JP2008031156A - Antiviral agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain an antiviral agent that has a strong adsorption inhibitory activity and infection inhibitory activity against viruses and is inexpensively produced. <P>SOLUTION: The sialyl-poly-N-acetyllactosamine-containing artificial polypeptide is obtained by modifying the sialosugar chain part of a sialyl-N-acetyllactosamine-containing artificial polypeptide. The sialyl-N-acetyllactosamine-containing artificial sugar chain polypeptide is obtained by changing an α polyglutamic acid having a backbone of a sialyl-N-acetyllactosamine-containing artificial polypeptide into a γ-polyglutamic acid being a natural substance and inexpensive and changing a linker part. The sialyl-poly-N acetyllactosamine-containing artificial sugar chain polypeptide is obtained by changing a sialosugar chain part to a sialyl-poly-N-acetyllactosamine. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to an antiviral agent containing a sialosugar chain-containing polymer as an active ingredient.

  Influenza virus is one of the pathogens for which no effective treatment or protection method has been found. Influenza viruses are extremely infectious, repeat antigenic mutations, and new species that differ from the antigenicity so far appear, so that there is a problem that it is difficult to establish effective protective measures with vaccines.

  Recently, there are concerns about the emergence of new human influenza viruses originating from avian influenza virus, and development of influenza preventive / therapeutic drugs is expected.

  Infection of influenza virus into host cells is considered to be caused by hemagglutinin (hemagglutinin: HA), a virus surface protein, which recognizes and binds sialic acid-containing complex sugar chains on the cell surface as receptors. For example, influenza virus type A isolated from avian species preferentially binds to a sugar chain having N-acetylneuraminic acid α2,3 galactose (NeuAc (α2,3) Gal) at its terminal, It has been reported that the human influenza virus similar to the above shows high binding affinity for the terminal NeuAc (α2,6) Gal structure.

  Because there are various subtypes of influenza virus due to mutations, the development of anti-influenza virus agents that inhibit receptor binding widely regardless of the virus subtype is desired as a more effective anti-influenza virus agent . Under such circumstances, for example, it is expected that sialic acid-containing sugar chain (sialog sugar chain) compounds can function as infection inhibitors that inhibit the binding to viral receptors, which is the first step of influenza virus infection. The

  Specifically, although influenza virus adhesion inhibition experiments using hemagglutination inhibition activity as an index were conducted on various sialoglycan compounds, the virus adhesion inhibition activity was low, or the synthesis method of sialoglycan compounds For reasons such as being complicated and impractical, an influenza virus infection inhibitor containing a sialo-glycan compound as an active ingredient has not yet been developed (Non-patent Document 1, Non-patent Document 2, Patent Document 1, Patent Document 2, Patent Document 3).

  Among them, Kobayashi et al. Reported that sialyl lactose (NeuAc (α2,6 or α2,3) Gal (β1,4) GlcNAc), which is a receptor for influenza A virus, has a low anti-adhesion effect on influenza virus, but polystyrene. It has been found that a polymer obtained by binding sialyl lactose to a derivative has a strong binding ability to influenza virus (Patent Document 4). However, the polymer has a manufacturing problem that sialyl lactose must be prepared from milk, and cannot be said to be a practical compound.

  In addition, Toya et al. Introduced α-polyglutamic acid as a skeleton and N-acetyllactosamine into the side chain of α-polyglutamic acid as a p-aminophenyl group as a linker, and added sialic acid to the non-reducing end of the oligosaccharide chain. Sialyl-N-acetyllactosamine-containing artificial sugar chain polypeptide having a molecular weight of 2,000 to 1,000,000 was synthesized, and it was found that this compound has infection inhibitory activity against a wide range of influenza virus isolates (Patent Document 5, Non-patent document 3).

Chemistry Letter, 1259-1260 (1999) Glycoconj. J., 7, 349-359 (1990) Glycobiology, 13, 315-326 (2003) Special table 2002-514186 Special table 2003-535965 WO2003 / 074570 JP 10-310610 A JP 2003-73397 A

  However, the above-mentioned sialyl-N-acetyllactosamine-containing artificial sugar chain polypeptide does not necessarily have a high infection inhibitory activity against influenza virus, and α-polyglutamic acid and N-acetylglucosamine derivatives which are raw materials for polypeptides It has been pointed out that (P-nitrophenyl glycoside) and the like are expensive and the production method is complicated.

  Therefore, it has been desired to develop a novel artificial sialo-glycan polymer that has strong antiviral activity such as adsorption inhibitory activity and infection inhibitory activity against influenza virus and can be produced at a low production cost.

  As a result of intensive studies to solve the above problems, the present inventors have modified (1) the sialo-glycan part of the sialyl-N-acetyllactosamine-containing artificial polypeptide of Toya et al. -By having an acetyllactosamine-containing artificial polypeptide, it has several tens to several hundreds of times of influenza infection inhibitory activity, and (2) α which is a skeleton of a sialyl-N-acetyllactosamine-containing artificial polypeptide (3) the sialo, in which the polyglycamic acid is changed to natural and inexpensive γ-polyglutamic acid and the sialyl-N-acetyllactosamine-containing artificial sugar chain polypeptide having a modified linker moiety has high influenza virus infection inhibitory activity; Sialyl-poly N-acetyllactosami with sugar chain part converted to sialyl-poly N-acetyllactosamine -Containing artificial sugar chain polypeptide has higher influenza virus infection inhibitory activity, and (4) lactose is introduced between the reducing end of sialyl-N-acetyllactosamine and the linker part of the sialosugar chain part. The present inventors have found that a sialyl-N-acetyllactosamine-lactose-containing artificial sugar chain polypeptide also has high influenza virus infection inhibitory activity, and completed the present invention. Accordingly, the present invention is as follows.

（Ｉ）

（ＩＩ）
（式（Ｉ）中、Ｚは水酸基又は式（ＩＩ）で表されるシアロ糖鎖部であり、ｎは１０以上の整数を示す。式（ＩＩ）中、Ｙ_１及びＹ_２は水酸基又はＮ−アセチルノイラミン酸残基、Ｘは水酸基又はアセチルアミノ基、Ｒは炭化水素又はオリゴ糖を示す。ただし、Ｙ_１とＹ_２は同一ではない。） [1] An antiviral agent comprising, as an active ingredient, a sialo-sugar chain-containing polymer represented by the formula (I) and having sialic sugar chains bonded to γ-polyglutamic acid.

(I)

(II)
(In formula (I), Z is a hydroxyl group or a sialosugar chain represented by formula (II), and n represents an integer of 10 or more. In formula (II), Y ₁ and Y ₂ are a hydroxyl group or N. - acetylneuraminic acid residue, X is a hydroxyl group or an acetylamino group, R represents a hydrocarbon or oligosaccharide, however, Y ₁ and Y ₂ are not the same)..

（ＩＩＩ）

（ＩＶ）
（式中、Ｌは炭化水素、Ｘ_１及びＸ_２は水酸基又はアセチルアミノ基を示す。ただし、Ｘ_１とＸ_２は同一であっても相違していてもよい） [2] The antiviral agent according to [1] above, wherein the oligosaccharide represented by R is the following formula (III) or (IV).

(III)

(IV)
(In the formula, L represents a hydrocarbon, and X ₁ and X ₂ represent a hydroxyl group or an acetylamino group. However, X ₁ and X ₂ may be the same or different.)

（Ｖ）

（ＩＩ´）
（式（Ｖ）中、Ｚは水酸基又は式（ＩＩ´）で示されるシアロ糖鎖部であり、ｎは１０以上の整数を示す。式（ＩＩ´）中、Ｙ_１及びＹ_２は水酸基又はＮ−アセチルノイラミン酸残基、Ｘは水酸基又はアセチルアミノ基、Ｒ’はオリゴ糖を示す。ただし、Ｙ_１とＹ_２は同一ではない。） [3] An antiviral agent comprising, as an active ingredient, a sialosugar chain-containing polymer represented by the formula (V) and having an α-polyglutamic acid bound to a sialosugar chain.

(V)

(II ')
(In Formula (V), Z is a hydroxyl group or a sialo-sugar chain part represented by Formula (II ′), and n represents an integer of 10 or more. In Formula (II ′), Y ₁ and Y ₂ are hydroxyl groups or N-acetylneuraminic acid residue, X represents a hydroxyl group or acetylamino group, R ′ represents an oligosaccharide, provided that Y ₁ and Y ₂ are not the same.

（ＩＩＩ）

（ＩＶ）
（式中、Ｌは炭化水素、Ｘ_１及びＸ_２は水酸基又はアセチルアミノ基を示す。ただし、Ｘ_１とＸ_２は同一でも相違していてもよい） [4] The antiviral agent according to [3] above, wherein the oligosaccharide represented by R ′ is the following formula (III) or (IV).

(III)

(IV)
(In the formula, L represents a hydrocarbon, and X ₁ and X ₂ represent a hydroxyl group or an acetylamino group. However, X ₁ and X ₂ may be the same or different.)

[5] The antiviral agent according to any one of [1] to [4] above, which is an anti-influenza virus agent.
[6] The antiviral agent according to any one of [1] to [5], wherein the molecular weight is 2 to 5 million.
[7] The antiviral agent according to any one of [1] to [6] above, wherein the degree of polymerization of glutamic acid units is 10 to 10,000.
[8] The antiviral agent according to any one of [1] to [7] above, wherein the introduction rate of the sialosugar chain to the glutamic acid residue is 10 to 80%.

  The present invention provides a conventional sialo-glycan-containing polypeptide by using as an active ingredient a compound in which either or both of the sialo-glycan part and the polypeptide part of a conventional sialo-glycan-containing polypeptide are modified. An antiviral agent exhibiting an influenza virus infection inhibitory activity several tens to several hundred times greater than the above is provided. In particular, among the active ingredients of the present invention, a sialosugar chain-containing polymer having γ-polyglutamic acid as a polypeptide part can be produced from an inexpensive raw material and can be stably supplied.

  Therefore, the antiviral agent of the present invention can be applied in various means and places for preventing influenza virus infection and influenza epidemic. Specifically, it can be applied to nasal sprays, oral sprays, etc. as an antiviral agent for the prevention and treatment of influenza virus infection, as well as to remove influenza virus from the air by filling a filter, and to a mask. It can be used to prevent the spread of viruses and prevent infection by filling, and its application range is extremely wide.

  Furthermore, if the sialic acid imparted to the non-reducing end of the sugar chain is α2,6, it can be used as an antiviral agent for inhibiting infection against human-infected influenza viruses. If the sialic acid is added to α2,3 type, it can be used as an antiviral agent to inhibit infection against avian infectious influenza virus, and α2,6 and α2,3 types are used in combination. By doing so, it is possible to cope with a mutation from an avian infectious type to a human infectious type.

  The active ingredient of the antiviral agent of the present invention is a sialo-sugar chain-containing polymer compound represented by the following (I) or (V). However, in Formula (I) and (V), Z is a hydroxyl group or a sialo-sugar chain part represented by Formula (II) or Formula (II ′), and n represents an integer of 10 or more. In the sialo-sugar chain-containing polymer, the sialo-sugar chain-substituted glutamic acid residue and the unsubstituted glutamic acid residue are mixed in an arbitrary ratio, and the ratio is represented by the degree of sugar residue substitution (Degree of substitution (DS)).

（Ｉ）

（ＩＩ）

（Ｖ）

（ＩＩ´）

(I)

(II)

(V)

(II ')

  In Formula (I) or Formula (V), Z is a hydroxyl group or a sialo-sugar chain part represented by Formula (II) or (II ′). When Z is a hydroxyl group, an unsubstituted glutamic acid residue, Formula (II) ) Or a sialic acid sugar chain part represented by the formula (II ′) is a sialo sugar chain-substituted glutamic acid residue. As shown in Formula (I) or Formula (V), the number and position of the sialo-glycan-substituted glutamine in the polyglutamic acid polymer are not particularly limited.

In formula (II) or formula (II ′), Y ₁ and Y ₂ represent a hydroxyl group or an N-acetylneuraminic acid residue. However, Y ₁ and Y ₂ are not the same, and if one is a hydroxyl group, the other is an N-acetylneuraminic acid residue. Specifically, if α2,6 type Y ₂ is a hydroxyl group in Y ₁ is N- acetylneuraminic acid residue, it is an antiviral agent for inhibiting infection to human infective influenza virus, Y ₁ is If it is α2,3 type in which Y ₂ is a hydroxyl group and N ₂ is an N-acetylneuraminic acid residue, it can be used as an antiviral agent for inhibiting infection against avian infectious influenza virus. In the N-acetylneuraminic acid residue, the hydroxyl group, carboxyl group, and amide group may be chemically modified with a halogen group, an alkyl group, an acyl group, or the like, the same or different.

  In the formula, the hydrocarbon represented by R is preferably one having 1 to 20 carbon atoms, and may be either a saturated hydrocarbon group or an unsaturated hydrocarbon group, specifically an alkyl group, an alkenyl group, an alkynyl group. Cycloalkyl group, aryl group, aralkyl group, cycloalkyl-substituted alkyl group, and the like.

  Here, as an alkyl group, an alkenyl group, and an alkynyl group, a C1-C20 linear or branched thing is mentioned. Specific examples of the alkyl group include methyl group, ethyl group, n-propyl group, n-butyl group, n-pentyl group, n-hexyl group, n-octyl group, n-decyl group, n-dodecyl group, n -Straight chain alkyl groups such as tetradecyl group; branched alkyl groups such as isopropyl group, isobutyl group, t-butyl group, 2-ethylhexyl group and the like.

