JP6324660B2 - Method for producing novel (2 → 3) -linked sialosugar chain - Google Patents

Description

  The present invention relates to a novel sialosugar chain, a production method thereof, and a production apparatus thereof.

  A sialic sugar chain is a general term for a sugar chain having sialic acid at its non-reducing end, and is an important component of glycolipids, gangliosides and various glycoprotein sugar chains. The functions of sialoglycans vary widely and govern essential phenomena in life activities such as intercellular recognition, differentiation, and proliferation, and are also involved in canceration, virus infection, and the like. For example, in cancer-related sugar chain antigens, it has been elucidated that there are many sialo sugar chains, and the sialylgalactose moiety (disaccharide combined with sialic acid and galactose) that is expressed on the host cell surface It has been elucidated to infect as a scaffold.

  Thus, since sialo-sugar chains are involved in various phenomena, their application range can be very diverse. Therefore, development of a sialo-glycan having a structure that can be used in many fields is demanded.

  Further, since the production and purification methods of sialo-sugar chains are usually complicated, the production and purification of sialo-sugar chains require extremely advanced techniques, expensive equipment and reagents, and harmful reagents. Therefore, development of a method for producing a large amount of sialo-glycan simply and efficiently has been demanded.

  As an example of producing a sialo-sugar chain, there is known an example produced by reacting lactose and sialic acid in the presence of sialidase derived from Arthrobacter ureafaciens (Non-patent Document 1). However, this production example is insufficient to produce a sialo-glycan simply and efficiently. As a specific example, a plurality of sialo-glycan chains with different non-reducing ends in lactose to which sialic acid in the sialo-sugar chain binds are produced. Moreover, the sialosugar chain produced by this production example is simply a sialic acid and lactose linked together, and is difficult to apply in many fields.

  As another production example of a sialic sugar chain, an example in which various sugar derivatives and a sialic acid donor are reacted in the presence of sialidase is known (Non-patent Document 2). However, since the yield of this production example is about 10 to 20%, improvement of the yield is required.

Biosci. Biotech. Biochem., 56 (10), 1557-1561, 1992 Journal of Organic Chemistry, 2000, Vol.65, No.25, p.8518-8526

  An object of the present invention is to provide a novel sialosugar chain, a method for producing the sialosugar chain, and an apparatus for producing the sialosugar chain.

  As a result of intensive studies to achieve the above-mentioned problems, the present inventors, as a result, a sugar in which the reducing end hydroxyl group is substituted with a reactive group (hereinafter also referred to as “reactive group-introducing sugar”), a sial It has been found that by reacting with an acid donor in the presence of sialidase, a sialo-sugar chain in which sialic acid is bound to a specific non-reducing end of a reactive group-introduced sugar can be easily and efficiently produced.

  Furthermore, the present inventors have found that the sialo sugar chain produced by the above production method (hereinafter sometimes referred to as a reactive group-introduced sialo sugar chain) can be used very easily in many fields.

  Furthermore, it has been found that a reactive group-introduced sialo-sugar chain can be more easily produced by using a column containing a carrier on which a sialic acid-introducing enzyme is immobilized.

  That is, the present invention includes the following configurations.

  Item 1. General formula (5);

[Wherein, X represents a single bond or a sugar residue obtained by removing a hydroxyl group from a hydroxyl group at the reducing end of a monosaccharide or oligosaccharide and removing hydrogen from a hydroxyl group at the non-reducing end; Y represents an ethynyl group, vinyl; A group, an azido group, an epoxy group, an aldehyde group, or an oxylamino group, R ⁵ and R ⁶ are different from each other and represent hydrogen or a hydroxyl group, R ⁷ represents a hydroxyl group or an acetylamino group, and R ⁸ represents a hydrogen or amino group. And R ⁹ is the same or different and represents a hydrogen or hydroxyl protecting group, and n is an integer of 1 to 10.
A compound represented by

  Item 2. Item 2. The compound according to Item 1, wherein in General Formula (5), X is a sugar residue obtained by removing a hydroxyl group from a hydroxyl group at the reducing end of glucose and removing hydrogen from a hydroxyl group at the non-reducing end.

  Item 3. General formula (7a);

[Wherein, X represents a single bond or a sugar residue obtained by removing a hydroxyl group from a hydroxyl group at the reducing end of a monosaccharide or oligosaccharide and removing hydrogen from a hydroxyl group at the non-reducing end; Y represents an ethynyl group, vinyl; A group, an azido group, an epoxy group, an aldehyde group, or an oxylamino group, R ⁵ and R ⁶ are different from each other and represent hydrogen or a hydroxyl group, R ⁷ represents a hydroxyl group or an acetylamino group, and R ⁸ represents a hydrogen or amino group. A group that acylates a group, R ⁹ is the same or different and represents a hydrogen or hydroxyl protecting group, n is an integer of 1 to 10, and R ¹ represents an organic group]
Or general formula (7b);

[Wherein, X represents a single bond or a sugar residue obtained by removing a hydroxyl group from a hydroxyl group at the reducing end of a monosaccharide or oligosaccharide and removing hydrogen from a hydroxyl group at the non-reducing end; Y represents an ethynyl group, vinyl; A group, an azido group, an epoxy group, an aldehyde group, or an oxylamino group, R ⁵ and R ⁶ are different from each other and represent hydrogen or a hydroxyl group, R ⁷ represents a hydroxyl group or an acetylamino group, and R ⁸ represents a hydrogen or amino group. A group that acylates a group, R ¹⁰ is the same or different and represents a hydrogen or hydroxyl protecting group, n is an integer of 1 to 10, and R ¹ represents an organic group]
A compound represented by

  Item 4. General formula (5);

[Wherein, X represents a single bond or a sugar residue obtained by removing a hydroxyl group from a hydroxyl group at the reducing end of a monosaccharide or oligosaccharide and removing hydrogen from a hydroxyl group at the non-reducing end; Y represents an ethynyl group, vinyl; A group, an azido group, an epoxy group, an aldehyde group, or an oxylamino group, R ⁵ and R ⁶ are different from each other and represent hydrogen or a hydroxyl group, R ⁷ represents a hydroxyl group or an acetylamino group, and R ⁸ represents a hydrogen or amino group. And R ⁹ is the same or different and represents a hydrogen or hydroxyl protecting group, and n is an integer of 1 to 10.
A process for producing a compound represented by
A sialic acid donor;
General formula (3);

[Wherein, X represents a single bond or a sugar residue obtained by removing a hydroxyl group from a hydroxyl group at the reducing end of a monosaccharide or oligosaccharide and removing hydrogen from a hydroxyl group at the non-reducing end; Y represents an ethynyl group, vinyl; A group, an azido group, an epoxy group, an aldehyde group, or an oxylamino group, R ⁵ and R ⁶ are different from each other and represent hydrogen or a hydroxyl group, R ⁷ represents a hydroxyl group or an acetylamino group, and n is an integer of 1 to 10 Is]
A compound represented by
A production method comprising reacting in the presence of sialidase.

  Item 5. Item 5. The production method according to Item 4, wherein the sialidase is derived from an organism belonging to the genus Salmonella.

  Item 6. Item 6. The production method according to Item 4 or 5, wherein X is a sugar residue obtained by removing a hydroxyl group from a hydroxyl group at the reducing end of glucose and removing hydrogen from a hydroxyl group at the non-reducing end.

  Item 7. General formula (5);

[Wherein X, Y, R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , and n are the same as above]
The manufacturing method in any one of claim | item 4 -6 of the compound represented by these,
(Step 1) General formula (1);

[Wherein, X represents a single residue, or a sugar residue obtained by removing a hydroxyl group from a hydroxyl group at the reducing end of a monosaccharide or oligosaccharide and removing hydrogen from a hydroxyl group at the non-reducing end, and R ⁵ and R ⁶ are different. Represents hydrogen or a hydroxyl group, and R ⁷ represents a hydroxyl group or an acetylamino group]
A compound represented by
General formula (2);

[Wherein Y represents an ethynyl group, a vinyl group, an azide group, an epoxy group, an aldehyde group, or an oxylamino group, and n is an integer of 1 to 10]
A compound represented by
Reacting in the presence of glycosidase, general formula (3);

[Wherein, X, Y, R ⁵ , R ⁶ , R ⁷ and n are the same as above]
A step of obtaining a compound represented by: and (Step 2) a sialic acid donor,
General formula (3);

[Wherein, X, Y, R ⁵ , R ⁶ , R ⁷ and n are the same as above]
A compound represented by
The manufacturing method characterized by including the process made to react in presence of sialidase.

  Item 8. Item 8. The production method according to any one of Items 4 to 7, wherein the sialidase is a sialidase immobilized on a carrier.

  Item 9. The sialic acid donor has the general formula (4);

[Wherein, R ² is the same or different and represents a substituent, R ⁸ represents hydrogen or a group for acylating an amino group, R ⁹ is the same or different and represents a hydrogen or hydroxyl protecting group, m is It is an integer of 1-3. ]
Item 9. The production method according to any one of Items 4 to 8, which is at least one selected from the group consisting of a compound represented by: and colominic acid.

  Item 10. An apparatus comprising a column containing glycosidase immobilized on a carrier (hereinafter referred to as column A) having a supply port and a discharge port.

Item 11. (A) Column A, and (B) a reverse phase column (hereinafter referred to as column B) having a supply port and a discharge port,
The discharge port of column A and the supply port of column B are connected by a flow path b,
Item 11. The apparatus according to Item 10, wherein the channel b includes a channel switching device b ′.

  Item 12. General formula (3);

[Wherein, X represents a single bond or a sugar residue obtained by removing a hydroxyl group from a hydroxyl group at the reducing end of a monosaccharide or oligosaccharide and removing hydrogen from a hydroxyl group at the non-reducing end; Y represents an ethynyl group, vinyl; A group, an azido group, an epoxy group, an aldehyde group, or an oxylamino group, R ⁵ and R ⁶ are different from each other and represent hydrogen or a hydroxyl group, R ⁷ represents a hydroxyl group or an acetylamino group, and n is an integer of 1 to 10 Is]
Item 12. The device according to Item 10 or 11, which is a device for producing a compound represented by the formula:

  Item 13. An apparatus comprising a column containing sialidase immobilized on a carrier and having a supply port and a discharge port.

Item 14. (C) a column containing sialidase immobilized on a carrier having a supply port and a discharge port (hereinafter referred to as column C),
(D) a reverse phase column (hereinafter referred to as column D) having a supply port and a discharge port, and (E) an ion exchange column (hereinafter referred to as column E) having a supply port and a discharge port,
The discharge port of column C and the supply port of column D are connected by a flow path d,
The discharge port of column D and the supply port of column E are connected by a flow path e.
The channel d includes a channel switching device d ′,
Item 14. The apparatus according to Item 13, wherein the channel e includes a channel switching device e ′.

Item 15. The apparatus according to item 10 (hereinafter referred to as apparatus 1) and the apparatus according to item 13 (hereinafter referred to as apparatus 2),
A device characterized in that the discharge port of the device 1 and the supply port of the device 2 are connected by a flow path c.

Item 16. The apparatus according to item 11 (hereinafter referred to as apparatus 1a) and the apparatus according to item 14 (hereinafter referred to as apparatus 2a),
The discharge port of the device 1a and the supply port of the device 2a are connected by a flow path c,
The channel c is provided with a channel switching device c ′.

