JP2005213195A - New sialic acid donor and method for producing the same and method for introducing sialic acid using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for introducing a sialic acid into a sugar chain in high efficiency by a simple means without using any sulfur compound emitting intense offensive odor. <P>SOLUTION: A new sialic acid donor is provided, being represented by formula(I)( wherein, R is a 6-15C long-chain alkyl; and R' is a protective group ). The method for introducing a sialic acid involves using the donor. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The invention of this application relates to a sialic acid donor for introducing sialic acid into a sugar derivative or the like. More specifically, the invention of this application relates to a method for introducing sialic acid into a sugar chain with high efficiency by a simple method without using a sulfur compound having a strong malodor, and a sialic acid donor for the method.

  The invention of this application relates to a novel branched sialoglycomolecule. More specifically, the invention of this application relates to pharmaceuticals and viruses that can cope with variations in the host range and antigenic variation of influenza viruses and can prevent infection of all human and animal-derived influenza A and B viruses. The present invention relates to a novel branched sialoglycomolecule useful as an adsorbent for removal filters and the like.

  Sialic acid is a generic name for 2-keto-3-deoxynononic acid with a carboxyl group consisting of nine skeletal carbons. N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), deaminoneuron Three major molecular species of laminic acid (KDN) and their modified sialic acids such as acetylation, lactylation and sulfation are known. Such diversity of molecular species of monosaccharides is a feature not found in other monosaccharides, and is one basis on which sialic acid is considered to have a unique biological function.

  In addition, sialic acid is an important component of various functional sugar chains located in the peripheral part of glycoproteins and glycolipids. By binding to lactose and N-acetyllactosamine, cancer, aging, etc. It is known to be a receptor for markers and viruses involved in Therefore, it is considered an extremely important sugar from the biochemical or biomedical viewpoint.

  For example, in Japan, infection with influenza, which occurs in several hundred thousand to over one million patients every year, binds hemagglutinin (HA), a viral glycoprotein, to sialic acid-containing sugar chains present on the host cell surface. It is known that this occurs (Non-Patent Documents 1 to 3). Therefore, this sialic acid-containing sugar chain has been extensively studied, and various sialic acid-containing sugar chain (sialog sugar chain) compounds have been reported as anti-influenza virus agents (Non-Patent Documents 4 to 5, Patent Document 1).

However, since the amount of sialic acid-containing sugars that can be extracted from nature is limited, it is necessary to perform chemical synthesis in order to supply these compounds in large quantities to the market. Several methods for constructing sugar chains into which sialic acid has been introduced have been reported so far, but the actual situation is that further improvements relating to the efficiency of synthesis and derivatization are desired. In addition, sialic acid donors obtained by glycosylation utilizing thioglycosides have been used in the conventionally reported methods for introducing sialic acid into sugar chains, but mercaptans used in such glycosylation reactions have been used. There was a problem that the kind had a strong odor. Furthermore, since the yield and stereoselectivity of the product are low, there is a problem that the purification process becomes complicated.
Suzuki, Y .: Progress in Lipid Research, 33, 429-457 (1994) Paulson, JC: The Receptors, Vol. 2. Academic Press, Orlando, pp. 131-219 (1985) Wiley, DC and Skehel, JJ: Annual Review of Biochemistry, 56, 365-394 (1987) Fujimoto, K., Hayashida, O., Aoyama, Y., Guo, CT, Hidari, KIPJ, and Suzuki, Y .: Chemistry Letter, 1259-1260 (1999) Suzuki, Y., Sato, K., Kiso, M., and Hasegawa, A .: Glycoconj J. 7, 349-54 (1990) Marra, A. and Sinay, P., Carbohydrate Research, 190 (1989) 317-322. Japanese Patent Application 2001-75081

  Therefore, the invention of this application has been made in view of the circumstances as described above, solves the problems of the prior art, and does not use a sulfur compound having a strong malodor and is highly efficient by a simple method. It is an object to provide a method for introducing sialic acid into a chain.