  Specific examples of the alkenyl group include a vinyl group, a propenyl group, and an allyl group. Specific examples of the alkynyl group include ethynyl group, propynyl group, butynyl group and the like. Examples of the cycloalkyl group include those having 3 to 10 carbon atoms, particularly those having 3 to 8 carbon atoms, such as a cyclopropyl group, a cyclopentyl group, and a cyclohexyl group.

  Examples of the aryl group include those having 6 to 14 carbon atoms, such as a phenyl group, a tolyl group, and a naphthyl group. Examples of the aralkyl group include an aralkyl group having 7 to 14 carbon atoms, specifically a benzyl group and a phenethyl group. Cycloalkyl-substituted alkyl groups include C3-C8 cycloalkyl-substituted C1-C10 alkyl groups such as cyclopropylmethyl group, cyclopentylmethyl group, cyclohexylmethyl group, cyclopropylethyl group, cyclopentylethyl group, cyclohexylethyl group, and cyclopropylpropyl group. , Cyclopentylpropyl group, cyclohexylpropyl group and the like.

  The hydrocarbon may have a substituent, and examples of such a substituent include a hydroxyl group, an azide group, a cyano group, an alkoxy group, a cycloalkyloxy group, an aryloxy group, and an esterified group. And a good carboxyl group.

（ＩＩＩ）

（ＩＶ）
（式中、Ｌは炭化水素、Ｘ１及びＸ２は水酸基又はアセチルアミノ基を示す。ただし、Ｘ１とＸ２は同一であっても相違していてもよい） In the formula, the oligosaccharide represented by R or R ′ refers to an oligosaccharide composed of disaccharide to hexasaccharide, and specifically, those represented by the following formula (III) or (IV) are exemplified. be able to.

(III)

(IV)
(In the formula, L represents a hydrocarbon, and X1 and X2 represent a hydroxyl group or an acetylamino group. However, X1 and X2 may be the same or different.)

  X and L in the above formula (III) or (IV) are as defined above, and examples of the hydrocarbon represented by L include the same hydrocarbons as R.

  The sialo-sugar chain-containing polymer as the active ingredient of the present invention may be either a salt type or a free acid type. Examples of the salt type include alkali metal salts (for example, sodium salts, potassium salts, etc.); Examples include earth metal salts (for example, calcium salts and magnesium salts); organic base salts (for example, trimethylamine salts, triethylamine salts, pyridine salts, picoline salts, dicyclohexylamine salts and the like). Further, it may be a hydrate or a solvate with alcohol or the like.

  Moreover, the molecular weight of the sialo-sugar chain-containing polymer that is the active ingredient of the present invention is, for example, in the range of 2000 to 5 million, and the glutamic acid unit polymerization degree (n) is, for example, 10 to 10000, preferably 100 to 6000. The sugar residue substitution degree (DS), which is the range and is the introduction rate of sialyl oligosaccharides with respect to glutamic acid residues, is in the range of 10 to 80%, preferably 30 to 70%.

Among these active ingredients of the present invention, preferable examples include those that satisfy the following conditions.
(A) A compound belonging to formula (I).
(B) A compound belonging to formula (I), wherein R is aminoalkyl.
(C) A compound belonging to formula (I), wherein R is aminoalkyl, Y ₁ is an N-acetylneuraminic acid residue, and Y ₂ is a hydroxyl group.
(D) A compound belonging to formula (I), wherein R is aminoalkyl, Y ₁ is an N-acetylneuraminic acid residue, Y ₂ is a hydroxyl group, and X is acetylamino.

(E) A compound belonging to formula (I), wherein R is an oligosaccharide of formula (III).
(F) A compound belonging to formula (I), wherein R is an oligosaccharide of formula (III), Y ₁ is an N-acetylneuraminic acid residue, and Y ₂ is a hydroxyl group.
(G) A compound belonging to formula (I), wherein R is an oligosaccharide of formula (III), Y ₁ is an N-acetylneuraminic acid residue, Y ₂ is a hydroxyl group, and X and X ₁ are acetylamino. A compound.
(H) a compound belonging to formula (I), wherein R is an oligosaccharide of formula (III), Y ₁ is an N-acetylneuraminic acid residue, Y ₂ is a hydroxyl group, X is acetylamino, X ₁ Wherein is a hydroxyl group.

(I) A compound belonging to formula (I), wherein R is an oligosaccharide of formula (IV).
(J) A compound belonging to formula (I), wherein R is an oligosaccharide of formula (IV), Y ₁ is an N-acetylneuraminic acid residue, and Y ₂ is a hydroxyl group.
(K) A compound belonging to formula (I), wherein R is an oligosaccharide of formula (IV), Y ₁ is an N-acetylneuraminic acid residue, Y ₂ is a hydroxyl group, and X, X ₁ and X ₂ are A compound that is acetylamino.
(L) a compound belonging to formula (I), wherein R is an oligosaccharide of formula (IV), Y ₁ is an N-acetylneuraminic acid residue, Y ₂ is a hydroxyl group, and X and X ₁ are acetylamino , X ₂ is a hydroxyl group.

(M) A compound belonging to formula (V), wherein R is an oligosaccharide of formula (III).
(N) A compound belonging to formula (V), wherein R is an oligosaccharide of formula (III), Y ₁ is an N-acetylneuraminic acid residue, and Y ₂ is a hydroxyl group.
(O) a compound belonging to formula (V), wherein R is an oligosaccharide of formula (III), Y ₁ is an N-acetylneuraminic acid residue, Y ₂ is a hydroxyl group, and X and X 1 are acetylamino Compound.

(P) A compound belonging to formula (V), wherein R is an oligosaccharide of formula (IV).
(Q) A compound belonging to formula (V), wherein R is an oligosaccharide of formula (IV), Y ₁ is an N-acetylneuraminic acid residue, and Y ₂ is a hydroxyl group.
(R) a compound belonging to formula (V), wherein R is an oligosaccharide of formula (IV), Y ₁ is an N-acetylneuraminic acid residue, Y ₂ is a hydroxyl group, and X, X ₁ and X ₂ are A compound that is acetylamino.

  The sialo-sugar chain-containing polymer used as the active ingredient of the present invention is prepared by the step 1 for synthesizing the asialo-sugar chain, the step 2 for condensing the asialog-sugar chain and polyglutamic acid, and the step 3 for sialylating the asialog-sugar chain polymer. Is possible. Steps 1 to 3 will be described in detail below.

(Step 1) Synthesis of asialo sugar chain Step 1 consists of sugar acceptor (eg, sugar-paranitrophenol, 5-aminoalkylated sugar, etc.) and sugar nucleotide (eg, uridine 5′-diline) which is a sugar donor. Glycosyltransferases (for example, β1,4-galactosyltransferase, β1,3-N-acetylglucosaminyltransferase, etc.) suitable for the reaction system containing acid galactose, uridine 5′-diphosphate N-acetylglucosamine, etc. ) To synthesize the target asialo sugar chain part.

  The glycosyltransferase added to the reaction system may be in any form as long as it has the desired enzyme activity. Enzymes produced by enzyme production using so-called DNA recombination technology, in which the enzyme gene is cloned, expressed in large quantities in microbial cells, and prepared in large quantities in order to increase preparation efficiency as well as the ease of enzyme preparation Is the most convenient.

  Specific examples of the enzyme preparation include microbial cells, processed products of the cells, enzyme preparations obtained from the processed products, and the like. Preparation of the microbial cells can be performed by a method of collecting the cells by centrifugation or the like after culturing by a conventional method using a medium in which the microorganism can grow. Specifically, taking bacteria belonging to Escherichia coli as an example, the medium is bouillon medium, LB medium (1% tryptone, 0.5% yeast extract, 1% salt) or 2 × YT medium. (1.6% tryptone, 1% yeast extract, 0.5% sodium chloride) can be used, and after inoculating the medium with inoculum, the mixture is stirred at 30-50 ° C. for about 10-50 hours as necessary. By culturing and collecting the microbial cells by centrifuging the obtained culture solution, microbial cells having the desired enzyme activity can be prepared.

  Processed microorganism cells include mechanical destruction (using a Waring blender, French press, homogenizer, mortar, etc.), freeze-thawing, self-digestion, drying (by freeze-drying, air drying, etc.), enzyme treatment ( Lysozyme), sonication, chemical treatment (by acid, alkali treatment, etc.), etc. be able to.

  As the enzyme preparation, the fraction having the enzyme activity from the above-mentioned processed bacterial cell product is purified by usual enzyme purification means (salting out treatment, isoelectric point precipitation treatment, organic solvent precipitation treatment, dialysis treatment, various chromatographic treatments, etc. A crude enzyme or a purified enzyme obtained by applying ().

  Commercially available products can be used for the sugar nucleotide and sugar receptor used in the reaction. The concentration used can be appropriately set within the range of 1 to 200 mM, preferably 5 to 50 mM. When a 5-aminoalkylated saccharide is used as the sugar acceptor, as shown in the examples described later, it is possible to alkylamination of the hydroxyl group of the saccharide using the reverse reaction of cellulase.

  In the synthesis of asialo sugar chain, glycosyltransferase is added to the reaction system containing the above sugar receptor and sugar nucleotide so that the amount of glycosyltransferase is 0.001 unit / ml or more, preferably about 0.01 to 10 unit / ml. It can be carried out by adding each and reacting at 5 to 50 ° C., preferably 10 to 40 ° C. for about 1 to 100 hours, with stirring if necessary.

  The asialo sugar chain thus produced may be isolated and purified by appropriately combining, for example, reverse phase ODS column chromatography, ion exchange column chromatography, or the like, using ordinary oligosaccharide separation and purification means.

(Step 2) Condensation of asialo sugar chain and polyglutamic acid Step 2 is a step of chemically condensing the asialo sugar chain synthesized in step 1 to the carboxyl group side chain of polyglutamic acid.

  When a sugar receptor having a p-nitrophenyl group is used as the sugar acceptor in Step 1, a 5-aminoalkylated sugar is used as the sugar acceptor after reducing the nitro group to an amino group. In this case, the protecting group of the amino group is deprotected by a conventional method, and then polyglutamic acid is treated with a condensing agent in the presence of a base such as triethylamine or tributylamine to produce an asialo sugar chain-containing polymer.

  The conditions used for the reduction reaction of the p-nitrophenyl group may be those usually applied to the reduction of the aromatic nitro group. Specific examples include hydrogen, formic acid in water, methanol, ethanol and other organic solvents. It can be carried out by treating with palladium carbon in the presence of a hydrogen donor such as ammonium or cyclohexene.

  The polyglutamic acid used as the raw material polymer may be either α-type or γ-type.

  In the condensation step, the activity of carboxyl groups such as p-nitrophenyl chloroformate, disuccimidyl carbonate, and carbonyldiimidazole in the presence of a base such as triethylamine or tributylamine in an organic solvent such as dimethylformamide or dimethylsulfoxide. After the treatment with the esterifying agent, the 5-aminoalkylated sugar fork is reacted with the product of the above reduction reaction.

  The amount of the 5-aminoalkylated sugar or the product of the above reduction reaction may be added according to the sugar substitution rate of the target sialo-sugar chain-containing polymer, and is generally 0.1% relative to the glutamic acid unit of polyglutamic acid. One equivalent or more is sufficient. Moreover, the usage-amount of the base used for a condensation reaction should just be 1 equivalent or more with respect to the glutamic acid unit of polyglutamic acid.

  The condensation reaction can be carried out at -10 ° C to 100 ° C. Further, a general acylation reaction catalyst such as 4-N, N-dimethylaminopyridine or 1-hydroxy-1H-benzotriazole may be added as necessary.

  The synthesized asialo sugar chain-containing polymer may be usually purified by a method used for protein purification. For example, it can be isolated and purified by appropriately combining dialysis and gel filtration.

(Step 3) Sialization of asialo sugar chain polymer Step 3 includes cytidine 5′-monophosphate N-acetylneuraminic acid (which is a sugar nucleotide at the non-reducing end of the sugar chain part of the asialo sugar chain polymer synthesized in step 2). This is a step of sialylation using CMP-NeuAc) as a substrate using sialyltransferase.

  As the sialyltransferase, α2,6-sialyltransferase is used for the production of an infection-inhibiting polymer against human-infected influenza virus, and for the production of avian-infectious influenza virus infection-inhibiting polymer. Uses α2,3-sialyltransferase. The sialyltransferase used for the reaction can be prepared by the same method as in Step 1, and the reaction can also be carried out by the same method as in Step 1.

  The synthesized sialo-sugar chain-containing polymer may be usually purified by a method used for protein purification. For example, it can be isolated and purified by appropriately combining dialysis and gel filtration. In addition to the steps described above, it is also possible to prepare a sialo-sugar chain-containing polymer by synthesizing a sialo-sugar chain in advance and condensing the sialo-sugar chain and polyglutamic acid.

  The antiviral agent of the present invention can be applied to various means for preventing viral infection and virus epidemic. For example, it can be used as a virus adsorbent in filters such as masks, air purifiers and air conditioners as well as pharmaceuticals. It is done.

  As the target virus, influenza virus is a suitable virus, and specific examples include highly pathogenic type A avian influenza virus, human type A influenza virus and human type B influenza virus.

  Moreover, it corresponds to the sialo-sugar chain-containing polymer used as an active ingredient and can be applied to various viruses other than influenza virus. Examples of such viruses include paramyxovirus group, parainfluenza virus group, rotavirus, adenovirus, coronavirus, polyomavirus and the like.