  Item 17. General formula (5);

[Wherein, X represents a single bond or a sugar residue obtained by removing a hydroxyl group from a hydroxyl group at the reducing end of a monosaccharide or oligosaccharide and removing hydrogen from a hydroxyl group at the non-reducing end; Y represents an ethynyl group, vinyl; A group, an azido group, an epoxy group, an aldehyde group, or an oxylamino group, R ⁵ and R ⁶ are different from each other and represent hydrogen or a hydroxyl group, R ⁷ represents a hydroxyl group or an acetylamino group, and R ⁸ represents a hydrogen or amino group. And R ⁹ is the same or different and represents a hydrogen or hydroxyl protecting group, and n is an integer of 1 to 10.
Item 17. The device according to any one of Items 13 to 16, which is a device for producing a compound represented by the formula:

  Item 18. General formula (3) using the apparatus according to item 10 (hereinafter referred to as apparatus 1);

[Wherein, X represents a single bond or a sugar residue obtained by removing a hydroxyl group from a hydroxyl group at the reducing end of a monosaccharide or oligosaccharide and removing hydrogen from a hydroxyl group at the non-reducing end; Y represents an ethynyl group, vinyl; A group, an azido group, an epoxy group, an aldehyde group, or an oxylamino group, R ⁵ and R ⁶ are different from each other and represent hydrogen or a hydroxyl group, R ⁷ represents a hydroxyl group or an acetylamino group, and n is an integer of 1 to 10 Is]
A process for producing a compound represented by
(Step a) General formula (1);

[Wherein X represents a sugar residue obtained by removing a hydroxyl group from the hydroxyl group at the reducing end of a single bond or monosaccharide or oligosaccharide and removing hydrogen from the hydroxyl group at the non-reducing end, and R ⁵ and R ⁶ are different. Represents hydrogen or a hydroxyl group, and R ⁷ represents a hydroxyl group or an acetylamino group]
And a compound represented by the general formula (2):

[Wherein Y represents an ethynyl group, a vinyl group, an azide group, an epoxy group, an aldehyde group, or an oxylamino group, and n is an integer of 1 to 10]
A production method comprising a step of introducing a compound represented by formula (1) from the supply port of the column A into the column A and reacting in the column A.

  Item 19. General formula (3) using the apparatus (apparatus 1a) of claim | item 11.

[Wherein, X, Y, R ⁵ , R ⁶ , R ⁷ and n are the same as above]
The production method according to Item 18, wherein the compound is represented by:
(Step a) General formula (1);

[Wherein, X, R ⁵ , R ⁶ and R ⁷ are the same as above]
And a compound represented by the general formula (2):

[Wherein Y and n are the same as above]
A step of introducing a compound represented by the following formula into the column A from the supply port of the column A and reacting in the column A:
(Step b) including a step of introducing a reactant in column A into a supply port of column B, and (step c) a step of purifying the compound represented by general formula (3) from column B. Manufacturing method.

  Item 20. General formula (5) using the apparatus according to item 13 (hereinafter, referred to as apparatus 2);

[Wherein, X represents a single bond or a sugar residue obtained by removing a hydroxyl group from a hydroxyl group at the reducing end of a monosaccharide or oligosaccharide and removing hydrogen from a hydroxyl group at the non-reducing end; Y represents an ethynyl group, vinyl; A group, an azido group, an epoxy group, an aldehyde group, or an oxylamino group, R ⁵ and R ⁶ are different from each other and represent hydrogen or a hydroxyl group, R ⁷ represents a hydroxyl group or an acetylamino group, and R ⁸ represents a hydrogen or amino group. And R ⁹ is the same or different and represents a hydrogen or hydroxyl protecting group, and n is an integer of 1 to 10.
A process for producing a compound represented by
(Step d) sialic acid donor and general formula (3);

[Wherein, X represents a single bond or a sugar residue obtained by removing a hydroxyl group from a hydroxyl group at the reducing end of a monosaccharide or oligosaccharide and removing hydrogen from a hydroxyl group at the non-reducing end; Y represents an ethynyl group, vinyl; A group, an azido group, an epoxy group, an aldehyde group, or an oxylamino group, R ⁵ and R ⁶ are different from each other and represent hydrogen or a hydroxyl group, R ⁷ represents a hydroxyl group or an acetylamino group, and n is an integer of 1 to 10 Is]
A compound represented by
A production method comprising a step of introducing into a column C from a supply port of the column C and reacting in the column C.

  Item 21. Item 20. The production method according to Item 20, wherein the compound represented by General Formula (5) is used using the apparatus according to Item 13, wherein the sialic acid donor is represented by General Formula (4);

[Wherein, R ² is the same or different and represents a substituent, R ⁸ represents hydrogen or a group for acylating an amino group, R ⁹ is the same or different and represents a hydrogen or hydroxyl protecting group, m is It is an integer of 1-3. ]
The manufacturing method which is at least 1 sort (s) selected from the group which consists of the compound represented by these, and colominic acid.

  Item 22. General formula (5) using the apparatus of claim | item 14 (henceforth the apparatus 2a);

[Wherein X, Y, R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , and n are the same as above]
The production method according to item 20, wherein the compound is represented by:
(Step d) sialic acid donor and general formula (3);

[Wherein, X, Y, R ⁵ , R ⁶ , R ⁷ and n are the same as above]
A compound represented by
Introducing into the supply port of column C in the apparatus 2 for reaction;
(Step e) A step of introducing the reactant in the column C into the supply port of the column D,
(Step f) Step of introducing the contents in column D into the supply port of column E (Step g) Production comprising the step of purifying the compound represented by general formula (5) from column E Method.

  Item 23. Item 23. The production method according to Item 22, wherein the sialic acid donor is represented by General Formula (4):

[Wherein, R ² is the same or different and represents a substituent, R ⁸ represents hydrogen or a group for acylating an amino group, R ⁹ is the same or different and represents a hydrogen or hydroxyl protecting group, m is It is an integer of 1-3. ]
The manufacturing method which is at least 1 sort (s) selected from the group which consists of the compound represented by these, and colominic acid.

  ADVANTAGE OF THE INVENTION According to this invention, the novel sialo-sugar chain, the manufacturing method of this sialo-sugar chain, and the manufacturing apparatus of this sialo-sugar chain can be provided.

  In the novel sialosugar chain of the present invention, the sugar is substituted with a reactive group as represented by the general formula (5). Therefore, it is possible to link to various materials or probes that can be used in various fields, for example, the biochemical field and the medical field, through this reactive group.

  Further, the production method of the present invention comprises reacting a saccharide having a reactive group substituted with a hydroxyl group at the reducing end and a sialic acid donor represented by the general formula (3) in the presence of sialidase. Sialize a specific non-reducing end of a reactive group-introduced sugar easily and efficiently without the need for complicated steps such as protection and deprotection, and without using expensive or dangerous reagents. A sialo-sugar chain to which an acid is bonded can be produced.

  Furthermore, by using a column containing a sialic acid-introducing enzyme immobilized on a carrier, a sialosugar chain can be produced more easily and at a low cost.

A schematic representation of the device 1a is shown. A schematic representation of the device 2a is shown. A schematic representation of the device 3a is shown. The schematic of the apparatus (equivalent to the apparatus 1a) manufactured in Example 8 is shown. The schematic of the apparatus manufactured in Example 9 (equivalent to the apparatus 2a) is shown.

  Hereinafter, the present invention will be described in detail.

1. A sialo-sugar chain and a sialo-sugar chain linked with an organic group are represented by the general formula (5):

[Wherein, X represents a single bond or a sugar residue obtained by removing a hydroxyl group from a hydroxyl group at the reducing end of a monosaccharide or oligosaccharide and removing hydrogen from a hydroxyl group at the non-reducing end; Y represents an ethynyl group, vinyl; A group, an azido group, an epoxy group, an aldehyde group, or an oxylamino group, R ⁵ and R ⁶ are different from each other and represent hydrogen or a hydroxyl group, R ⁷ represents a hydroxyl group or an acetylamino group, and R ⁸ represents a hydrogen or amino group. And R ⁹ is the same or different and represents a hydrogen or hydroxyl protecting group, and n is an integer of 1 to 10.
It is a compound represented by these.

  X represents a single bond or a sugar residue obtained by removing a hydroxyl group from the hydroxyl group at the reducing end of a monosaccharide or oligosaccharide and removing hydrogen from the hydroxyl group at the non-reducing end. X is preferably a single bond or a monosaccharide, and more preferably a monosaccharide.

  Y represents an ethynyl group, a vinyl group, an azide group, an epoxy group, an aldehyde group, or an oxylamino group. Among these, Preferably an ethynyl group is mentioned.

R ⁵ and R ⁶ are different and represent hydrogen or a hydroxyl group. It is preferred that R ⁵ is a hydroxyl group and R ⁶ is hydrogen.

R ⁷ represents a hydroxyl group or an acetylamino group. R ⁷ is preferably a hydroxyl group.

R ⁸ represents hydrogen or a group for acylating an amino group. Specific examples of R ⁸ include hydrogen, acetyl group, glycolyl group, levulinoyl group, chloroacetyl group, bromoacetyl group, trichloroacetyl group, trifluoroacetyl group, benzoyl group, pivaloyl group, and tlock group. it can. Among these, an acetyl group or a glycolyl group is preferable, and an acetyl group is more preferable.

R ⁹ may be the same or different and each represents a hydrogen or hydroxyl protecting group. Specific examples of R ⁹ include hydrogen, a sulfate group, and a phosphate group. Among these, hydrogen or a sulfate group is preferable, and hydrogen is more preferable.

  n is an integer of 1-10. Preferably it is 1-5, More preferably, it is 1-3, More preferably, it is 1 or 2, Especially preferably, it is 1.

  The monosaccharide is not particularly limited, and a known monosaccharide can be employed. For example, heptose sugar, hexose sugar, pentose sugar, tetrose sugar, or tricose sugar can be used, and among these, hexose sugar is preferred. Examples of the hexose include galactose, glucose, N-acetylglucosamine, N-acetylgalactosamine, mannose, fructose, allose, talose, gulose, altrose, idose, psicose, sorbose, and tagatose. Among these, galactose , Glucose, N-acetylglucosamine, and N-acetylgalactosamine are preferable, galactose or glucose is more preferable, and glucose is particularly preferable. These may be either D-form or L-form. In addition, in these, some of the hydroxyl groups may be reduced and substituted with hydrogen atoms, or some of the hydroxyl groups may be protected with a known protecting group, or may be substituted with a functional group such as a sulfate group. Also good.

  An oligosaccharide is a sugar in which two or more monosaccharides are linked to one molecule by a glycosidic bond. Examples of the number of monosaccharide molecules constituting the oligosaccharide include 2 to 20, preferably 2 to 10, more preferably 2 to 5, and still more preferably 2 to 3. The type of monosaccharide constituting the oligosaccharide is not particularly limited, and the monosaccharides shown above can be employed. Further, the combination of monosaccharides constituting the oligosaccharide is not particularly limited. Specific examples of oligosaccharides include oligosaccharides having two monosaccharide molecules (eg lactose, Galβ (1 → 3) GalNAc, Galβ (1 → 4) GlcNAc, Galβ (1 → 6) GlcNAc, sucrose , Maltose, trehalose, turanose, cellobiose, etc.), oligosaccharides having a monosaccharide molecule number of 3 (for example, raffinose, melezitose, maltotriose, etc.), oligos having a monosaccharide molecule number of 4 Examples include sugars (for example, acarbose or stachyose), oligosaccharides having 5 or more monosaccharide molecules, and oligosaccharides having 2 monosaccharide molecules are preferable among these, lactose, Galβ (1 → 3) GalNAc, Galβ (1 → 4) GlcNAc, or Galβ (1 → 6) GlcNAc is more preferable, and lactose is more preferable.

  The sugar residue is a divalent group obtained by removing the hydroxyl group from the hydroxyl group at the reducing end of the monosaccharide or oligosaccharide and removing hydrogen from the hydroxyl group at the non-reducing end.

  * The carbon atom indicated by 1 is a carbon atom bonded to the oxygen atom obtained by removing hydrogen from the hydroxyl group at the non-reducing terminal of X, and the oxygen atom indicated by * 2 is an anomeric carbon atom of X. Is an oxygen atom bonded to

  * As the non-reducing terminal bonded with the carbon atom represented by X and from which hydrogen of X is removed, for example, 3-position, 4-position, or 6-position can be mentioned, and 3-position or 4-position is preferred. More preferably.

