JPH11279192A - Production of new chromanol glycoside - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the new glycoside having pH stability, water solubility, resistance to oxidation and protecting actions on radiation, by subjecting a saccharide derivative in which an elimination group is introduced to the anomer site and other hydroxyl groups are protected with protecting groups with a 2-substituted chromanol to a condensation reaction. SOLUTION: This new chromanol glycoside is shown by formula I [R<1> to R<5> are each H, a lower alkyl or a lower acyl; X is a monosaccharide residue (H of hydroxyl group in the saccharide residue may be substituted with a lower alkyl or a lower acyl); (n) is an integer of 0-4] and is expected to be useful in various field since it has excellently protecting actions on radiation besides excellent heat stability, pH stability and antioxidative activity. The compound is obtained by subjecting a saccharide derivative in which an elimination group is introduced to the anomer site and other hydroxyl groups are protected with hydroxyl groups and a 2-substituted alcohol derivative of formula II to a condensation reaction.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a novel chromanol glycoside and a chromanol glycoside produced by the method. More specifically,
The present invention relates to a method for producing a chromanol glycoside using an organic synthesis technique and a chromanol glycoside produced by the method.

[0002]

2. Description of the Related Art The following general formula:

[0003]

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(Wherein, R ¹ , R ² , R ³ and R ⁴ represent the same or different hydrogen atoms or lower alkyl groups, R ⁵ represents a hydrogen atom, a lower alkyl group or a lower acyl group; Represents a monosaccharide residue or an oligosaccharide residue, wherein the hydrogen atom of the hydroxyl group of the sugar residue may be substituted with a lower alkyl group or a lower acyl group, and n is 0 to
Is an integer of 4 and m is an integer of 1 to 6).
No. 7 reports that the compound has excellent antioxidant activity, heat and pH stability, and water solubility.

[0005] In recent years, studies by the present inventors have revealed that chromanol glycosides represented by the above general formula have, in addition to the above-mentioned properties, a radioprotective effect and an inflammatory bowel disease preventive and therapeutic effect. Was.

[0006] Chromanol glycosides having such various excellent properties have heretofore been prepared by using an enzyme that catalyzes the transglycosylation by the following general formula:

[0007]

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(Wherein, R ¹ , R ² , R ³ and R ⁴ represent the same or different hydrogen atoms or lower alkyl groups, R ⁵ represents a hydrogen atom, a lower alkyl group or a lower acyl group, and n is an integer of 0 to 4.)
By reacting a 2-substituted alcohol represented by the formula with a saccharide in the presence of an organic solvent (JP-A-7-1182)
No. 87) or by reacting a 2-substituted alcohol represented by the above general formula with a sugar in the presence of an organic solvent using an immobilized enzyme in which α-glucosidase is immobilized on porous chitosan beads (Japanese Unexamined Patent Publication No. 9-249688
No.), has been manufactured.

However, both of the above methods
Although an enzyme is used, it is well applicable when an enzyme capable of efficiently transferring the target saccharide to the 2-substituted alcohol is available. However, depending on the type of saccharide such as fucose, saccharide can be used. An enzyme capable of producing the desired chromanol glycoside by transferring to a substituted alcohol has not yet been discovered, or is very expensive even if known, and even if the enzyme producing the desired chromanol glycoside is not available. Even so, if the reaction efficiency is very poor, there is a problem that the yield is poor and it cannot be economically adapted.

[0010] On the other hand, as described above, chromanol glycosides have various properties.
It naturally depends on the type of sugar that binds to the substituted alcohol.Furthermore, the sugar that binds to the stability of the glycosidic bond of the chromanol glycoside and the manner of incorporation into cells when the chromanol glycoside is administered to a living body. It is common sense that there is a difference depending on the type. Therefore, the desired properties (for example, antioxidant activity, heat stability, pH stability,
It is very useful to examine the solubility in water, the radioprotective effect or the effect of preventing or treating inflammatory bowel disease), the stability of glycosidic bonds and the ease of incorporation into cells.

Therefore, there is a strong demand for a method for producing a chromanol glycoside which can overcome the drawbacks such as the inability to transfer the sugar by the enzyme or the inefficient production of the desired chromanol glycoside even if the sugar can be transferred. Existing.

[0012]

Accordingly, an object of the present invention is to provide a method for producing a novel chromanol glycoside.

Another object of the present invention is to provide a chromanol derivative by organic synthesis which overcomes the drawbacks such as the inability to transfer sugar by an enzyme or the inefficient production of a desired chromanol glycoside even if transfer is possible. An object of the present invention is to provide a method for producing a saccharide.

A further object of the present invention is to provide a method for producing a chromanol glycoside in a high yield while preventing the decomposition of the chromanol glycoside produced.

Still another object of the present invention is to provide a chromanol glycoside produced by such a method.

[0016]

Means for Solving the Problems The present inventors have conducted intensive studies in view of the above circumstances, and as a result, have found that a sugar derivative having a leaving group introduced at the anomeric position and another hydroxyl group protected with a protecting group is obtained. −
It was discovered that the desired chromanol glycoside can be efficiently synthesized by condensation with a substituted alcohol derivative. In addition to the above, the present inventors have also conducted intensive studies on a method for deprotecting a protecting group after the condensation reaction, and as a result, deprotection of the protecting group was performed by acid decomposition, enzymatic decomposition, or metal reagents. It has been found that the use of the compound effectively prevents decomposition of the produced chromanol glycoside, thereby improving the yield of the chromanol glycoside. Based on these findings, the present invention has been completed.

That is, the above objects are achieved by the following (A) to (N).

(A) A sugar derivative in which a leaving group is introduced at the anomeric position and another hydroxyl group is protected with a protecting group, and a compound represented by the general formula (2):

[0019]

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[Wherein, R ¹ , R ² , R ³ and R ⁴ represent the same or different hydrogen atom, lower alkyl group or lower acyl group, and R ⁵ represents a hydrogen atom, lower alkyl group or lower acyl group. And n is an integer from 0 to 4. A condensation reaction of a 2-substituted alcohol derivative represented by the general formula (1):

[0021]

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[Wherein, R ¹ , R ² , R ³ and R ⁴ represent the same or different hydrogen atoms, lower alkyl groups or lower acyl groups, and R ⁵ represents a hydrogen atom, lower alkyl group or lower acyl group. X represents a monosaccharide residue (however, a hydrogen atom of a hydroxyl group in the sugar residue may be substituted with a lower alkyl group or a lower acyl group), and n is an integer of 0 to 4. . ] The manufacturing method of the chromanol glycoside represented by these.

(B) The sugar derivative is an L-fucose donor represented by the general formula (3), an L-rhamnose donor represented by the general formula (4), or a general formula (5) The method according to (a) above, which is a D-xylose donor, a D-galactose donor represented by the general formula (6), or a D-mannose donor represented by the general formula (7).

[0024]

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[0025]

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[0026]

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[0027]

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[0028]

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[However, equations (3), (4), (5),
In (6) and (7), Y represents a halogen atom, a sulfur compound or a trichloroacetimide group, and preferentially takes either α-form or β-form depending on the type thereof, and R represents the same or different acetyl group. Benzoyl group, pivaloyl group, chloroacetyl group, levulinoyl group, benzyl group, p-methoxybenzyl group, allyl group, t-butyldimethylsilyl group, t-butyldiphenylsilyl group, trimethylsilyl group or trityl group. (C) The method according to (a) or (b) above, wherein the deprotection of the protecting group after the condensation reaction is carried out by acid decomposition.

(D) The method according to (c) above, wherein the acid decomposition is carried out in the presence of a solvent obtained by adding hydrochloric acid to methanolic hydrochloric acid or alcohol.

(E) The method according to (a) or (b) above, wherein the deprotection of the protecting group after the condensation reaction is carried out by enzymatic decomposition.

(F) The method according to (e) above, wherein the enzyme used for the enzymatic decomposition is esterase or lipase.

(G) The deprotection of a protecting group after the condensation reaction is carried out using a metal reagent.
Or the method described in (b).

(H) The method according to (g), wherein the metal reagent is lithium aluminum hydride or diisobutylaluminum hydride.