  In order to solve the above problems, the invention of this application includes, firstly, the following formula (I):

(However, R is a long-chain alkyl group having 6 to 15 carbon atoms, and R ′ represents a protecting group.)
A novel sialic acid donor represented by the formula:

  The invention of this application is, secondly, the following formula (II)

(Where R ′ represents a protecting group)
Is reacted with an alkanethiol having a long-chain alkyl group having 6 to 15 carbon atoms to convert the anomeric R′O group in the fully protected sialic acid methyl ester into an alkylthio group. And a method for producing a novel sialic acid donor.

  The third aspect of the invention of this application is the following formula (I):

(However, R is a long-chain alkyl group having 6 to 15 carbon atoms, and R ′ represents a protecting group.)
A method for introducing sialic acid into a sugar derivative is provided, wherein the activated alkylthioglycoside and a compound to which sialic acid is to be introduced are reacted in the presence of molecular sieves. .

  Since the novel sialic acid donor of the first invention is efficiently and easily synthesized by reacting a long-chain alkanethiol with a low odor and a complete protector of sialic acid, it is compared with the conventional sialic acid donor. Workability is high. Further, if the RHN group at the 5-position of such a novel sialic acid donor is converted to an amino group, an azide can be obtained.

  Further, the novel sialic acid donor produced by the method of the second invention can be obtained as an anomeric mixture, but since these can be easily separated, a complicated purification step is not required.

  Furthermore, in the method for introducing sialic acid according to the third aspect of the invention, a glycosylation reaction of a sugar chain or the like is performed using a novel sialic acid donor, and any anomers (α-form, β-form) are used. , Α form can be obtained with high stereoselectivity. Therefore, it can be said that this is a highly convenient sialic acid introduction method.

  The novel sialic acid donor of the invention of this application has the following formula (I)

It is represented by At this time, R is a long-chain alkyl group, specifically, a long-chain alkyl group having 6 to 15 carbon atoms. R 'represents a protecting group, and examples thereof include ester-based protecting groups such as Ac (acetyl) and Bz (benzoyl) and alkyl-based protecting groups such as Bn (benzyl). Of these, ester protecting groups are preferred.

  Such a novel sialic acid donor is a complete protector of sialic acid, that is, the following formula (II)

It is obtained by reacting acetosialic acid methyl ester represented by the formula (I) with an alkanethiol having an alkyl group length of C6-15. Examples of the alkanethiol include hexanethiol, heptanethiol, octanethiol, nonanethiol, decanethiol, undecanethiol, dodecanethiol, tridecanethiol, tetradecanethiol, and pentadecanethiol. At this time, the alkyl group may be linear or branched.

  In such a reaction, the concentration of the reaction substrate, the reaction temperature, the atmosphere of the reaction system and the like are not particularly limited. For example, an excess of alkanethiol, specifically 1.2 to 4 equivalents, per equivalent of acetosialic acid methyl ester In addition, it can be ice-cooled. At this time, you may use organic solvents, such as chloroform, a dichloromethane, and toluene, as a reaction solvent. A Lewis acid such as trimethylsilyl trifluoromethanesulfonate or boron trifluoride diethyl ether complex can be added in order to activate the anomeric R′O group and facilitate attack by alkanethiol. Among these, boron trifluoride diethyl ether complex is preferably used because it is inexpensive and easily available.

  By the reaction as described above, the anomeric R′O group at the 2-position in acetosialic acid methyl ester is converted to an alkylthio group, and the novel sialic acid donor is obtained. In such a reaction, since the alkyl chain length of the alkanethiol used is as long as C6 to 15 and there is little odor, the workability is better than the conventional sialic acid donor using a sulfur compound having a bad odor.

  In addition, the novel sialic acid donor of the invention of this application is produced as an anomeric mixture, but it can be easily subjected to α-alkylthioglycoside and β-alkylthioside through general purification steps such as washing, extraction, drying and column chromatography. Can be isolated to glycosides. The production ratio of α-alkylthioglycoside and β-alkylthioglycoside varies slightly depending on the reaction conditions and the like, but usually a β-alkylthio form is selectively produced.