Specific examples of such sugar residues include
General formula (8a);

[Wherein, R ^7a represents a hydroxyl group or an acetylamino group. ]
A sugar residue represented by the general formula (8b);

[Wherein, R ^7a represents a hydroxyl group or an acetylamino group. ]
And the like. Among these, Preferably the sugar residue represented by general formula (8a) is mentioned.

  In General Formulas (8a) and (8b), any one of (3), (4), and (6) represents a bonding site with the carbon atom indicated by * 1 in General Formula (5). And the other two represent hydrogen. Preferably (3) or (4) represents the bonding site with the carbon atom indicated by * 1 in general formula (5), more preferably (4) is the carbon atom indicated by * 1 in general formula (5). The binding site is shown.

  In the general formulas (8a) and (8b), (* 2) represents a bonding site with the oxygen atom indicated by * 2 in the general formula (5).

In the general formula (5), as a combination of X, Y, R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , and n,
For example, X is a single bond, glucose, galactose, N-acetylglucosamine, or N-acetylgalactosamine, Y is an ethynyl group, R ⁵ and R ⁶ are different from each other, and hydrogen or a hydroxyl group, and R ⁷ is hydrogen. And R ⁸ is an acetyl group or a glycolyl group, R ⁹ is the same or different and is hydrogen or a sulfate group, and n is 1 to 3 in combination.
Preferably, X is a single bond or glucose, Y is an ethynyl group, R ⁵ is a hydroxyl group, R ⁶ is hydrogen, R ⁷ is hydrogen, and R ⁸ is an acetyl group, Examples include a combination in which R ⁹ is hydrogen and n is 1.

Further, in the general formula ^{(5), X, Y,} as another combination of ^{R 5, R 6, R 7} , R 8, R 9, and n is
For example, X is a single bond, a sugar residue represented by the general formula (8a), or a sugar residue represented by the general formula (8b). In the general formula (8a) and the general formula (8b), (3 ) Or (4) is different from hydrogen or a bonding site to the carbon atom indicated by * 1 in the general formula (5), (6) is hydrogen, R ^7a is a hydroxyl group or an acetylamino group, Y Is an ethynyl group, R ⁵ and R ⁶ are different hydrogen or hydroxyl group, R ⁷ is hydrogen, R ⁸ is acetyl group or glycolyl group, and R ⁹ is the same or different, hydrogen or sulfuric acid A group (preferably all hydrogen) and a combination in which n is 1 to 3,
Preferably, X is a single bond or a sugar residue represented by the general formula (8a), and in the general formula (8a), (4) is the bonding site to the carbon atom indicated by * 1 in the general formula (5). (3) and (6) are hydrogen, R ^7a is a hydroxyl group, is an ethynyl group, R ⁵ is a hydroxyl group, R ⁶ is hydrogen, R ⁷ is hydrogen, R ^{A combination in which 8} is an acetyl group, R ⁹ is hydrogen, and n is 1 is mentioned.

  The compound represented by the general formula (5) has a structure in which the second carbon of sialic acid and the third carbon of sugar are linked by a glycosidic bond. When the sugar is galactose, this structure is Neu5Acα (2 → 3) Gal structure. Here, it is known that the avian influenza virus specifically binds to a sialo-sugar chain having a Neu5Acα (2 → 3) Gal structure existing outside the avian cell membrane. Therefore, the compound represented by the general formula (5) having a Neu5Acα (2 → 3) Gal structure or a similar structure is excellent in that it has a property of specifically binding to avian influenza virus.

  The compound represented by the above general formula (5) has a reactive group (ethynyl group, vinyl group, azide group, epoxy group, aldehyde group, or oxylamino group). An alkynyl group is known to form a 1,2,3-triazole ring by a 1,3-dipolar cycloaddition reaction with an azide group. The vinyl group reacts with the thiol group to form a bond. Epoxy groups react with amino groups and thiol groups to form bonds. The aldehyde group reacts with the amino group to form a Schiff base, which is reduced to form a bond. The oxylamino group reacts with a ketone group or an aldehyde group to form an oxime. An azide group is known to form a 1,2,3-triazole ring by a 1,3-dipolar cycloaddition reaction with an alkynyl group. And using these properties, a compound represented by the general formula (5) having a reactive group and a group that reacts with the reactive group (for example, an azide group, an amino group, a thiol group, a hydroxy group, a ketone) Group and an aldehyde group) can be easily linked. Thus, the compound represented by the general formula (5) can be linked to any compound as long as it has a group that reacts with a reactive group.

  Examples of the compound represented by the general formula (5) linked to the organic group include the general formula (7a);

[Wherein R ¹ represents an organic group, and X, Y, R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , and n are the same as defined above]
Or general formula (7b);

[Wherein R ¹ represents an organic group, and X, Y, R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , and n are the same as defined above]
The compound represented by these is mentioned.

  * The oxygen atom indicated by 3 is an oxygen atom bonded to the anomeric carbon atom of X.

The organic group represented by R ¹ is not particularly limited as long as one hydrogen is removed from the organic compound. The organic compound is not particularly limited as long as it has a group that reacts with a reactive group, and examples thereof include a hydrocarbon, a synthetic polymer, and a natural polymer that may have a substituent.

  As the hydrocarbon which may have a substituent, either a straight chain or a branched chain can be mentioned, and for example, it has 1 to 40 carbon atoms, preferably 5 to 30 carbon atoms, more preferably 8 to carbon atoms. 25, more preferably a hydrocarbon which may have a substituent having 10 to 20 carbon atoms. Examples of the hydrocarbon include saturated hydrocarbon, unsaturated hydrocarbon, chain hydrocarbon, cyclic hydrocarbon, aromatic hydrocarbon, heteroaromatic hydrocarbon and the like. Examples of the substituent include an alkyl group, vinyl group, allyl group, aryl group, phenyl group, naphthyl group, araalkyl group, benzyl group, cycloalkyl group, alkoxy group, methoxy group, and ethoxy group.

  Synthetic polymers include polyethylene glycol, polyvinyl chloride, phenolic resin, silicone rubber, silicone oil, nylon, vinylon, polyester, or polyethylene terephthalate viscose fiber, copper ammonia fiber, acetate fiber, promix fiber, nylon fiber, Vinylon fiber, polyvinylidene chloride synthetic fiber, polyvinyl chloride synthetic fiber, polyester synthetic fiber, polyacrylonitrile synthetic fiber, polyethylene synthetic fiber, polypropylene synthetic fiber, polyurethane synthetic fiber, polyclar synthetic fiber, carbon fiber Etc.

  Examples of natural polymers include proteins, nucleic acids, lipids, sugars, cotton, hemp, linen, silk, and hair. Proteins and nucleic acids include those artificially synthesized by known genetic engineering techniques. Examples thereof include a protein obtained by introducing foreign DNA into a host cell and expressing the DNA in the host cell.

Specific examples of the organic group represented by R ¹ include, for example, the general formula (9);

[Wherein q is an integer of 1 to 40]
The group represented by these is mentioned. The group represented by the general formula (9) may be linear or branched.

  q is preferably 5 to 30, more preferably 8 to 25, and still more preferably 10 to 20.

  As a preferable aspect of General formula (9), for example, General formula (9a);

[Wherein r is an integer of 1 to 39]
The group represented by these is mentioned.

  r is preferably 4 to 29, more preferably 7 to 24, and still more preferably 9 to 19.

Another specific example of the organic group represented by R ¹ is, for example, the general formula (10);

Wherein W is the formula (11a);

(Wherein R ⁴ represents a substituent and u is an integer of 1 to 3)
A group represented by formula (11b);

(Wherein R ⁴ and u are the same as above)
Or a group represented by formula (11c);

(Wherein v is an integer of 1 to 8)
S is an integer of 1 to 4, t is an integer of 1 to 30, and R ³ is a substituent. ]
The group represented by these is mentioned.

R ³ is a substituent, and examples thereof include a methyl group, an acetyl group, an ethyl group, an allyl group, a phenyl group, a benzoyl group, a benzyl group, an acyl protecting group, an ether protecting group, and a methacryl group.

R ⁴ is a substituent, and examples thereof include an alkyl group such as a methyl group or an ethyl group, a hydroxy group, a methoxyl group, or an ethoxyl group.

  s is an integer of 1-4, Preferably it is an integer of 2-3.

  t is an integer of 1 to 30, preferably 4 to 20, and more preferably 8 to 16.

  u is an integer of 1 to 3.

  v is an integer of 1 to 8, preferably 1 to 3.

  Taking the compound represented by the general formula (7a) or (7b) as an example, the compound represented by the general formula (5) linked to the organic group is used in various fields according to the kind of the organic group. I can do it. For example, in the biochemical field or the medical field, more specifically in sugar chain vaccines, production of monoclonal antibodies, drug delivery systems, TLC / virus binding assays, intermolecular interaction analysis, air cleaner filters, masks, etc. Can be used.

  Organic group is nylon, _ viscose fiber, copper ammonia fiber, acetate fiber, promix fiber, nylon fiber, vinylon fiber, polyvinylidene chloride synthetic fiber, polyvinyl chloride synthetic fiber, polyester synthetic fiber, polyacrylonitrile series Synthetic fiber, polyethylene-based synthetic fiber, polypropylene-based synthetic fiber, polyurethane-based synthetic fiber, polyclar-based synthetic fiber, carbon fiber, cotton, hemp, linen, silk, or a group in which one hydrogen is removed from the hair, or a general formula ( In the case of a group that is a material of a fibrous material, such as a group represented by 10), for example, a substance having a property of binding to a sialo-sugar chain (for example, a virus such as influenza virus) can be removed. It can be used for an intended air cleaning filter or mask. In addition, when an organic group is a group obtained by removing one hydrogen from a suitable organic compound, an organic group linked with a sialosugar chain is attached to a fiber (specifically, for example, an organic group linked with a sialosugar chain). An aqueous solution containing a group is sprayed onto the fiber and then dried), whereby an air cleaning filter or a mask to which a difference in sialo or the like is attached can be produced.

  When the organic group is a hydrocarbon group as in the compound represented by the general formula (9), for example, by utilizing the improved immunogenicity of the sialo-glycan by adding a hydrocarbon group, It can be used for antibody production.

In the general formulas (7a) and (7b), as a combination of X, R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , n, and R ¹ ,
For example, X is a single bond, glucose, galactose, N-acetylglucosamine, or N-acetylgalactosamine, R ⁵ and R ⁶ are different from each other and are hydrogen or a hydroxyl group, R ⁷ is hydrogen, and R ⁸ is an acetyl group Or a glycolyl group, R ⁹ is the same or different and is hydrogen or a sulfate group, n is 1 to 3, and R ¹ is a hydrocarbon, a synthetic polymer, or a natural polymer. The combination of
Preferably, X is a single bond or glucose, R ⁵ is a hydroxyl group, R ⁶ is hydrogen, R ⁷ is hydrogen, R ⁸ is an acetyl group, and R ⁹ is hydrogen , N is 1, and R ¹ is nylon, viscose fiber, copper ammonia fiber, acetate fiber, promix fiber, nylon fiber, vinylon fiber, polyvinylidene chloride synthetic fiber, polyvinyl chloride synthetic fiber, polyester synthetic Fiber, polyacrylonitrile synthetic fiber, polyethylene synthetic fiber, polypropylene synthetic fiber, polyurethane synthetic fiber, polyclar synthetic fiber, carbon fiber, cotton, hemp, linen, silk, or hair-removed group The combination that is.

2. Method for producing sialo-sugar chain and sialo-sugar chain linked with organic group The sialo-sugar chain of the present invention and the sialo-sugar chain linked with an organic group are represented by the following reaction formulas, for example, step 1, step 2, and step 3 can be manufactured.