(I) General formula (1) produced by the method according to any one of (A) to (H):

[0036]

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Wherein R ¹ , R ² , R ³ and R ⁴ represent the same or different hydrogen atoms, lower alkyl groups or lower acyl groups, and R ⁵ represents a hydrogen atom, lower alkyl group or lower acyl group. X represents a monosaccharide residue (however, a hydrogen atom of a hydroxyl group in the sugar residue may be substituted with a lower alkyl group or a lower acyl group), and n is an integer of 0 to 4. . A chromanol glycoside represented by the formula:

(N) The following general formulas (8), (9) and (1)
0), (11) or (12), wherein
2. The chromanol glycoside according to item 1.

[0039]

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[0040]

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[0041]

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[0042]

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[0043]

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[Equations (8), (9), (10),
In (11) and (12), R ¹ , R ² , R ³ and R ⁴
Represents the same or different hydrogen atom, lower alkyl group or lower acyl group, R ⁵ represents a hydrogen atom, lower alkyl group or lower acyl group, n is an integer of 0 to 4, and R is the same or different acetyl Group, benzoyl group,
Represents a pivaloyl group, a chloroacetyl group, a levulinoyl group, a benzyl group, a p-methoxybenzyl group, an allyl group, a t-butyldimethylsilyl group, a t-butyldiphenylsilyl group, a trimethylsilyl group or a trityl group. ]

[0045]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention provides the following general formula (2):

[0046]

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[Wherein, R ¹ , R ² , R ³ and R ⁴ represent the same or different hydrogen atom, lower alkyl group or lower acyl group, and R ⁵ represents a hydrogen atom, lower alkyl group or lower acyl group. And n is an integer from 0 to 4. A derivative of a sugar in which a leaving group is introduced at the anomeric position by using a 2-substituted alcohol derivative represented by the following formula (hereinafter referred to as “sugar acceptor”) and another hydroxyl group is protected by a protecting group (hereinafter referred to as “sugar acceptor”) , Referred to as “sugar donor”) to cause a condensation reaction to give the following general formula (1):

[0048]

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[Wherein, R ¹ , R ² , R ³ and R ⁴ represent the same or different hydrogen atoms, lower alkyl groups or lower acyl groups, and R ⁵ represents a hydrogen atom, lower alkyl group or lower acyl group. X represents a monosaccharide residue (however, a hydrogen atom of a hydroxyl group in the sugar residue may be substituted with a lower alkyl group or a lower acyl group), and n is an integer of 0 to 4. . ] The chromanol glycoside represented by this is manufactured.

The present invention will be described in detail below.

In the present invention, the sugar donor is a sugar derivative in which a leaving group is introduced at the anomeric position of a sugar residue, and another hydroxyl group is protected with a protecting group. At this time, examples of the leaving group introduced at the anomeric position include halogen atoms such as chlorine, bromine and fluorine, sulfur compounds such as thiomethyl group, thioethyl group and thiophenyl group, and trichloroacetimide group. Of these leaving groups, bromine, chlorine, thiomethyl, thioethyl, thiophenyl and trichloroacetimide groups are preferably used.

In the present invention, the protecting group for protecting a hydroxyl group other than the anomeric position includes an acyl protecting group such as an acetyl group, a benzoyl group, a pivaloyl group, a chloroacetyl group and a levulinoyl group; a benzyl group; Examples include ether-based protecting groups such as methoxybenzyl, allyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trimethylsilyl, and trityl. Among these protecting groups, an acyl protecting group, particularly an acetyl group, is preferably used.

The sugar moiety in the sugar donor according to the present invention is
Although it depends on the type of the desired chromanol glycoside, specifically, D, L-glucose, D, L-galactose, D, L-fucose, D, L-xylose, D,
L-mannose, D, L-rhamnose, D, L-arabinose, D, L-lyxose, D, L-ribose, D,
L-allose, D, L-altrose, D, L-idose, D, L-talose, D, L-deoxyribose,
D, L-2-deoxyribose, D, L-quinobose and D, L-avequose. Of these, D, L-glucose, D, L-galactose, D,
L-fucose, D, L-xylose, D, L-mannose, D, L-rhamnose, D, L-arabinose, D,
L-lyxose, D, L-ribose, D, L-talose, D, L-deoxyribose and D, L-2-deoxyribose are preferably used, and particularly, L-lyxose represented by the following general formula (3). Fucose donor, L-rhamnose donor represented by the following general formula (4), D-xylose donor represented by the following general formula (5), D-galactose donor represented by the following general formula (6) Or a D-mannose donor represented by the following general formula (7) is preferably used as a sugar donor.

[0054]

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[0055]

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[0056]

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[0057]

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[0058]

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[However, expressions (3), (4), (5),
In (6) and (7), Y represents a halogen atom, a sulfur compound or a trichloroacetimide group, and preferentially takes either α-form or β-form depending on the type thereof, and R represents the same or different acetyl group. Benzoyl group, pivaloyl group, chloroacetyl group, levulinoyl group, benzyl group, p-methoxybenzyl group, allyl group, t-butyldimethylsilyl group, t-butyldiphenylsilyl group, trimethylsilyl group or trityl group. According to the method of the present invention, when the hydroxyl group at the 2-position of the sugar donor is condensed by protecting it with the acyl protecting group, a chromanol glycoside having a 1,2-trans-type glycoside bond due to an adjacent group effect Is obtained. For example, a gluco-type sugar in which the 2-position hydroxyl group such as glucose, galactose, fucose and xylose is coordinated in equatorial form is bonded to an aglycone in β-form, and the 2-position hydroxyl group such as mannose or rhamnose is axially conformed. Manno-type sugars are α-type and bind to aglycone.

Further, in the above general formulas (1) and (2), which represent the chromanol glycoside and the sugar acceptor in the present invention, respectively, R ¹ , R ² , R ³ and R
⁴ represents the same or different hydrogen atom or lower alkyl group. When representing a lower alkyl group, the number of carbon atoms is preferably 1 to 8, more preferably 1 to 8.
And lower alkyl groups such as methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, pentyl group, isopentyl group, hexyl group, heptyl group and octyl group. home,
Methyl and ethyl groups are preferred. Similarly, R
⁵ represents a hydrogen atom, a lower alkyl group or a lower acyl group. Of these, when representing a lower alkyl group, it is the same as in R ¹ , R ² , R ³ and R ⁴ above. Yes, when representing a lower acyl group, preferably a lower acyl group having 1 to 10, more preferably 1 to 8 carbon atoms, for example, acetyl, propionyl, butyryl, isobutyryl, Examples thereof include a valeryl group, an isovaleryl group, a pivaloyl group, a hexanoyl group, and an octanoyl group. Of these, an acetyl group and a propionyl group are preferable. Further, in the above general formula (1), X represents glucose, galactose,
Represents a monosaccharide residue such as fucose, xylose, mannose, rhamnose, arabinose, lyxose, ribose, allose, altrose, idose, talose, deoxyribose, 2-deoxyribose, quinobose and avequose; The hydrogen atom of the hydroxyl group therein may be substituted with a lower alkyl group, preferably a lower alkyl group having 1 to 8 carbon atoms or a lower acyl group, preferably a lower acyl group having 1 to 10 carbon atoms.
Of these, when representing a lower alkyl group, the above R
¹ , R ² , R ³ and R ⁴ ,
Furthermore, when representing a lower acyl group is the same as in the above R ^5. Among the monosaccharide residues, glucose,
Galactose, fucose, xylose, mannose, rhamnose, arabinose, lyxose, ribose, talose, deoxyribose and 2-deoxyribose are represented by X
And fucose, xylose and rhamnose are particularly preferably used. Further, n is 0
To 4, preferably an integer of 1 to 3.

In the present invention, the sugar acceptor is
An acetyl group, a benzoyl group, a pivaloyl group, a chloroacetyl group, a levulinoyl group, a benzyl group,
It is preferable to protect with a protecting group such as a p-methoxybenzyl group, an allyl group, a t-butyldimethylsilyl group, a t-butyldiphenylsilyl group, a trimethylsilyl group and a trityl group, preferably an acetyl group or a benzoyl group.