  When the antiviral agent of the present invention is used as a pharmaceutical, it is not particularly limited to its dosage form, but it is directly applied to the nasal cavity and pharynx such as nasal solutions and gels and aerosols such as oral sprays. Preparations for nasal application or preparations for percutaneous absorption such as ointments, emulsions and creams are exemplified as preferred dosage forms, and they can be prepared by conventional methods.

  For example, as a nasal preparation, it is preferably used as a dosage form such as a liquid, a gel, a suspension, and an aerosol. The base component for producing the dosage form will be described. The base component for producing a liquid agent, gel agent, suspension agent and aerosol agent includes higher alcohols such as octyldodecanol; isopropyl myristate, adipine Fatty acid esters such as diisopropyl acid and isopropyl palmitate; suspending agents such as polysorbate 80 and polyoxyethylene hydrogenated castor oil; polymer compounds such as carboxyvinyl polymer, hydroxypropylcellulose, polyvinylpyrrolidone and sodium hyaluronate; glycerin, propylene Polyhydric alcohols such as glycol and 1,3-butylene glycol; pH regulators such as diisopropanolamine, sodium hydroxide, potassium dihydrogen phosphate and sodium hydrogen phosphate; sodium hydrogen phosphate, sodium chloride , Sodium thiosulfate, stabilizers such as sodium sulfite and sodium edetate; methylparaben, propylparaben, preservatives such as benzalkonium chloride and benzethonium chloride.

  When producing a liquid agent, a gel agent, a suspension agent, and an aerosol agent, it is produced by mixing a medium such as a sialo-sugar chain-containing polymer and water, which is an active ingredient of the present invention, but the mixing ratio is not particularly limited, For example, water (medium) 50 to 99% by weight, higher alcohol 0 to 25% by weight, fatty acid ester 0 to 20% by weight, suspending agent 0 to 5% by weight, polymer compound 0 to 10% by weight, polyhydric alcohol 0 -30% by weight, pH regulator 0-10% by weight, stabilizer 0-10% by weight and preservative 0-5% by weight.

  Base ingredients for the production of ointments, emulsions and creams include hydrocarbons such as white petrolatum, liquid paraffin, paraffin, microcrystalline wax, squalane and plastibase; higher alcohols such as cetanol, stearyl alcohol and behenyl alcohol; isostearin Higher fatty acids such as acids and oleic acids; fatty acid esters such as medium chain fatty acid triglycerides, isopropyl myristate, diisopropyl adipate and isopropyl palmitate; surfactants such as polysorbate 80 and polyoxyethylene hydrogenated castor oil; carboxyvinyl polymer, hydroxy Polymer compounds such as propylcellulose, polyvinylpyrrolidone and sodium hyaluronate; glycerin, propylene glycol, 1,3-butylene glycol Polyhydric alcohols; pH regulators such as diisopropanolamine, sodium hydroxide, potassium dihydrogen phosphate and sodium hydrogen phosphate; stability such as sodium hydrogen phosphate, sodium chloride, sodium thiosulfate, sodium sulfite and sodium edetate Examples of the agent include antiseptics such as methylparaben, propylparaben, benzalkonium chloride, and benzethonium chloride.

  In the case of producing an ointment, emulsion and cream, it is produced by mixing a sialo-glycan-containing polymer, which is an active ingredient of the present invention, and a medium such as water, but there is no particular limitation on the blending ratio, for example, water (medium ) 0 to 80 wt%, hydrocarbons 0 to 90 wt%, higher alcohols 0 to 25 wt%, higher fatty acids 0 to 25 wt%, fatty acid esters 0 to 20 wt%, surfactants 0.01 to 10 wt%, The polymer compound is 0 to 10% by weight, the polyhydric alcohol is 0 to 30% by weight, the pH regulator is 0 to 10% by weight, the stabilizer is 0 to 10% by weight, and the preservative is 0 to 5% by weight.

  In addition, when the antiviral agent of the present invention is used by filling a filter or a mask for preventing the spread of viruses or preventing infection, for example, the antiviral agent of the present invention can be carried on a sheet. .

  The sheet used is a nonwoven fabric or woven fabric made of synthetic fiber or natural fiber, and examples of the material include polyester, polyamide, polyacryl, polypropylene, rayon, cotton, wood pulp and the like. In particular, melt blown nonwoven fabrics are preferable as filters for masks, air purifiers and air conditioners because the fiber diameter can be reduced and the pore size can be reduced. In addition, a method for supporting the sialo-sugar chain-containing polymer, which is an active ingredient, on the sheet is arbitrary. For example, a method of supporting the sheet using an adhesive can be employed.

  Next, Examples (Synthesis Examples, Test Examples, Formulation Examples) of the present invention will be described. In addition, this invention is not restrict | limited at all by the following Example.

<Material preparation method and analysis method>
(1) High performance liquid chromatography (HPLC)
All samples were analyzed after filtering through a 0.45 μl filter. The analysis conditions shown below were used.
Column: Mightysil Si60 (φ4.6 × 250 mm)
Column temperature: 40 ° C
Flow rate: 1.0 ml / min
Detection wavelength: 210 nm
Solvent: 90% CH ₃ CN
Or
Column: YMC Pro C18RS (φ6.0 × 150 mm)
Column temperature: 40 ° C
Flow rate: 1.0 ml / min
Detection wavelength: 300 nm
Solvent: 20% MeOH-50 mM TEAA

(2) Nuclear magnetic resonance apparatus (NMR)
Analytical instrument: JEOL EX-270 NMR spectrometer,
JEOL lamda 500FT NMR spectrometer
Bruker AV-500 NMR spectrometer
External standard: TPS [sodium3- (trimethylsilyl) -propionate]
Solvent: D ₂ O
Temperature: 25 ° C or 60 ° C
Sample tube: φ3 or 5mm

(3) Preparation of enzyme (3-1) Preparation of β1,4-galactosyltransferase (β1,4-GalT) Preparation of β1,4-GalT is described in the method of Noguchi et al. (Japanese Patent Application Laid-Open No. 2002-335988). The expression plasmid pTGF-A is used. E. coli JM109 holding pGTF-A was inoculated into 50 ml of 2 × YT medium containing 100 μg / ml ampicillin and cultured with shaking at 30 ° C. When reaching 4 × 10 ⁸ cells / ml, IPTG was added to the culture solution to a final concentration of 0.1 mM, and the shaking culture was continued at 30 ° C. for 16 hours. After completion of the culture, the cells were collected by centrifugation (9,000 × g, 20 minutes) and suspended in 5 ml of a buffer solution (10 mM Tris-HCl (pH 8.0), 1 mM EDTA). The microbial cells were crushed by sonication, and the microbial cell residues were removed by centrifugation (20,000 × g, 10 minutes), and the resulting supernatant fraction was used as an enzyme solution. The β1,4-GalT activity in the enzyme solution was measured by the method described in JP-A-2005-335988.

(3-2) Preparation of Neisseria meningitidis β1,3-N-acetylglucosaminyltransferase (β1,3-GlcNAcT) (3-2-1) Cloning of lgtA gene encoding β1,3-N-acetylglucosaminyltransferase

  Using the Neisseria meningitidis MC58 chromosomal DNA (ATCC BAA-335D) as a template, the following two types of primer DNAs were synthesized according to a conventional method, and the Neisseria meningitidis lgtA gene (EMBL / GENEBANK / DDBJ DATA BANKS, No. AF53681) was amplified.

Primer (A): 5'-ttccatggcgtgtgaagccttcagaggggcatcg-3 '
Primer (B): 5'-ttaagcttaacgggtttttcagcaatcgggtcaaaa-3 '

  Amplification of the lgtA gene by PCR was performed in 50 μl of reaction solution (50 mM potassium chloride, 10 mM Tris-HCl (pH 8.3), 1.5 mM magnesium chloride, 0.001% gelatin, 0.2 mM dATP, 0.2 mM dGTP, 0. 2 mM dCTP, 0.2 mM dTTP, template DNA 0.1 μg, primer DNA (A) (B) 0.2 mM each, AmpliTaq DNA Polymerase 2.5 unit (manufactured by Takara Bio Inc.), DNA Thermal Cycler (94 ° C., 30 seconds), annealing (55 ° C., 45 seconds), extension reaction (72 ° C., 2 minutes 30 seconds) were repeated 30 times.

  After gene amplification, the reaction solution was treated with a phenol / chloroform (1: 1) mixture, and 2 volumes of ethanol was added to the water-soluble fraction to precipitate DNA. The DNA collected by precipitation was separated by agarose gel electrophoresis according to the method described in the literature (Molecular cloning, edited by Maniatis et al., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1982)), and a DNA fragment corresponding to 1.0 kb was purified. The DNA was cleaved with restriction enzymes NcoI and HindIII, and ligated with TpDNA ligase and plasmid pTrc99A (currently obtained from Amersham Biosciences) digested with restriction enzymes NcoI and HindIII. (E. coli) JM109 bacteria were transformed, and plasmid pTrc-NMGnT was isolated from the resulting ampicillin resistant transformant.

  pTrc-NMGnT is obtained by inserting an NcoI-HindIII DNA fragment containing a Neisseria meningitidis lgtA structural gene into the NcoI-HindIII cleavage site downstream of the trc promoter of pTrc99A.

(3-2-2) Preparation of enzyme solution having β1,3-N-acetylglucosamine transfer activity Inoculation of E. coli K12 strain JM109 carrying plasmid pTrc-NMGnT in 50 ml of 2 × YT medium containing 100 μg / ml ampicillin And cultured with shaking at 37 ° C. When the concentration reached 2 × 10 ⁸ cells / ml, IPTG was added to the culture solution to a final concentration of 0.5 mM, and the shaking culture was continued at 20 ° C. for 20 hours. After completion of the culture, the cells were collected by centrifugation (9,000 × g, 10 minutes), and 5 ml of buffer solution (50 mM Tris-HCl (pH 7.5), 0.5 M sodium chloride, 5 mM 2-mercap and ethanol, 10 % (W / v) glycerol, containing 0.2% (v / v) Triton X-100). The microbial cells were crushed by sonication, and the microbial cell residues were removed by centrifugation (20,000 × g, 10 minutes).

  The supernatant fraction thus obtained was used as an enzyme solution, and the results of measuring the β1,3-N-acetylglucosamine transfer activity in the enzyme solution together with the control bacterium (E. coli K12 strain JM109 holding pTrc99A) are shown in Table 1 below. Show. The unit of β1,3-acetylglucosamine transferase activity in the present invention is a measurement of the synthetic activity of lacto N-triose (LNT-2) from UDP-N-acetylglucosamine and lactose by the method shown below. , Calculated.

N. measurement of meningitidis N-acetylglucosamine transferase activity and unit calculation method;
Β1,3-N-acetylglucosamine transferase is added to 100 mM Tris-HCl buffer (pH 7.5) containing 10 mM magnesium chloride, 10 mM manganese chloride, 20 mM lactose, and UDP-N-acetylglucosamine, and 30 ° C. is added. Reacted for 1 minute. In addition, a similar reaction was performed using a cell disruption solution of large intestine JM109 strain carrying pTrc99A in place of β1,3-N-acetylglucosaminyltransferase, and this was used as a control.

  The reaction was stopped by adding 1/10 volume of 1M sodium hydroxide to the reaction solution, which was diluted and then analyzed by a Dionex DX-500 apparatus. For separation, a CarboPacPA1 column manufactured by Nippon Dionex Co., Ltd. was used, and deionized water, 0.2 M aqueous sodium hydroxide and 1 M aqueous sodium acetate were used as eluents. The amount of LNT-2 in the reaction solution was calculated in terms of lactose from the DX-500 analysis result, and β1,3-N was defined as the activity of synthesizing 1 μmole of N-acetyllactosamine per minute at 30 ° C. -Acetylglucosamine transferase activity was calculated.

(3-3) Preparation of human β1,3-N-acetylglucosaminyltransferase (β3GnTII) Prepared according to the literature (Protein Expr. Purif., 35, 54-61 (2004)).

(3-4) Sialyltransferase α2,3-sialyltransferase (Rat, Recombinant, Spodoptera frugiperda) and α2,6-sialyltransferase (Rat, Recombinant, Spodoptera frugiperda) were purchased from Calbio.

(3-5) Preparation of Trichoderma reesei-derived cellulase Trichoderma reesei-derived cellulase (cellulase XL-522) was purchased from Nagase ChemteX and used after partial purification described below.

  T.A. A crude enzyme solution of reesei cellulase (1000 ml, 875 kU) was treated with 25% saturated ammonium sulfate, and then centrifuged at 4 ° C. using a high-speed microcentrifuge (KUBOTA 1720; using RA-200J rotor, manufactured by KUBOTA) (6010 g). X 20 min) The supernatant was recovered. This was treated with 75% saturated ammonium sulfate, centrifuged under the same conditions, and the resulting precipitate was dissolved in 10 mM sodium phosphate buffer (pH 6.0). After desalting using an ultrafiltration membrane (PM-30, Millipore Corp.) having a molecular weight cut off of 30,000, freeze drying was performed to obtain 7.8 g of enzyme powder. Of these, 1.0 g was dissolved in 10 mM sodium phosphate buffer (pH 6.0) and subjected to DEAE-Sepharose Fast Flow column chromatography (φ2.6 × 18 cm) equilibrated in advance with the same buffer. After washing the column with 1000 ml of the same buffer, stepwise elution was performed with the same buffer containing 600 ml of 500 mM NaCl. The column adsorption part was desalted and concentrated by ultrafiltration, and then freeze-dried to obtain a partially purified enzyme (0.7 g, 0.7 U / mg).