[Wherein, X, Y, n, R ¹ , R ⁵ , R ⁶ , R ⁷ , R ⁹ , and R ¹⁰ are the same as above]
Hereinafter, steps 1 to 3 will be described.

2-1. Step 1 ((1) + (2) → (3))
Step 1 is a method for producing a compound represented by the general formula (3), in which the compound represented by the general formula (1) and the compound represented by the general formula (2) are combined in the presence of glycosidase. It is the process made to react by.

  * The hydrogen atom indicated by 4 is a hydrogen atom bonded to the oxygen atom obtained by removing hydrogen from the hydroxyl group at the non-reducing end of X, and the oxygen atom indicated by * 5 is an anomeric carbon atom of X. Is an oxygen atom bonded to

  What can be used as a glycosidase is not specifically limited, Well-known glycosidase is mentioned regardless of an exo type or an endo type, Preferably an exo type glycosidase is mentioned. Examples include cellulase, glucosidase, lactase, amylase, chitinase, sucrase, xylanase, maltase, invertase, or hyaluronidase.

  The glycosidase is selected depending on the type of sugar residue having an anomeric carbon atom to which the oxygen atom represented by * 5 is bonded in the compound represented by the general formula (1). In addition, when X is a monosaccharide residue, an enzyme that recognizes X is selected. As a specific example, in the compound represented by the general formula (1), when the terminal is a glucose residue, the sugar residue having an anomeric carbon atom to which an oxygen atom is bonded as indicated by * 5 is a glucose residue. Therefore, a glycosidase that recognizes a glucose residue, such as cellulase or glucosidase, is preferably selected. As another specific example, when the sugar residue on the reducing end side is a galactose residue, galactosidase is selected, when it is a mannose residue, mannosidase is selected, and when it is a xylose residue, xylanase is selected. Select N-acetylglucosaminidase for N-acetylglucosamine residues, select N-acetylgalactosaminidase for N-acetylgalactosamine residues, and select cellulase for N-acetyllactosamine residues. .

  When X is an oligosaccharide, it is preferable that sugars other than the reducing end side do not contain the sugar so that cleavage by glycosidase does not occur in the middle of the sugar chain.

  The glycosidase is preferably a glycosidase immobilized on a carrier. As a method for immobilizing glycosidase on a carrier, a known method can be employed. The carrier is not particularly limited as long as glycosidase can be immobilized, and examples thereof include a carrier having a carboxyl group capable of amide bonding with an amino group contained in glycosidase. As a specific example, the glycosidase is immobilized on the carrier by amide bonding the activated ester group of Sepharose (carrier) esterified with N-hydroxysuccinimide (NHS) to the amino group of glycosidase. Can do. Other specific examples include an immobilization method in which a carrier is activated with cyanogen bromide and an amide bond is formed with an amino group of glycosidase.

  The reaction is carried out in a suitable solvent. The solvent is not particularly limited as long as it can dissolve the compound represented by the general formula (1) and the compound represented by the general formula (2) and does not deactivate the glycosidase. Examples of such solvents include water, acetate buffer, phosphate buffer, citrate buffer, borate buffer, tartrate buffer, Tris buffer, and / or phosphate buffered saline. Preferably, an acetate buffer is used.

  The reaction temperature is not particularly limited as long as the glycosidase is not inactivated, and is appropriately selected according to the optimum reaction temperature for glycosidase. For example, 5 degreeC-70 degreeC, Preferably it is 30-50 degreeC, More preferably, 34-40 degreeC is mentioned.

  The reaction time is not particularly limited as long as the glycosidase activity can be sufficiently exerted. For example, 1 to 100 hours, preferably 50 to 80 hours, more preferably 65 to 75 hours.

  The reaction pH is not particularly limited as long as glycosidase activity is sufficiently exerted. For example, pH 3 to 10, preferably pH 4 to 6, more preferably pH 4.5 to 5.5 can be mentioned.

  The molar ratio of the compound represented by the general formula (1) and the compound represented by the general formula (2) in the reaction is not particularly limited as long as the reaction by glycosidase occurs, but for example, represented by the general formula (1) The compound represented by the general formula (2) is 0.1 to 100, preferably 1 to 50, more preferably 3 to 30, and still more preferably 5 to 15 mol per 1 mol of the compound.

  The above reaction is preferably performed in the presence of glycosidase immobilized on a carrier. In this case, the compound represented by the general formula (1) and the compound represented by the general formula (2) can be brought into contact with the glycosidase immobilized on the carrier, and the column is placed under an appropriate temperature condition. The reaction may be performed according to

  The amount of glycosidase immobilized on the carrier is 0.1-10 mg, preferably 0.5-3 mg, per 1 mL of carrier.

  After the reaction, a solution containing the compound represented by the general formula (3) obtained by the reaction may be further subjected to a purification step. As a purification means, a known method (separation, distillation, chromatography, recrystallization, etc.) can be employed.

  By the said process 1, the compound represented by General formula (3) can be manufactured in large quantities simply and efficiently.

  The compound represented by the general formula (3) thus obtained can be used in the following step 2.

2-2. Step 2 ((3) + sialic acid donor → (5))
Step 2 is a method for producing a compound represented by the general formula (5), in which the compound represented by the general formula (3) is reacted with a sialic acid donor in the presence of sialidase. . By employing the compound represented by the general formula (3), the compound represented by the general formula (5) can be efficiently produced.

  The sialidase is not particularly limited, and may be a known sialidase regardless of exo-type or endo-type, and preferably an exo-type sialidase. As sialidase, sialidases derived from various organisms can be used. Examples include sialidases derived from organisms belonging to the genus Salmonella, Newcastle disease virus, organisms belonging to the genus Vibrio, organisms belonging to the genus Arthrobacter, or organisms belonging to Clostridium. Among these, from the viewpoint that the compound represented by the general formula (5) can be more efficiently produced, an organism belonging to the genus Salmonella or a sialidase derived from Newcastle disease virus is preferable, and a genus Salmonella is more preferable. Sialidase derived from the organism to which it belongs is mentioned, more preferably sialidase derived from Salmonella typhimurium. A sialidase may be used individually by 1 type and may be used in combination of 2 or more type.

  The sialidase is preferably a sialidase immobilized on a carrier. The method for immobilizing sialidase on a carrier is the same as that for immobilizing glycosidase on the carrier.

  The sialic acid donor is a compound having a sialic acid skeleton, for example, the general formula (4):

[Wherein, R ² is the same or different and represents a substituent, m is an integer of 1 to 3, and R ⁸ and R ⁹ are the same as defined above]. ]
A compound represented by the formula: colominic acid or general formula (12):

[Wherein, R ⁸ and R ⁹ are the same as defined above. ]
The compound represented by these is mentioned.

R ² is the same or different and represents a substituent. The substituent is not particularly limited, and a known substituent can be employed. For example, a nitro group, fluorine, chlorine, bromine, iodine, a hydroxy group, an alkoxy group, or a lower alkyl group (having 1 to 6 carbon atoms) can be mentioned, and a nitro group, fluorine, chlorine, bromine, or iodine is more preferable. .

m represents the number of R ² substituted, and is an integer of 1 to 3, preferably 1.

The substitution position of R ² may be para, ortho, or meta with respect to the oxygen atom bonded to the benzene ring, but is preferably para.

In the general formula (4), the substitution positions of R ² , m and R ² and the combination of R ⁸ and R ⁹ are as follows:
For example, R ² is a nitro group, fluorine, chlorine, bromine, or iodine, m is 1, the substitution position of R ² is para to the oxygen atom bonded to the benzene ring, and R ⁸ is A combination of acetyl group or glycolyl group, and R ⁹ is the same or different and is hydrogen or sulfate group,
Preferably, R ² is a nitro group, m is 1, the substitution position of R ² is para to the oxygen atom bonded to the benzene ring, R ⁸ is an acetyl group, and R ⁹ is A combination of hydrogen is mentioned.

In the general formula (12), as a combination of R ⁸ and R ⁹ ,
For example, a combination in which R ⁸ is an acetyl group or a glycolyl group, and R ⁹ is the same or different and is hydrogen or a sulfate group is exemplified.
Preferably, R ⁸ is acetyl group, and a combination of R ⁹ is hydrogen.

  Colominic acid is a polymer compound obtained by polymerizing sialic acid or a salt thereof, and is not particularly limited. As sialic acid, substances in which the amino group or hydroxy group of neuraminic acid is substituted can be widely used. Specific examples of sialic acid include N-acetylneuraminic acid and N-glycolylneuraminic acid. The sialic acid salt is not particularly limited, and examples thereof include sodium sialic acid and potassium sialic acid. Specific examples of colominic acid include:

[Wherein w is 50 to 100]
Examples thereof include sodium colominate.

  The sialic acid donor is preferably a compound represented by the general formula (4) or colominic acid. A sialic acid donor may be used individually by 1 type, and may be used in combination of 2 or more type.

  The reaction is carried out in a suitable solvent. The solvent is not particularly limited as long as it can dissolve the compound represented by the general formula (3) and the sialic acid donor and can exhibit the sialic acid introduction activity of sialidase. The solvent can be appropriately selected according to the desired reaction pH. Examples of the solvent include water, acetate buffer, phosphate buffer, citrate buffer, borate buffer, tartrate buffer, Tris buffer, and / or phosphate buffered saline. Tris buffer may be mentioned.

  The reaction temperature is not particularly limited as long as the sialic acid introduction activity of sialidase is exhibited, and is appropriately selected, and examples thereof include 5 to 60 ° C. From a viewpoint that the compound represented by General formula (5) can be manufactured more efficiently, Preferably it is 20-40 degreeC, More preferably, it is 25-35 degreeC, More preferably, 27-33 degreeC is mentioned.

  The reaction time is not particularly limited as long as the sialic acid introduction activity of sialidase can be exhibited. For example, 1 to 48 hours, preferably 12 to 36 hours, more preferably 20 to 28 hours.

  The reaction pH is not particularly limited as long as the sialic acid introduction activity of sialidase is exhibited, and examples thereof include pH 3 to 10. From the viewpoint that the compound represented by the general formula (5) can be more efficiently produced, the pH is preferably 6 to 9, more preferably pH 7.5 to 8.5, and still more preferably 7.8 to 8.2. It is done.

  The molar ratio of the compound represented by the general formula (3) and the sialic acid donor in the reaction is not particularly limited as long as the sialic acid introduction reaction by sialidase occurs. For example, the molar ratio is represented by the general formula (3). The sialic acid residue (group obtained by removing two hydroxyl groups from sialic acid) contained in the sialic acid donor is 0.001 mol to 3 mol with respect to 1 mol of the compound. The number of moles of the sialic acid residue (group obtained by removing two hydroxyl groups from sialic acid) contained in the sialic acid donor is generally 1 mole per mole of the compound represented by the general formula (3). From the viewpoint that the compound represented by the formula (5) can be produced more efficiently, it is preferably 0.001 mol to 0.5 mol, more preferably 0.05 to 0.4 mol, more preferably 0.01. -0.3 mol, More preferably, it is 0.01-0.2 mol, More preferably, it is 0.01-0.1 mol, Most preferably, 0.01-0.05 mol is mentioned.

  The above reaction is preferably performed in the presence of sialidase immobilized on a carrier. In this case, the reaction is carried out by bringing the compound represented by the general formula (3) and the sialic acid donor into contact with the sialidase immobilized on the carrier and placing the column under an appropriate temperature condition. Good. This reaction may be a batch process or a continuous process.

  By carrying out the reaction in the presence of sialidase immobilized on a carrier, sialidase can be easily separated by removing the carrier by centrifugation or the like after completion of the reaction, and the stability of sialidase is improved. The sialidase immobilized on the carrier can be reused. Thus, by using sialidase immobilized on a carrier, the production of the compound represented by the general formula (5) can be carried out more simply and efficiently at a low cost.

