoseamine, cedoheptuloseamine, coriooseamine, psicoseamine, fructoseamine, sorboseamine In an anomeric position selected from tagatose amine, ribulose amine, xylulose amine, erythrulose amine, and sialic acid amine Monosaccharide amine Min is bound; and the a sugar chain composed monosaccharide corresponding to the monosaccharide amine, sugar amine anomer of reducing terminal is linked amines;
A sugar amine in which at least one of a hydroxyl group, an amino group, and a carboxy group of the monosaccharide amine or sugar chain amine is modified by acylation, esterification, sulfate esterification, and / or phosphate esterification; and glucosamine, mannosamine , A method according to any one of [1] to [6] above, which is a compound selected from the group consisting of sugars having an amino group selected from galactosamine and sialic acid.
[8] The above [1] to [7], wherein the protein (P) is avidin, streptavidin, human serum albumin, cyclooxygenase-1, nicotinic acetylcholine receptor, casein, α ₁ -acid glycoprotein or phosphorylated protein. ] The method as described in any one of.
[9] N-type sugar streptavidin introduced with a monosaccharide amine or a sugar chain amine.
[10] The N-type sugar streptavidin according to [9] above, wherein a monosaccharide amine or a sugar chain amine is introduced into the side chain carboxy group of the 51st glutamic acid residue from the N-terminus of streptavidin.
[11] The N-type sugar streptavidin according to [9] or [10] above, wherein the monosaccharide amine is N-acetylglucosamine amine.
[12] The N-type sugar streptavidin according to the above [9] or [10], wherein the sugar chain amine is lactose amine.

  Since the present invention is an organic synthetic chemistry method, an N-type sugar chain having an arbitrary uniform structure can be efficiently introduced into an amino acid residue (side chain carboxy group) of a protein. Therefore, according to the present invention, a simple and precise method for synthesizing various asparagine or glutamine-linked glycoproteins including N-type glycoproteins to which non-naturally occurring (non-natural) functional sugar chains are bound is provided. be able to. In addition, if there is a ligand that binds in the vicinity of an aspartic acid residue or glutamic acid residue to which a sugar amine (sugar chain) is to be introduced, the N-type sugar can be selected in any position in principle by design of the ligand compound. Chains can be introduced. Furthermore, since a protein in which any amino acid residue is converted into an aspartic acid residue or a glutamic acid residue by genetic recombination technology can be mass-produced by E. coli culture, any N type can be combined with the method of the present invention. A method for mass-producing glycoproteins at low cost can be provided. This has the advantage that the production cost of N-type glycoprotein drugs having various functions currently used in clinical practice such as antibody drugs and erythropoietin can be greatly reduced.

Furthermore, since glutamine-linked glycoproteins cannot be synthesized by cell culture or the like, synthesis of glutamine-linked glycoproteins has become possible for the first time by the method of the present invention.
The natural N-type sugar chain is introduced only into asparagine in the peptide chain region of asparagine-X-serine / threonine (X is any amino acid other than proline), but there is no such limitation in the method of the present invention. An N-type sugar chain can be introduced into any aspartic acid and / or glutamic acid.
In the method using culture or an enzyme, the structure of the sugar chain that can be introduced is considered to be limited. On the other hand, in the method of the present invention, any sugar chain can be introduced if an amino group is bonded to the sugar. Therefore, glycoproteins that cannot be synthesized by conventional methods using enzymes or living organisms can be synthesized.
For example, (1) sugar chains such as sugar chains and glycolipids of different species of organisms can be applied to drug delivery systems and the like because their functions and pharmacokinetics vary greatly depending on the structure. (2) Alkynes -Sugar chains with functional functional groups such as azides, fluorescent substances, and radioisotopes can be applied to chemical biology and in-vivo imaging (eg, sugar chain target proteins (unknown Search for lectins).
Thus, creation of functional neoglycoprotein can also be expected by the method of the present invention.

It is a schematic diagram which shows an example of the manufacturing method of N type glycoprotein of this invention. It is a schematic diagram which shows an example of the manufacturing method of N type glycoprotein of this invention. It is a schematic diagram which shows an example of the manufacturing method of N type glycoprotein of this invention. It is a figure which shows the detection of the introduction | transduction of the lactose amine to streptadipine by SDS-polyacrylamide gel electrophoresis. (Example 2) It is a figure which shows the detection of introduction | transduction of N-acetylglucosamine amine (GlcNAc amine) to streptadipine by SDS-polyacrylamide gel electrophoresis. (Example 3)

Hereinafter, the method for producing the N-type glycoprotein of the present invention will be described.
In the present specification, a protein that is an object of N-type sugar introduction is indicated as (P), and a sugar amine compound introduced into the protein is indicated as (A).
The ligand compound of the present invention refers to a compound capable of interacting with the protein (P), which is denoted as (L), and a modified ligand compound in which the ligand compound (L) is modified with a tertiary amine (L ′ ).
Furthermore, the 1,3,5-triazine compound used as a condensing agent shall be denoted as (T).
Two or more selected from the above (P), (A), (L), (L ′) and (T) react to form a covalent bond, a hydrogen bond, an ionic bond, a van der Waals bond, a coordination bond, etc. Alternatively, a compound bonded by a combination of two or more of these is connected by “+” and displayed. For example, an N-type glycoprotein in which the sugar amine compound (A) is introduced into the protein (P) is displayed as (P + A).

In the present invention, the protein (P) to which N-type sugar is introduced has an acidic amino acid side chain having a carboxy group such as an aspartic acid residue or a glutamic acid residue, and / or a C-terminal carboxy group. If it does not specifically limit. A protein that does not contain a sugar chain is preferred, but even a protein containing a sugar chain can be used in the present invention as long as it has a carboxy group for introducing a sugar chain.
Specifically, those known to have a ligand compound (L) that interacts with the protein (P) are preferable. For example, avidin, streptavidin, serum albumin, cyclooxygenase-1, nicotinic acetylcholine receptor, casein , Α1-acid glycoprotein, phosphorylated protein, and the like can be used, and any other ligand compounds that interact can be used without limitation. Furthermore, even if the interacting ligand compound is a protein not found at the time of filing of the present application, it can be used for the protein (P) of the present invention as long as a known or novel ligand compound is found thereafter.

In the present invention, the “ligand compound (L)” required in the process of producing the N-type glycoprotein is not particularly limited as long as it is a substance that can interact with the protein (P).
The interaction between the protein (P) and the ligand compound (L) means that the ligand compound (L) is bonded to any position of the protein (P), such as a hydrogen bond, an ionic bond, a van der Waals bond, or a coordinate bond. Means reversible binding with an arbitrary affinity such as a combination of two or more of the above, and there is a side chain and / or a C-terminal carboxy group in the vicinity of the site where the ligand compound (L) binds to the protein (P) The aspect which is doing is preferable.
Moreover, two or more types of ligand compounds (L) may exist in one protein (P), and each ligand compound (L) may bind to a different part of the protein (P). In that case, by selecting the ligand compound (L), it is possible to introduce N-type sugar chains regioselectively into different carboxy groups of the protein (P).
Specifically, when the protein (P) is serum albumin, warfarin, phenytoin, phenylbutazone, bucolome, acetylsalicylic acid, ibuprofen, ketoprofen, flurbiprofen, N-acetyl-L-tryptophan, naproxen, pheno Any drug selected from prophen, diclofenac sodium, diazepam, and digitoxin or a derivative thereof; biotin or a derivative thereof in the case of avidin or streptavidin; indomethacin, ibuprofen or a derivative thereof in the case of cyclooxygenase-1 In the case of nicotinic acetylcholine receptors, hexamethonium, decamethonium, acetylcholine or derivatives thereof, in the case of casein and phosphorylated proteins, Phos-tag R) or derivatives thereof.