  Further, the partially purified enzyme (50 mg, Lacβ-pNP hydrolyzing activity 35 U, Galβ-pNP hydrolyzing activity 19 U) was dissolved in 50 mM sodium phosphate buffer pH 6.0 (1.0 ml) and equilibrated in advance with the same solvent. The resulting solution was subjected to a modified Gal-amidine affinity column chromatography (φ1.2 × 1.7 cm). 1 ml was dispensed into each Eppendorf tube at a flow rate of 10 ml / h, and the non-adsorbed part was washed with 50 mM sodium phosphate buffer pH 6.0 (30 ml). The adsorbed part was eluted with 50 mM sodium phosphate buffer pH 6.0 (20 ml) containing 1.0 M NaCl, and further eluted with 50 mM sodium acetate buffer pH 4.0 (10 ml) containing 0.5 M methyl β-Gal. The protein was detected by measuring the absorbance at 280 nm, and the hydrolysis activity of Lacβ-pNP and Galβ-pNP was measured. Each fraction was concentrated using an ultrafiltration membrane (PM-30, Millipore Corp.) having a molecular weight cut off of 30000, freeze-dried, and partially purified enzyme (β-D-galactosidase was removed from the non-adsorbed part ( Lacβ-pNP hydrolysis activity 32 U, Galβ-pNP hydrolysis activity 0.3 U). In the subsequent reactions, a partially purified enzyme from which β-D-galactosidase was removed was used. The method for measuring the hydrolysis activity of Lacβ-pNP and Galβ-pNP is as follows.

(Lacβ-pNP hydrolysis activity)
T.A. The enzyme activity of the reesei cellulase was measured by quantifying the amount of pNP released from Lacβ-pNP. 10 mM Lacβ-pNP (25 μl) and 50 mM sodium acetate buffer pH 5.0 (70 μl) were mixed, an appropriate amount of enzyme was added to make a total volume of 100 μl, and the reaction was carried out at 40 ° C. for 20 minutes. Take 10 μl from the reaction solution over time and mix with 1.0 M sodium carbonate solution (190 μl) dispensed in advance into a 96-well microplate. Immediately after stopping the reaction, the absorbance at 405 nm is measured using a plate reader, Liberated pNP was quantified. Enzyme activity 1U was defined as the amount of enzyme that liberates 1 μmol of pNP per minute.

(Galβ-pNP hydrolysis activity)
T.A. The β-D-galactosidase activity measurement method contaminated in reesei cellulase was carried out by quantifying the amount of pNP released from Galβ-pNP. 10 mM Galβ-pNP (25 μl) and 50 mM sodium acetate buffer pH 5.0 (25 μl) were mixed, an appropriate amount of enzyme was added to make a total volume of 100 μl, and the reaction was carried out at 40 ° C. for 20 minutes. Take 10 μl from the reaction solution over time and mix with 1.0 M sodium carbonate solution (190 μl) dispensed in advance into a 96-well microplate. Immediately after stopping the reaction, the absorbance at 405 nm is measured using a plate reader, Liberated pNP was quantified. Enzyme activity 1U was defined as the amount of enzyme that liberates 1 μmol of pNP per minute.

(4) Materials: Reaction substrates and reagents Lactose Monohydrate and 5-amino-1-pentanol were purchased from Wako Pure Chemical Industries, Ltd. γ-PGA was purchased from Meiji Seika Co., Ltd., sugar nucleotides (UDP-GlcNAc, UDP-Gal, CMP-NeuAc) were purchased from Yamasa Shoyu Co., Ltd., and LacNAc was purchased from Yaizu Suisan Co., Ltd. Trifluoroacetic Anhydride and MnCl ₂ .4H ₂ O were purchased from Wako Pure Chemical Industries, Ltd. α-PGA, BOP, HOBt, and BSA were purchased from Sigma-Aldrich.

Abbreviations for the materials used are as follows.
UDP-GlcNAc: UDP-Glucose N-acetylglucosamine
UDP-Gal: UDP-Galactose
CMP-NeuAc: CMP-N-acetylneuraminic acid
pNP: p-nitrophenol
pAP: p-aminophenol
Lac: Lactose (Gal (β1,4) Glc)
LacNAc: N-acetyllactosamine (Gal (β1,4) GlcNAc)
NeuAc: N-acetylneuraminic acid
α-PGA: α-polyglutamic acids
γ-PGA: γ-polyglutamic acids
BOP: Benzotriazol-1-yloxytris- (dimethylamino) phosphonium hexafluorophosphate
HOBt: 1-hydroxybenzotriazole hydrate
PBS: 10 mM Phosphate buffered saline (pH 7.4)
TPS: Sodium 3- (trimethylsilyl) -propionate
BSA: Bovine Serum Albumin

(5) Synthesis Example [Synthesis Example 1]
3'-SialylLacNAc / α-PGA (Poly (Neu5Ac (α2,3) Gal (β1,4) GlcNAc β-p-aminophenyl / α-polyglutamic acid)) and 6'-SialylLacNAc / α-PGA (Poly (NeuAc (Α2,6) Gal (β1,4) GlcNAc β-p-aminophenyl / α-polyglutamic acid))

[1-1] Preparation of 3′-SialylLacNAc / α-PGA According to the method of the literature (Glycobiology, 13, 315-326 (2003)), 3′-SialylLacNAc / α having a sugar substitution degree of 40% and a sialylation rate of 97% -PGA was obtained in a yield of 9.4 mg.

[1-2] Preparation of 6′-SialylLacNAc / α-PGA According to the method of the literature (Glycobiology, 13, 315-326 (2003)), 6′-SialylLacNAc / α having a sugar substitution degree of 40% and a sialylation ratio of 90% -PGA was obtained in a yield of 13.2 mg.

[Synthesis Example 2]
3′-Sialyl (LacNAc) ₂ / α-PGA (Poly (Neu5Ac (α2,3) Gal (β1,4) GlcNAc (β1,3) Gal (β1,4) GlcNAc β-p-aminophenyl / α-polyglutamic acid )), And 6′-Sialyl (LacNAc) ₂ / α-PGA (Poly (Neu5Ac (α2,6) Gal (β1,4) GlcNAc (β1,3) Gal (β1,4) GlcNAc β-p-aminophenyl / Preparation of α-polyglutamic acid))

[2-1] Preparation of (LacNAc) ₂ β-pNP To 150 mM Tris-HCl buffer (pH 6.8, 9.2 ml), 101 mg (0.2 mmol) of LacNAc β-pNP as an acceptor substrate and UDP as a donor substrate -After 260 mg (0.4 mmol) of GlcNAc, 2Na, 32 mg (0.16 mmol) of MnCl ₂ .4H ₂ O as a cofactor were dissolved, and 0.3 ml of 1% (w / v) NaN ₃ was added as a preservative. , 66 mU of β3GnTII was added to carry out the reaction. After 435 hours, when the GlcNAc transfer had progressed 83%, the reaction was stopped by boiling the reaction solution for 5 minutes. Next, 238 mg (0.4 mmol) of UDP-Gal, 2Na was added to the reaction solution, and β1,4-GalT400 mU was added to perform a Gal transfer reaction. The reaction was followed by HPLC as in the GlcNAc transfer reaction. After 483 hours, it was confirmed that the peak of GlcNAc (β1-3) LacNAc β-pNP disappeared on HPLC, and the reaction was stopped by boiling the reaction solution for 5 minutes.

To the reaction solution, 10.7 ml of 150 mM Tris-HCl buffer (pH 6.8) and 0.3 ml of 1% (w / v) NaN ₃ were added. Thereto, 130 mg (0.2 mmol) of UDP-GlcNAc, 2Na was dissolved, and finally 472 mU of β3GnTII was added to initiate the GlcNAc transfer reaction. The reaction was followed by HPLC, and 556 hours after the reaction stopped, another β3GnTII 472 mU was added. 648 hours after the reaction stopped progressing again, the reaction was stopped by boiling the reaction solution for 10 minutes. Subsequently, 119 mg (0.2 mmol) of UDP-Gal, 2Na was added to the reaction solution, and 400 mU of β1,4-GalT was added to perform a Gal transfer reaction. After 813 hours, it was confirmed that the peak of GlcNAc (β1-3) (LacNAc) ₂ β-pNP almost disappeared, and the reaction was stopped by boiling the reaction solution for 10 minutes.

Next, the reaction solution was applied to a Toyopearl HW-40S column pre-equilibrated with 25% MeOH (φ 6.0 × 100 cm; Flow rate, 1.0 ml / ml; 25% MeOH; Fraction size, 10 ml / tube; Detection; 260 nm & 300 nm). As a result, three peaks showing absorption at 260 nm and 300 nm appear, and F-1 (No. 28-46), F-2 (No. 52-70), F-3 (No. 71-91), F -4 (No. 92-124). After concentration with a rotary evaporator and lyophilization, analysis was performed with ¹ H-NMR and HPLC. F-1 is (LacNAc) ₄ β-pNP, F-2 is (LacNAc) ₃ β-pNP, F-3 is GlcNAc (β1-3) (LacNAc) ₂ β-pNP and (LacNAc) ₃ β-pNP. Each included. F-4 was (LacNAc) ₂ β-pNP, and the yield was 30 mg.

[2-2] Preparation of (LacNAc) ₂ β-pAP To 30 ml of H ₂ O, 105 mg (0.12 mmol) of (LacNAc) ₂ β-pNP prepared by the method of (1) above, 210 mg (3.3 mmol) of ammonium formate ) Was completely dissolved. 13.6 mg of 10% Pd / C was added thereto and reacted while performing sonication (ULTRASONIC TRANSDUCER). The reaction was followed by HPLC, and after confirming that the peak of (LacNAc) ₂ β-pNP disappeared, the mixture was centrifuged at 8,000 × g for 20 minutes. The supernatant was collected, and the remaining Pd / C was further removed with a 0.45 μm filter. Next, the reaction solution was concentrated (30 ° C.) with a rotary evaporator and replaced with H ₂ O so as not to dry. Then, it was applied to a Sep-pak C18 column (φ2.5 × 5.5 cm, Waters) previously washed with 100% MeOH and equilibrated with H ₂ O. While flowing H ₂ O, the liquid passing through the column was sampled occasionally, and the absorbance at 210 nm derived from ammonium formate was measured. After confirming that ammonium formate had completely eluted, the solvent was changed to 10% MeOH, and (LacNAc) ₂ β-pAP was eluted while confirming the absorbance at 300 nm derived from the p-aminophenyl group. The eluate was concentrated with a rotary evaporator (30 ° C.), replaced with H ₂ O, and lyophilized to obtain 83.3 mg of (LacNAc) ₂ β-pAP.

Preparation of [2-3] (LacNAc) ₂ / α-PGA (Poly ((LacNAc) ₂ β-pAP / α-PGA)) α-PGA (MW: 11,000) 6.5 mg, BOP 74.5 mg, HOBt8 After dissolving 4 mg in 500 μl of DMSO, 52.5 mg of (LacNAc) ₂ β-pAP was finally dissolved, and the reaction was carried out at room temperature with stirring overnight (20 hours). After completion of the reaction, PBS was added to 1.4 ml, and centrifuged at 6,500 × g for 15 minutes to collect the supernatant. Thereafter, the precipitate was suspended in about 500 μl of PBS and centrifuged again to recover the supernatant. After performing the same operation again, the supernatants were combined, and PBS was added so that the total amount was 2.5 ml.

Then, it was applied to PD-10 (Sephadex G-25, Pharmacia) previously equilibrated with PBS, and (LacNAc) ₂ / α-PGA was eluted with 3.5 ml of PBS. Next, this fraction was boiled and placed in a cellulose tube from which glycerin had been removed, and dialyzed against H ₂ O for 6 days. Meanwhile, the exchange of H ₂ O was performed 5 times. After dialysis, the mixture was concentrated and freeze-dried with a rotary evaporator, and ¹ H-NMR structural analysis was performed. From ¹ H-NMR, the sugar residue substitution degree is determined by integration ratio (a) of sugar chain-derived pAP, integration ratio of β-position and γ-position proton of polyglutamic acid and methyl proton derived from N-acetyl group of GlcNAc. (B) was calculated by fitting to the following equation. As a result, (LacNAc) ₂ / α-PGA having a sugar substitution degree of 37% was obtained in a yield of 20.3 mg.

  Sugar residue substitution rate (%) = ((a / 4) / ((b-6a / 4) / 4)) × 100

[2-4] Synthesis of 3′-Sialyl (LacNAc) ₂ / α-PGA (Poly (Neu5Ac (α2,3) (LacNAc) ₂ β-pAP / α-PGA)) Synthesized in [2-3] above. (LacNAc) ₂ / α-PGA (6.1 mg) and CMP-NeuAc / sodium salt were dissolved in 50 mM MOPS buffer (pH 7.4) to final concentrations of 8 mM and 16 mM, respectively. MnCl ₂ , BSA, and alkaline phosphatase were added to final concentrations of 2.5 mM, 1.0 mg / ml, and 10 U / ml, respectively, and finally 2,3-sialyltransferase was adjusted to 40 mU / ml. The mixture was added and reacted at 37 ° C. for 48 hours. The reaction was boiled for 5 minutes to stop the reaction, centrifuged at 5,500 × g for 5 minutes, and the supernatant was collected.