  The amount of sialidase immobilized on the carrier is preferably 0.01 mg to 10 mg with respect to 1 mL of the carrier, and 0.1 to 3 mg is preferable.

  After the reaction, a solution containing the compound represented by the general formula (5) obtained by the reaction may be further subjected to a purification step. As a purification means, a known method (separation, distillation, chromatography, recrystallization, etc.) can be employed.

  As in the above step 2, in the condensation reaction between a saccharide and a compound having a sialic acid skeleton, the compound represented by the general formula (3) is employed as the saccharide, and the reaction is carried out in the presence of sialidase. In addition, a large amount of the compound represented by the general formula (5) can be produced.

2-3. Step 3 ((5) + (6) → (7a) and (7b))
Step 3 is an example of a method for producing the compounds represented by the general formulas (7a) and (7b), which is an example of the compound represented by the general formula (5) linked to an organic group. In this step, the compound represented by 5) is reacted with the compound represented by the general formula (6). The compound represented by the general formula (5) linked to an organic group can be produced according to Step 3 and a known method. * The oxygen atom indicated by 3 is an oxygen atom bonded to the anomeric carbon atom of X.

  The compound represented by the general formula (6) can be produced by a known method.

  The reaction is a 1,3-dipole addition reaction. The reaction can be performed in the presence or absence of a solvent, but when performed in the presence of a solvent, the solvent is not particularly limited as long as it does not adversely affect the reaction. For example, distilled water, methanol, tetrahydrofuran, dioxane, and / or dimethyl sulfoxide can be used. The reaction is preferably carried out in the presence of a suitable catalyst. An example of the catalyst is a copper (I) catalyst. In the case of using a copper (I) catalyst, for example, a method in which divalent copper such as copper sulfate and a reducing agent (for example, hydroquinone or sodium ascorbate) are introduced into the system and monovalent copper is reacted. It is done.

  Although reaction temperature will not be specifically limited if it is below the boiling point of a solvent, For example, 15-80 degreeC, Preferably it is 20-40 degreeC, More preferably, 27-33 degreeC is mentioned.

  Although reaction time is not specifically limited, For example, 1 to 24 hours, Preferably it is 6 to 20 hours, More preferably, 10 to 14 hours is mentioned.

  The molar ratio of the compound represented by the general formula (5) and the compound represented by the general formula (6) in the reaction is not particularly limited. For example, 1 mol of the compound represented by the general formula (5) In contrast, 1 to 5 mol, preferably 1 to 3 mol, more preferably 1.25 to 1.75 mol of the compound represented by the general formula (6) may be mentioned.

  After the reaction, a solution containing the compound represented by the general formula (5) obtained by the reaction may be further subjected to a purification step. As a purification means, a known method (separation, distillation, chromatography, recrystallization, etc.) can be employed.

3. Production device The present invention comprises a device 1 (a device comprising a column containing glycosidase (hereinafter referred to as column A) having a supply port and a discharge port, which is immobilized on a carrier), and a device 2 (supply port) And a device comprising a column containing sialidase immobilized on a carrier (hereinafter referred to as column C) having a discharge port, and a device 3 (equipped with the device 1 and the device 2, and the discharge of the device 1). An apparatus is provided in which the outlet and the supply port of the apparatus 2 are connected by a flow path c). This will be specifically described below.

3-1. Device 1
The apparatus 1 is an apparatus characterized by including a column (column A) containing glycosidase immobilized on a carrier and having a supply port and a discharge port as an essential component. Therefore, the apparatus 1 is suitable for the production of the compound represented by the general formula (3) according to Step 1 described above, in which the reaction is performed in the presence of glycosidase. That is, the apparatus 1 is preferably an apparatus for producing a compound represented by the general formula (3).

  On the supply port side of the column A, a flow path switching device a ′, for example, a three-way cock or an injector may be provided.

  Furthermore, the apparatus 1 preferably includes a reverse phase column (hereinafter referred to as column B) having a supply port and a discharge port. Further, the discharge port of the column A and the supply port of the column B are preferably connected by a flow path b, and the flow path b more preferably includes a flow path switching device b '. The device 1 including the column B and the flow path switching device b 'is shown as a device 1a (FIG. 1).

  The flow path switching device b ′ is configured so that the flow path in the flow path b includes the column A side (“1” side in FIG. 1), the column B side (“2” side in FIG. 1), and the outside of the apparatus 1a (FIG. 1). Middle, "3" side) function that can be switched in three directions, ie, the function that closes the flow path on the 3 side and connects the flow path on the 1 side and the flow path on the 2 side, and closes the flow path on the 2 side and closes the 1 side And a function of connecting the three-side flow path, and a function of closing the one-side flow path and connecting the two-side flow path and the three-side flow path.

  In addition to the channel switching device b ′, various detectors such as an absorbance detector, an optical rotation detector, or a fluorescence detector may be provided on the outlet side of the channel b and the column B. .

  A step of introducing the compound represented by the general formula (1) and the compound represented by the general formula (2) into the column A from the supply port of the column A and causing the reaction in the column A using the apparatus 1 (hereinafter referred to as “a”). By performing step a), the compound represented by the general formula (3) can be produced. In this case, in column A, the compound represented by general formula (1) and the compound represented by general formula (2) react in the presence of glycosidase contained in column A and immobilized on a carrier. Thereby, the above-mentioned process 1 is performed. Accordingly, conditions such as the reaction temperature in step a can be set in the same manner as in step 1 above. The reaction product in the column A, that is, the reaction product containing the compound represented by the general formula (3) is eluted from the column A by eluting with a suitable solvent, for example, the solvent in which the reaction in the step (a) is performed. Can be eluted.

  Furthermore, it is preferable to purify the reaction product containing the compound represented by the general formula (3) by the step a by a column (for example, column B) connected to the outlet side of the column A. For example, following step a, the step of introducing the reactant in column A into the supply port of column B (hereinafter referred to as step b) and the compound represented by general formula (3) from column B are purified. It is preferable to carry out purification by carrying out a step (hereinafter referred to as step c).

  The method for introducing the reactant in the column A into the supply port of the column B is not particularly limited, and a known method can be adopted. For example, by supplying a solvent (preferably, the solvent in which the reaction of step (a) was performed) is supplied from the supply port of column A, the reaction solution of step (a) existing in column A can be obtained. And a method of extruding from the outlet of the column A and introducing it into the supply port of the column B connected to the outlet of the column A via the flow path b.

  The method for purifying the compound represented by the general formula (3) from the column B is not particularly limited, and according to a known method, the necessary types can be purified by changing the type and mixing ratio of the solvent. it can. For example, the solvent is present in the column B by passing the solvent into the column B from the supply port of the column A or from the supply port of the column B, preferably from the outside of the device 1 through the flow switching device b ′. The compound represented by the general formula (3) is eluted and the fraction containing the compound is fractionated. The solvent used in this case is not particularly limited as long as the compound represented by the general formula (3) can be eluted from the column B. Examples thereof include water, acetonitrile, methanol, ethanol, and / or tetrahydrofuran. It is done.

  At this time, in the column B, there are mainly the unreacted compound represented by the general formula (1) and the compound represented by the general formula (3). When water is used as the solvent, the unreacted compound represented by the general formula (1) is eluted first, and then the compound represented by the general formula (3) is eluted.

  The compound represented by the general formula (3) obtained using the apparatus 1 may be further subjected to a known purification means (chromatography, etc.) and a drying step (lyophilization, etc.).

  As described above, by using the apparatus 1, an excellent effect that the compound represented by the general formula (3) can be easily and efficiently produced in large quantities is exhibited. Specifically, by using a column (column A) containing glycosidase immobilized on a carrier, the enzyme activity of glycosidase is less likely to decrease, and the enzyme can be reused, and column A is used. This is excellent in that it is not necessary to remove the enzyme after the reaction. Further, when column B is employed, the compound represented by the general formula (1) obtained by the purification by the column B and the point that the purification after the reaction can be performed very easily are again represented by the general formula (3 It is excellent in that it can be reused in the production of the compound represented by

3-2. Device 2
The apparatus 2 is an apparatus characterized by including a column (column C) containing sialidase immobilized on a carrier and having a supply port and a discharge port as an essential component. Therefore, the apparatus 2 is suitable for the production of the compound represented by the general formula (5) according to the above-mentioned step 2 in which the reaction is performed in the presence of sialidase. That is, the device 2 is preferably a device for producing a compound represented by the general formula (5).

  On the supply port side of the column C, a flow path switching device c ′, for example, a three-way cock or an injector may be provided.

  The apparatus 2 includes a reverse-phase column (hereinafter referred to as column D) having a supply port and a discharge port, and / or an ion exchange column (hereinafter referred to as column E) having a supply port and a discharge port. preferable. Further, the discharge port of the column C and the supply port of the column D are preferably connected by a flow channel d, and the flow channel d more preferably includes a flow channel switching device d '. Furthermore, the discharge port of the column D and the supply port of the column E are preferably connected by a flow channel e, and the flow channel e more preferably includes a flow channel switching device e ′. The apparatus 2 including the column D, the column E, the flow path switching device d ', and the flow path switching device e' is referred to as a device 2a (FIG. 2).

  The flow path switching device d ′ is configured such that the flow path in the flow path d is the column C side (“4” side in FIG. 2), the column D side (“5” side in FIG. 2), and the outside of the apparatus 2a (FIG. 2). Middle, "6" side) function that can be switched to three directions, that is, the function that closes the 6-side flow path and connects the 4-side flow path to the 5-side flow path, and closes the 5-side flow path to the 4-side And the function of connecting the 6-side channel and the function of connecting the 5-side channel and the 6-side channel.

  The flow path switching device e ′ includes a flow path in the flow path e on the column D side (“7” side in FIG. 2), the column E side (“8” side in FIG. 2), and the outside of the device 2a (FIG. 2). Middle, “9” side), a function that can be switched to three directions, that is, a function that closes the 9 side flow path and connects the 7 side flow path to the 8 side flow path, and closes the 8 side flow path to the 7 side. This function has a function of connecting the 9th flow path and the 9th flow path, and a function of closing the 7th flow path and connecting the 8th flow path and the 9th flow path.

  On the supply port side of the column C, a flow path switching device c, for example, an injector may be provided. Various detectors such as an absorbance detector, an optical rotation detector, or a fluorescence detector may be provided on the outlet side of the channel d, the channel e, and the column E.

  A step of introducing the compound represented by the general formula (3) and the sialic acid donor into the column C from the supply port of the column C and reacting in the column C using the apparatus 2 (hereinafter referred to as step d). ), The compound represented by the general formula (5) can be produced. In this case, in the column C, the compound represented by the general formula (3) and the sialic acid donor are reacted in the presence of sialidase contained in the column C and immobilized on the carrier, whereby the above-mentioned step 2 Is done. Accordingly, conditions such as the reaction temperature in step d can be set in the same manner as in step 1 above. The reaction product in the column C, that is, the reaction product containing the compound represented by the general formula (5) is eluted from the column C by eluting with an appropriate solvent, for example, the solvent in which the reaction in the step (d) was performed. Can be eluted.

  Furthermore, it is preferable to purify the reaction product containing the compound represented by the general formula (5) in step d by a column (for example, column D or column E) connected to the outlet side of column A. For example, following the step d, a step of introducing the reactant in the column C into the supply port of the column D (hereinafter referred to as step e), a step of introducing the contents in the column D into the supply port of the column E ( Hereinafter, it is preferable to carry out the purification by carrying out a step (hereinafter referred to as step g) and a step of purifying the compound represented by the general formula (5) from the column E (hereinafter referred to as step g).

  The method for introducing the reactant in the column C into the supply port of the column D is not particularly limited, and a known method can be adopted. For example, by supplying a solvent (preferably exemplified by the solvent in which the reaction of step (d) was performed) from the supply port of the column C, the reaction solution according to the step (d) existing in the column C can be obtained. There is a method of extruding from the outlet of the column C and introducing it into the supply port of the column D connected to the outlet of the column C via the flow path d.