  In particular, for serum albumin, three binding sites (sites 1 to 3) are known. For example, site 1 of serum albumin is warfarin, phenytoin, phenylbutazone, bucolome, acetylsalicylic acid, and site 2 is ibuprofen. , Ketoprofen, flurbiprofen, N-acetyl-L-tryptophan, naproxen, fenoprofen, diclofenac sodium, diazepam, and site3 are known to bind digitoxin, etc. A sugar chain can be introduced in a regioselective manner.

The “sugar amine compound (A)” introduced into the protein (P) in the present invention is not particularly limited as long as it is a sugar having at least one amino group in its structure, and may be a monosaccharide or a polysaccharide. , D-form or L-form.
Without being particularly limited by the origin and origin of the sugar amine compound (A), those obtained from nature, those genetically engineered by animal cells, plant cells, microorganisms, etc., those produced enzymatically, fermentation Those manufactured by the above, or artificially chemically synthesized may be included.
For example, N-acetylglucosamine amine, N-acetylgalactosamine amine, N-acetylmannosamine amine, allose amine, talose amine, growth amine, glucose amine, altrose amine, mannose amine, galactose amine, glucuronic acid amine, galacturonic acid amine , Deoxyriboseamine, fucoseamine, rhamnoseamine, idoseamine, riboseamine, lyxoseamine, xyloseamine, arabinoseamine, erythroseamine, threoseamine, cedheptuloseamine, coriousamine, psicoseamine, fructoseamine, Monosaccharide amines such as sorbose amine, tagatose amine, ribulose amine, xylulose amine, erythrulose amine, sialic acid amine;
A sugar chain composed of monosaccharides corresponding to these monosaccharide amines, wherein the amine is bonded to the anomeric position of the reducing end (the upper limit of the sugar chain length is not particularly limited, and the desired function is What can be expected can be used without any particular limitation, and can be appropriately selected from, for example, sugar chain amines composed of 2 to 30 monosaccharides, such as sucrose amine, lactulose amine, lactose amine, N-acetyl lactose amine, maltose Disaccharide amines such as amine, isomaltoseamine, trehaloseamine, cellobioseamine, melibioseamine; nigerotrioseamine, maltotrioseamine, melezitoseamine, maltotriuroseamine, raffinoseamine, kestoseamine, etc. Trisaccharide amine; nistosamine, nigerotetraoseamine, Tetrasaccharide amines such as chiose amines; usually composed of 2 to 30 monosaccharides, typically oligosaccharide amines composed of 2 to 20 monosaccharides, which are glucose, galactose, mannose, Homo-oligosaccharide amine or hetero-oligosaccharide amine composed of 2 to 30 monosaccharides selected from glucosamine, N-acetylglucosamine, fructose and the like (for example, maltooligosaccharide amine, isomaltooligosaccharide amine, lactoligosaccharide amine, lactosamine oligosaccharide) Amine, N-acetyllactosamine oligosaccharide amine, cellooligosaccharide amine, merbiooligosaccharide amine, N-acetylchitotrioseamine, N-acetylchitotetraoseamine, N-acetylchitopentaoseamine, etc.);

And functional sugar chains such as sialylglycanamines represented by the formula: )
At least one of the hydroxyl group, amino group, and carboxy group of the monosaccharide amine or sugar chain amine is acylated (eg, acetylated, benzoylated), esterified (methyl esterified, ethyl esterified, benzyl esterified, etc.) ), Sugar ester modified by sulfate esterification and / or phosphate esterification;
Functional functional groups (for example, functional groups having bioorthogonal properties such as alkynes, azides, ketones, and aldehydes), fluorescent materials such as DBD-ED and cascade blue, and light such as diazirine and benzophenone. A sugar group in which an isotope ( ³ H, ¹⁸ O, ¹⁵ N, ¹⁴ C, ¹¹ C, ¹²³ I, ²⁰¹ Tl, ⁶⁷ Ga, etc.) is introduced into any atom constituting a sensitive functional group or sugar chain;
Sugars having amino groups such as glucosamine, mannosamine, galactosamine, sialic acid;
Examples include, but are not limited to, sugars obtained by converting any hydroxyl group of a sugar corresponding to the monosaccharide amine or sugar chain amine to an amino group (in this case, the amine may not be bonded to the anomeric position). Is not to be done.
The sugar amine compound (A) may be specifically introduced one by one into each protein (P), or when there are two or more ligand binding sites of the protein (P), a plurality of different ones are used. It may be introduced.

Production method of N-type glycoprotein (P + A) The N-type glycoprotein (P + A) of the present invention can be produced by a method according to a so-called modular affinity labeling method (MoAL method: see FIGS. 1 and 2). JP 2009-175139 A).
That is, the N-type glycoprotein (P + A) of the present invention can be produced by a method including the following steps 1) to 4) (see FIGS. 1 and 2).
1) a step of modifying a ligand compound (L) with a tertiary amine to prepare a modified ligand compound (L ′);
2) A step of mixing the modified ligand compound (L ′) and the protein (P);
3) A step of reacting the protein (L ′ + P) interacting with the modified ligand compound (L ′) with a 1,3,5-triazine compound (T) to synthesize a complex (P + L ′ + T);
4) A step of further reacting the sugar amine compound (A) to synthesize an N-type glycoprotein (P + A).

Alternatively, the labeled polymer compound (P + A) can also be produced by a method including the following steps 1), 2 ′), 3 ′) and 4).
1) a step of modifying a ligand compound (L) with a tertiary amine to prepare a modified ligand compound (L ′);
2 ′) reacting the modified ligand compound (L ′) with the 1,3,5-triazine compound (T) to synthesize a dehydrating condensing agent (L ′ + T);
3 ′) the step of mixing the dehydrating condensing agent (L ′ + T) and the protein (P) of the above 2 ′) to synthesize a complex (P + L ′ + T);
4) A step of further reacting the sugar amine compound (A) to synthesize an N-type glycoprotein (P + A).

When the modified ligand compound (L ′) reacts with the protein (L ′ + P) ((b) in FIG. 2) interacting with the protein (P), the 1,3,5-triazine compound (T) is modified. The tertiary amine in the ligand compound (L ′) reacts with the 1,3,5-triazine compound (T) to form a dehydrating condensing agent ((c) in FIG. 2), and then the protein (P) Lead to a complex (P + L ′ + T) of protein (L ′ + P) and 1,3,5-triazine compound (T) by reacting with a carboxy group present in the vicinity of the binding site of the ligand compound (L). ((D) in FIG. 2). In the complex (P + L ′ + T), the carboxy group is activated by the 1,3,5-triazine compound (T). By reacting with the sugar amine compound (A), the ligand compound (P) The sugar amine compound (A) can be selectively introduced into the carboxy group present in the vicinity of the binding site of L) ((e) in FIG. 2).
In addition, as in steps 2 ′) and 3 ′), after the modified ligand compound (L ′) and the 1,3,5-triazine compound (T) are reacted, the dehydrating condensing agent (L ′ + T) is first synthesized. A complex (P + L ′ + T) can also be synthesized by mixing protein (P).