An embodiment of the method for preparing a sugar donor according to the present invention will be described below. 5 to 2 pyridine per gram of sugar
0 ml, preferably 10 to 15 ml, is added and dissolved. Further, acetic anhydride was added to this solution for 10 to 20 m.
l, preferably 15-20 ml, and add 3-8 at room temperature.
Stir for hours, preferably 5 to 7 hours. Next, 20 ml of methanol was added to the stirred solution to destroy excess acetic anhydride, and then a sugar in which all hydroxyl groups were protected with acetyl groups (hereinafter referred to as “acetyl sugar derivative”) was methylene chloride, chloroform, Extract with an organic solvent such as ethyl acetate, benzene, toluene and ether, preferably methylene chloride. The obtained extract is concentrated under reduced pressure, and then purified by silica gel column chromatography (ethyl acetate: hexane = 1: 1 (v / v)) to obtain an acetyl sugar derivative. Further, 5 g of methylene chloride is added to 1 g of the acetyl sugar derivative thus obtained.
ml, preferably 10-15 ml. Further, to this solution was added a solution of hydrogen bromide in acetic acid (25%
(W / w)) is 0.5 to 2 ml, preferably 1 to 1.5 ml.
5 to 30 ° C., preferably 10 to 25 ° C.
And stir for 15 to 60 minutes, preferably 20 to 30 minutes. Next, 5-30 m of distilled water was added to the stirred solution on ice.
l, preferably 15 to 20 ml, to decompose hydrogen bromide. The reaction solution is extracted with an organic solvent such as methylene chloride, chloroform, ethyl acetate, benzene, toluene and ether, preferably methylene chloride. By concentrating the obtained extract under reduced pressure, a sugar donor (hereinafter referred to as "1-bromo sugar donor") in which bromine is introduced at the 1-position and other hydroxyl groups are protected with an acetyl group is obtained. Can be

Next, one embodiment of the method for preparing a sugar receptor according to the present invention will be described. Pyridine is added to and dissolved in 5 to 20 ml, preferably 10 to 15 ml, per 1 g of the 2-substituted alcohol. Further, 0.7-1.5 g, preferably 0.9-1.0 g of t-butyldimethylsilyl chloride is added to this solution, and the mixture is stirred at room temperature for 3-8 hours, preferably 4-5 hours. Next, 0.8 to 2 ml, preferably 1 to 1.5 ml of acetic anhydride is added to this stirring solution, and the mixture is added at 5 to 30 ° C, preferably 10 to 25 ° C, for 3 to 8 ml.
Stir for hours, preferably 4-5 hours. Excess acetic anhydride is destroyed by adding 2 ml of methanol to the reaction solution, and then a 2-substituted alcohol in which the hydroxyl group at the 6-position is protected with an acetyl group and the hydroxyl group at the 2-position is protected with a t-butyldimethylsilyl group. . The 2-substituted alcohol thus protected is extracted with an organic solvent such as methylene chloride, chloroform, ethyl acetate, benzene, toluene and ether, preferably methylene chloride. The obtained extract is concentrated under reduced pressure, and the obtained compound is acidified, for example, in an aqueous acetic acid solution (80% (w / w)) at 5 to 30 ° C, preferably at 10 to 25 ° C. 36-54 hours, preferably 40-
Stir for 48 hours to deprotect the t-butyldimethylsilyl group. The deprotected compound is subjected to silica gel column chromatography (ethyl acetate: hexane = 1: 1 (v /
By purifying in v)), 6 of the 2-substituted alcohol is obtained.
Thus, a sugar acceptor in which only the hydroxyl group at the 1-position is protected with an acetyl group is obtained.

Further, one embodiment of the condensation reaction of the sugar donor and the sugar acceptor according to the present invention will be described below for the sugar donor and the sugar acceptor obtained above. 1.0 to 1.5 equivalents in molar ratio to 1 g of sugar receptor and 1 g of sugar receptor,
Preferably, 1.2 equivalents of a 1-bromo sugar donor is dissolved in a non-polar solvent such as methylene chloride or benzene, and 2 to 4 g, preferably 2.5 to 4 g of molecular sieve 4A (4 °) is dissolved.
3.0 g is added, and 5 to 30 ° C., preferably 10 to 25 ° C.
Stir at 1 ° C. for 1-5 hours, preferably 3 hours. An activator is added to the reaction solution under anhydrous conditions, and
Stir at 12 ° C., preferably 10-25 ° C., for 12-48 hours, preferably 20-30 hours. The resulting reaction solution was filtered through celite to remove molecular sieve 4A (4 °) and silver perchlorate, and the product was subjected to silica gel column chromatography (ethyl acetate: hexane = 1: 1 (v / v).
By purifying in v)), a chromanol glycoside in which all hydroxyl groups are protected with acetyl groups (hereinafter referred to as "chromanol glycoside acetyl derivative") is obtained.

The activator used in the present invention includes boric acid trifluoride / ether complex, silver perchlorate, silver trifluoromethanesulfonate, mercury bromide, mercury cyanide, N-iodosuccinimide-trifluorofluoride Methanesulfonic acid, dimethylmethylthiosulfonium triflate and p-toluenesulfonic acid.
When bromine is used as a leaving group of a sugar donor, it is preferable to use a heavy metal salt such as silver perchlorate.

In the above embodiment, purification was performed by silica gel column chromatography, but the present invention is not limited to this purification method, and a known purification method may be used.

Then, the acetyl derivative of chromanol glycoside is deprotected as follows.

In the present invention, a condensate obtained after the above condensation reaction between a sugar donor and a sugar acceptor, that is, an acetyl derivative of a chromanol glycoside in which a hydroxyl group is protected by a protecting group such as an acetyl group. The deprotection method for removing the protecting group is not particularly limited. For example, various methods such as an alkali decomposition method, an acid decomposition method, an enzymatic decomposition method and a deprotection method using a metal reagent are adopted. it can.

However, chromanol glycosides are very unstable compounds under alkaline conditions due to their chemical properties. Therefore, deprotection of the compounds obtained by condensation is carried out in the presence of pyrogallol, an antioxidant. Alkaline decomposition methods such as alkali decomposition have the disadvantage that chromanol glycosides are decomposed and the yield is reduced. For this reason, considering the synthesis of a large amount of chromanol glycoside, the deprotection method using an acid decomposition method, enzymatic decomposition method, and using a metal reagent, especially using a metal reagent, is compared with the case using the alkali decomposition method. The deprotection method used is preferably used in the present invention.

First, when deprotection of a protecting group such as an acetyl group of a condensate is performed by acid decomposition, decomposition of chromanol glycoside is prevented, and the deprotection is performed by alkali decomposition as compared with the case of deprotection by alkali decomposition. Chromanol glycosides can be obtained in good yield. For example, the acid decomposition method of dissolving the compound obtained by condensation in methanolic hydrochloric acid can obtain about 7 times the amount of glycosides as compared to the alkali decomposition method. Becomes possible. However, in the above-described acid-decomposition deprotection method, depending on the type of sugar bonded to the 2-substituted alcohol, there is a glycoside in which a glycoside bond is cleaved simultaneously with deprotection of an acetyl group under acidic conditions, In such a case, it becomes difficult to obtain a glycoside in a good yield. For this reason, it is preferable to use an acid-stable glycoside for the deprotection treatment by acid decomposition.In the case of an acid-labile glycoside, enzymatic decomposition or metal reagent is used instead of acid decomposition. It can be said that a method of performing deprotection using the above method is preferable.

Next, when a protecting group such as an acetyl group of the compound obtained by condensation is hydrolyzed with an enzyme such as esterase to perform a deprotection treatment, the decomposition of the chromanol glycoside is performed in the same manner as described above. Chromanol glycosides can be obtained in good yield compared to the case where deprotection is carried out by alkali decomposition.For example, a method of performing a deprotection treatment by hydrolyzing a compound obtained by condensation with esterase can be obtained. It becomes possible to obtain about 7 times the amount of glycosides as compared with the alkali decomposition method. However, the enzymatic deprotection method has a problem in that the enzyme used is expensive, the cost of the produced chromanol glycoside increases, and it is not economical.

When the deprotection of the acetyl group or the like of the condensate is carried out reductively by using a metal reagent for these deprotection methods, degradation of the chromanol glycoside is prevented in the same manner as described above. Chromanol glycosides can be obtained in a higher yield than in the case of deprotection by alkali decomposition.For example, the compound obtained by condensation is deprotected using lithium aluminum hydride or diisobutylaluminum hydride. Is carried out at about 8.25 in comparison with the alkaline decomposition method.
It is possible to obtain double the amount of glycoside. In addition to these advantages, the deprotection method using a metal reagent can be applied to deprotection of all glycosides regardless of the type of sugar to be bound, and can be performed in a high yield. It also has the advantage that the body can be manufactured.