Next, the precipitate was washed several times with H ₂ O, and the supernatant was further collected to make the total volume 2.5 ml. This was 2.5ml charge against previously _{H 2} O equilibrated with Sephadex G-25M column (PD-10), it was eluted fractions containing the desired product with _{H 2} O of 3.5 ml. This fraction was dialyzed against H ₂ O and then applied to a Dowex AG 50W-X8 (Na ⁺ form) column, which is an ion exchange resin. Thereafter, concentration and freeze-drying were performed, and the structure was confirmed by ¹ H-NMR (FIG. 1). The sialylation ratio was calculated from ¹ H-NMR by applying the integral ratio (a) of pAP protons derived from sugar chains and the integral ratio (c) of 3-position equatorial protons characteristic of Neu5Ac to the following formulas. As a result, 3′-Sialyl (LacNAc) ₂ / α-PGA having a sialylation rate of 94% was obtained in a yield of 5.5 mg.

  In the figure, the structural formula of the sialo-sugar chain-containing polymer shows only the structure of the unsubstituted glutamic acid residue and the sialo-sugar chain-substituted glutamic acid residue in the polymer, and the unsubstituted glutamic acid residue and the sialo-sugar chain-substituted glutamic acid are shown. The number and position of the residues are not limited to those shown in the figure. Further, the wavy line in the figure means that the unsubstituted glutamic acid residue and the sialo-glycan-substituted glutamic acid residue are mixed in an arbitrary ratio. Hereinafter, the same applies to the sialo-sugar chain-containing polymers synthesized in other synthesis examples.

Sialylation rate (%) = ((c / 1) / (a / 4)) × 100

[2-5] Synthesis of 6′-Sialyl (LacNAc) ₂ / α-PGA (Poly (NeuAc (α2,6) (LacNAc) ₂ β-pAP / α-PGA)) LacNAc) ₂ / α-PGA 6.0 mg and CMP-NeuAc · sodium salt were dissolved in 50 mM MOPS buffer (pH 7.4) to a final concentration of 8 mM and 16 mM, respectively, and MnCl ₂ , BSA, alkaline phosphatase were added thereto. Were added to final concentrations of 2.5 mM, 1.0 mg / ml, and 10 U / ml, respectively, and finally 2,6-sialyltransferase was added to 50 mU / ml, and the reaction was allowed to proceed at 37 ° C. for 48 hours. made. the reaction mixture was treated in the same manner as in [2-3], its structure was confirmed by ¹ H-NMR (Fig. 2 . Sialylation ^rate, 1 from H-NMR, integration ratio of protons pAP from sugar (a), the integral ratio of the three characteristic of equatorial proton Neu5Ac (c) and 3-position Aki integration ratio of sialic protons ( d) was calculated by applying the following formula, and as a result, 6'-Sialyl (LacNAc) ₂ / α-PGA having a sialylation rate of 100% was obtained in a yield of 6.3 mg.

Sialylation rate = (((c + d) / 2) / (a / 4)) × 100

[Synthesis Example 3]
3′-Sialyl (LacNAc) ₃ / α-PGA (Poly (Neu5Ac (α2,3) Gal (β1,4) GlcNAc (β1,3) Gal (β1,4) GlcNAc (β1,3) Gal (β1,4 ) GlcNAc β-p-aminophenyl / α-polyglutamic acid))), and 6′-Sialyl (LacNAc) ₃ / α-PGA (Poly (Neu5Ac (α2,6) Gal (β1,4) GlcNAc (β1,3) Gal (Β1,4) GlcNAc (β1,3) Gal (β1,4) GlcNAc β-p-aminophenyl / α-polyglutamic acid))

Preparation of [3-1] (LacNAc) ₃ β-pNP The fraction (F-3) collected on the Toyoperal HW-40S column of Synthesis Example 2 [2-1] was applied to a Toyopearl HW-50S column, and (LacNAc ) ₃ fractions were collected containing the beta-pNP, it could be obtained 88mg of _{(LacNAc) 3} β-pNP.
Preparation of [3-2] (LacNAc) ₃ β-pAP To 55 ml of H ₂ O, 180 mg (0.15 mmol) of (LacNAc) ₃ β-pNP prepared by the method of [3-1] above, 360 mg of ammonium formate (5 0.7 mmol) was completely dissolved. Thereto, 23.4 mg of 10% Pd / C was added and reacted while performing sonication (ULTRASONIC TRANSDUCER). The reaction was followed by HPLC, and after confirming that the peak of (LacNAc) ₃ β-pNP disappeared, the mixture was centrifuged at 8,000 × g for 20 minutes. The supernatant was collected, and the remaining Pd / C was further removed with a 0.45 μm filter. Next, the reaction solution was concentrated (30 ° C.) with a rotary evaporator and replaced with H ₂ O so as not to dry. Then, it was applied to a Sep-pak C18 column (φ2.5 × 5.5 cm, Waters) previously washed with 100% MeOH and equilibrated with H ₂ O. While flowing H ₂ O, the liquid passing through the column was sampled occasionally, and the absorbance at 210 nm derived from ammonium formate was measured. After confirming that ammonium formate had completely eluted, the solvent was changed to 20% MeOH, and (LacNAc) ₃ β-pAP was eluted while confirming the absorbance at 300 nm derived from the p-aminophenyl group. The eluate was concentrated with a rotary evaporator (30 ° C.), replaced with H ₂ O, and freeze-dried to obtain 152.3 mg of (LacNAc) ₃ β-pAP.

[3-3] Preparation of (LacNAc) ₃ / α-PGA (Poly ((LacNAc) ₃ β-pAP / α-PGA)) α-PGA (MW: 11,000) 5.2 mg, BOP 59.6 mg, HOBt6 After dissolving 8 mg in 600 μl of DMSO, 60.3 mg of (LacNAc) ₃ β-pAP was finally dissolved, and the reaction was carried out with stirring at room temperature all day and night (20 hours). After completion of the reaction, PBS was added to 1.4 ml, and centrifuged at 6,500 × g for 15 minutes to collect the supernatant. Thereafter, the precipitate was suspended in about 500 μl of PBS and centrifuged again to recover the supernatant. After performing the same operation again, the supernatants were combined, and PBS was added so that the total amount was 2.5 ml.

It was applied to PD-10 (Sephadex G-25, Pharmacia) previously equilibrated with PBS, and Poly (LacNAc) ₃ β-pAP / α-PGA) was eluted with 3.5 ml of PBS. Next, this fraction was boiled and placed in a cellulose tube from which glycerin had been removed, and dialyzed against H ₂ O for 6 days. Meanwhile, the exchange of H ₂ O was performed 5 times. After dialysis, the mixture was concentrated and freeze-dried with a rotary evaporator, and ¹ H-NMR structural analysis was performed. From ¹ H-NMR, the sugar residue substitution degree is determined by integration ratio (a) of sugar chain-derived pAP, integration ratio of β-position and γ-position proton of polyglutamic acid and methyl proton derived from N-acetyl group of GlcNAc. (B) was calculated by fitting to the following equation. As a result, (LacNAc) ₂ / α-PGA having a sugar residue substitution degree of 36% was obtained in a yield of 20.9 mg.

  Sugar residue substitution degree (%) = ((a / 4) / ((b-9a / 4) / 4)) × 100

[3-4] 3'-Sialyl (LacNAc ) 3 / α-PGA synthesized synthesized above [3-3] of _{(LacNAc) 3 / α-PGA} 6.0mg respectively final concentration CMP-NeuAc · sodium salt It dissolved in 50 mM MOPS buffer (pH 7.4) so that it might become 8 mM and 16 mM. MnCl ₂ , BSA, and alkaline phosphatase were added to final concentrations of 2.5 mM, 1.0 mg / ml, and 10 U / ml, respectively, and finally 2,3-sialyltransferase was adjusted to 40 mU / ml. The mixture was added and reacted at 37 ° C. for 48 hours. The reaction was boiled for 5 minutes to stop the reaction, centrifuged at 5,500 × g for 5 minutes, and the supernatant was collected.

Next, the precipitate was washed several times with H ₂ O, and the supernatant was further collected to make a total volume of 2.5 ml. This was 2.5ml charge against previously _{H 2} O equilibrated with Sephadex G-25M column (PD-10), it was eluted fractions containing the desired product with _{H 2} O of 3.5 ml. This fraction was dialyzed against H ₂ O and then applied to a Dowex AG 50W-X8 (Na ⁺ form) column, which is an ion exchange resin. Thereafter, concentration and freeze-drying were performed, and the structure was confirmed by ¹ H-NMR (FIG. 3). The sialylation ratio was calculated from ¹ H-NMR by applying the integral ratio (a) of pAP protons derived from sugar chains and the integral ratio (c) of 3-position equatorial protons characteristic of Neu5Ac to the following formulas. As a result, 3′-Sialyl (LacNAc) ₃ / α-PGA having a sialylation rate of 100% was obtained in a yield of 6.0 mg.

Sialylation rate (%) = ((c / 1) / (a / 4)) × 100

[3-5] Synthesis of 6′-Sialyl (LacNAc) ₃ / α-PGA (Poly (NeuAc (α2,6) (LacNAc) ₃ β-pAP / α-PGA)) LacNAc) ₃ / α-PGA 6.9 mg and CMP-NeuAc · sodium salt were dissolved in 50 mM MOPS buffer (pH 7.4) to a final concentration of 8 mM and 16 mM, respectively, and then MnCl ₂ , BSA, alkaline phosphatase. Were added to final concentrations of 2.5 mM, 1.0 mg / ml, and 10 U / ml, respectively, and finally 2,6-sialyltransferase was added to 50 mU / ml, and the reaction was allowed to proceed at 37 ° C. for 48 hours. made. the reaction mixture was treated in the same manner as in [3-3], its structure was confirmed by ^{1 H-NMR} (Fig. 4). Allylation ratio ^is 1 than H-NMR, integration ratio of protons pAP from sugar (a), the integral ratio of the three characteristic of equatorial proton Neu5Ac (c) and 3-position Aki integration ratio of sialic protons (d ) Was applied to the following formula, and as a result, 6′-Sialyl (LacNAc) ₃ / α-PGA having a sialylation rate of 100% was obtained in a yield of 6.9 mg.

Sialylation rate = (((c + d) / 2) / (a / 4)) × 100

[Synthesis Example 4]
3′-SialylLacNAc / γ-PGA (Poly (Neu5Ac (α2,3) Gal (β1,4) GlcNAc β-5-aminopentyl / γ-PGA)) and 6′-SialylLacNAc / γ-PGA (Poly (Neu5Ac (α2 , 6) Preparation of Gal (β1,4) GlcNAc β-5-aminopentyl / γ-PGA))

[4-1] Chemical synthesis of 5-Trifluoroacetamido-1-pentanol First, 5-amino-1-pentanol (10 g, 97 mmol) was dissolved by adding pyridine (20 ml). This was ice-cooled and stirred, and trifluoroacetic anhydride (25 ml, 180 mmol) was added dropwise thereto to initiate the reaction. The reaction was confirmed using phosphomolybdic acid color by TLC (developing solvent: chloroform: acetone = 8: 2) every 5 minutes from the start of the reaction. After 1 hour, after confirming disappearance of the raw material by TLC, the reaction was stopped by adding about the same amount of crushed ice as the reaction liquid, and then 200 ml of saturated aqueous sodium hydrogen carbonate solution was added to neutralize the reaction liquid. .

  After the reaction solution was concentrated, an appropriate amount of acetone was added and the mixture was concentrated again. After this operation was repeated about 3 times, the reaction solution was dissolved with acetone to precipitate a large amount of sodium hydrogen carbonate. This was filtered, concentrated, and subjected to silica gel column chromatography (φ4.5 × 35 cm) equilibrated with chloroform: acetone = 8: 2 (10 ml / min). The moving bed that passed through the column was sampled about every 25 ml. The elution fraction was TLC (developing solvent chloroform: acetone = 8: 2), and the product was confirmed using phosphomolybdic acid color. The fraction containing the target product was concentrated to obtain 18 g of the target product, 5-Trifluoroacetamido-1-pentanol, in a yield of 94%.

[4-2] Synthesis of 5-Trifluoroacetamidopentyl β-N-acetyllactosamineide As substrates, N-acetyllactosamine (20.0 g, 52.2 mmol), 5-Trifluoroacetamido-1-pentanol (15.6 g, 78.4 mmol) and 100-mM acetic acid. The galactosidase was dissolved in T. plutonium dissolved in sodium buffer pH 4.0 (52.2 ml). reesei-derived cellulase (6200 U) was added to initiate the reaction. To trace the reaction, 10 μl of the reaction solution was collected over time, 190 μl of demineralized water was added, the reaction was stopped by boiling for 10 minutes at 100 ° C., filtered through a 0.45 μm filter, and analyzed by HPLC. did. The reaction solution was vigorously shaken (200 rpm) and reacted at 40 ° C. for 144 hours. Thereafter, the reaction was stopped by boiling at 100 ° C. for 10 minutes.

  The reaction solution was concentrated and then subjected to activated carbon-celite chromatography (φ4.5 × 100 cm) equilibrated with water (5.0 ml / min). First, LacNAc used as a substrate was eluted by an ethanol linear concentration gradient method of 0% (5.0 L) to 25% (5.0 L). After fractionation by 60 ml / tube, each fraction was measured by absorbance at 210 nm derived from N-acetyl group. By concentrating the fraction containing LacNAc, LacNAc was recovered in an amount of 17.2 g and a recovery rate of 86%. Next, the adsorption part was eluted by switching to 80% ethanol (5.0 L). After fractionation by 60 ml / tube, each fraction was measured by absorbance at 210 nm. Subsequently, the fraction containing the target fraction was concentrated and applied to Silica Gel 60N column chromatography (φ4.5 × 50 cm) equilibrated with chloroform: methanol: water = 7: 3: 0.5 (10 ml / min). The sample was eluted with the same solvent, fractionated by 28 ml / tube, and analyzed by TLC (chloroform: methanol: water = 7: 3: 0.5). The fraction containing the objective fraction was concentrated to obtain 5-Trifluoroacetamidopentyl β-N-acetyllactosaminide in a yield of 322 mg and a yield of 1.1%.