In the method of the present invention, as long as the protein (P) interacts with the modified ligand compound (L ′), even if the protein (P) has a secondary and / or tertiary structure, it is denatured by heat or the like and is secondary and / or tertiary. The structure may be destroyed.
For example, the binding of Phos-tag (registered trademark) to casein and phosphorylated protein utilizes affinity for a phosphate ester, regardless of whether the protein has a three-dimensional structure or is denatured. It is thought to interact. In this case, if the protein has a three-dimensional structure, a sugar chain is introduced into a carboxy group that is spatially close to the binding site, and if it is denatured (if the peptide chain is fully extended), the primary protein It is considered that a sugar chain is introduced into a structurally close carboxy group (see FIG. 3). In other words, once a protein is denatured, a sugar chain can be selectively introduced into a carboxy group close to the primary structure and then refolded to synthesize a glycoprotein. The width of the position design is greatly expanded.

  Each said process can be performed in a suitable solvent. Examples of the solvent include water or a buffer set to an appropriate pH. In order to dissolve the modified ligand compound (L ′), 1,3,5-triazine compound (T), dehydrating condensing agent (L ′ + T) and sugar amine compound (A), a small amount of methanol, ethanol or acetonitrile, acetone A solvent miscible with water may be used, but it is necessary that the solvent does not denature protein (P). Even when using an organic solvent, a concentration of less than 5% (volume percent) is preferred.

  In the present invention, the “1,3,5-triazine compound (T)” is a compound having a 1,3,5-triazine ring as a basic skeleton, preferably a compound represented by the following formula (I) It is. The 1,3,5-triazine compound (T) is preferably water-soluble, but may be amphiphilic or fat-soluble.

[Wherein, R ¹ and R ² are each independently a hydrogen atom, an alkyl group, a hydroxyalkyl group, a sulfo group, a phospho group, —R ³ —R ⁴ (where R ³ represents — (C _m H _2m Q) _n , Q is O or NR ¹³ , R ⁴ is a hydrogen atom, an alkyl group, a sulfo group, a phospho group, or an alkyl group having an amino group, an ammonio group, a sulfo group, or a phospho group R ¹³ is an alkyl group or an alkyl group having an amino group, an ammonio group, a sulfo group, or a phospho group, m is an integer of 1 to 30, and n is an integer of 1 to 120.) And a substituent selected from the group consisting of an amino group, an ammonio group, a sulfo group, or an alkyl group having a phospho group, and X has a chlorine atom, a fluorine atom, a bromine atom, an iodine atom, and a substituent. Also There alkylsulfonyloxy group, an optionally substituted aryl sulfonyloxy group, or an N- methylmorpholinium (NMM) group. ]

  In the above, the alkyl group can have 1 to 30 carbon atoms, more preferably 1 to 20 carbon atoms, and particularly preferably 1 to 6 carbon atoms such as a methyl group, an ethyl group, and an n-propyl group. , Isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, n-pentyl group, isopentyl group, tert-pentyl group, neopentyl group, n-hexyl group, isohexyl group, 2-hexyl group , 3-hexyl group and the like.

  The aryl group is an aromatic group and may be composed of hydrocarbons alone or may contain heteroatoms such as oxygen, nitrogen and sulfur, and the ring size may be a 5-membered or 6-membered ring. However, it may be a 7-membered ring or other sizes. Some rings may be condensed. For example, a phenyl group, 1-naphthyl group, 2-naphthyl group, pyridyl group, imidazolyl group and the like can be mentioned.

  The amino group, ammonio group, sulfo group and phospho group may have a substituent, and examples of the substituent include the above alkyl group and aryl group. In the amino group, ammonio group, and phospho group, when there are two or more substituents, the substituents may be the same or different.

  Examples of the substituent of the “optionally substituted alkylsulfonyloxy group” include a halogen atom (chlorine atom, fluorine atom, bromine atom, iodine atom). The number of substituents is 1 to 5, preferably 1 to 3. When there are two or more substituents, the substituents may be the same or different.

  Examples of the substituent of the “arylsulfonyloxy group which may have a substituent” include an alkyl group, a halogen atom (a chlorine atom, a fluorine atom, a bromine atom, an iodine atom), a haloalkyl group, and the like. The number of substituents is 1 to 5, preferably 1 to 3. When there are two or more substituents, the substituents may be the same or different.

Preferably, R ¹ and R ² each independently represent a hydrogen atom, a methyl group, an ethyl group, a hydroxyalkyl group having 2 to 5 carbon atoms, — (CH ₂ CH ₂ O) _n R ¹² (where n Is an integer of 1 to 120, and R ¹² is a hydrogen atom, a methyl group, an ethyl group, a propyl group, a sulfo group, a phospho group or an alkyl group having an amino group, an ammonio group, a sulfo group, or a phospho group. And — (CH ₂ CH ₂ NR ^{13 ′} ) _n H (where n is an integer of 1 to 120, and R ^{13 ′} is an alkyl group having 2 to 5 carbon atoms, N, N-dialkyl) An aminoalkyl group, or —CH ₂ CH ₂ N ⁺ (CH ₃ ) ₃ ), —CH ₂ CH ₂ SO ₃ ^— , —CH ₂ CH ₂ N ⁺ (CH ₃ ) ₃ , X is a chlorine atom, Fluorine atom, bromine atom, iodine Child, trifluoromethanesulfonyloxy group, an optionally substituted aryl sulfonyloxy group, or a NMM group.
Specific examples of the 1,3,5-triazine compound include 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) and 4- (4,6-dimethoxy which is a quaternary ammonium salt. -1,3,5-triazin-2-yl) -4-methylmorpholinium chloride (DMT-MM), most preferably CDMT.

  The 1,3,5-triazine compound (T) can be synthesized by a method known per se, for example, by the method described in International Publication No. 2000/053544 pamphlet, International Publication No. 2005/074442 pamphlet or the like. be able to. The obtained 1,3,5-triazine compound (T) can be separated and purified by means usually used by those skilled in the art. For example, after completion of the reaction, an organic solvent is added to the reaction solution, and the resulting 1,3,5-triazine compound (T) is extracted into an organic layer or purified by a fractionation method such as chromatography known per se. Can do.

  In the present invention, the “tertiary amine” is used to modify the ligand compound (L) and prepare the modified ligand compound (L ′). The structure of the tertiary amine is not particularly limited as long as it has a functional group for forming a bond with the ligand compound (L). The modification can be performed by introducing a tertiary amine via a covalent bond at an appropriate site in the molecular structure of the ligand compound (L).

  Specific examples of the tertiary amine include compounds represented by the following formula (II).