The deprotection according to the present invention is divided into the cases of alkali decomposition, acid decomposition, enzymatic decomposition and the use of metal reagents, and one embodiment thereof will be described below by using the acetyl derivative of chromanol glycoside obtained above. Shown in

First, when deprotection of an acetyl group by alkali decomposition is carried out, for example, 1 g of the obtained acetyl derivative of chromanol glycoside is mixed with a distilled water / methanol mixed solution (1: 1 (v / v)) 20 to 50 ml, preferably 25
Dissolve in ３０30 ml, add 10 g of pyrogallol, and at 5-30 ° C., preferably 10-25 ° C., for 30 minutes-
Stir for 2 hours, preferably 1 hour to 1 hour 30 minutes.
Sodium hydroxide 1 was added to the reaction solution thus obtained.
5 to 30 g, preferably 8 to 12 g.
C., preferably 10 to 25.degree. C., for 1 to 4 hours, preferably 2 to 3 hours, to perform deprotection of the acetyl group to obtain the desired chromanol glycoside.

Next, when deprotecting an acetyl group by acid decomposition, first, 20 to 100 g of sodium chloride,
Preferably 40 g to 50 g of concentrated sulfuric acid 50 ml to 200 m
l, preferably 80 ml to 100 ml, is dropped and hydrogen chloride gas generated is absorbed in methanol (dehydrated with a molecular sieve in advance) to prepare methanolic hydrochloric acid. At this time, the concentration of hydrochloric acid is preferably 5 to 15%. And 1 g of chromanol glycoside acetyl derivative
The methanolic hydrochloric acid prepared as described above 10
The acetyl group is dissolved in 50 ml, preferably 20 to 30 ml, and stirred at room temperature for 2 to 8 hours, preferably 4 to 6 hours to acid-decompose the acetyl group. Thereafter, the desired chromanol glycoside is separated and purified from the reaction solution.

Further, in the case of deprotection of an acetyl group by enzymatic decomposition, the deprotection is performed using a hydrolase in a buffer solution. At this time, the enzyme used for deprotecting the acetyl group includes an esterase such as Sigma (S
Esterases and lipases from pig liver and rabbit liver manufactured by IGMA), such as lipase from pig pancreas.

In the above embodiment, the amount of the enzyme to be added varies depending on the type of the enzyme used, the reaction conditions, and the like. For example, 2 g of chromanol glycoside can be obtained by adding SIGMA-derived esterase derived from pig liver. When adding to 500 ml of the reaction solution containing the acetyl derivative,
0000 to 50,000 U, preferably 20,000
0 to 40,000 U. 10,000U enzyme
If it is less than 1, the catalytic action of the enzyme is not sufficient, which is not preferable. On the other hand, when the amount of the enzyme exceeds 50,000 U, it is uneconomical because the effect corresponding to excessive addition cannot be obtained. The “1U” of the esterase derived from pig liver used above refers to ethyl butyrate as a substrate at pH 8.0 and 25 ° C. for 1 minute per minute.
1 means the amount of enzyme that hydrolyzes ethyl butyrate.

In the deprotection reaction by the enzymatic decomposition method, it is desirable to dissolve the chromanol glucoside acetyl derivative obtained by condensation in the reaction solution. Therefore, the chromanol glucoside acetyl derivative having a very low solubility in water is desirable. It is necessary to add an organic solvent capable of dissolving the compound. At the same time, the added organic solvent must be capable of efficiently expressing the esterase hydrolysis activity. Examples of the organic solvent satisfying such conditions include dimethyl sulfoxide, N, N-dimethylformamide, methanol, ethanol, and acetonitrile. For example, when an esterase derived from pig liver is used in an enzymatic degradation reaction, it is desirable to use methanol, ethanol or dimethyl sulfoxide in order to enhance the esterase hydrolysis activity. The concentration of the organic solvent to be added varies depending on the type of the enzyme to be used. For example, when an esterase is used, the concentration of the organic solvent to be added is preferably 10 to 25%. ~ 20%. When the organic solvent concentration is less than 10%, it is difficult to dissolve the desired chromanol glycoside in the reaction solution, and when it exceeds 25%, the stability of the enzyme is reduced and the hydrolysis efficiency is reduced. It is not preferable because it becomes worse.

Furthermore, in the embodiment using the enzymatic decomposition method, the reaction conditions vary depending on the type and amount of the enzyme and the chromanol glycoside acetyl derivative used. For example, when using a pig liver-derived esterase, the pH of the reaction system is 6.5 to 8.5, preferably 7.5 to 8.0.
It is. When the pH is out of the above range, the above-mentioned enzyme is inactivated, which is not preferable. The reaction temperature is, for example, 20 to 60 ° C., preferably 30 to 40 ° C. when an esterase derived from pig liver is used.
If the reaction temperature is below the lower limit of the above range, the activity of the enzyme used is reduced, it is not economical because it takes a long time to secure a sufficient hydrolysis rate, and the reaction temperature is in the above range. Exceeding the upper limit is not preferred because the enzyme is inactivated. Further, the reaction time is, for example, 2 to 60 hours, preferably 15 to 50 hours when using esterase derived from pig liver. At this time, if the reaction time is less than 2 hours, the reaction has not reached near equilibrium, so that a sufficient hydrolysis rate cannot be obtained. It is not preferable because no further effect can be expected.

Finally, the deprotection of an acetyl group using a metal reagent is a simple method in which a compound obtained by condensation is added to an organic solvent in which a metal reagent is dissolved and reacted. . More specifically, first, 1 g of the acetyl derivative of chromanol glycoside obtained by condensation was added to 50 to 1
Dissolve in 00 ml of non-polar solvent. This solution is added to a non-polar solvent 50 to 100 ml in which 0.5 to 3 g, preferably 0.75 to 1.25 g of a metal reagent is previously dissolved with respect to 1 g of the sugar acetyl derivative, while cooling, for 1 to 2 hours. Drip over. After the addition, the mixture was allowed to stand at room temperature for 1
The acetyl group is deprotected by stirring for 6 hours, preferably 2 to 4 hours. The desired chromanol glycoside is separated and purified from the reaction solution.

The metal reagent used in this reaction includes lithium aluminum hydride (LiAlH ₄ ) and its trialkoxy derivative (LiAlH (OR) ₃ ,
In this case, R represents an alkyl group having 1 to 4 carbon atoms),
Diisobutylaluminum hydride _{((i-C 4 H 9} ) 2
AlH), sodium borohydride (NaBH ₄ ), lithium borohydride (LiBH ₄ ), and the like.
Of these, lithium aluminum hydride (LiAL
H ₄ ) and diisobutylaluminum hydride ((i-C
₄ H ₉ ) ₂ AlH) is preferably used.

The non-polar solvent used in the above embodiment includes, for example, ether, methylene chloride,
Examples include tetrahydrofuran, toluene, benzene and hexane. Of these, tetrahydrofuran is preferably used as the non-polar solvent.

After the reaction is completed as described above, the desired chromanol glycoside obtained by deprotection is separated and purified from the reaction solution to obtain a high-purity chromanol glycoside.

Thus, the sugar donor and the sugar acceptor are dissolved in the non-polar solvent, and the condensation reaction of the sugar donor and the sugar acceptor is carried out in the presence of an activating agent under anhydrous conditions to obtain a sugar donor. The chromanol glycoside of the present invention can be produced in a high yield by a simple step of purifying the resulting compound and deprotecting the protecting group.

[0085]

EXAMPLES Next, the present invention will be described in more detail by way of examples, but it goes without saying that the scope of the present invention is not limited by these examples.

Example 1 1) Production of fucose donor 1150 mg of L-fucose was dissolved in 15 ml of pyridine, 15 ml of acetic anhydride was added, and the mixture was stirred at room temperature for 6 hours.
Excess acetic anhydride was destroyed by adding 15 ml of methanol to the reaction solution, and the product was extracted with 30 ml of methylene chloride. Next, the extract was washed sequentially with 30 ml of 2N hydrochloric acid, 30 ml of a saturated sodium carbonate solution and 30 ml of a saturated saline solution to remove pyridine and acetic acid. This solution was dried under reduced pressure, and purified by silica gel column chromatography (ethyl acetate: hexane = 1: 1 (v / v)).