  The method for introducing the contents in the column D into the supply port of the column E is not particularly limited, and a known method can be adopted. For example, by supplying a solvent (for example, water, acetonitrile, methanol, ethanol, and / or tetrahydrofuran, from the supply port of the column D, the contents of the step (d) existing in the column D are removed from the column D. There is a method of elution and introduction to the supply port of the column E connected to the discharge port of the column D through the flow path e. By purifying using the column D, unreacted general formula (3) And the compound represented by the general formula (5) can be separated from the paranitrophenol produced when the compound represented by the general formula (4) is used (paranitrophenol is a column). D).

  The method of purifying the compound represented by the general formula (5) from the column E is not particularly limited, and according to a known method, the necessary types can be purified by changing the type and mixing ratio of the solvent. it can. A known method can be employed. For example, by passing the solvent into the column E from the supply port of the column C, from the supply port of the column D, or from the supply port of the column B, preferably from the outside of the device 2 through the flow switching device e ′. The compound represented by the general formula (E) present in the column E is eluted, and the fraction containing the compound is fractionated. The solvent used in this case is not particularly limited as long as the compound represented by the general formula (5) can be eluted from column E. For example, a sodium chloride aqueous solution, a potassium chloride aqueous solution, and / or a calcium chloride aqueous solution, etc. Is mentioned. When these solvents are used, the salt concentration is preferably set to about 0.5 to 2M, for example.

Preferably, the compound represented by the general formula (3) in the column E is eluted by introducing water into the column E, and then the compound represented by the general formula (5) is eluted with the solvent. The compound may be purified. The obtained compound represented by the general formula (3) is represented by the general formula (5) obtained using the apparatus 1 that can be reused for the production of the compound represented by the general formula (5). Further, the compound may be subjected to known purification means (chromatography and the like) and drying step (lyophilization and the like).

  As described above, by using the apparatus 2, an excellent effect that the compound represented by the general formula (5) can be easily and efficiently produced in large quantities is exhibited. Specifically, by using a column containing sialidase immobilized on a carrier (column C), the enzyme activity of sialidase is less likely to decrease, and the enzyme can be reused, and column C is used. This is excellent in that it is not necessary to remove the enzyme after the reaction. Furthermore, when column D and / or column E are employed, the compound represented by the general formula (3) obtained by the purification by column E and the point that purification after the reaction can be performed very easily are performed again. This is excellent in that it can be reused in the production of the compound represented by the general formula (3).

3-3. Device 3
As described above, the device 3 includes the device 1 and the device 2, and the discharge port of the device 1 and the supply port of the device 2 are connected by the flow path c (hereinafter referred to as the device 3). ). Therefore, the device 3 is suitable for the production of the compound represented by the general formula (5). That is, the device 3 is preferably a device for producing a compound represented by the general formula (5).

  The discharge port of the device 1 means a column provided at the most discharge port side of the device 1 or the discharge port of the device. For example, when the device 1 is composed only of the column A, the discharge port of the device 1 is the discharge port of the column A. When the device 1 is an outlet and the device 1a includes the column A, the column B, and the flow path switching device b ′, the outlet of the device 1 means the outlet of the column B.

  The supply port of the device 2 means a column provided on the most supply port side of the device 2, that is, a supply port of the column C.

  The channel c preferably includes a channel switching device c ', for example, a three-way cock.

  The device 3 preferably includes the device 1a and the device 2a.

  The device 3 including the device 1a, the device 2a, and the flow path switching device c 'is referred to as a device 3a (FIG. 3).

  The flow path switching device c ′ includes a flow path in the flow path c on the column B side (“10” side in FIG. 3), the column C side (“11” side in FIG. 3), and the apparatus 3a outside (FIG. 3). (12) side), that is, the function of closing the 12-side flow path to connect the 10-side flow path and the 11-side flow path, and closing the 11-side flow path to the 10-side And the 12 side channel and the 10 side channel are closed and the 11 side channel and the 12 side channel are connected.

  Various detectors such as an absorbance detector, an optical rotation detector, or a fluorescence detector may be provided in the channel c.

  By using the apparatus 1 as described in “3-1. Apparatus 1”, a compound represented by the general formula (3) is obtained, and the apparatus as described in “3-2. Apparatus 2” is obtained. By using 2, the compound represented by the general formula (5) can be produced. For example, the compound represented by the general formula (5) can be produced by performing the steps (a) to (g) using the device 3a included in the device 3.

  Thus, by using the apparatus 3, it was possible to produce a large amount of the compound represented by the general formula (5) more simply and efficiently than using the apparatus 1 and the apparatus 2 alone. An effect is produced.

  EXAMPLES The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

Example 1. Preparation of Column Containing Cellulase Immobilized on Carrier Cellulase (manufactured by Wako Co., Ltd.) was prepared on the carrier in HiTrap NHS-activated HP column (GE Healthcare Biosciences) by the following steps (1) to (10). (Product name: Cellulase, derived from Aspergillus niger) was immobilized.

  (1) Ice-cooled 1 mM HCl (5 mL) was passed through the column using a syringe pump at a flow rate of 0.5 ml / min.

  (2) A 1.8 U (unit) cellulase solution [50 mM sodium acetate buffer, pH = 5 (1 mL) dissolved in cellulase] was passed through the column at a flow rate of 0.5 ml / min using a syringe pump. The column was then left at 4 ° C. for 12 hours.

  (3) 0.2 M sodium hydrogen carbonate solution, containing 0.5 M NaCl, pH = 8.3 (3 mL) It was passed through the column at a flow rate of 0.5 ml / min using a syringe pump.

  (4) 0.5 M monoethanolamine solution, containing 0.5 M NaCl, pH = 8.3 (6 mL) It was passed through the column at a flow rate of 1 ml / min using a syringe pump.

  (5) 0.1 M sodium acetate buffer, containing 0.5 M NaCl, pH = 4 (6 mL) It was passed through the column at a flow rate of 1 ml / min using a syringe pump.

  (6) 0.5 M monoethanolamine solution, containing 0.5 M NaCl, pH = 8.3 (6 mL) It was passed through the column at a flow rate of 1 ml / min using a syringe pump. The column was then left at 4 ° C. for 4 hours.

  (7) 0.1 M sodium acetate buffer, containing 0.5 M NaCl, pH = 4 (6 mL) It was passed through the column at a flow rate of 1 ml / min using a syringe pump.

  (8) 0.5 M monoethanolamine solution, containing 0.5 M NaCl, pH = 8.3 (6 mL) It was passed through the column at a flow rate of 1 ml / min using a syringe pump.

  (9) 0.1 M sodium acetate buffer, containing 0.5 M NaCl, pH = 4 (6 mL) It was passed through the column at a flow rate of 1 ml / min using a syringe pump.

  (10) 50 mM sodium acetate buffer, pH = 5 (2 mL) was passed through the column using a syringe pump at a flow rate of 1 ml / min.

  The column obtained in the above steps (1) to (10) is referred to as “a column containing cellulase immobilized on a carrier” as “Example 3. Production of propargyl group-introduced lactose” and “Example 8. Reaction. It was used in “Production of sex group-introducing sugar production apparatus 1”.

Example 2 Preparation of Column Containing Sialidase Immobilized on Carrier The sialidase (New England Biolabs) was prepared on the carrier in the HiTrap NHS-activated HP column (GE Healthcare Biosciences) by the following steps (11) to (20). (Salmonella typhimurium) was immobilized.

  (11) Ice-cooled 1 mM HCl (5 mL) was passed through the column at a flow rate of 0.5 ml / min using a syringe pump.

  (12) 1 U (unit) of sialidase solution (commercially prepared) (1 mL) was passed through the column using a syringe pump at a flow rate of 0.5 ml / min. The column was then left at 4 ° C. for 12 hours.

  (13) 0.2 M sodium hydrogen carbonate solution, containing 0.5 M NaCl, pH = 8.3 (3 mL) It was passed through the column at a flow rate of 0.5 ml / min using a syringe pump.

  (14) 0.5 M monoethanolamine solution, containing 0.5 M NaCl, pH = 8.3 (6 mL) It was passed through the column at a flow rate of 1 ml / min using a syringe pump.

  (15) 0.1 M sodium acetate buffer, 0.5 M NaCl contained, pH = 4 (6 mL) It was passed through the column at a flow rate of 1 ml / min using a syringe pump.

  (16) 0.5 M monoethanolamine solution, containing 0.5 M NaCl, pH = 8.3 (6 mL) It was passed through the column at a flow rate of 1 ml / min using a syringe pump. The column was then left at 4 ° C. for 4 hours.

  (17) 0.1 M sodium acetate buffer, containing 0.5 M NaCl, pH = 4 (6 mL) It was passed through the column at a flow rate of 1 ml / min using a syringe pump.

  (18) 0.5 M monoethanolamine solution, containing 0.5 M NaCl, pH = 8.3 (6 mL) It was passed through the column at a flow rate of 1 ml / min using a syringe pump.

  (19) 0.1 M sodium acetate buffer, containing 0.5 M NaCl, pH = 4 (6 mL) The column was passed through the column at a flow rate of 1 ml / min using a syringe pump.

  (20) 100 mM sodium acetate buffer, pH = 5.5 (2 mL) was passed through the column at a flow rate of 1 ml / min using a syringe pump.

  The column obtained in the above steps (11) to (20) is referred to as “a column containing sialidase immobilized on a carrier” as “Example 5. Production of propargyl group-introduced sialyl lactose” (column) and “Implementation”. Example 9 “Production of reactive group-introduced sialosugar production apparatus 2”.

Example 3 FIG. Production of Propargyl Group-Introduced Lactose Propargyl group-introduced lactose (Compound 3) was produced according to the following reaction formula. Details will be described below.

  A mixed solution of lactose (compound 1) (1 M, 500 μL), propargyl alcohol (compound 2) (250 μL), and sodium acetate buffer (50 mM, pH = 5.0, 250 μL) was prepared in Example 1. The column was passed through a column containing cellulase immobilized on a carrier.

  Both ends of the column were capped, and the column was placed in a thermostatic bath heated to 37 ° C. and incubated for 70 hours.

  Next, sodium acetate buffer (50 mM, pH = 5.0) (2 mL) was passed through the column, and the reaction solution was allowed to flow out of the column.

  The reaction solution was applied to a reverse phase column (BONDESIL-C18, L = 6 cm) and eluted with water to obtain Compound 3 (49.5 mg). NMR data is shown below.

¹ H-NMR (D ₂ O) δ4.96 (d, 1 H, J = 3.6 Hz), 4.54 (d, 1 H, J = 8.4 Hz), 4.34 (s, 2 H), 4.32 (d, 1 H, J = 8.0 Hz), 3.86 (d, 1 H, J = 11.6 Hz), 3.69-3.52 (m, 4 H), 3.42 (dd, 1 H, J = 8.4, J = 9.2 Hz), 3.22 (br-t, 1 H), 2.78 (s, 1 H).

  In the above method, various reactive groups are introduced by using a compound in which the ethynyl group of compound 2 is substituted with another reactive group (for example, vinyl group or azide group) instead of compound 2. Lactose can be made.

Example 4 Production of Propargyl Group-Introduced Sialyl (2 → 3) Lactose According to the following reaction formula, propargyl group-introduced sialyl (2 → 3) lactose (Compound 5) was produced. Details will be described below.

  Compound 3, paranitrophenyl sialic acid (compound 4) prepared according to Volker Eschenfelder et al. (Cabohydr. Res. 162, 294-297, 1987), and sialidase (0.25 U) from Salmonella typhimurium (New England Biolabs) A reaction solution was prepared by dissolving in sodium acetate buffer (100 mM sodium acetate, pH = 5.5), and incubated at 30 ° C. for 20 hours. The amounts of Compound 3 and Compound 4 dissolved in the reaction solution are as follows.