  The novel sialic acid donor of the present invention as described above can be used for simply introducing sialic acid into an organic compound or sugar chain. Specifically, sialic acid can be introduced into the compound by reacting an alkylthioglycoside with the compound into which sialic acid is to be introduced in the presence of molecular sieves. At this time, the molecular sieves act as removing water present in the reaction system.

  Moreover, in such a reaction, although it is necessary to activate thioglycoside, the reagent for that is not specifically limited. For example, a combination of N-iodosuccinimide and trimethylsilyl triflate is applicable.

  The alkyl glycoside may be either α-form or β-form, or an anomeric mixture.

  Moreover, the compound made to react with alkylthioglycoside is not specifically limited, Various things are applicable. The introduction of sialic acid occurs by a condensation reaction with an alkylthioglycoside with an unprotected OH group. That is, the sialic acid residue is bonded to the compound via the O atom. Therefore, it is desirable that the compound to be reacted with the alkylthioglycoside has a hydroxyl group. Specific examples include sugar chains such as glucose OH derivatives and lactose OH derivatives, and alcohols such as cyclohexanol. When the compound for introducing sialic acid is a sugar chain, it may be a monosaccharide, an oligosaccharide or a polysaccharide, and in the case of an alcohol, its molecular weight and structure are not particularly limited.

  Hereinafter, examples will be shown, and the embodiments of the present invention will be described in more detail. Of course, the present invention is not limited to the following examples, and it goes without saying that various aspects are possible in detail.

Example 1 Methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-laurylthio-α / β-D-glycero-D-galacto-2-nonuropyranoside ) Onate (Methyl (5-acetoamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-lauryl thio-α / β-D-glycero-D-galacto-2-nonulopyranoside) onate) (Synthesis of Compound 2α and Compound 2β) Compound 2α and Compound 2β were synthesized according to the following reaction formula (A).

  First, acetosialic acid methyl ester (methyl 5-acetamido-2,4,7,8,9-penta-O-acetyl-3,5-dideoxy-D-glycero-D-galacto-2-nonuropyranosonate Compound 1: synthesized by the method of Non-Patent Document 6 (5.6 g, 10.5 mmol) was dissolved in dichloromethane (60 mL), dodecanethiol (10.1 mL, 42.0 mmol, 4 eq) was added, and the mixture was ice-cooled.

  Next, boron trifluoride diethyl ether complex (Tokyo Chemical Industry) (4.0 mL, 31.5 mmol, 3 eq) was added dropwise and stirred for 30 minutes. The mixture was warmed to room temperature and further stirred for 3 hours.

  The reaction solution was diluted with chloroform, and the organic layer was washed successively with cold water, a cold saturated aqueous sodium hydrogen carbonate solution and saturated brine, and then the organic layer was dried over anhydrous magnesium sulfate. The organic layer was filtered and the filtrate was concentrated. The residue was purified by silica gel chromatography [toluene-ethyl acetate (1: 2), 600 mL] to obtain a β-laurylthio compound (compound 2β) (4.3 g, 59.2%) and an α-laurylthio compound (compound 2α) (1.9 g). , 26.2%).

  The identification results of compounds 2α and 2β are shown in Table 1.

Example 2 Methyl O- (Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl 3,5-deoxy-D-glycero-D-galacto-2-nonuropyranosylate)- (2 → 6) -2,3,4-Tri-O-benzyl-α-D-glycopyranoside (Methyl O- (Methyl 5-acetoamido-4,7,8,9-tetra-O-acetyl3,5-deoxy -D-glycero-D-galacto-2-nonulopyranosylonate)-(2 → 6) -2,3,4-tri-O-benzyl-α-D-glcopyranoside) (compounds 7α and 7β) Compound 7α and Compound 7β were synthesized according to the reaction formula (B).