The compound thus obtained was dissolved in 20 ml of methylene chloride, and a solution of hydrogen bromide in acetic acid (25% (w
/ W)) 1.5 ml was added and stirred at room temperature for 30 minutes.
20 ml of distilled water was added to the reaction solution to destroy the reagent, and a sugar donor in which bromine was introduced at the first position and the other hydroxyl groups were protected with an acetyl group was extracted with 20 ml of methylene chloride. The extract was washed with 20 ml of a saturated sodium carbonate solution to remove residual acetic acid. After adding 10 g of sodium sulfate to remove water and drying under reduced pressure, 2,3,4-tri-O-acetyl-α-L-fucopyranosyl bromide (2 , 3,4-tri-O-acetyl-α-L-fucop
2100 mg of yranosyl bromide (hereinafter referred to as "fucose donor").

[0088]

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2) Production of sugar acceptor 2-hydroxymethyl-2,5,7,8-tetramethylchroman-6-ol (2-hydroxymethyl-2,5,7,8-tetra
methylchroman-6-ol) 1000mg to pyridine 10ml
950m t-butyldimethylsilyl chloride
g was added and stirred at room temperature for 5 hours. 1 ml of acetic anhydride was added to this solution and stirred at room temperature for 5 hours. While cooling on ice, 2 ml of methanol was added to the reaction solution to destroy excess acetic anhydride, and 2-hydroxymethyl-2,5,7,8-
A compound in which the 6-position hydroxyl group of tetramethylchroman-6-ol was protected with an acetyl group and the 2-position hydroxyl group was protected with a t-butyldimethylsilyl group was extracted with 20 ml of methylene chloride and dried under reduced pressure. This compound is dissolved in 100 ml of an aqueous acetic acid solution (80% (w / w)) and
Stirred for hours. Ethanol was added to the reaction solution, and acetic acid and distilled water were removed while azeotropic distillation under reduced pressure. The yellow syrupy compound thus obtained was subjected to silica gel column chromatography (ethyl acetate: hexane = 1: 1).
1 (v / v)) to protect only the hydroxyl group at the 6-position of 2-hydroxymethyl-2,5,7,8-tetramethylchroman-6-ol represented by the following general formula with an acetyl group. 2-hydroxymethyl-2,5
7,8-tetramethylchroman-6-acetate (2-hyd
roxymethyl-2,5,7,8-tetramethylchroman-6-acetate)
1100 mg (hereinafter referred to as "sugar receptor") was obtained.

[0090]

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3) Production of glycoside 2100 mg of fucose donor obtained in 1) and 1100 mg of sugar acceptor obtained in 2) were dissolved in 10 ml of methylene chloride, and 3 g of molecular sieve 4A (4 °) was added. After stirring for 3 hours, 1200 mg of silver perchlorate
And 1600 mg of silver carbonate were added, and the mixture was stirred at room temperature for 24 hours to carry out a condensation reaction. After the condensation, the reaction solution was filtered through celite to remove molecular sieve 4A (4 °), silver perchlorate and silver carbonate, and then subjected to silica gel column chromatography (ethyl acetate: hexane = 1: 1 (v /
Purified in v)). This compound was dissolved in 50 ml of a 50% methanol solution, 25 g of pyrogallol was added, and the mixture was stirred for 1 hour. Then, 25 g of sodium hydroxide was added, and the mixture was stirred at room temperature for 3 hours. The reaction solution was neutralized by adding 10 ml of concentrated hydrochloric acid, and then applied to a column (carrier: XAD-4) equilibrated with a 30% methanol solution. 3 as eluent
After eluting the non-adsorbed material using a 0% methanol solution,
Chromanol glycosides were eluted using a methanol solution of 10% as an eluent. After drying the eluate under reduced pressure, silica gel column chromatography (chloroform: methanol =
8: 1 (v / v)) to give 2- (β-L-fucopyranosyl) methyl-2,5,7,8-tetramethylchroman- having the following physical properties and represented by the following general formula: 6-ol (2- (β-L-fucopyranosyl) methyl-2,5,7,8-
200 mg of tetramethylchroman-6-ol) were obtained.

[0092]

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FIG. 1 shows the infrared absorption spectrum of this compound.
Shown in

The results of ¹³ C-NMR, mass spectrometry and specific rotation of the above compound are as follows.

¹³ C-NMR δ (75 MHz, DMSO
-D _6, proton decoupling): 11.8 11.8 12.7 16.6 19.9 22.4 and 22.6 28.0 70.0 70.2 71.1 73.1 73.5 74 1.0 and 74.0 103.8 and 104.0 116.8 120.3 120.9 122.6 144.3 145.2 Mass spectrum (FAB) m / z 382 (molecular ion peak) Specific rotation

[0096]

[Outside 1]

Example 2 2100 mg of fucose donor obtained in 1) of Example 1
Then, 1100 mg of the sugar receptor obtained in 2) of Example 1 was dissolved in 10 ml of methylene chloride, and molecular sieve 4 was dissolved.
After adding 1 g of A (4 °) and stirring at room temperature for 2 hours, 1200 mg of silver perchlorate and 1600 mg of silver carbonate were added, and the mixture was stirred at room temperature for 24 hours to conduct a condensation reaction.
After condensation, the reaction solution was filtered through celite to remove molecular sieve 4A (4 °), silver perchlorate and silver carbonate, and then purified by silica gel column chromatography (ethyl acetate: hexane = 1: 1 (v / v)). did.

The obtained compound was dissolved in 80 ml of methanol, and then dissolved in a 50 mM phosphate buffer (pH 7.5).
ml. Next, esterase 20,
000 U was added thereto and reacted at 35 ° C. for 48 hours. The hydrolysis rate at this time was 90%. After completion of the reaction, the reaction solution was centrifuged (12000 rpm, 4 ° C., 5 minutes), and the glycoside was extracted from the supernatant with ethyl acetate. The extract was dried under reduced pressure, and then purified by silica gel column chromatography (chloroform: methanol = 8: 1 (v / v)) to obtain a compound of the same general formula as that obtained in Example 1; 2- (β-L- having the results of absorption spectrum, ¹³ C-NMR, mass spectrometry, and specific rotation
Fucopyranosyl) methyl-2,5,7,8-tetramethylchroman-6-ol (2- (β-L-fucopyranosyl) methy
l) -2,5,7,8-tetramethylchoman-6-ol) 1400 mg was obtained.

From this, deprotection is carried out by enzymatic decomposition using esterase (Example 2, yield: 1400 mg).
As a result, a 7-fold amount of chromanol glycoside was obtained as compared with the case where deprotection was performed by alkali decomposition (Example 1, yield: 200 mg).

Example 3 2100 mg of fucose donor obtained in 1) of Example 1
And 1100 mg of the sugar acceptor obtained in 2) of Example 1 were dissolved in 10 ml of methylene chloride, and molecular sieve 4A was dissolved.
(4 °) After adding 3 g and stirring at room temperature for 3 hours, 1200 mg of silver perchlorate and 1600 mg of silver carbonate were added, and the mixture was stirred at room temperature for 24 hours to perform a condensation reaction. After the condensation, the reaction solution was filtered through celite to remove molecular sieve 4A (4 °), silver perchlorate and silver carbonate, and then subjected to silica gel column chromatography (ethyl acetate: hexane = 1: 1 (v / v)). Purified. This compound was dissolved in 50 ml of tetrahydrofuran, and added dropwise with cooling to a solution of 2 g of lithium aluminum hydride in 50 ml of tetrahydrofuran. After stirring this mixture at room temperature for 3 hours, 50 ml of methanol was added to the reaction solution while cooling, thereby terminating the reaction. After adding 100 ml of distilled water to the reaction solution, glycosides were extracted with ethyl acetate. After the extract was dried under reduced pressure, silica gel column chromatography (chloroform: methanol =
8: 1 (v / v)), the analysis results of the same general formula, infrared absorption spectrum, ¹³ C-NMR, mass spectrometry, and specific rotation as those obtained in Example 1 were obtained. Having 2- (β-L-fucopyranosyl) methyl-
2,5,7,8-tetramethylchroman-6-ol (2
-(β-L-fucopyranosyl) methyl) -2,5,7,8-tetramethylch
oman-6-ol) was obtained.

The deprotection is carried out using lithium aluminum hydride (Example 3, yield: 1650 mg).
As a result, 8.25 times the amount of chromanol glycoside was obtained as compared with the case where deprotection was performed by alkali decomposition (Example 1, yield: 200 mg).