Wherein one or two of R ⁵ , R ⁶ and R ⁷ can form a bond to the ligand compound (L) that can interact with the protein (P) and modify it It is a substituent containing a functional group, and the ligand compound (L) may be directly bonded to a tertiary amine via the functional group, or a linker having an appropriate length composed of an alkyl group, ethylene glycol, or the like. You may put a chain called. Examples of the bond formed between the ligand compound (L) and the tertiary amine via the functional group include, for example, carbon chain, ester bond, thioester bond, amide bond, thioamide bond, ether bond, thioether bond, aromatic Examples thereof include a ring, a carbamate bond, a urea bond, a phosphate ester bond, a phosphate amide bond, a sulfone ester bond, and a sulfonamide bond. Therefore, as the functional groups for forming these bonds, sulfur-containing functional groups such as hydroxyl groups, carboxy groups, amino groups, carbonyl groups, halogen atoms, mercapto groups, sulfo groups, azido groups, alkynyl groups, alkenyl groups, aryls Group, a phosphorus-containing functional group such as a phosphine group and a phospho group, and a carbon-hydrogen bond having high acidity. These functional groups can be bonded to the nitrogen atom of the tertiary amine via an alkyl group, an aryl group, or a group composed of two or more thereof.
The remaining R ⁵ , R ⁶ and R ⁷ are each independently an alkyl group, alkenyl group, alkynyl group, aryl group, arylalkyl group, —CH ₂ COOR ⁸ , CH ₂ CONR ⁹ R ¹⁰ (where, R ⁸ , R ⁹ , and R ¹⁰ are each independently a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, an arylalkyl group, or — (C _y H _2y O) _z R ¹⁴ , and R ¹⁴ Is a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, or an arylalkyl group, y is an integer of 1 to 30, and z is an integer of 1 to 120).

  Here, the alkyl group can have 1 to 30 carbon atoms, more preferably 1 to 20, and particularly preferably 1 to 6, for example, a methyl group, an ethyl group, or an n-propyl group. , Isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, n-pentyl group, isopentyl group, tert-pentyl group, neopentyl group, n-hexyl group, isohexyl group, 2-hexyl group , 3-hexyl group and the like.

  The alkenyl group can have 2 to 30 carbons, more preferably 2 to 20 and particularly preferably 2 to 6, for example, vinyl group, allyl group, isopropenyl group, 1-butenyl group. 2-butenyl group, 2-methyl-2-propenyl group, 1-methyl-2-propenyl group, 2-methyl-1-propenyl group, pentenyl group, hexenyl group and the like.

  The alkynyl group can have 2 to 30 carbons, more preferably 2 to 20 and particularly preferably 2 to 6, for example, ethynyl group, 1-propynyl group, 2-propynyl group, 1 -Butynyl group, 2-butynyl group, 3-butynyl group, 3-methyl-1-propynyl group, 2-methyl-3-propynyl group, pentynyl group, hexynyl group and the like can be mentioned.

  Alkyl, alkenyl, and alkynyl groups are halogen atoms (chlorine, fluorine, bromine, iodine), oxygen functional groups (eg, hydroxy groups), nitrogen functional groups (eg, nitro groups, amino groups), etc. It may be replaced with. The number of substituents is 1 to 5, preferably 1 to 3. When there are two or more substituents, the substituents may be the same or different.

  The aryl group is an aromatic group, and may be composed of only a hydrocarbon such as a phenyl group, or may contain a heteroatom such as oxygen, nitrogen or sulfur, and the ring size is five-membered. It may be a ring, a 6-membered ring, a 7-membered ring, or any other size. Some rings may be condensed. For example, a phenyl group, 1-naphthyl group, 2-naphthyl group, pyridyl group, imidazolyl group and the like can be mentioned. The aryl group may be substituted with an alkyl group, a halogen atom (a chlorine atom, a fluorine atom, a bromine atom, an iodine atom), a haloalkyl group, or the like. The number of substituents is 1 to 5, preferably 1 to 3. When there are two or more substituents, the substituents may be the same or different.

Another example of the tertiary amine is a compound having a morpholine ring having a low basicity as shown in the formula (III). Here, R ¹¹ is a substituent including a functional group for forming a bond with the ligand compound (L) and modifying the bond, and R ¹⁵ is an alkyl group, an alkenyl group, an alkynyl group, an aryl group, or an arylalkyl group. It is.
The substituent, the alkyl group, the alkenyl group, the alkynyl group, the aryl group, and the arylalkyl group containing a functional group that can form a bond with the ligand compound (L) are as defined in the formula (II).

  Hereinafter, each process in the manufacturing method of N type glycoprotein (P + A) of this invention is explained in full detail. In the above, further necessary processing steps may be provided before and after each step. Further, the steps 3) and 4) or the steps 3 ') and 4) may be reversed in order as necessary, or may be performed simultaneously.

1) Step of modifying a ligand compound (L) with a tertiary amine to prepare a modified ligand compound (L ′) The modification of the ligand compound (L) with a tertiary amine involves the introduction site and introduction method depending on the chemical structure. It is determined appropriately.
For example, carbon chain, ester bond, thioester bond, amide bond, thioamide bond, ether bond, thioether bond, aromatic ring, carbamate ester bond, urea bond, phosphate ester bond, phosphate amide bond, sulfone ester bond, sulfonamide Examples of methods for forming bonds include, for example, Experimental Chemistry Course (5th edition, 2007, edited by The Chemical Society of Japan, Maruzen Co., Ltd.) Volume 14 (Synthesis of Organic Compounds II Alcohol / Amine), Volume 15 (Organic Compounds) Synthetic III aldehydes / ketones / quinones), Volume 16 (Synthesis of organic compounds IV Carboxylic acid / Amino acid / peptide), Volume 18 (Synthesis of organic compounds VI Organic synthesis using metals) It can be performed according to the method to do.
When the ligand compound (L) and the tertiary amine are bound via one or more linkers, the ligand compound (L) and / or the tertiary amine is first bound to the linker by the above-described bond formation method, and then the same. This may be performed by repeating the bond forming method.

2) Step of mixing protein (P) and modified ligand compound (L ′) In the present invention, the step of mixing protein (P) and modified ligand compound (L ′) comprises modifying ligand in an aqueous solution containing protein (P). By mixing the compound (L ′) and allowing the both compounds to be in an equilibrium state of binding or dissociation, they are allowed to interact for a certain period of time by stirring, leaving, or incubation. The solvent of the aqueous solution, the concentration ratio of the protein (P) and the modified ligand compound (L ′), the temperature and time of this step can be appropriately determined for each compound.

3) Step of reacting 1,3,5-triazine compound (T) with protein (L ′ + P) interacting with modified ligand compound (L ′) to synthesize complex (P + L ′ + T) 1,3,5-triazine compound (T) is reacted with protein (L ′ + P) obtained by the interaction of protein (P) and modified ligand compound (L ′) in 2) above, , 5-Triazine compound (T) binds to the tertiary amine in the modified ligand compound (L ′) to form a dehydrating condensing agent. The presence of the carboxy group on the protein (P) in the vicinity of the 1,3,5-triazine compound attacks the triazine ring, and the triazine moves from the tertiary amine to the carboxy group. As a result, the carboxy group is activated in the form of an active ester (referred to as acyloxytriazine or triazinyl ester). Finally, in the step 4) described later, the amino group of the sugar amine compound (A) attacks this active ester. And carboxy group and amide bond (see FIGS. 1 and 2).

  The 1,3,5-triazine compound (T) can be added after allowing the protein (P) and the modified ligand compound (L ′) to interact to synthesize a complex (P + L ′ + T).

  The step 3) can be performed by adding a solution of an organic solvent such as an aqueous solution of 1,3,5-triazine compound (T) or 1,3,5-triazine compound to the obtained aqueous solution. The concentration of the triazine compound, the reaction temperature in this step, and the reaction time can be appropriately determined.