  Compound 2β (390 mg, 0.58 mmol) and glucose 6-OH derivative (compound 3) (130 mg, 0.28 mmol) are dissolved in acetonitrile (2.6 ml), and MS3A (260 mg) is added thereto, and several times at room temperature. Stir for hours. The reaction mixture was cooled to −35 ° C., N-iodosuccinimide (260 mg, 1.15 mmol) and trimethylsilyl triflate (21 μL, 0.12 mmol) were sequentially added, and the mixture was stirred at the same temperature for 3 hours.

  The reaction solution was diluted with chloroform and filtered through celite. The filtrate was washed successively with a cold saturated aqueous sodium hydrogen carbonate solution, a 1 M aqueous sodium thiosulfate solution and saturated brine, and the organic layer was dried over anhydrous magnesium sulfate. After filtration, the filtrate was concentrated, and the residue was purified by silica gel column chromatography [toluene-ethyl acetate (1: 2), 60 mL], and α- (2 → 6) linked disaccharide derivative (compound 7α). (160 mg, 61.1%) and β- (2 → 6) linked disaccharide derivative (compound 7β) (85 mg, 32.4%) were isolated, respectively.

  The identification results are shown in Table 2.

Example 3 Methyl (cyclohexyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-D-galacto-2-nonuropyranoside) oneate -acetoamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-D-galacto-2-nonulopyranoside) onate) (hereinafter referred to as compound 8α and compound 8β) Compound 8α and Compound 8β were synthesized according to the following reaction formula (C).

  Compound 2β (180 mg, 0.27 mmol) was dissolved in acetonitrile (1.5 mL), MS3A (120 mg) and cyclohexanol (compound 4) (14 μL, 0.13 mmol) were further added, and the mixture was stirred for several hours. The reaction mixture was cooled to −35 ° C., N-iodosuccinimide (Tokyo Chemical Industry) (120 mg, 0.53 mmol) and trimethylsilyl triflate (9 μL, 0.05 mmol) were sequentially added, and the mixture was stirred at the same temperature for 3 hours.

  The reaction solution was diluted with chloroform and filtered through celite. The filtrate was washed successively with a cold saturated aqueous sodium hydrogen carbonate solution, a 1 M aqueous sodium thiosulfate solution and saturated brine, and the organic layer was dried over anhydrous magnesium sulfate. After filtration, the filtrate was concentrated, and the residue was purified by silica gel column chromatography [toluene-ethyl acetate (1: 2) 30 mL], α-cyclohexyl glycoside (Compound 8α) (40 mg, 52%) , And β-cyclohexyl glycoside (Compound 8β) (30 mg, 39%) were isolated, respectively.

  The identification results are shown in Table 3.

Example 4 Benzyl [methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-D-galacto-2-nonuropyranosyl) onate]-(2 → 6) -O- (2,3,4-Tri-O-benzyl-β-D-galactopyranosyl)-(1 → 4) -2,3,6-tri-O-benzyl-β-D -Glucopyranoside (Benzyl [Methyl (5-acetamido-4,7,8,9-tetra-O-acethyl-3,5-dideoxy-D-glycero-D-galacto-2-nonulopyranosyl) onate]-(2 → 6 ) -O- (2,3,4-tri-O-benzyl-β-D-galactopyranosyl)-(1 → 4) -2,3,6-tri-O-benzyl-β-D-glucopyranoside (hereinafter, Synthesis of Compound 9α and Compound 9β) According to the following reaction formula (D), Compound 9α and Compound 9β were synthesized.

  Compound 2β (206 mg, 0.30 mmol) and lactose 6′-OH derivative (compound 5) (157 mg, 0.16 mmol) were dissolved in acetonitrile (1.5 mL), and MS3A (150 mg) was added thereto at room temperature. Stir for several hours. The reaction mixture was cooled to −35 ° C., N-iodosuccinimide (137 mg, 0.61 mmol) and trimethylsilyl triflate (11 μL, 0.06 mmol) were sequentially added, and the mixture was stirred at the same temperature for 3 hours.