  The reaction solution after the incubation was analyzed by NMR. As a result, a compound (propargyl group-introduced sialyl (2) having a carbon atom at the 2-position of sialic acid and a carbon atom at the 3-position of propargyl group-introduced lactose (compound 3) dissolved through an oxygen atom) was obtained. → 3) Lactose (compound 5)) was produced. In addition, the compound which sialic acid couple | bonded with carbons other than the 3rd-position of propargyl group introduction | transduction lactose (compound 3) was not produced | generated. NMR data is shown below.

¹ H-NMR (D ₂ O) δ 5.03 (d, 1 H, J 3.6 Hz), 4.48 (d, 1 H, J 8.4 Hz), 4.34 (d, 1 H,
J 7.6 Hz), 3.93 (m), 3.78−3.36 (m), 3.11 (t, 1 H, J 9.2 Hz), 2.80 (br-d, 1 H), 2.57 (dd, 1 H, J 4.4, J 12.0 Hz), 1.83 (s, 3 H), 1.63 (t, 1 H, J 12.0 Hz).

The reaction mixture after incubation was analyzed by HPLC (column: YMC-Pack Polyamin II (φ4.6 × 250 mm), column temperature: 40 ° C, flow rate: 1.0 ml / min, detection wavelength: 210 nm, elution solvent: 65 % MeCN-20 mM KH ₂ PO ₄ aqueous solution) and propargyl group-introduced sialyl (2 → 3) lactose (compound 5) peak area was measured. Based on the measured amount, the yield of Compound 5 was determined according to the following formula. As a result, the yield was 40% in Example 4-1, 63% in Example 4-2, 78% in Example 4-3, and 90% in Example 4-4.

Example 5 FIG. Production of propargyl group-introduced sialyl lactose (column)
Propargyl group-introduced sialyl lactose (Compound 5) was produced according to the reaction formula described in Example 4. Details will be described below.

  Compound 3 (223.5 nmol) and paranitrophenylsialic acid (compound 4) (6.9 nmol) prepared according to Volker Eschenfelder et al. (Cabohydr. Res. 162, 294-297, 1987) were mixed with Tris-HCl buffer (100 mM). A reaction solution (540 μL) obtained by dissolving in Tris-HCl, pH = 8.0) was passed through a column immobilized with sialidase.

  Both ends of the column were capped, and the column was placed in a constant temperature bath heated to 30 ° C. and incubated for 24 hours.

  Subsequently, 100 mM sodium acetate buffer, pH = 5.5 (2 mL) was passed through the column, and the reaction solution was allowed to flow out of the column.

  The reaction solution was applied to a reverse phase cartridge column Sep pack C18, and unreacted compound 3, sialic acid, and compound 5 were obtained as a mixture in the water fraction (4 mL).

  Then, the water fraction was applied to an ion exchange cartridge column (Acrosep Q Ceramic Hyper DF), and compound 3 was recovered in the water fraction (5 mL), and sialic acid and the target were collected in the 2 M NaCl fraction (1 mL). Compound 5 was obtained as a mixture. In addition, the compound which sialic acid couple | bonded with carbons other than the 3rd-position of propargyl group introduction | transduction lactose (compound 3) was not produced | generated. NMR data is shown below.

¹ H-NMR (D ₂ O) δ 5.03 (d, 1 H, J 3.6 Hz), 4.48 (d, 1 H, J 8.4 Hz), 4.34 (d, 1 H,
J 7.6 Hz), 3.93 (m), 3.78−3.36 (m), 3.11 (t, 1 H, J 9.2 Hz), 2.80 (br-d, 1 H), 2.57 (dd, 1 H, J 4.4, J 12.0 Hz), 1.83 (s, 3 H), 1.63 (t, 1 H, J 12.0 Hz).

  Further, the yield of compound 5 was determined in the same manner as in Example 4. As a result, the yield was 92%.

Example 6 Production of propargyl group-introduced sialyl (2 → 3) lactose into which polyethylene glycol has been introduced Propargyl group-introduced sialyl (2 → 3) lactose into which polyethylene glycol has been introduced (compound 7a) is produced according to the following reaction formula. Details will be described below.

  According to KB Sharpless et al. (Angew. Chem. Int. Ed. 41, 2596-2599, 2002), an aqueous solution of compound 5 (50 μL) was added to M. Yamaguchi et al. (Tetrahedron Lett. 47 , 7455-7458, 2006), compound 6a (30 μL; 90 μM in DMSO), 10 mM aqueous copper sulfate solution (50 μL), and 10 mM aqueous sodium ascorbate solution (50 μL) were added in that order at 30 ° C. Incubate for 12 hours.

  The reaction solution was concentrated under reduced pressure, the residue was dissolved in distilled water, applied to a reverse phase cartridge column Sep pack C18, and sialic acid and unreacted compound 5 were eluted and removed using water (2 mL). Using a mixed solvent (2 mL) of water: methanol = 1: 1 as an elution solvent, an eluate containing the target compound 7a is obtained, and this is concentrated under reduced pressure to obtain the target compound 7a.

Production Example 1 Production of lipid having azide group A lipid having azide group (compound 6b) was produced according to the following reaction formula. Details will be described below.

  l-Tetradecanol (1 g, 4.66 mmol) was dissolved in pyridine (3 mL). P-Toluenesulfonyl chloride (1.3 g, 6.82 mmol) was added thereto, and the reaction was carried out with stirring at room temperature for 4 hours.

  Thereafter, the reaction solution was extracted with chloroform, washed with a 2 M aqueous hydrochloric acid solution, dried by adding magnesium sulfate, and concentrated under reduced pressure.

  The obtained syrup was subjected to silica gel column chromatography and purified with ethyl acetate: hexane = 1: 10 as an elution solvent to obtain paratoluenesulfonyltetradecanol (1.7 g, yield 99%).

  Next, the obtained paratoluenesulfonyltetradecanol (1.7 g, 4.61 mmol) was dissolved in N, N-dimethylformamide (3 mL).

  Sodium azide (1.4 g, 21.5 mmol) was added thereto at room temperature, and the reaction was carried out with stirring at 40 ° C. for 24 hours.

  Thereafter, the reaction solution was extracted with chloroform, washed with distilled water, dried by adding magnesium sulfate, and concentrated under reduced pressure.

  The obtained syrup was subjected to silica gel column chromatography, and purified with ethyl acetate: hexane = 1: 10 as an elution solvent to obtain Compound 6b (1.1 g) quantitatively. NMR data is shown below.

¹ H-NMR (CDCl ₃ ) δ 3.25 (t), 1.63-1.56 (m), 1.37-1.26 (m), 0.88 (t).

Example 7 Production of Propargyl Group-Introduced Sialyl (2 → 3) Lactose Introduced With Lipid Having Azide Group Propargyl group-introduced sialyl lactose (compound 7b) into which a lipid having azide group is introduced is produced according to the following reaction formula. Details will be described below.

  According to KB Sharpless et al. (Angew. Chem .. Int. Ed. 41, 2596-2599, 2002), Compound 5b (30 μL; 90 μM in DMSO) was added to Compound 5 aqueous solution (50 μL) under a nitrogen stream, Add 10 mM aqueous copper sulfate solution (50 μL) and 10 mM aqueous sodium ascorbate solution (50 μL) in this order, and incubate at 30 ° C. for 12 hours.

  The reaction solution was concentrated under reduced pressure, the residue was dissolved in distilled water, applied to a reverse phase cartridge column Sep pack C18, and sialic acid and unreacted compound 5 were eluted and removed using water (2 mL). Using a mixed solvent (2 mL) of water: methanol = 1: 1 as an elution solvent, an eluate containing the target compound 7b is obtained, and this is concentrated under reduced pressure to obtain the target compound 7b.

Example 8 FIG. General formula (3) represented by compound 3 produced in Production Example 3 of the reactive group-introducing sugar production apparatus 1 ;

[Wherein, X, Y, R ⁵ , R ⁶ , R ⁷ and n are the same as above]
The apparatus suitable for manufacture of the compound represented by these was manufactured. Details are given below with reference to FIG.

  The three-way cock a on the supply port side (upstream side) and the three-way cock b on the discharge port side (downstream side) of the column containing cellulase immobilized on the carrier (cellulase-immobilized column A) manufactured in Example 1 Attached.

  Further, a reverse phase column (BONDESIL-C18, L = 6 cm) (reverse phase column B) was attached to the downstream side of the three-way cock b.

Example 9 General formula (5) represented by compound 5 produced in Production Examples 4 and 5 of the reactive group-introduced sialosugar production apparatus 2 ;

[Wherein X, Y, R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , and n are the same as above]
The apparatus suitable for manufacture of the compound represented by these was manufactured. Details are given below with reference to FIG.

  The three-way cock c on the supply port side (upstream side) and the three-way cock d on the discharge port side (downstream side) of the column containing sialidase immobilized on the carrier (sialidase-immobilized column C) produced in Example 2 Attached.

  Then, two reverse-phase cartridge columns (Sep pack C18) connected to the downstream side of the three-way cock d (shown as reverse-phase column D1 and reverse-phase column D2 from the upstream side) (reverse-phase column D1 and reverse-phase column D2 are connected) The connected product is shown as reverse phase column D).

  Further, a three-way cock e was attached to the downstream side of the reverse phase column D2.

  Further, an ion exchange cartridge column (Acrosep Q Ceramic Hyper DF) (ion exchange column E) was attached to the downstream side of the three-way cock e.

Example 10 Production of Propargyl Group-Introduced Lactose (Compound 3) Using Reactive Group-Introduced Sugar Production Apparatus 1 According to the following reaction formula, the apparatus for producing propargyl group-introduced lactose (compound 3) in Example 8 (FIG. 4) was used. Manufactured. Details will be described below.

  A mixed solution of lactose (compound 1) aqueous solution (1 M, 500 μL), propargyl alcohol (compound 2) (250 μL), and sodium acetate buffer (50 mM, pH = 5.0, 250 μL) is added to the three-way cock using a syringe. A was passed through the cellulase immobilization column A through a.

  At that time, the flow path of the three-way cock b is outside the apparatus so that the solution eluted from the outlet of the cellulase-immobilized column A is discharged out of the apparatus through the three-way cock b without passing through the reverse-phase column B. I was able to communicate.

  The supply port and the discharge port of the cellulase-immobilized column A were closed, and the cellulase-immobilized column A portion was placed in a thermostatic bath heated to 37 ° C. and incubated for 70 hours (implementation of sugar transfer reaction in the column).

  2 mL of sodium acetate buffer (50 mM, pH = 5.0) was passed through the three-way cock a. The eluate at this time has a role of pushing the reaction product out of the column and a meaning of substituting the column with a buffer for stabilizing cellulase.

  At that time, the flow path of the three-way cock b was connected to the reverse phase column B so that the solution eluted from the cellulase-immobilized column A column was introduced into the reverse phase column B.

  Unreacted lactose (compound 1) and propargyl group-introduced lactose (compound 3) are separated from the three-way cock b into the reverse phase column B through distilled water (6 mL). Unreacted lactose (compound 1) was eluted in the first half (3 mL) of the distilled water that passed through. This can be reused in the next reaction after vacuum concentration. In the latter half (3 mL), propargyl group-introduced lactose (compound 3) was recovered. The aqueous solution of compound 3 was concentrated under reduced pressure.

  After recovering the target product, the reverse-phase column is regenerated in order from the three-way cock through water: methanol = 1: 1 solution (5 mL), methanol (5 mL), and distilled water (5 mL) in order to prepare for the next reaction. be able to.

NMR data: ¹ H-NMR (D ₂ O) δ4.96 (d, 1 H, J = 3.6 Hz), 4.54 (d, 1 H, J = 8.4 Hz), 4.34 (s, 2 H), 4.32 (d, 1 H, J = 8.0 Hz), 3.86 (d, 1 H, J = 11.6 Hz), 3.69-3.52 (m, 4 H), 3.42 (dd, 1 H, J = 8.4, J = 9.2 Hz), 3.22 (br-t, 1 H), 2.78 (s, 1 H).