2 ′) a step of reacting the modified ligand compound (L ′) with the 1,3,5-triazine compound (T) to synthesize a dehydrating condensing agent (L ′ + T), and 3 ′) a dehydrating condensing agent (L ′ + T). ) And protein (P) to synthesize a complex (P + L ′ + T) The modified ligand compound (L ′) is reacted with the 1,3,5-triazine compound (T) to form a dehydrating condensing agent (L After synthesizing '+ T), the complex (P + L' + T) can also be synthesized by interacting with protein (P).
Steps 2 ′) and 3 ′) can be carried out in the same manner as 3) and 2), respectively.

4) A step of reacting the sugar amine compound (A).
In the present invention, the step of reacting the sugar amine compound (A) can be performed by adding the aqueous solution of the sugar amine compound (A) to the aqueous solution obtained by the step 3) or 3 ′). The concentration of the sugar amine compound (A), the reaction temperature in this step, and the reaction time can be appropriately determined. This step may be performed after the step 3) or 3 ′) as necessary, or the step 3) or 3 ′) and this step may be performed simultaneously.
After completion of the reaction, the modified ligand compound (L ′), unreacted 1,3,5-triazine compound (T) and sugar amine compound (A) contained in the reaction solution, and 1,3,5- Low molecular weight compounds such as compounds derived from the triazine compound (T) and other by-products can be purified by repeating purification processes such as dialysis, ultrafiltration, ultracentrifugation, and gel filtration chromatography. Can be separated and removed.

  The protein (P) is dehydrated from the tertiary amine on the modified ligand compound (L ′) and the 1,3,5-triazine compound (T) when the modified ligand compound (L ′) interacts. Using a condensing agent, a carboxy group on the protein (P) and an amino group on the sugar amine compound (A) are subjected to a dehydration condensation reaction, so that the carboxy group on the protein (P) and the sugar amine compound (A) Are bound to produce N-type glycoprotein (P + A).

  In the present invention, the method for detecting N-type glycoprotein (P + A) is not particularly limited. For example, N-type glycoprotein (P + A) can be detected by means known per se, such as gel filtration, SDS-polyacrylamide gel electrophoresis (SDS- Analysis by a method such as PAGE), and measuring each obtained fraction by chemiluminescence after reacting with a labeled lectin (for example, Horseradish Peroxidase (HRP) labeled lectin) that binds to the introduced sugar chain Can be detected.

Although the entire desired N-type sugar chain can be directly introduced by the method of the present invention, when the sugar amine compound (A) is N-acetylglucosamine (GlcNAc), the introduced GlcNAc is converted into a sugar receptor ( Acceptor), and a sugar chain of GlcNAc whose reducing end is converted to oxazoline is used as a sugar donor (donor) to conduct a transglycosylation reaction, thereby leading to a desired N-type glycoprotein (for example, Non-Patent Document 2). : M. Umekawa et al., Biochimica et Biophysica Acta, 2010, 1800, 1203).
For example, as shown in the following reaction scheme, first, GlcNAc is introduced into the side chain carboxy group of any aspartic acid residue of protein (P) by the method of the present invention to obtain a sugar acceptor, and then the reducing end is When a sugar chain (hereinafter simply referred to as a sugar donor) converted to oxazoline and a glycosyltransferase are allowed to act, the sugar chain of the sugar donor with respect to the 4-position hydroxyl group of GlcNAc introduced into the protein (P) Can be granted.

The sugar chain that is GlcNAc in which the reducing end used as a sugar donor is converted to oxazoline can be prepared according to a method known per se, for example, as described in International Publication No. 2008/111526 and Non-Patent Document 2. it can.
Further, the reducing end of the sugar donor to be converted into oxazoline may be other than GlcNAc as long as it has an acetamide group at the 2-position. For example, N-acetylgalactosamine or N-acetylmann having the reducing end converted to oxazoline. A sugar chain such as nosamine can also be used.

Examples of glycosyltransferases include those obtained from animals including humans, plants, microorganisms, recombinant enzymes produced by genetic engineering techniques, mutant enzymes, and the like. Examples of the glycosyltransferase include chitinase, mutant chitinase, endoglycosidase such as endo-β-N-acetylglucosaminidase, hyaluronidase, chondroitinase, and the like.
Chitinases and mutant chitinases include those derived from Bacillus sp. Shoda et al. , Helvetica Chemical Acta, Vol. 85, pp. 3919-3936 (2002), for example, Bacillus circulans WL-12-derived chitinase A1 and mutant chitinases thereof, that is, E204Q, D202N, D200N, Y279F, D280N, W433F, etc. .
Examples of endoglycosidase include endo-β-N-acetylglucosaminidase, for example, endo-β-N-acetylglucosaminidase M (Endo-M) derived from Mucor hiemalis (Yamamoto, K. et al., Biochem. Biophys. Commun., 203, pp. 244-252 (1994); non-patent document 2 mutant enzyme Endo-M-N175Q), endo-β-N-acetylglucosaminidase A (Endo-A) derived from Arthrobacter protophormiae (Takegawa, K. et al., Biochem. Int., 24, pp. 849-855 (1991)).
Hyaluronidase is derived from mammals, for example, testis, semen, skin, spleen from higher animals, leeches, bee venom, snake venom, pneumococci, streptococci, staphylococci, gas It may be obtained from microorganisms such as gangrene, and representative examples include bovine testicular hyaluronidase and sheep testicular hyaluronidase.
Examples of the chondroitinase include those derived from Flavobacterium heparinum, those derived from Proteus vulgaris, those derived from Arthrobacter aurescens, and the like, such as chondroitinase ABC (Proteus vulgaris), chondroitinase ACII Arthro (Arthrobacter aurescens), Commercial products such as itinase B (Flavobacterium heparinum) (manufactured by Seikagaku Corporation) can be used.
Glycosyltransferases can be used alone, or if necessary, two or more types can be mixed and used at an appropriate ratio. The glycosyltransferase can be used as it is or in a form immobilized on a resin or the like.
As the glycosyltransferase, Endo-M is preferable, and Endo-M-N175Q is more preferable.

  The glycosyltransferase reaction can be performed by allowing glycosyltransferase to act on a sugar donor and a sugar acceptor. For example, the reaction can be initiated by mixing a buffer solution or an aqueous solution containing an enzyme with an aqueous solution of a sugar donor and a sugar acceptor that are substrates. The reaction is usually carried out in water or a mixed system of water and a water-miscible organic solvent (for example, ethanol, methanol, dioxane, dimethyl sulfoxide, etc.), or an organic solvent that is substantially insoluble or hardly soluble in water. Although it can be carried out in a liquid two-phase system (for example, hexane, heptane, toluene, methylene chloride, etc.) and water, it is generally preferably carried out in an aqueous system.

The reaction conditions can be appropriately determined as long as the reaction is not impaired by the substrate and enzyme used. For example, the concentration of the sugar donor as a substrate and the sugar acceptor is preferably 0.001 to 20%, more preferably 0.01 to 10%. The pH of the reaction solution is preferably 5 to 13, more preferably 6 to 10, and the reaction temperature is preferably 10 to 50 ° C, more preferably 20 to 40 ° C. Buffers (eg, phosphate buffer, citrate buffer, Tris buffer, etc.) can also be used to stabilize the pH. Furthermore, in order to adjust pH, it can also adjust using an acid and a base. The reaction time is 1 minute to 200 hours, preferably 20 minutes to 150 hours, and is appropriately determined depending on the enzyme concentration, the sugar donor used and the sugar acceptor.
For example, in chitinase A1 and its mutant chitinase, Endo-A, Endo-M, etc., a pH at which the enzyme is stable can be adopted, for example, pH is about 4-7, preferably pH 5.5-6, For example, the reaction may be performed at a temperature of 50 ° C. or lower, preferably around 37 ° C.
Alternatively, the glycosyltransferase reaction may be performed in the form of a bioreactor in which an immobilized enzyme is packed in a column and an aqueous solution of the substrate is continuously flowed.