  The reaction solution was diluted with chloroform and filtered through celite. The filtrate was washed successively with a cold saturated aqueous sodium hydrogen carbonate solution, a 1 M aqueous sodium thiosulfate solution and saturated brine, and the organic layer was dried over anhydrous magnesium sulfate. After filtration, the filtrate was concentrated, and the residue was purified by silica gel column chromatography [toluene-ethyl acetate (1: 1), 40 mL] to obtain an α- (2 → 6) -linked trisaccharide derivative (Compound 9α). ) (110 mg, 47.2%) and a β- (2 → 6) linked trisaccharide derivative (Compound 9β) (84 mg, 36.1%) were isolated.

  The identification results are shown in Table 4.

Example 5 2- (Trimethylsilyl) ethyl [Methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2- Nonuropyranosyl) onate]-(2 → 3) -O- (2,6-di-O-benzyl-β-D-galactopyranosyl)-(1 → 4) -2,3,6-tri-O- Benzyl-β-D-glucopyranoside (2- (Trimethylsilyl) ethyl [Methyl (5-acetamido-4,7,8,9-tetra-O-acethyl-3,5-dideoxy-D-glycero-α-D-galacto -2-nonulopyranosyl) onate]-(2 → 3) -O- (2,6-di-O-benzyl-β-D-galactopyranosyl)-(1 → 4) -2,3,6-tri-O- benzyl-β-D-glucopyranoside) (hereinafter compound 10)
Compound 10 was synthesized according to the following reaction formula (E).

  MS3A (6.4 g) was added to compound 2β (5.0 g, 7.2 mmol) and lactose 3 ′, 4′-OH derivative (3.2 g, 3.6 mmol), dissolved in acetonitrile (64 mL), and stirred for several hours. The reaction solution was cooled to −35 ° C., N-iodosuccinimide (3.3 g, 14.5 mmol) and trimethylsilyl triflate (265 mL, 1.5 mmol) were sequentially added, and the mixture was stirred for 3 hours.

The reaction solution was diluted with chloroform and filtered through celite. The filtrate was extracted in the order of cold saturated aqueous sodium hydrogen carbonate solution, 1 M aqueous sodium thiosulfate solution and saturated brine, and the organic layer was dried over magnesium sulfate. The filtrate was concentrated and purified by silica gel column chromatography [toluene-ethyl acetate (1: 1 → 0: 1), 500 mL], and α- (2 → 3) -linked trisaccharide derivative (2.7 g, 55.0% )
<Example 6>
The same reaction as in Examples 2 to 5 was performed using Compound 2α synthesized in Example 1. The yield and stereoselectivity of the obtained product are shown in Table 5.

  From the above, it was confirmed that by using the novel sialic acid donor of the invention of this application, the introduction of sialic acid into the compound proceeds easily and with high yield and stereoselectivity.

  As described above in detail, the present invention provides a method for introducing sialic acid into a sugar chain with high efficiency by a simple method without using a sulfur compound having a strong malodor.

Claims (3)

Formula (I)

(However, R is a long-chain alkyl group having 6 to 15 carbon atoms, and R ′ represents a protecting group.)
A novel sialic acid donor represented by the formula: A process for producing the novel sialic acid donor according to claim 1, wherein the formula (II)

(Where R ′ represents a protecting group)
Is reacted with an alkanethiol having a long-chain alkyl group having 6 to 15 carbon atoms to convert the anomeric R′O group in the fully protected sialic acid methyl ester into an alkylthio group. And a method for producing a novel sialic acid donor. Formula (I)

(However, R is a long-chain alkyl group having 6 to 15 carbon atoms, and R ′ represents a protecting group.)
A method for introducing sialic acid into a sugar derivative, wherein the activated alkylthioglycoside and a compound to which sialic acid is to be introduced are reacted in the presence of molecular sieves.