[4-3] Synthesis of 5-aminopentyl β-N-acetyllactosaminide Add 1.0 M NaOH (1.2 ml) to 5-Trifluoroacetamide β-N-acetyllactosamine (100 mg, 0.18 mmol), and react at room temperature. Started. The reaction was confirmed by TLC (developing solvent chloroform: methanol: water = 6: 4: 1) using orcinol sulfate color development and phosphomolybdic acid color development every 30 minutes from the start of the reaction. After 1 hour, after confirming disappearance of the raw material by TLC, the reaction solution was subjected to Sephadex G-25 column chromatography (φ2.5 × 55 cm) equilibrated with water (1.0 ml / min). The moving bed that passed through the column was sampled about every 2.0 ml. The eluted fraction was confirmed to be a product using absorbance at 210 nm derived from the N-acetyl group and phosphomolybdic acid color development by TLC (developing solvent chloroform: methanol: water = 6: 4: 1). The fraction containing the desired product was concentrated to obtain the desired product, 5-aminopentyl β-N-acetyllactosamine, in a yield of 82 mg and a yield of 99%.

[4-4] Synthesis of Poly (5-aminopentyl β-N-acetyllactosaminide / γ-PGA) γ-PGA (MW: 990000, 15.0 mg) was added to 100 mM Na ₂ CO ₃ / NaHCO ₃ aqueous solution (1.3 ml, pH 10 0.0), BOP (119 mg) and HOBt (15 mg) previously dissolved in DMSO (3.5 ml) were added, and the mixture was stirred with a stirrer. Finally, 5-aminopentyl β-N-acetyllactosaminide (140 mg) was dissolved in 100 mM Na ₂ CO ₃ / NaHCO ₃ aqueous solution (0.9 ml, pH 10.0), and reacted at room temperature for 24 hours with stirring. After completion of the reaction, PBS was added so that the reaction solution became 7.5 ml. Thereafter, 2.5 ml of the reaction solution per PD-10 column was applied to a PD-10 (φ1.7 × 5.0 cm, Sephadex G-25) column equilibrated with PBS, and Poly ( 5-aminopentyl β-N-acetyllactosamine / γ-PGA) was eluted.

Next, this fraction was dialyzed against 2.5 L of ultrapure water for 3 days. Meanwhile, the exchange of ultrapure water was performed 6 times. After dialysis, it was concentrated and lyophilized. The sugar residue substitution degree (%) was calculated from ¹ H-NMR based on the integration ratio (A) of β- and γ-position protons and N-acetyl groups of γ-PGA and the aglycon site of 5-aminopentyl β-N-acetyllactosaminide. The integral ratio (B) for six protons was calculated by fitting into the following equation. As a result, Poly (5-aminopentyl β-N-acetyllactosaminide / γ-PGA) having a sugar residue substitution degree of 43% was obtained in a yield of 20.7 mg.

  Sugar residue substitution degree (%) = (4 × 100) / (A− (B / 6 × 3))

[4-5] Synthesis of 3′-SialylLacNAc / γ-PGA Poly (5-aminopentyl β-N-acetyllactosaminide / γ-PGA) _{[43%, 2200 kDa]} as a receptor substrate _{, 8.} 5 mg per LacNAc-unit. 0mM, CMP-Neu5Ac 16.0mM as the donor _{substrate, MnCl 2 2.5mM, BSA 0.1%} , was adjusted to be MOPS buffer (pH7.4) 50mM. Next, 10 U / ml alkaline phosphatase (Boehringer Mannheim) and 40 mU / ml α2,3-sialyltransferase were added to the reaction solution, and the reaction was performed at 37 ° C. for 48 hours. Subsequently, this reaction solution was treated in the same manner as in “2-4” of Synthesis Example 2. From ¹ H-NMR, the sialylation rate was determined from the integration ratio (A) of 8 protons at the aglycon site of 5-aminopentyl β-N-acetyllactosaminide, the integration ratio of the 3-position equatorial and axial protons characteristic of Neu5Ac (B ) Was calculated according to the following formula. As a result, 3′-SialylLacNAc / γ-PGA having a sialylation rate of 100% was obtained in a yield of 6.2 mg (FIG. 5).

Sialylation rate (%) = ((B / 2) × 100) / (A / 8)

[4-6] Synthesis of 6'-SialylLacNAc / γ-PGA Poly (5-aminopentyl β-N-acetyllactosaminide / γ-PGA) _{[43%, 2200 kDa]} as a receptor substrate 8 mg of LacNAc-unit .0mM, CMP-Neu5Ac 16.0mM as the donor _{substrate, MnCl 2 2.5mM, BSA 0.1%} , was adjusted to be MOPS buffer (pH7.4) 50mM. Next, 10 U / ml alkaline phosphatase and 40 mU / ml α2,6-sialyltransferase were added to the reaction solution and reacted at 37 ° C. for 48 hours. Subsequently, this reaction solution was treated in the same manner as in [2-5] of Synthesis Example 2. From the ¹ H-NMR, the sialylation rate was determined from the integration ratio (A) of 8 protons at the aglycon site of 5-aminopentyl β-N-acetyllactosaminide, the integration ratio of the 3-position equatorial and axial protons characteristic of Neu5Ac (B ) Was calculated according to the following formula. As a result, 6′-SialylLacNAc / γ-PGA having a sialylation rate of 100% was obtained in a yield of 6.8 mg (FIG. 6).

Sialylation rate (%) = ((B / 2) × 100) / (A / 8)

[Synthesis Example 5]
3′-Sialyl (LacNAc) ₂ / γ-PGA (Poly (Neu5Ac (α2,3) Gal (β1,4) GlcNAc (β1,3) Gal (β1,4) GlcNAc β-5-aminopentyl / γ-polyglutamic acid )), And 6′-Sialyl (LacNAc) ₂ / γ-PGA (Poly (Neu5Ac (α2,6) Gal (β1,4) GlcNAc (β1,3) Gal (β1,4) GlcNAc β-5-aminopentyl / Preparation of γ-polyglutamic acid))

[5-1] Synthesis of 5-Trifluoroacetamidopentyl β- (LacNAc) ₂ 5-Trifluoroacetamidopentyl β-N- prepared in “4-2” of Synthesis Example 4 in 150 mM Tris-HCl buffer (pH 6.8, 44.6 ml) 300 mg (0.53 mmol) of aceticylactosamine, 692 mg (1.06 mmol) of UDP-GlcNAc · 2Na as a donor substrate, 84.1 mg (0.42 mmol) of MnCl ₂ · 4H ₂ O as a cofactor were dissolved and used as a preservative. After adding 0.53 ml of 1% (w / v) NaN ₂ , 7.9 ml (600 mU) of purified β3GnTII was added and the reaction was carried out at 37 ° C. After 97 hours, when the GlcNAc transfer progressed 100%, the reaction was stopped by boiling the reaction solution for 5 minutes.

Next, 671 mg (1.10 mmol) of UDP-Gal · 2Na was added to the reaction solution, and 300 μl (6000 mU) of β4GalTI was added to perform a Gal transfer reaction. After 162 hours, the reaction was stopped by boiling the reaction solution for 5 minutes. The reaction solution was concentrated and subjected to ODS column chromatography (φ2.5 × 50 cm) equilibrated with 10% MeOH (1.5 ml / min). After fractionation by 22 ml / tube, each fraction was measured by absorbance at 210 nm. After the raw materials and the like were completely eluted, the solvent was switched to 20% MeOH and the fraction containing the target fraction was concentrated to obtain 5-Trifluoroacetamidopentyl β- (LacNAc) ₂ in a yield of 355 mg and a yield of 72%.

[5-2] Synthesis of 5-aminopentyl β- (LacNAc) ₂ Add 1.0M NaOH (1.0 ml) to 5-Trifluoroacetamidopentyl β- (LacNAc) ₂ (154 mg, 0.17 mmol) and dissolve at room temperature. Reacted for 1 hour. Using TLC (developing solvent: chloroform: methanol: water = 6: 4: 1) using orcinol sulfate color development and phosphomolybdic acid color development, the reaction solution was equilibrated with water (0. 5 ml / min) was subjected to Sephadex G-25 column chromatography (φ2.5 × 55 cm). The eluted fraction was confirmed to be a product using absorbance at 210 nm derived from the N-acetyl group and phosphomolybdic acid coloring by TLC (developing solvent chloroform: methanol: water = 6: 4: 1). The fraction containing the desired product was concentrated to obtain the desired product, 5-Trifluoroacetamidopentyl β- (LacNAc) ₂ , in a yield of 136 mg and a yield of 98%.

[5-3] Synthesis of Poly (5-aminopentyl β- (LacNAc) ₂ / γ-PGA) γ-PGA (MW: 990000, 15.0 mg) was added to 100 mM Na ₂ CO ₃ / NaHCO ₃ aqueous solution (2 (0.0 ml, pH 10.0), BOP (118 mg) and HOBt (15 mg) previously dissolved in DMSO (5.5 ml) were added, and the mixture was stirred with a stirrer. Finally, 5-aminopentyl β- (LacNAc) ₂ (83 mg) was dissolved in 100 mM Na ₂ CO ₃ / NaHCO ₃ aqueous solution (1.0 ml, pH 10.0) and then added and reacted at room temperature for 24 hours with stirring. It was. After completion of the reaction, PBS was added so that the reaction solution was 10.0 ml.

Thereafter, 2.5 ml of the reaction solution per PD-10 column was applied to a PD-10 (φ1.7 × 5.0 cm, Sephadex G-25) column equilibrated with PBS, and Poly ( 5-aminopentyl β-N-acetyllactosamine / γ-PGA) was eluted. Next, this fraction was dialyzed against 2.5 L of ultrapure water for 3 days. Meanwhile, the exchange of ultrapure water was performed 6 times. After dialysis, it was concentrated and lyophilized. The sugar residue substitution degree (%) was calculated from ¹ H-NMR, based on the integration ratio (A) of β- and γ-position protons and N-acetyl groups of γ-PGA and the aglycon site of 5-aminopentyl β- (LacNAc) ₂ The integral ratio (B) for six protons of was calculated by fitting into the following equation. As a result, Poly (5-aminopentyl β- (LacNAc) ₂ / γ-PGA) having a sugar residue substitution degree of 48% was obtained in a yield of 31.8 mg.

  Sugar residue substitution degree (%) = (4 × 100) / (A− (B / 6 × 6))

[5-4] Synthesis of 3′-Sialyl (LacNAc) ₂ / γ-PGA Poly (5-aminopentyl β- (LacNAc) ₂ / γ-PGA) of the above “5-3” _{[48%, 3500 kDa]} 8 mg was adjusted to be 8.0 mM per (LacNAc) ₂ -unit, CMP-Neu5Ac 16.0 mM, MnCl ₂ 2.5 mM, BSA 0.1%, MOPS buffer (pH 7.4) 50 mM as a donor substrate. Next, 10 U / ml alkaline phosphatase and 40 mU / ml α2,3-sialyltransferase were added to the reaction solution and reacted at 37 ° C. for 48 hours. Subsequently, this reaction solution was treated in the same manner as in [2-4] of Synthesis Example 2. From the ¹ H-NMR, the sialylation rate was determined from the integration ratio (A) of 8 protons at the aglycon site of 5-aminopentyl β- (LacNAc) ₂ and the integration ratio of the 3-position equatorial and axial protons characteristic of Neu5Ac ( B) was calculated by fitting into the following equation. As a result, 3′-Sialyl (LacNAc) ₂ / γ-PGA having a sialylation rate of 100% was obtained in a yield of 6.6 mg (FIG. 7).

Sialylation rate (%) = ((B / 2) × 100) / (A / 8)

[5-5] Synthesis of 6′-Sialyl (LacNAc) ₂ / γ-PGA Poly (5-aminopentyl β- (LacNAc) ₂ / γ-PGA) _{[48%, 3500 kDa] of the} above “5-3”. 9 mg was adjusted to be 8.0 mM per (LacNAc) ₂ -unit, CMP-Neu5Ac 16.0 mM as donor substrate, MnCl ₂ 2.5 mM, BSA 0.1%, MOPS buffer (pH 7.4) 50 mM. Next, 10 U / ml alkaline phosphatase and 40 mU / ml α2,6-sialyltransferase were added to the reaction solution and reacted at 37 ° C. for 48 hours. Subsequently, this reaction solution was treated in the same manner as in [2-5] of Synthesis Example 2. From the ¹ H-NMR, the sialylation rate was determined from the integration ratio (A) of 8 protons at the aglycon site of 5-aminopentyl β- (LacNAc) ₂ and the integration ratio of the 3-position equatorial and axial protons characteristic of Neu5Ac ( B) was calculated by fitting into the following equation. As a result, 6′-Sialyl (LacNAc) ₂ / γ-PGA having a sialylation rate of 100% was obtained in a yield of 6.5 mg (FIG. 8).