  All of the sugar chains of natural N-type glycoproteins have a basic structure represented by the following formula in which GlcNAc is directly bonded to the side chain of asparagine. From this structure, more sugars bind and have various functions. It is expressed.

Since GlcNAc can be introduced into the side chain carboxy group of any aspartic acid residue of a protein by the method of the present invention, a method for introducing an entire N-type sugar chain having the above basic structure into a protein by combining with the above-mentioned glycosyl transfer reaction Can be said to be established.
Furthermore, since GlcNAc can be introduced into the side chain carboxy group of any glutamic acid residue of the protein by the method of the present invention, it is considered that an unnatural N-type glycoprotein having the above basic structure can also be produced.

Furthermore, novel proteins having various functions and utilities can be synthesized by the method of the present invention.
For example, streptavidin is a protein used as a biochemical research tool that specifically binds to a biotin derivative, but has a problem of low water solubility. It is considered that streptavidin into which a sugar chain has been introduced by the method of the present invention has an advantage that water solubility increases and handling becomes easy.

  The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1 Synthesis of Modified Ligand Compound The modification of the tertiary amine of the ligand compound (L) will be specifically illustrated and described. Here, the protein (P) is streptavidin, the ligand compound is biotin, and the tertiary amine is N-methylmorpholine.

  According to the following method, 6-amino-N- [2- (4-methylmorpholin-2-yl) ethyl] hexanoic acid amide trifluoroacetate (Compound 7) was synthesized using Compound 1 as a starting material. Next, compound 7 and compound 8 were subjected to dehydration condensation to synthesize a modified ligand compound (compound 9).

1) Synthesis of Compound 2 As compound 2, (4-methylmorpholin-2-yl) methyl paratoluenesulfonate was synthesized by the following method.

Compound 1 (Briks, RS et al., J. Org. Chem. 52, 5247-5254) (2.24 g, 17.2 mmol) known in the literature is dissolved in methylene chloride (50 mL) and cooled to 0 ° C. After that, paratoluenesulfonyl chloride (3.60 g, 18.9 mmol) and triethylamine (3.6 mL, 25.8 mmol) were added. After removing the ice bath and stirring overnight, the reaction mixture was extracted with methylene chloride, and the organic layer was washed with saturated sodium carbonate (Na ₂ CO ₃ ) and then with saturated brine. After drying over sodium sulfate, the mixture was concentrated and purified by silica gel column chromatography (AcOEt: MeOH = 80: 20) to obtain Compound 2 (3.4 g, yield 70%).
¹ H NMR (CDCl ₃ ): δ 7.79 (d, J = 8Hz, 2H), 7.75 (d, J = 8Hz, 2H), 4.03 (dd, J = 10, 6Hz, 1H), 3.97 (dd, J = 10, 5 Hz, 1H), 3.82 (ddd, J = 11, 3, 2Hz, 1H), 3.72 (m, 1H), 3.59 (dt, J = 11, 3 Hz, 1H), 2.68 (dt, J = 11, 2Hz, 1H), 2.58 (dq, J = 12, 4, 2Hz, 1H), 2.45 (s, 3H), 2.26 (s, 3H), 2.08 (dt, J = 11, 3Hz, 1H), 1.86 (dd, J = 11, 10Hz, 1H)
LRMS (ESI): 286 (M + H) ⁺

2) Synthesis of Compound 3 As compound 3, (4-methylmorpholin-2-yl) acetonitrile was synthesized by the following method.

Compound 2 (3.4 g, 12.0 mmol) was dissolved in dimethyl sulfoxide (6 mL), potassium cyanide (1.57 g, 24.1 mmol) was added, and the mixture was reacted at 120 ° C. for 4 hours. Water (6 mL) was added to the reaction mixture, and the mixture was extracted with AcOEt: EtOH = 9: 1 and concentrated. Purification by silica gel column chromatography (CHCl ₃ : MeOH = 95: 5 + triethylamine 1%) gave Compound 3 (952 mg, yield 56%).
¹ H NMR (CDCl ₃ ): δ 4.22 (dd, J = 7.2, 14.5Hz, 1H), 3.90 (ddd, J = 11.5, 3.5, 1.8Hz, 1H), 3.80 (m, 1H), 3.69 (td, J = 11.5, 2.8Hz, 1H), 2.81 (m, 1H), 2.64 (ddd, J = 11.5, 3.8, 1.8Hz, 1H), 2.55 (d, J = 6.0Hz, 2H), 2.31 (s, 3H ), 2.16 (td, J = 11.5, 3.8Hz, 1H), 1.97 (dd, J = 10.0, 11.5Hz, 3H)
LRMS (ESI): 141 (M + H) ⁺

3) Synthesis of Compound 4 As compound 4, 2- (4-methylmorpholin-2-yl) ethylamine was synthesized by the following method.

  To a diethyl ether suspension (3 mL) of lithium aluminum hydride (81.2 mg, 2.14 mmol), a diethyl ether solution (1 mL) of compound 3 (100 mg, 0.71 mmol) was added dropwise at 0 ° C. After 30 minutes, diethyl ether (8 mL) was added, and then water (81.2 μL), 15% aqueous sodium hydroxide solution (81.2 μL), and water (162.4 μL) were sequentially added. The resulting solid was removed by celite filtration and concentrated. The obtained compound 4 (66.2 mg) was used in the next reaction without purification.

4) Synthesis of Compound 6 As compound 6, tert-butyl 6- [2- (4-methylmorpholin-2-yl) ethylamino] -6-oxohexyl] carbamate was synthesized by the following method.

To a solution of compound 4 (50 mg, 0.35 mmol) and compound 5 (74.6 mg, 0.35 mmol) in methanol (1.5 mL) was added 4- (4,6-dimethoxy-1,3,5-triazine-2. -Il) -4-methylmorpholinium chloride (DMT-MM: 115 mg, 0.42 mmol) was added, and the mixture was stirred at room temperature for 1 hour. A portion of the solvent methanol was distilled off under reduced pressure, chloroform was added, the mixture was washed with 1M aqueous sodium hydroxide solution and saturated brine in that order, dried over sodium sulfate and concentrated. Silica gel column chromatography (CHCl ₃ : MeOH = 95 : 5 + triethylamine 1%) to obtain Compound 6 (87.5 mg, yield 71%).
¹ H NMR (CDCl ₃ ): δ 6.24 (s, 1H), 4.77 (s, 1H), 3.88 (dd, J = 12.0, 3.0Hz, 1H), 3.66 (td, J = 11.5, 1.8Hz, 1H) , 3.59 (m, 1H), 3.50 (m, 1H), 3.26 (m, 1H), 3.11 (dd, J = 14.0, 7.2Hz, 2H), 2.68 (t, J = 11.5Hz, 2H), 2.28 ( s, 3H), 2.16 (t, J = 7.2Hz, 2H), 2.10 (td, J = 11.5, 3.0Hz, 1H), 1.85 (t, J = 11.5Hz, 1H), 1.73-1.57 (m, 4H ), 1.56-1.40 (m, 2H), 1.44 (s, 9H), 1.40-1.25 (m, 2H)
LRMS (ESI): 358 (M + H) ⁺

5) Synthesis of Compound 7 As compound 7, 6-amino-N- [2- (4-methylmorpholin-2-yl) ethyl] hexanoic acid trifluoroacetate was synthesized by the following method.