Example 4 A glycoside was produced in the same manner as in Example 3 except that diisobutylaluminum hydride was used instead of lithium aluminum hydride, to obtain a glycoside similar to that obtained in Example 1. General formula, infrared absorption spectrum, ¹³
2- (β-L-fucopyranosyl) methyl-2,5, which has the results of C-NMR, mass spectrometry, and specific rotation.
7,8-tetramethylchroman-6-ol (2- (β-Lf
ucopyranosyl) methyl) -2,5,7,8-tetramethylchoman-6-o
l) 1650 mg were obtained.

The deprotection is carried out using diisobutylaluminum hydride (Example 4, yield: 1650 m).
g) As a result, 8.25 times the amount of chromanol glycoside was obtained as compared with the case of performing deprotection by alkali decomposition (Example 1, yield: 200 mg).

Example 5 A condensation reaction and separation and recovery were carried out in the same manner as in Example 1 except that L-rhamnose was used instead of L-fucose. 2- (α-L-rhamnopyranosyl) methyl-2,5,7,8-tetramethylchroman-6-ol (2- (α-L-rhamnopyranosyl) methyl-2,5,7, 8-tetram
(ethylchroman-6-ol) 160 mg was obtained.

[0105]

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The infrared absorption spectrum of this compound is shown in FIG.
Shown in

The results of ¹³ C-NMR, mass spectrometry and specific rotation of the above compound are as follows.

¹³ C-NMR δ (75 MHz, DMSO
-D _6, proton decoupling): 11.7 11.8 12.7 17.7 and 17.9 19.8 21.9 and 22.0 28.4 68.4 and 68.5 70.4 70. 7 70.9 and 71.1 71.3 73.7 100.1 and 100.2 116.6 120.2 121.0 122.6 144.2 145.2 Mass spectrum (FAB) m / z 382 (Molecule Ion peak) Specific rotation

[0109]

[Outside 2]

Example 6 A condensation reaction and separation and recovery were carried out in the same manner as in Example 1 except that D-xylose was used instead of L-fucose. 2- (β-D-xylopyranosyl) methyl-2,5,7,8-tetramethylchroman-6-ol (2- (β-D-xylopyranosyl) methyl-2,5,7, 8-tetramet
(Hylchroman-6-ol) 250 mg was obtained.

[0111]

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The infrared absorption spectrum of this compound is shown in FIG.
Shown in

The results of ¹³ C-NMR, mass spectrometry and specific rotation of the above compound are as follows.

¹³ C-NMR δ (75 MHz, DMSO
-D _6, proton decoupling): 11.7 11.8 12.7 19.7 and 19.8 20.7 22.2 and 22.3 28.0 and 28.2 65.6 69.5 73. 273.9 and 74.0 76.5 103.9 and 104.1 116.8 120.2 120.9 122.6 144.2 145.2 Mass spectrum (FAB) m / z 368 (molecular ion peak) Specific rotation

[0115]

[Outside 3]

Example 7 1) Production of Mannose Donor 1150 mg of D-mannose was dissolved in 15 ml of pyridine, 15 ml of acetic anhydride was added, and the mixture was stirred at room temperature for 6 hours.
Excess acetic anhydride was destroyed by adding 15 ml of methanol to the reaction solution, and the product was diluted with 30 ml of methylene chloride.
Extracted. Next, the extract was washed sequentially with 2N hydrochloric acid, 30 ml of saturated sodium carbonate solution and 30 ml of saturated saline to remove pyridine and acetic acid. This solution was dried under reduced pressure, and purified by silica gel column chromatography (ethyl acetate: hexane, 1: 1 (v / v)).

The resulting compound was dissolved in 20 ml of methylene chloride, and a solution of hydrogen bromide in acetic acid (25% (w / w)) was added.
Then, the mixture was stirred at room temperature for 30 minutes. 20 ml of distilled water was added to the reaction solution to destroy the excess reagent, and a sugar donor in which bromine was introduced at the first position and the other hydroxyl groups were protected with an acetyl group was extracted with 20 ml of methylene chloride. The extract was washed with 20 ml of a saturated sodium carbonate solution to remove acetic acid. After adding 10 g of sodium sulfate to remove water and drying under reduced pressure, 2,3,4,6 represented by the following formula:
-Tetra-O-acetyl-α-D-mannopyranosyl bromide (2,3,4,6-tetra-O-acetyl-α-D-mannopyranosyl
bromide) (hereinafter referred to as “mannose donor”) 21
00 mg was obtained.

[0118]

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2) Production of sugar acceptor 1000 mg of 2-hydroxymethyl-2,5,7,8-tetramethylchroman-6-ol was added to 10 m of pyridine.
1) and dissolved in 950 t-butyldimethylsilyl chloride.
mg was added and stirred at room temperature for 5 hours. 1 ml of acetic anhydride was added to this solution and stirred at room temperature for 5 hours. While cooling on ice, 2 ml of methanol was added to the reaction solution to destroy excess acetic anhydride, and the 6-hydroxyl group of 2-hydroxymethyl-2,5,7,8-tetramethylchroman-6-ol was protected with an acetyl group. The compound in which the hydroxyl group at the 2-position was protected with a t-butyldimethylsilyl group was extracted with 20 ml of methylene chloride and dried under reduced pressure. This compound was treated with an acetic acid solution (80%
(W / w)) dissolved in 100 ml and stirred at room temperature for 24 hours. Ethanol was added to the reaction liquid, and acetic acid and distilled water were removed while azeotropic distillation under reduced pressure. The thus obtained yellow syrupy compound was subjected to silica gel column chromatography (ethyl acetate: hexane = 1: 1 (v
/ V)) to give 2-hydroxymethyl-2,5,7,8-tetramethylchroman-6-acetate (2-hydroxymethyl-2,5,7,8) represented by the following general formula:
-tetramethylchroman-6-acetate) (hereinafter, "sugar receptor")
1100 mg).

[0120]

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3) Production of Glycoside [0120] 2100 mg of the mannose donor obtained in 1) and 1100 mg of the sugar acceptor obtained in 2) were mixed with 10 ml of methylene chloride.
, And 1 g of molecular sieve 4A (4 °) was added, and the mixture was stirred at room temperature for 2 hours. Then, 1200 mg of silver perchlorate and 1600 mg of silver carbonate were added, and the mixture was stirred at room temperature for 24 hours to perform a condensation reaction. After the condensation, the reaction solution was filtered through celite to remove molecular sieve 4A (4 °), silver perchlorate and silver carbonate, and then subjected to silica gel column chromatography (ethyl acetate: hexane = 1: 1 (v /
Purification was performed in v)) to obtain a compound in which all hydroxyl groups of the target glycoside were protected with an acetyl group. The obtained compound was dissolved in 50 ml of a 50% methanol solution, 25 g of pyrogallol was added, and the mixture was stirred at room temperature for 1 hour. Then, 25 g of sodium hydroxide was added, and the mixture was stirred at room temperature for 3 hours. The reaction solution was neutralized by adding 10 ml of concentrated hydrochloric acid, and then applied to a column (carrier: XAD-4) equilibrated with a 30% methanol solution. After eluting the non-adsorbed substance using a 30% methanol solution as an eluent, chromanol glycoside was eluted using a 100% methanol solution as an eluent. After the eluate was dried under reduced pressure, it was purified by silica gel column chromatography (chloroform: methanol = 6: 1 (v / v)) and had the following physical properties and was represented by the following general formula, 2- (α-D) -Mannopyranosyl) methyl-2,5,7,8-tetramethylchroman-6-ol (2- (α-D-mannopyranosyl) meth)
yl-2,5,7,8-tetramethylchroman-6-ol) 200 mg was obtained.

[0122]

Embedded image

The infrared absorption spectrum of this compound is shown in FIG.
Shown in

Further, ¹ H-NMR, ¹³ C-
The results of NMR, mass spectrometry and specific rotation are as follows.