Sialylation rate (%) = ((B / 2) × 100) / (A / 8)

[Synthesis Example 6]
3′-Sialyl (LacNAc) ₃ / γ-PGA (Poly (Neu5Ac (α2,3) Gal (β1,4) GlcNAc (β1,3) Gal (β1,4) GlcNAc (β1,3) Gal (β1,4 ) GlcNAc β-5-aminopentyl / γ-polyglutamic acid))) and 6′-Sialyl (LacNAc) ₃ / γ-PGA (Poly (Neu5Ac (α2,6) Gal (β1,4) GlcNAc (β1,3) Gal ( β1,4) GlcNAc (β1,3) Gal (β1,4) GlcNAc β-5-aminopentyl / γ-polyglutamic acid))

[6-1] Synthesis of 5-Trifluoroacetamidopentyl β- (LacNAc) ₃ 5-Trifluoroacetamidopentyl β- (LacNAc) prepared by the method of Synthesis Example 5 (1) in 150 mM Tris-HCl buffer (pH 6.8, 8.8 ml). ₂ ) 197 mg (0.21 mmol), UDP-GlcNAc · 2Na as a donor substrate, 276 mg (0.42 mmol), MnCl ₂ · 4H ₂ O as a cofactor 33.6 mg (0.17 mmol) are dissolved, and a preservative Then, 0.21 ml of 1% (w / v) NaN ₂ was added, and then 12.2 ml (345 mU) of purified β3GnTII was added and the reaction was performed at 37 ° C. After 48 hours, when the GlcNAc transfer progressed 100%, the reaction was stopped by boiling the reaction solution for 5 minutes. Next, 259 mg (0.42 mmol) of UDP-Gal · 2Na was added to the reaction solution, and 200 μl of β4GalTI (2000 mU) was added to perform a Gal transfer reaction. After 288 hours, the reaction was stopped by boiling the reaction solution for 5 minutes when the Gal transition advanced 100%. The reaction solution was concentrated and subjected to ODS column chromatography (φ3.5 × 60 cm) equilibrated with 10% MeOH (1.5 ml / min). After the raw materials and the like were completely eluted, the solvent was switched to 20% MeOH and the fraction containing the target substance was concentrated to obtain 5-Trifluoroacetamidopentyl β- (LacNAc) ₃ in a yield of 232 mg and a yield of 85%.

Synthesis of [6-2] 5-aminopentylβ- (LacNAc) ₃ Add 1.0 M NaOH (1.3 ml) to 5-Trifluoroacetamidopentylβ- (LacNAc) ₃ (197 mg, 0.15 mmol) and dissolve at room temperature for 1 hour. Reacted. The reaction was confirmed using TLC (developing solvent: chloroform: methanol: water = 6: 4: 1) using orcinol sulfate coloration and phosphomolybdic acid coloration. After confirming the disappearance of the raw materials, the reaction solution was equilibrated with water. (1.0 ml / min) and subjected to Sephadex G-25 column chromatography (φ2.5 × 55 cm). The eluted fraction was confirmed to be a product using absorbance at 210 nm derived from the N-acetyl group and phosphomolybdic acid coloring by TLC (developing solvent chloroform: methanol: water = 6: 4: 1). The fraction containing the desired product was concentrated to obtain the desired product, 5-aminopentyl β- (LacNAc) ₃ , in a yield of 145 mg and a yield of 81%.

[6-3] Synthesis of Poly (5-aminopentyl β- (LacNAc) ₃ / γ-PGA) γ-PGA (MW: 990,000, 15.0 mg) was added to 100 mM Na ₂ CO ₃ / NaHCO ₃ aqueous solution (2. (0 ml, pH 10.0), BOP (118 mg) and HOBt (15 mg) previously dissolved in DMSO (5.5 ml) were added, and the mixture was stirred with a stirrer. Finally, 5-aminopentyl β- (LAcNAc) ₃ (119 mg) was dissolved in 100 mM Na ₂ CO ₃ / NaHCO ₃ aqueous solution (1.0 ml, pH 10.0), followed by reaction at room temperature for 24 hours with stirring. After completion of the reaction, PBS was added so that the reaction solution was 10.0 ml. Thereafter, 2.5 ml of the reaction solution per PD-10 column was applied to a PD-10 (φ1.7 × 5.0 cm, Sephadex G-25) column equilibrated with PBS, and Poly ( 5-aminopentyl β-N-acetyllactosamine / γ-PGA) was eluted. Next, this fraction was dialyzed against 2.5 L of ultrapure water for 3 days. Meanwhile, the exchange of ultrapure water was performed 6 times. After dialysis, it was concentrated and lyophilized. The sugar residue substitution degree (%) was calculated from ¹ H-NMR based on the β and γ-position proton and N-acetyl group integration ratio (A) of γ-PGA and the aglycon site of 5-aminopentyl β- (LAcNAc) ₃ The integral ratio (B) for six protons of was calculated by fitting into the following equation. As a result, Poly (5-aminopentyl β- (LacNAc) ₃ / γ-PGA) having a sugar residue substitution degree of 44% was obtained in a yield of 38.6 mg.

  Sugar residue substitution degree (%) = (4 × 100) / (A− (B / 6 × 9))

[6-4] Synthesis of 3′-Sialyl (LacNAc) ₃ / γ-PGA Poly (5-aminopentyl β- (LacNAc) ₃ / γ-PGA) synthesized in [6-3] above as a receptor substrate _{[44 %, 4300 kDa]} 5.6 mg (LacNAc) ₃ -8.0 mM per unit, CMP-Neu5Ac 16.0 mM as donor substrate, MnCl ₂ 2.5 mM, BSA 0.1%, MOPS buffer (pH 7.4) 50 mM It adjusted so that it might become. Next, 10 U / ml alkaline phosphatase and 40 mU / ml α2,3-sialyltransferase were added to the reaction solution and reacted at 37 ° C. for 48 hours. Subsequently, this reaction solution was treated in the same manner as in “3-4” of Synthesis Example 3. From the ¹ H-NMR, the sialylation rate was determined from the integration ratio (A) of 8 protons at the aglycon site of 5-aminopentyl β- (LacNAc) ₃ and the integration ratio of the 3-position equatorial and axial protons characteristic of Neu5Ac ( B) was calculated by fitting into the following equation. As a result, 3′-Sialyl (LacNAc) ₃ / γ-PGA having a sialylation rate of 100% was obtained in a yield of 6.0 mg (FIG. 9).

Sialylation rate (%) = ((B / 2) × 100) / (A / 8)

[6-5] Synthesis of 6′-Sialyl (LacNAc) ₃ / γ-PGA Poly (5-aminopentyl β- (LacNAc) ₃ / γ-PGA) synthesized in the above [6-3] as a receptor substrate _{[44 %, 4300 kDa]} 5.5 mg (LacNAc) ₃ -8.0 mM per unit, CMP-Neu5Ac 16.0 mM as donor substrate, MnCl ₂ 2.5 mM, BSA 0.1%, MOPS buffer (pH 7.4) 50 mM It adjusted so that it might become. Next, 10 U / ml alkaline phosphatase and 40 mU / ml α2,6-sialyltransferase were added to the reaction solution and reacted at 37 ° C. for 48 hours. Subsequently, this reaction solution was treated in the same manner as in [3-5] of Synthesis Example 3. From the ¹ H-NMR, the sialylation rate was determined from the integration ratio (A) of 8 protons at the aglycon site of 5-aminopentyl β- (LacNAc) ₃ and the integration ratio of the 3-position equatorial and axial protons characteristic of Neu5Ac ( B) was calculated by fitting into the following equation. As a result, 6′-Sialyl (LacNAc) ₃ / γ-PGA having a sialylation rate of 100% was obtained in a yield of 5.4 mg (FIG. 10).

Sialylation rate (%) = ((B / 2) × 100) / (A / 8)

[Synthesis Example 7]
6′-SialylLacNAc-Lac / γ-PGA (Poly (Neu5Ac (α2,6) Gal (β1,4) GlcNAc (β1,3) Gal (β1,4) GlcNAc (β1,3) Gal (β1,4) Glc Preparation of β-5-aminopentyl / γ-polyglutamic acid))

[7-1] Synthesis of 5-Trifluoroacetamidopentyl β-lactoside (Lac-5AP-TFA) As substrates, lactose (54.3 g, 151 mmol), 5-Trifluoroacetamido-1-pentanol (30.0 g, 151 mmol) and 50 mM sodium acetate Dissolved in buffer pH 5.0 (151 ml), galactosidase was removed therefrom. reesei-derived cellulase (4500 U) was added to initiate the reaction. The reaction solution was vigorously shaken (200 rpm), reacted at 40 ° C. for 120 hours, and then stopped by boiling at 100 ° C. for 10 minutes. After concentrating the reaction mixture, it was applied to Silica Gel 60N column chromatography (φ4.5 × 50 cm) equilibrated with chloroform: methanol: water = 7: 3: 0.5 (10 ml / min) and eluted with the same solvent, 23 ml / Tube was fractionated and analyzed by TLC (chloroform: methanol: water = 7: 3: 0.5). The fraction containing the target fraction was concentrated, dissolved in heavy water, and structurally analyzed by ¹ H-NMR. As a result, 5-Trifluoroacetamidopentyl β-lactoside was obtained in a yield of 849 mg and a yield of 1.0%.

[7-2] Synthesis of LacNAc-Lac-5AP-TFA 10 mM Lac-5AP-TFA (100 mg), 200 mM Tris-HCl buffer (pH 7.0), 20 mM manganese chloride, 15 mM UDP-GlcNAc, 3 U / mL CIAP, 20 ml of a solution containing 0.3 U / mL β1,3-GlcNAc transferase was incubated at 20 ° C. for 42 hours. After stopping the reaction by boiling for 3 minutes, manganese chloride and UDP-galactose were added to the reaction stop solution so that the final concentrations were 20 mM and 12 mM, respectively, and 0.1 U / mL β1,4-galactose transferase was present. The reaction was carried out at 37 ° C. for 20 hours. After boiling for 3 minutes, the residue was removed by centrifugation (20,000 × g, 10 minutes), and the obtained supernatant was applied to a chromatolex column (200 ml, Fuji Silysia Chemical Co., Ltd.) equilibrated with distilled water. did. After washing with 400 ml distilled water, the target substance was eluted with 20% methanol, and this was concentrated by an evaporator and vacuum dried (50 ° C.) to obtain 108 mg of LacNAc-Lac-5AP-TFA powder.

[7-3] Preparation of LacNAc-Lac-5AP 98.9 mg of LacNAc-Lac-5AP-TFA was dissolved in 400 ml of 1M sodium hydroxide and stirred at room temperature for 30 minutes. The completion of the reaction was confirmed by TLC (CHCl ₃ : MeOH: H ₂ O = 4: 5: 1, colored with anisaldehyde), and then adjusted to pH 5.5 with 0.5 M hydrogen chloride. The reaction solution was adjusted to 5 ml with distilled water, and then purified using a Sephadex G-25 column (GE Healthcare Biosciences, 150 ml) equilibrated with distilled water. The elution position of the target product was confirmed by TLC (CHCl ₃ : MeOH: H ₂ O = 4: 5: 1, colored with anisaldehyde), then the target fraction was collected, concentrated in an evaporator, and vacuum dried (50 ° C.) To collect 103 mg of LacNAc-Lac-5AP powder.

[7-4] Synthesis of LacNAc-Lac / γ-PGA (Poly (Gal (β1,4) GlcNAc (β1,3) Gal (β1,4) Glc β-5-aminopentyl / γ-PGA) LacNAc-Lac- 5AP 92.7 mg, 1-hydroxybenzotriazole anhydrous (HOBt) 15 mg, γ-PGA 13 mg, triethylamine 15 μl were completely dissolved in 1 ml of distilled water and 0.8 ml of DMF, and 1-ethyl-3- ( 58 mg of 3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) was added and stirred overnight at room temperature, 1 ml of distilled water was added to the reaction solution, and then 1 ml of 0.5 M sodium hydroxide solution was added to adjust the pH to 11. The base treatment solution was placed in a dialysis tube and 400 ml distilled water was stirred. After collecting the dialyzed solution, it was applied to a Sephadex G-50 column (8 ml) equilibrated with distilled water, and the collected fraction was further dialyzed three times with 400 ml distilled water and three times with 400 ml ultrapure water. The dialyzed internal solution was collected, replaced with sodium salt using an AG50W resin column (6 ml, sodium type, Bio-Rad), and then concentrated by an evaporator, and the resulting concentrated solution was lyophilized to obtain LacNAc. 44.4 mg of -Lac-5AP / γ-PGA was obtained, and the sugar residue substitution rate (DS) was 55% by ¹ H-NMR analysis.

[7-5] Synthesis of 6′-SialylLacNAc-Lac / γ-PGA 50 mM cacodylate buffer (pH 6.0), 2.5 mM manganese chloride, 20 mg LacNAc-Lac-5AP / γPGA, 20 mM CMP-NeuAc, 0. 2 ml of a solution containing 1% (W / V) BSA, 20 U / mL CIAP, 0.02 U / mL α2,6-sialyltransferase (Calbiochem) was incubated at 37 ° C. for 44 hours and then boiled for 1 minute. After removing the residue by centrifugation (20,000 × g, 10 minutes), the supernatant was applied to an ultrapure water equilibrated gel filtration column (Sephadex G-50, 8 ml). The target fraction was collected and dialyzed three times with 400 ml distilled water and three times with 400 ml ultrapure water. After recovering the dialyzed solution, it was replaced with a sodium salt using an AG50W resin column (6 ml, sodium type), and then concentrated by an evaporator. The obtained concentrated solution was freeze-dried to obtain 22.1 mg of 6′-SialylLacNAc-Lac / γPGA. In addition, the sialylation rate was 94% by ¹ H-NMR analysis (FIG. 11).