  In the above method, various reactive groups are introduced by using a compound in which the ethynyl group of compound 2 is substituted with another reactive group (for example, vinyl group or azide group) instead of compound 2. Lactose can be made.

Example 11 Production of Propargyl Group-Introduced Sialyl Lactose (Compound 5) Using Reactive Group-Introduced Sialo Sugar Chain Production Device 2 An apparatus for producing propargyl group-introduced sialyl lactose (compound 5) in Example 8 according to the following reaction formula (Fig. 5). Details will be described below.

Compound 3 (223.5 nmol) and paranitrophenol sialic acid (compound 4) (6.9 nmol) or sodium colominate (10 mg) (average polymerization degree: 100) represented by the following formula were mixed with Tris-HCl buffer (100 mM Tris -HCl, 0.5 mM, containing CaCl ₂ , pH = 8.0, 1 mL), and is passed to sialidase-immobilized column C through a three-way cock c using a syringe. At that time, the flow path of the three-way cock d is outside the apparatus so that the solution eluted from the outlet of the sialidase-immobilized column C is discharged outside the apparatus through the three-way cock d without passing through the reverse phase column D. Keep in touch.

  A thermostatic bath in which the supply port and the discharge port of the sialidase-immobilized column C are closed, the flow paths of the three-way cock c and the three-way cock d are directed to the sialidase-immobilized column C side, and the sialidase-immobilized column C portion is heated to 30 ° C. And incubate for 24 hours (perform transglycosylation in the column).

  Pass the 3-way cock c through 100 mM sodium acetate buffer, pH = 5.5 (2 mL). The eluate at this time has the role of pushing the reaction product out of the column and the meaning of replacing the column with a buffer stabilized by sialidase. At that time, the flow path of the three-way cock d is connected to the reverse phase column D so that the solution eluted from the sialidase-immobilized column C is introduced into the reverse phase column D.

  Separating sugar chains from hydrophobic components (paranitrophenol released during the reaction) from three-way cock d to reversed-phase column D through distilled water (3 mL) (* Reverse-phase column when colominic acid is used as a donor) Step can be omitted). The sugar chain containing the target compound is completely eluted from the reverse phase column D with the distilled water (3 mL) passed through. At this time, the flow path of the three-way cock e installed below the reverse phase column is connected to the ion exchange column E so that the eluate flows directly into the ion exchange column E connected next.

  In order from the three-way cock d, water: methanol = 1: 1 solution (10 mL), methanol (10 mL), distilled water (10 mL) are passed in order to regenerate the reverse phase column D and prepare for the next reaction. . At that time, the flow path of the three-way cock e is connected to the outside of the apparatus so that all the solution eluted from the reverse phase column D is discharged from the three-way cock e to the outside of the apparatus.

  Compound 3 is recovered by passing distilled water (5 mL) from the three-way cock e to the ion exchange column E. This can be reused in the next reaction after concentration under reduced pressure. Next, a propargyl group-introduced sialyl lactose (compound 5) and free sialic acid are obtained as a mixture through a 2 M sodium chloride aqueous solution (2 mL) from the three-way cock e to the ion exchange column E.

  The ion exchange column can be regenerated and prepared for the next reaction by passing distilled water (10 mL) from the three-way cock e.

Claims (7)

General formula (5);

[In the formula, X represents a single bond or a sugar residue obtained by removing a hydroxyl group from a hydroxyl group at the reducing end of glucose, galactose, N-acetylglucosamine, or N-acetylgalactosamine, and removing a hydrogen from a hydroxyl group at the non-reducing end. Y represents an ethynyl group, a vinyl group, an azide group, an epoxy group, an aldehyde group, or an oxylamino group; R ⁵ and R ⁶ are different from each other and represent hydrogen or a hydroxyl group; and R ⁷ represents a hydroxyl group or an acetylamino group. R ⁸ represents hydrogen or a group for acylating an amino group, R ⁹ is the same or different and represents a hydrogen or hydroxyl protecting group, and n is an integer of 1 to 10.
A process for producing a compound represented by
(Step 1) General formula (1);

[In the formula, X represents a single bond or a sugar residue obtained by removing a hydroxyl group from a hydroxyl group at the reducing end of glucose, galactose, N-acetylglucosamine, or N-acetylgalactosamine, and removing a hydrogen from a hydroxyl group at the non-reducing end. The left side of X represents the non-reducing end side, the right side of X represents the reducing end side, R ⁵ and R ⁶ are different and represent hydrogen or a hydroxyl group, and R ⁷ represents a hydroxyl group or an acetylamino group]
A compound represented by
General formula (2);

[Wherein Y represents an ethynyl group, a vinyl group, an azide group, an epoxy group, an aldehyde group, or an oxylamino group, and n is an integer of 1 to 10]
A compound represented by
Reacting in the presence of glycosidase, general formula (3);

[In the formula, X represents a single bond or a sugar residue obtained by removing a hydroxyl group from a hydroxyl group at the reducing end of glucose, galactose, N-acetylglucosamine, or N-acetylgalactosamine, and removing a hydrogen from a hydroxyl group at the non-reducing end. Y represents an ethynyl group, a vinyl group, an azide group, an epoxy group, an aldehyde group, or an oxylamino group; R ⁵ and R ⁶ are different from each other and represent hydrogen or a hydroxyl group; and R ⁷ represents a hydroxyl group or an acetylamino group. N is an integer from 1 to 10]
A step of obtaining a compound represented by:
(Step 2) a sialic acid donor;
General formula (3);

[ Wherein , X, Y, R ⁵ , R ⁶ , R ⁷ and n are the same as above ]
A compound represented by
A production method comprising reacting in the presence of sialidase.   The production method according to claim 1, wherein the sialidase is derived from an organism belonging to the genus Salmonella. The method according to claim 1 or 2 , wherein the sialidase is a sialidase immobilized on a carrier. The sialic acid donor has the general formula (4);

[Wherein, R ² is the same or different and represents a substituent, R ⁸ represents hydrogen or a group for acylating an amino group, R ⁹ is the same or different and represents a hydrogen or hydroxyl protecting group, m is It is an integer of 1-3. ]
At least one a method according to any one of claims 1 to 3, which is selected in the compounds and the group consisting of colominic acid represented. General formula (7a);

[In the formula, X represents a single bond or a sugar residue obtained by removing a hydroxyl group from a hydroxyl group at the reducing end of glucose, galactose, N-acetylglucosamine, or N-acetylgalactosamine, and removing a hydrogen from a hydroxyl group at the non-reducing end. R ⁵ and R ⁶ are different from each other and represent hydrogen or a hydroxyl group, R ⁷ represents a hydroxyl group or an acetylamino group, R ⁸ represents hydrogen or a group that acylates an amino group, and R ⁹ is the same or different. N represents an integer of 1 to 10, and R ¹ is obtained by removing one hydrogen from an optionally substituted hydrocarbon, synthetic polymer, or natural polymer. Indicates a group
Or general formula (7b);

[In the formula, X represents a single bond or a sugar residue obtained by removing a hydroxyl group from a hydroxyl group at the reducing end of glucose, galactose, N-acetylglucosamine, or N-acetylgalactosamine, and removing a hydrogen from a hydroxyl group at the non-reducing end. R ⁵ and R ⁶ are different from each other and represent hydrogen or a hydroxyl group, R ⁷ represents a hydroxyl group or an acetylamino group, R ⁸ represents hydrogen or a group that acylates an amino group, and R ⁹ is the same or different. N represents an integer of 1 to 10, and R ¹ is obtained by removing one hydrogen from an optionally substituted hydrocarbon, synthetic polymer, or natural polymer. Indicates a group
A process for producing a compound represented by
(Step 1) General formula (1);

[In the formula, X represents a single bond or a sugar residue obtained by removing a hydroxyl group from a hydroxyl group at the reducing end of glucose, galactose, N-acetylglucosamine, or N-acetylgalactosamine, and removing a hydrogen from a hydroxyl group at the non-reducing end. The left side of X represents the non-reducing end side, the right side of X represents the reducing end side, R ⁵ and R ⁶ are different and represent hydrogen or a hydroxyl group, and R ⁷ represents a hydroxyl group or an acetylamino group]
A compound represented by
General formula (2);

[Wherein Y represents an ethynyl group, a vinyl group, an azide group, an epoxy group, an aldehyde group, or an oxylamino group, and n is an integer of 1 to 10]
A compound represented by
Reacting in the presence of glycosidase, general formula (3);

[In the formula, X represents a single bond or a sugar residue obtained by removing a hydroxyl group from a hydroxyl group at the reducing end of glucose, galactose, N-acetylglucosamine, or N-acetylgalactosamine, and removing a hydrogen from a hydroxyl group at the non-reducing end. Y represents an ethynyl group, a vinyl group, an azide group, an epoxy group, an aldehyde group, or an oxylamino group; R ⁵ and R ⁶ are different from each other and represent hydrogen or a hydroxyl group; and R ⁷ represents a hydroxyl group or an acetylamino group. N is an integer from 1 to 10]
Obtaining a compound represented by:
(Step 2) a sialic acid donor;
General formula (3);

[Wherein, X, Y, R ⁵ , R ⁶ , R ⁷ and n are the same as above]
A compound represented by
Reacting in the presence of sialidase, general formula (5);

[In the formula, X represents a single bond or a sugar residue obtained by removing a hydroxyl group from a hydroxyl group at the reducing end of glucose, galactose, N-acetylglucosamine, or N-acetylgalactosamine, and removing a hydrogen from a hydroxyl group at the non-reducing end. Y represents an ethynyl group, a vinyl group, an azide group, an epoxy group, an aldehyde group, or an oxylamino group; R ⁵ and R ⁶ are different from each other and represent hydrogen or a hydroxyl group; and R ⁷ represents a hydroxyl group or an acetylamino group. R ⁸ represents hydrogen or a group for acylating an amino group, R ⁹ is the same or different and represents a hydrogen or hydroxyl protecting group, and n is an integer of 1 to 10.
A step of obtaining a compound represented by:
(Process 3)
General formula (5);

[Wherein X, Y, R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , and n are the same as above]
A compound represented by general formula (6);

[Wherein, R ¹ represents a group obtained by removing one hydrogen from an optionally substituted hydrocarbon, synthetic polymer, or natural polymer. ]
The manufacturing method characterized by including the process with which the compound represented by these is made to react . A general formula (5) comprising a column containing sialidase immobilized on a carrier (hereinafter referred to as column C) having a supply port and a discharge port ;

[In the formula, X represents a single bond or a sugar residue obtained by removing a hydroxyl group from a hydroxyl group at the reducing end of glucose, galactose, N-acetylglucosamine, or N-acetylgalactosamine, and removing a hydrogen from a hydroxyl group at the non-reducing end. Y represents an ethynyl group, a vinyl group, an azide group, an epoxy group, an aldehyde group, or an oxylamino group; R ⁵ and R ⁶ are different from each other and represent hydrogen or a hydroxyl group; and R ⁷ represents a hydroxyl group or an acetylamino group. R ⁸ represents hydrogen or a group for acylating an amino group, R ⁹ is the same or different and represents a hydrogen or hydroxyl protecting group, and n is an integer of 1 to 10.
The apparatus for manufacture of the compound represented by these . (A) Column C,
(B) a reverse phase column (hereinafter referred to as column D) having a supply port and a discharge port, and (C) an ion exchange column (hereinafter referred to as column E) having a supply port and a discharge port,
The discharge port of column C and the supply port of column D are connected by a flow path d,
The discharge port of column D and the supply port of column E are connected by a flow path e.
The channel d includes a channel switching device d ′,
The apparatus according to claim 6 , wherein the channel e includes a channel switching device e ′.

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