¹ H-NMR δ (300 MHz, DM
SO-d _6): 1.17 and 1.21 (s, 3H) 1.65 from 1.74 (m, 1H) 1.77 from 1.91 (m, 1H) 1.97 ( s, 3H) 2.01 (s, 3H) 2.04 (s, 3H) 2.50 (broad t, 2H) 3.16 to 4.69 (m, 13H) 7.39 (s, 1H) ¹³ C-NMR δ (75MHz, DMSO-d _6, proton decoupling spectrum): 11.7 11.8 12.7 19.7 and 19.8 21.9 and 22.1 28.2 and 28.5 61.0 and 61. 2 66.7 and 66.9 70.2 and 70.3 71.0 73.8 73.9 74.1 100.1 and 100.2 116.7 120.2 and 120.3 120.9 122.6 144.1 and 144.2 145.2 Quality Spectrum (FAB): m / z 398 (molecular ion peak) Specific optical rotation:

[0126]

[Outside 4]

Example 8 The mannose donor 2100 m obtained in 1) of Example 7)
g and the sugar receptor 1100 mg obtained in Example 7-2)
Was dissolved in 10 ml of methylene chloride, 1 g of molecular sieve 4A (4 °) was added, and the mixture was stirred at room temperature for 2 hours. Then, 1200 mg of silver perchlorate and 1600 mg of silver carbonate were added, and the mixture was stirred at room temperature for 24 hours to perform a condensation reaction. Was. After condensation, the reaction solution was filtered through celite to remove molecular sieve 4A (4 °), silver perchlorate and silver carbonate, and then subjected to silica gel column chromatography (ethyl acetate: hexane = 1: 1 (v / v)). A compound in which all hydroxyl groups of the glycoside were protected with an acetyl group was purified.

The obtained compound was dissolved in 50 ml of 10% methanolic hydrochloric acid and stirred at room temperature for 4 hours. A column in which the reaction solution was equilibrated with a 30% methanol solution (carrier: XA
D-4). After eluting the non-adsorbed substance using a 30% methanol solution as an eluent, chromanol glycoside was eluted using a 100% methanol solution as an eluent. After the eluate was dried under reduced pressure, silica gel column chromatography (chloroform: methanol = 6: 1 (v /
By purifying in v)), 2- (α-)-which exhibits the same infrared absorption spectrum, ¹³ C-NMR, mass spectrometry, and specific rotation analysis results as those obtained in Example 7.
1400 mg of (D-mannopyranosyl) methyl-2,5,7,8-tetramethylchroman-6-ol were obtained.

From this, deprotection is carried out by acidolysis using methanolic hydrochloric acid (Example 8, yield: 1400 m).
g), a 7-fold amount of chromanol glycoside was obtained as compared with deprotection by alkali decomposition (Example 7, yield: 200 mg).

Example 9 1) Production of galactose donor 1150 mg of D-galactose was dissolved in 15 ml of pyridine, 15 ml of acetic anhydride was added, and the mixture was stirred at room temperature for 6 hours. Excess acetic anhydride was destroyed by adding 15 ml of methanol to the reaction solution, and the product was treated with 30 m of methylene chloride.
Extracted with l. Next, the extract was washed sequentially with 30 ml of 2N hydrochloric acid, 30 ml of saturated sodium carbonate solution and 30 ml of saturated saline to remove pyridine and acetic acid. This solution was dried under reduced pressure, and purified by silica gel column chromatography (ethyl acetate: hexane = 1: 1 (v / v)).

The compound thus obtained was dissolved in 20 ml of methylene chloride, and a solution of hydrogen bromide in acetic acid (25% (w
/ W)) and stirred at room temperature for 30 minutes. After the reaction, 20 ml of distilled water was added to destroy the excess reagent, and a sugar donor in which bromine was introduced at the first position and the other hydroxyl groups were protected with an acetyl group was extracted with 20 ml of methylene chloride.
The extract was washed with 20 ml of a saturated sodium carbonate solution to remove residual acetic acid. After adding 10 g of sodium sulfate to remove water and drying under reduced pressure,
2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl bromide (2,3,4,6
-tetra-O-acetyl-α-D-galactopyranosylbromide) (hereinafter, referred to as “galactose donor”) 2100 mg was obtained.

[0132]

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2) Production of sugar acceptor 2-hydroxymethyl-2,5,7,8-tetratylchroman-6-ol (2-hydroxymethyl-2,5,7,8-tetrame)
thylchroman-6-ol) 100mg
950 mg of -butyldimethylsilyl chloride was added, and the mixture was stirred at room temperature for 5 hours. 2 ml of acetic anhydride was added to this solution and stirred for 5 hours. While cooling on ice, 2 ml of methanol was added to the reaction solution to destroy excess acetic anhydride, and the hydroxyl group at the 6-position of 2-hydroxymethyl-2,5,7,8-tetramethylchroman-6-ol was replaced with an acetyl group. Protect
The compound in which the hydroxyl group at the 2-position was protected with a t-butyldimethylsilyl group was extracted with 20 ml of methylene chloride and dried under reduced pressure. This compound was treated with an aqueous acetic acid solution (80% (w / w)).
Dissolved in 100 ml and stirred at room temperature for 48 hours. Ethanol was added to the reaction solution, and acetic acid and distilled water were removed while azeotropic distillation under reduced pressure. The yellow syrupy compound thus obtained is purified by silica gel column chromatography (ethyl acetate: hexane = 1: 1 (v / v)) to give 2-hydroxymethyl- 2,5,7,8-tetramethylchroman-6
2 wherein only the hydroxyl group at position 6 of the ol is protected with an acetyl group
-Hydroxymethyl-2,5,7,8-tetramethylchroman-6-acetate (2-hydroxymethyl-2,5,7,8-tetra
1100 mg of methylchroman-6-acetate (hereinafter referred to as “sugar receptor”) was obtained.

[0134]

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3) Production of glycoside 2100 mg of galactose donor obtained in 1) and 2)
1100 mg of the sugar receptor obtained in
, and 3 g of molecular sieve 4A (4 °) was added. After stirring at room temperature for 3 hours, 1200 mg of silver perchlorate was added.
And 1600 mg of silver carbonate were added, and the mixture was stirred at room temperature for 24 hours to carry out a condensation reaction. After the condensation, the reaction solution was filtered through celite to remove molecular sieve 4A (4 °), silver perchlorate and silver carbonate, and then subjected to silica gel column chromatography (ethyl acetate: hexane = 1: 1 (v
/ V)). This compound was dissolved in 50 ml of a 50% methanol solution, and 25 g of pyrogallol was added.
After stirring for 25 hours, 25 g of sodium hydroxide was added, and the mixture was stirred at room temperature for 3 hours. The reaction solution was neutralized by adding 10 ml of concentrated sulfuric acid, and then applied to a column (carrier: XAD-4) equilibrated with a 30% methanol solution. After eluting non-adsorbed substances using 30% methanol solution as eluent,
Chromal glycosides were eluted using a 0% methanol solution as an eluent. After drying the eluate under reduced pressure, silica gel column chromatography (methyl acetate: methanol =
By purifying at 5: 1 (v / v)), 2- (β-D-galactopyranosyl) methyl-2,5,7,8-tetrafluoroethylene having the following physical properties and represented by the following general formula: Methylchroman-6-ol (2- (β-D-galactopyranosyl) methyl-
200 mg of (2,5,7,8-tetramethylchroman-6-ol) was obtained.

[0136]

Embedded image

The infrared absorption spectrum of this compound is shown in FIG.
Shown in

The ¹ H-NMR, ¹³ C-
The results of NMR, mass spectrometry and specific rotation are as follows.

¹ H-NMR δ (270 MHz, DM
SO-d _6): 1.21 and 1.24 (s, 3H) 1.67 from 1.73 (m, 1H) 1.90 from 1.93 (m, 1H) 1.98 ( s, 3H) 2.02 (s, 3H) 2.05 (s, 3H) 2.50 (broad t, 2H) 3.17 to 4.78 (m, 13H) 7.38 (s, 1H) ¹³ C-NMR δ (67.8 MHz, DMSO-d ₆ ,
Proton decoupling): 11.7 11.8 12.6 19.7 and 19.8 22.2 and 22.5 28.0 and 28.1 60.3 and 60.4 68.1 70.5 73. 1 and 73.2 73.4 73.9 and 74.0 75.1 and 75.2 104.0 and 104.2 116.8 120.2 120.8 122.5 144.2 145.2 Mass spectrum ( FAB): m / z 398 (molecular ion peak)

[0140]

[Outside 5]

Example 10 Galactose donor 2100 obtained in 1) of Example 9
mg and the sugar receptor 1100 mg obtained in Example 9-2)
Was dissolved in 10 ml of methylene chloride, 3 g of molecular sieve 4A (4 °) was added, and the mixture was stirred at room temperature for 3 hours.
A condensation reaction was carried out by adding 1200 mg of silver perchlorate and 1600 mg of silver carbonate and stirring the mixture at room temperature for 24 hours. After condensation, the reaction solution was filtered through celite to remove molecular sieve 4A (4 °), silver perchlorate and silver carbonate, and then purified by silica gel column chromatography (ethyl acetate: hexane = 1: 1 (v / v)). did. This compound was dissolved in 50 ml of tetrahydrofuran, and added dropwise with cooling to a solution of 2 g of lithium aluminum hydride in 50 ml of tetrahydrofuran. After stirring this mixture at room temperature for 3 hours, 50 ml of methanol was added to the reaction solution while cooling, thereby terminating the reaction. The filtrate obtained by filtering the precipitate was dried under reduced pressure. The obtained residue was purified by silica gel column chromatography (ethyl acetate: methanol = 5: 1 (v / v)) to obtain the same general formula as that obtained in Example 9.
2- (β-NMR) having the results of infrared absorption spectrum, ¹ H-NMR, ¹³ C-NMR, mass spectrometry, and specific rotation.
D-galactopyranosyl) methyl-2,5,7,8-tetramethylchroman-6-ol (2- (β-D-galactopyra
(nsosyl) methyl-2,5,7,8-tetramethylchroman-6-ol) 16
50 mg were obtained.