  Compound 6 (87 mg, 0.24 mmol) was dissolved in methylene chloride (1 mL), trifluoroacetic acid (181 μL) was added at 0 ° C., the temperature was returned to room temperature, and the mixture was reacted for 3 hours. The compound 7 obtained after concentration was used for the next reaction without purification.

6) Synthesis of modified ligand compound by amidation of compound 7 and compound 8 (compound 9)

To a solution of compound 7 and compound 8 known in the literature (JP 2009-175139 A, 87 mg, 0.24 mmol) in methanol (1 mL), sodium hydroxide (9.7 mg, 0.24 mmol) is added to water (0.4 mL). And then added 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium chloride (DMT-MM: 115 mg, 0.42 mmol), Stir overnight at room temperature. A portion of the solvent methanol was distilled off under reduced pressure, chloroform was added, the mixture was washed with 1M aqueous sodium hydroxide solution and saturated brine in that order, dried over sodium sulfate and concentrated. Silica gel column chromatography (CHCl ₃ : MeOH = 95 : 5 + triethylamine 1%) to obtain Compound 9 (88 mg, 61% yield).
¹ H NMR (CD ₃ OD): δ 4.49 (dd, J = 7.5, 4.0Hz, 1H), 4.30 (dd, J = 7.5, 4.0Hz, 1H), 3.86 (dd, J = 11.5, 3.0Hz, 1H ), 3.61 (td, J = 11.5, 2.0Hz, 1H), 3.29-3.10 (m, 7H), 2.92 (dd, J = 12.5, 5.0Hz, 1H), 2.76 (m, 2H), 2.70 (d, J = 12.5Hz, 1H), 2.32 (s, 3H), 2.25-2.10 (m, 7H), 1.90 (t, J = 11.0Hz, 1H), 1.80-1.20 (m, 20H)
LRMS (ESI): 598 (M + H) ⁺

(Example 2) Introduction of lactose amine into streptavidin Streptavidin, a protein having no sugar chain, was used.

(Glycosylation reaction)
The phosphate buffer solution (2 μL, 800 μM) of the biotin derivative obtained in Example 1 was added to the aqueous streptavidin solution (5 μL, 55 μM) and allowed to stand for 30 minutes. Sodium phosphate buffer (11 μL, 50 mM, pH 8.0, NaCl: 150 mM), CDMT 5% methanol-containing phosphate buffer (6 μL, 10 mM), lactose amine phosphate buffer (6 μL, 10 mM) And allowed to stand at room temperature for 8 hours (final concentrations were streptavidin: 10 μM, biotin derivative: 40 μM, CDMT: 2 mM, lactose amine: 2 mM). After completion of the reaction, a dimethylethylenediamine solution (4.5 μL, 500 mM) was added to stop the reaction.
A system in which biotin (3 μL, 20 mM) is added as an inhibitor to the above reaction system; a system in which N-methylmorpholine (2 μL, 800 mM) is added in place of the biotin derivative; streptavidin and lactoseamine are added without the biotin derivative. A mixed system was performed.

(analysis)
Each reaction solution was run on SDS-PAGE (18% separation gel), then transferred to a PVDF membrane, soaked in 5% BSA / TBS-T (0.5% tween) for 3 hours and blocked to recognize galactose Blotting was performed using a 100,000-fold diluted solution of HRP-labeled lectin (PNA-HRP), and lactose introduced into streptavidin was detected by chemiluminescence. The results are shown in FIG.

  From Lane 1, it was confirmed that lactoseamine was introduced into streptavidin. In addition, it was found from Lane 2 that the introduction of lactoseamine into streptavidin is inhibited by inhibiting the binding of the biotin derivative (modified ligand) to streptavidin. From Lanes 3 and 4, it was found that when no biotin derivative (modified ligand) was present, introduction of lactoseamine was inhibited. From the above results, it was confirmed that the introduction of lactoseamine was a reaction through the binding of biotin derivative (modified ligand) and streptavidin.

In the MoAL method, it is known that amidation proceeds to the 51st glutamic acid of streptavidin (Shuichi Nakanishi, doctoral dissertation, Kanazawa University). Therefore, it is considered that lactoseamine was introduced into the side chain carboxy group of the 51st glutamic acid of streptavidin. Since glutamine-linked glycoprotein cannot be synthesized by cell culture or the like, it can be said that the present invention can be synthesized for the first time.
Further, the N-type sugar chain is introduced only into asparagine in the peptide chain region of asparagine-X-serine / threonine (X is an arbitrary amino acid other than proline), but there is no such limitation in the method of the present invention. An N-type sugar chain can be introduced into any aspartic acid / glutamic acid.
Furthermore, although there is no natural N-type sugar chain having lactose amine bound thereto, Example 2 proved that a non-natural N-type sugar chain can be introduced into any carboxy group of a protein. . Thus, since the method of the present invention does not use an enzyme, the structure of the sugar chain that can be introduced is not limited, and any sugar chain can be introduced if an amino group is bonded to the sugar. Glycoproteins that cannot be synthesized by the conventional method used can also be synthesized.

(Example 3) Introduction of N-acetylglucosamine amine (GlcNAc amine) into streptavidin (sugar chain introduction reaction)
The phosphate buffer solution (2 μL, 800 μM) of the biotin derivative obtained in Example 1 was added to the aqueous streptavidin solution (5 μL, 55 μM) and allowed to stand for 30 minutes. Sodium phosphate buffer (11 μL, 50 mM, pH 8.0, NaCl: 150 mM), CDMT 5% methanol-containing phosphate buffer (6 μL, 10 mM), N-acetylglucosamine amine (GlcNAc amine) phosphate Buffer solution (6 μL, 10 mM) was added, allowed to stand, and allowed to react at room temperature for 8 hours (final concentrations were streptavidin: 10 μM, biotin derivative: 40 μM, CDMT: 2 mM, GlcNAc amine: 2 mM). After completion of the reaction, a dimethylethylenediamine solution (4.5 μL, 500 mM) was added to stop the reaction.
A system in which biotin (3 μL, 20 mM) was added as an inhibitor to the above reaction system; a system in which N-methylmorpholine (2 μL, 800 mM) was added in place of the biotin derivative; streptavidin and GlcNAc amine were added without the biotin derivative A mixed system was performed.

(analysis)
Each of the above reaction solutions was run on SDS-PAGE (18% separation gel), transferred to a PVDF membrane, blocked by immersion in 5% BSA / TBS-T (0.5% tween) for 3 hours, and GlcNAc was recognized. Blotting was performed using a 100,000-fold diluted solution of HRP-labeled lectin (WGA-HRP), and GlcNAc introduced into streptavidin was detected by chemiluminescence. The results are shown in FIG.

  From Lane 1, it was confirmed that GlcNAc amine was introduced into streptavidin. Moreover, it was found from Lane 2 that the introduction of GlcNAc amine into streptavidin is inhibited by inhibiting the binding of a biotin derivative (modified ligand) to streptavidin. From Lanes 3 and 4, it was found that the introduction of GlcNAc amine is inhibited when no biotin derivative (modified ligand) is present. From the above results, it was confirmed that the introduction of GlcNAc amine was a reaction through the binding of a biotin derivative (modified ligand) and streptavidin.