Test example 1
3′-SialylLacNAc / α-PGA, 3′-Sialyl (LacNAc) ₂ / α-PGA, and 3′-Sialyl (LacNAc) ₃ / α-PGA and 6′-SialylLacNAc / α-PGA, 6′-Sialyl ( In vitro measurement of influenza virus infection inhibitory activity of LacNAc) ₂ / α-PGA and 6′-Sialyl (LacNAc) ₃ / α-PGA As human influenza A / WSN / 33 (H1N1) (infectivity titer 2 ^{-6 HAU), Human strain A /} Aichi / 2/68 (H3N2) ( infectivity ^{2 -6 HAU), Avian strain A} / Duck / HongKong / 313/4/76 (H5N3) ( infectivity 2 ^-3 HAU) Infect Using the Mardin-Darby canine kidney cells (MDCK) as vesicles.

MDCK cells prepared in a cell suspension of 1.5 × 10 ⁵ cells / ml in a 10% FCS-containing MEM medium were seeded in 0.1 ml each in a 96-well plate and cultured at 37 ° C. overnight. On the next day, the influenza virus solution was diluted with Serum free MEM (SF-MEM) to achieve the above infectivity. The diluted virus solution and the serially diluted inhibitor solution were mixed in equal amounts on another microplate and preincubated for 1 hour at 4 ° C.

  After rinsing the cell culture well three times with SF-MEM, the cells were infected with the preincubation solution. Infection was carried out at 34.5 ° C. for 30 minutes. After removing the virus-inhibitor solution, the cells were washed 3 times with SF-MEM, Overlay medium (0.5% tragagant gum, 0.2% BSA-containing MEM) was added to the well, and 20-24 at 34.5 ° C. Incubate for hours.

  After completion of the culture, the overlay medium was removed, and the cells were fixed with 100% MeOH at room temperature for 10 minutes. After rinsing with PBS three times, a mouse anti-NP monoclonal antibody (clone H16: L10-4R5) was reacted as a primary antibody for 1 hour at room temperature. After rinsing with PBS three times, a HRP-labeled goat anti-mouse Ig antibody was reacted as a secondary antibody for 1 hour at room temperature. After rinsing three times with PBS, Konica Immunostain (HRP-1000) reagent was added to detect infected cells. Infected cells (blue cell population, focus) were counted under a light microscope. Triplicate measurements were taken for each sample and each concentration. The ratio of the relative focus number at the written inhibitor concentration when the average of the focus number when no inhibitor was added was taken as 100% was determined.

The results of the human strain A / WSN / 33 (H1N1) virus infection inhibition experiment are shown in FIG. 12, the human strain A / Aichi / 2/68 (H3N2) virus infection inhibition experiment is shown in FIG. 13, and the Avian strain A / Duck / The result of HongKong / 313/4/76 (H5N3) is shown in FIG. In addition, the density | concentration of the sialo-sugar chain containing polymer which is a compound is shown by the molar concentration of polymer itself. In Test 1, as a control, three types of asialogyl sugar chain-containing α-polyglutamic acid polymers having different repeating units of LacNAc (LacNAc / α-PGA, (LacNAc) ₂ / α-PGA, α-PGA, (LacNAc) ₃ / α-PGA) was used. As is apparent from FIG. 12 to FIG. 14, the α2,6-linked type sialo-sugar chain-containing polymer specifically inhibits human influenza virus infection, while the α2,3-linked type sialo-sugar chain-containing polymer. The polymer was confirmed to specifically inhibit infection against avian influenza virus.

In addition, polylactosamine-type sialosugar chain-containing polymers (Sialyl (LacNAc) ₂ / α-PGA and Sialyl (LacNAc) ₃ / α-PGA) are 1 / L of conventional SialylLacNAc type polymers (SialylLacNAc / α-PGA). Infection was inhibited at a concentration of 10 or 1/100, and it was confirmed that the polylactosamine-type sialosugar chain-containing polymer has 10 to 100 times higher infection-inhibiting activity than the conventional SialylLacNAc type polymer.

Test example 2
Measurement of In Vivo Animal Infection Inhibitory Activity of 6′-SialylLacNAc / α-PGA and 6′-Sialyl (LacNAc) ₃ / α-PGA Influenza Virus Infected viruses include Human strain, A / WSN / 33 (H1N1 ), BALB / c mice (female, 5weeks) were used as target animals, and 6 mice were used as one administration group. After the sialo-glycan chain-containing polymer solution and the virus solution were mixed well in equal amounts (the virus titer was final and 2 × 10 ⁴ pfu / ml, and the final sialo-glycan-containing polymer concentration (sugar chain unit) was 15 μM), then the nasal cavity of the mouse 50 μl per mouse was administered in a volume of 25 μl per side.

When the control polymers containing asialo sugar chains (LacNAc / α-PGA and (LacNAc) ₃ / α-PGA) and 6′-SialylLacNAc / α-PGA were used, all mice died on the 16th day after infection. On the other hand, in the group of mice administered with 6′-Sialyl (LacNAc) ₃ / α-PGA, only one mouse died and the remaining five survived.

Test example 3
Measurement of Anti-Human Influenza Virus Activity of Sialo Sugar Chain-Containing Polymers In Vitro Anti-influenza virus activity was tested by the CPE suppression method (Antivital Chemistry & Chemotherapy, 2, 243-248 (1991)). However, viruses that infect uses a human influenza virus (A strain Ishikawa (H3N2)) , cells to be infected using the above MDCK cells, also studied in two series of viral load 50TCID ₅₀ and ¹⁰ 2 TCID ₅₀ to infect did. Ribavirin was used as a positive control.

  As a result, as shown in Table 2, the anti-influenza virus activity of the conventional 6′-SialylLacNAc / α-PGA is weak, whereas the α-PGA skeleton polylactosamine-type sialo-sugar chain-containing polymer and γ-PGA in the present invention It was confirmed that the polymer containing a skeletal sialo-sugar chain has high anti-influenza virus activity.

Test example 4
3′-SialylLacNAc / γ-PGA, 3′-Sialyl (LacNAc) ₂ / γ-PGA, 3′-Sialyl (LacNAc) ₃ / γ-PGA, and 6′-SialylLacNAc / α-PGA, 6′-Sialyl ( 1. Measurement of influenza virus infection inhibitory activity of LacNAc) ₂ / α-PGA and 6′-Sialyl (LacNAc) ₃ / α-PGA in vitro Experimental procedure Measurement of influenza virus infectivity by focus assay method MDCK cells were seeded in a 96-well microplate for culturing so that the number of cells was 1.5 × 10 ⁵ cells / well, and cultured at 37 ° C. until confluent. . A solution in which a virus (human A / WSN / 33 (H1N1) strain) diluted with 0.1 μM zanamivir-containing serum free (SF) -MEM medium and a polymer was allowed to stand at 4 ° C. for 1 hour. After washing MDCK cells with serum free (SF) -MEM medium at a rate of 100 μl / well three times, the virus-polymer mixed solution was added to 50 μl / well and infected in a CO ₂ incubator at 34.5 ° C. for 30 minutes. I let you. After infection, each well was washed three times with SF-MEM medium at 100 μl / well, and 0.2% bovine serum albumin (BSA) and 0.5% tragacanthum-containing MEM medium were added to 50 μl / well. The cells were cultured at 34.5 ° C. in a CO ₂ incubator for about 20 hours. After completion of the culture, the medium was removed, ethanol was added at 100 μl / well, and the mixture was allowed to stand at room temperature for 10 minutes to immobilize the cells. After washing with 100 μl / well three times with phosphate-buffered saline (PBS), an anti-nucleoprotein monoclonal antibody solution diluted with 1% BSA-containing PBS solution was added to 50 μl / well and allowed to stand at room temperature for 1 hour. After washing with PBS 100 μl / well three times, an HRP-labeled goat anti-mouse immunoglobulin antibody solution diluted with 1% BSA-containing PBS solution was added to 50 μl / well and allowed to stand at room temperature for 1 hour. After the reaction, the plate was washed with PBS 100 μl / well three times, and OPD chromogenic substrate solution (100 mM Citrate-phosphate buffer, pH 5.5, 0.4 mg / ml o-phenylenediamine, 0.012% H ₂ O ₂ ) 50 μl / It was added to become a well and allowed to stand at room temperature. After color development, a reaction stop solution (1N HCl) was added to a concentration of 0 μl / well. Absorbance was measured with a microplate reader at a measurement wavelength of 492 nm and a control wavelength of 630 nm. The absorbance at the time of each polymer reaction when the absorbance of the well infected with the virus without reacting with the polymer was taken as 100% was determined as a relative infectivity value. The horizontal axis of the graph indicates the molar concentration of the polymer itself.

2. Experimental Results As shown in FIG. 15, it was observed that the effect of inhibiting influenza virus infection was enhanced by extending the internal sugar chain (LacNAc) in any polymer. In particular, the effect of the polymer having (LacNAc) repeated three times was the largest. In the figure, “3′SLN1” is 3′-SialylLacNAc / γ-PGA, “3′SLN2” is 3′-Sialyl (LacNAc) ₂ / γ-PGA, and “3′SLN3” is 3′-Sialyl (LacNAc). ) ₃ / γ-PGA. “6′SLN1” is 6′-SialylLacNAc / γ-PGA, “6′SLN2” is 6′-Sialyl (LacNAc) ₂ / γ-PGA, and “6′SLN3” is 6′-Sialyl (LacNAc) _3. / Γ-PGA.

Formulation Example 1
After dissolving the sialo-sugar chain-containing polymer, which is the active ingredient of the present invention, in purified water, citric acid, disodium hydrogen phosphate and the like are added as necessary to obtain the nasal preparation (liquid) of the present invention.

FIG. 1 shows an NMR chart of 3′-Sialyl (LacNAc) ₂ / α-PGA. FIG. 2 shows an NMR chart of 6′-Sialyl (LacNAc) ₂ / α-PGA. FIG. 3 shows an NMR chart of 3′-Sialyl (LacNAc) ₃ / α-PGA. FIG. 4 shows an NMR chart of 6′-Sialyl (LacNAc) ₃ / α-PGA. FIG. 5 shows an NMR chart of 3′-Sialyl (LacNAc) / γ-PGA. FIG. 6 shows an NMR chart of 6'-Sialyl (LacNAc) / γ-PGA. FIG. 7 shows an NMR chart of 3′-Sialyl (LacNAc) ₂ / γ-PGA. FIG. 8 shows an NMR chart of 6′-Sialyl (LacNAc) ₂ / γ-PGA. FIG. 9 shows an NMR chart of 3′-Sialyl (LacNAc) ₃ / γ-PGA. FIG. 10 shows an NMR chart of 6′-Sialyl (LacNAc) ₃ / γ-PGA. FIG. 11 shows an NMR chart of 6'-SialylLacNAc-Lac / γ-PGA. FIG. 12 shows the results of the human strain A / WSN / 33 (H1N1) virus infection inhibition experiment of Test Example 1. FIG. 13 shows the results of a human strain A / Aichi / 2/68 (H3N2) virus infection inhibition experiment in Test Example 1. FIG. 14 shows the results of Avian strain A / Duck / Hong Kong / 313/4/76 (H5N3) of Test Example 1. FIG. 15 shows the results of measurement of influenza virus infection inhibitory activity by the focus assay method of Test Example 4.

Claims (8)

An antiviral agent comprising, as an active ingredient, a sialo-sugar chain-containing polymer represented by the following formula (I) and having a sialo-sugar chain bonded to γ-polyglutamic acid.

(I)

(II)
(In formula (I), Z is a hydroxyl group or a sialo-glycan binding site represented by formula (II), and n represents an integer of 10 or more. In formula (II), Y ₁ and Y ₂ are a hydroxyl group or N-acetylneuraminic acid residue, X is a hydroxyl group or acetylamino group, R is a hydrocarbon or oligosaccharide, and n is an integer of 10 or more, provided that Y ₁ and Y ₂ are not the same. The antiviral agent according to claim 1, wherein the oligosaccharide represented by R is represented by the following formula (III) or (IV).

(III)

(IV)
(In the formula, L represents a hydrocarbon, and X ₁ and X ₂ represent a hydroxyl group or an acetylamino group. However, X ₁ and X ₂ may be the same or different.) An antiviral agent comprising, as an active ingredient, a sialo-sugar chain-containing polymer represented by the following formula (V) and having α-polyglutamic acid bonded to the sialo-sugar chain.

(V)

(II ')
(In Formula (V), Z is a hydroxyl group or a sialo-sugar chain part represented by Formula (II ′), and n represents an integer of 10 or more. In Formula (II ′), Y ₁ and Y ₂ are hydroxyl groups. Or an N-acetylneuraminic acid residue, X represents a hydroxyl group or an acetylamino group, and R ′ represents an oligosaccharide, provided that Y ₁ and Y ₂ are not the same. The antiviral agent according to claim 3, wherein the oligosaccharide represented by R 'is represented by the following formula (III) or (IV).

(III)

(IV)
(In the formula, L represents a hydrocarbon, and X ₁ and X ₂ represent a hydroxyl group or an acetylamino group. However, X ₁ and X ₂ may be the same or different.) The antiviral agent according to any one of claims 1 to 4, which is an anti-influenza virus agent. The antiviral agent according to any one of claims 1 to 5, having a molecular weight of 2 to 5 million. The antiviral agent according to any one of claims 1 to 6, wherein the degree of polymerization of glutamic acid units is 10 to 10,000. The antiviral agent according to any one of claims 1 to 7, wherein the introduction rate of the sialosugar chain to the glutamic acid residue is 10 to 80%.

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