  From Example 3, it was clarified that the GlcNAc amine can be introduced into a specific side chain carboxy group of the protein by the method of the present invention. A method for synthesizing a protein having a basic skeleton of an N-type sugar chain by providing a desired sugar chain by a glycosyl transfer reaction using a protein into which GlcNAc amine obtained by the method of the present invention is introduced as a raw material is established. It can be said that it was made.

  In addition, streptavidin into which sugar chains synthesized in Examples 2 and 3 are introduced is considered to have improved water solubility compared to streptavidin having no sugar chain, and thus a novel We were able to provide chemical research tools.

  INDUSTRIAL APPLICABILITY According to the present invention, an N-type glycoprotein in which an arbitrary uniform N-type sugar chain structure that has been difficult to obtain by a conventional production method using eukaryotic cell culture is selectively introduced into a protein is produced at low cost. A method is provided. The method of the present invention has made it possible to synthesize a novel N-type glycoprotein that cannot be synthesized by a method using culture or an enzyme.

Claims (12)

A method for producing an N-type glycoprotein comprising the following steps:
1) a step of modifying a ligand compound (L) with a tertiary amine to prepare a modified ligand compound (L ′);
2) A step of mixing the modified ligand compound (L ′) and the protein (P);
3) A step of reacting the protein (L ′ + P) interacting with the modified ligand compound (L ′) with a 1,3,5-triazine compound (T) to synthesize a complex (P + L ′ + T);
4) A step of further reacting the sugar amine compound (A) to synthesize an N-type glycoprotein (P + A).
Here, protein (P) is a compound having a carboxy group in the side chain and / or C-terminus,
The ligand compound (L) is a compound that can interact with the protein (P). A method for producing an N-type glycoprotein comprising the following steps:
1) a step of modifying a ligand compound (L) with a tertiary amine to prepare a modified ligand compound (L ′);
2 ′) reacting the modified ligand compound (L ′) with the 1,3,5-triazine compound (T) to synthesize a dehydrating condensing agent (L ′ + T);
3 ′) the step of mixing the dehydrating condensing agent (L ′ + T) and the protein (P) of the above 2 ′) to synthesize a complex (P + L ′ + T);
4) A step of further reacting the sugar amine compound (A) to synthesize an N-type glycoprotein (P + A).
Here, protein (P) is a compound having a carboxy group in the side chain and / or C-terminus,
The ligand compound (L) is a compound that can interact with the protein (P). The 1,3,5-triazine compound (T) is represented by the following formula (I):
[Wherein, R ¹ and R ² are each independently a hydrogen atom, an alkyl group, a hydroxyalkyl group, a sulfo group, a phospho group, —R ³ —R ⁴ (where R ³ represents — (C _m H _2m Q) _n , Q is O or NR ¹³ , R ⁴ is a hydrogen atom, an alkyl group, a sulfo group, a phospho group, or an alkyl group having an amino group, an ammonio group, a sulfo group, or a phospho group R ¹³ is an alkyl group or an alkyl group having an amino group, an ammonio group, a sulfo group, or a phospho group, m is an integer of 1 to 30, and n is an integer of 1 to 120.) And a substituent selected from the group consisting of an amino group, an ammonio group, a sulfo group, or an alkyl group having a phospho group, and X has a chlorine atom, a fluorine atom, a bromine atom, an iodine atom, and a substituent. Also There alkylsulfonyloxy group, an optionally substituted aryl sulfonyloxy group, or an N- methyl morpholinium group. The method of Claim 1 or 2 which is a compound represented by this.   The method according to claim 1 or 2, wherein the 1,3,5-triazine compound (T) is 2-chloro-4,6-dimethoxy-1,3,5-triazine.   The ligand compound (L) is biotin, warfarin, phenytoin, phenylbutazone, bucolome, acetylsalicylic acid, ketoprofen, flurbiprofen, N-acetyl-L-tryptophan, naproxen, fenoprofen, diclofenac sodium, diazepam, digitoxin, The method according to any one of claims 1 to 4, comprising a structure selected from indomethacin, ibuprofen, hexamethonium, decamethonium, and Phos-tag (R). A tertiary amine is represented by the following formula (II):
[Wherein, one or two of R ⁵ , R ⁶ and R ⁷ are substituents containing a functional group capable of forming a bond to the ligand compound (L), and the remaining R ⁵ , R ⁶ And R ⁷ are each independently an alkyl group, an alkenyl group, an alkynyl group, an aryl group, an arylalkyl group, —CH ₂ COOR ⁸ , or CH ₂ CONR ⁹ R ¹⁰ (where R ⁸ , R ⁹ , and R ¹⁰ is each independently a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, an arylalkyl group, or — (C _y H _2y O) _z R ¹⁴ , and R ¹⁴ is a hydrogen atom, an alkyl group , An alkenyl group, an alkynyl group, an aryl group, or an arylalkyl group, y is an integer of 1 to 30, and z is an integer of 1 to 120], or the following formula (III):
[Wherein R ¹¹ is a substituent containing a functional group capable of forming a bond to the ligand compound (L), and R ¹⁵ is an alkyl group, an alkenyl group, an alkynyl group, an aryl group, or an arylalkyl group. is there. The method of any one of Claims 1-5 which is a compound represented by this. The sugar amine compound (A) is
N-acetylglucosamineamine, N-acetylgalactosamineamine, N-acetylmannosamineamine, alloseamine, taloseamine, growthamine, glucoseamine, altroseamine, mannoseamine, galactoseamine, glucuronic acidamine, galacturonic acidamine, deoxy Riboseamine, fucoseamine, rhamnoseamine, idoseamine, riboseamine, lyxoseamine, xyloseamine, arabinoseamine, erythroseamine, threoseamine, cedoheptuloseamine, coriooseamine, psicoseamine, fructoseamine, sorboseamine In an anomeric position selected from tagatose amine, ribulose amine, xylulose amine, erythrulose amine, and sialic acid amine Monosaccharide amine Min is bound; and the a sugar chain composed monosaccharide corresponding to the monosaccharide amine, sugar amine anomer of reducing terminal is linked amines;
A sugar amine in which at least one of a hydroxyl group, an amino group, and a carboxy group of the monosaccharide amine or sugar chain amine is modified by acylation, esterification, sulfate esterification, and / or phosphate esterification; and glucosamine, mannosamine The method of any one of Claims 1-6 which is a compound selected from the group which consists of saccharide | sugar which has an amino group chosen from galactosamine and sialic acid. The protein (P) is avidin, streptavidin, human serum albumin, cyclooxygenase-1, nicotinic acetylcholine receptor, casein, α ₁ -acid glycoprotein, or phosphorylated protein. The method described in 1.   N-type sugar streptavidin into which a monosaccharide amine or a sugar chain amine has been introduced.   The N-type sugar streptavidin according to claim 9, wherein a monosaccharide amine or a sugar chain amine is introduced into the side chain carboxy group of the 51st glutamic acid residue from the N-terminus of streptavidin.   The N-type sugar streptavidin according to claim 9 or 10, wherein the monosaccharide amine is N-acetylglucosamine amine.   The N-type sugar streptavidin according to claim 9 or 10, wherein the sugar chain amine is lactose amine.