The deprotection is carried out using lithium aluminum hydride (Example 10, yield: 1650 m).
g) As a result, 8.25 times the amount of chromanol glycoside was obtained as compared with the case of performing deprotection by alkali decomposition (Example 9, yield: 200 mg).

Example 11 A glycoside was produced in the same manner as in Example 10 except that diisobutylaluminum hydride was used in place of lithium aluminum hydride, thereby obtaining a glycoside similar to that obtained in Example 9. General formula, infrared absorption spectrum,
2- (β-D-galactopyranosyl) methyl-2,5,7,8-tetramethylchroman- having the results of ¹ H-NMR, ¹³ C-NMR, mass spectrometry, and specific rotation.
6-ol (2- (β-D-galactopyransosyl) methyl-2,5,7,
1650 mg of 8-tetramethylchroman-6-ol) was obtained.

The deprotection is carried out using diisobutylaluminum hydride (Example 11, yield: 1650).
mg), which is 8.25 in comparison with deprotection by alkali decomposition (Example 9, yield: 200 mg).
A double amount of chromanol glycoside was obtained.

Comparative Example 1 Using a method similar to that described in Example 1 of JP-A-7-118,287, 2- (α-
D-glucopyranosyl) methyl-2,5,7,8-tetramethylchroman-6-ol (2- (α-D-glucopyranosy)
l) methyl-2,5,7,8-tetramethylchroman-6-ol)
0 mg was obtained.

[0146]

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Example 12: Evaluation of radioprotective action The chromanol glycosides obtained in Examples 1, 5 and 6 and Comparative Example 1 were each replaced with RPMI-1640 + 10
% Fetal bovine serum + HEDES buffer (25 mM) -based culture medium (hereinafter abbreviated as “complete culture medium”) at a final concentration of 1%.
The chromanol solution was dissolved in mM to prepare a chromanol glycoside solution in advance.

Next, mouse T lymphoma strain EL-4 cells were subcultured in a complete culture solution at 37 ° C. in a 5% CO ₂ atmosphere so that the cell density was 2 × 10 ⁵ cells / ml. It was adjusted. The supernatant of the EL-4 cell culture solution cultured in this manner was removed, and the same amount of the above chromanol glycoside solution was added. The cells were cultured under the same conditions as above for 30 minutes until X-ray irradiation. Culture was performed. After culturing in a culture solution containing chromanol glycoside for a predetermined time, 3 Gy radiation was irradiated at a dose rate of 0.92 Gy / min. Immediately after the end of irradiation, the cells were spun down (400 g × 5 minutes), and RPM
The cells were washed twice with 1-1640, resuspended in a complete culture solution, and cultured. To this, a DMSO solution of cytochalasin B (2 mg / ml concentration) was added to a final concentration of 3 μg / ml, and after culturing for 20 hours, the frequency of micronucleus-bearing cells in the binuclear cells (micronucleus induction frequency) was determined. It was measured and used as a measure of the frequency of radiation damage to cells. For each irradiated cell, the concentration of the chromanol glycoside solution was set to 0 μg.
The inhibition rate of micronucleus induction was calculated from the following formula based on the micronucleus induction frequency of the control obtained by repeating the above operation in the same manner except that the above procedure was used. At this time, the cells were 4 to 5 per group.
Irradiation experiments were performed in series, and the results were expressed as the average values. Table 1 shows the results.

[0149]

(Equation 1)

[0150]

[Table 1]

From Table 1, it can be seen that the frequency of micronucleus induction of the chromanol glycosides obtained in Examples 1, 5 and 6 is significantly smaller than the value of the chromanol glycoside obtained in Comparative Example 1,
This indicates that the chromanol glycoside according to the present invention effectively suppresses cell damage due to radiation exposure.

[0152]

As described above, the process for producing a chromanol glycoside according to the present invention provides a derivative of a saccharide in which a leaving group is introduced at the anomeric position and another hydroxyl group is protected with a protecting group, and a compound represented by the general formula (2). It comprises a condensation reaction of the represented 2-substituted alcohol derivative. Therefore, according to the method of the present invention, conventional enzymes such as fucose have
-The production of a chromanol glycoside using a sugar that cannot be transferred to a substituted alcohol derivative can be efficiently performed by a simple production and purification process.

In the process for producing a chromanol glycoside of the present invention, the deprotection of the protecting group after the condensation reaction is carried out by acid decomposition, enzymatic decomposition or by using a metal reagent, particularly by using a metal reagent. In addition, the decomposition of the chromanol glycoside is efficiently prevented, and therefore, the chromanol glycoside can be produced in a higher yield.

The chromanol glycoside of the present invention further comprises
In addition to heat and pH stability, water solubility and antioxidant activity, it is also excellent in radiation protection, and is expected to be used in various fields in the future.

[Brief description of the drawings]

FIG. 1 is an infrared absorption spectrum of 2- (β-L-fucopyranosyl) methyl-2,5,7,8-tetramethylchroman-6-ol obtained in Example 1.

FIG. 2 is an infrared absorption spectrum of 2- (α-L-rhamnopyranosyl) methyl-2,5,7,8-tetramethylchroman-6-ol obtained in Example 5.

FIG. 3 is an infrared absorption spectrum of 2- (β-D-xylopyranosyl) methyl-2,5,7,8-tetramethylchroman-6-ol obtained in Example 6.

FIG. 4 is an infrared absorption spectrum of 2- (α-D-mannopyranosyl) methyl-2,5,7,8-tetramethylchroman-6-ol obtained in Example 7.

FIG. 5 is an infrared absorption spectrum of 2- (β-D-galactopyranosyl) methyl-2,5,7,8-tetramethylchroman-6-ol obtained in Example 9.

Claims (6)

[Claims] 1. A sugar derivative in which a leaving group is introduced at the anomeric position and another hydroxyl group is protected with a protecting group, and a compound represented by the general formula (2): [Wherein, R ¹ , R ² , R ³ and R ⁴ represent the same or different hydrogen atoms, lower alkyl groups or lower acyl groups, R ⁵ represents a hydrogen atom, lower alkyl groups or lower acyl groups, And n are integers from 0 to 4. A condensation reaction of a 2-substituted alcohol derivative represented by the general formula (1): [Wherein, R ¹ , R ² , R ³ and R ⁴ represent the same or different hydrogen atoms, lower alkyl groups or lower acyl groups, R ⁵ represents a hydrogen atom, lower alkyl groups or lower acyl groups, X represents a monosaccharide residue (however, a hydrogen atom of a hydroxyl group in the sugar residue may be substituted with a lower alkyl group or a lower acyl group), and n is an integer of 0 to 4. ] The manufacturing method of the chromanol glycoside represented by these. 2. An L-fucose donor represented by the general formula (3), an L-rhamnose donor represented by the general formula (4), and a D represented by the general formula (5). The method according to claim 1, which is a xylose donor, a D-galactose donor represented by the general formula (6), or a D-mannose donor represented by the general formula (7). Embedded image Embedded image Embedded image Embedded image Embedded image [However, in the formulas (3), (4), (5), (6) and (7), Y represents a halogen atom, a sulfur compound or a trichloroacetimide group. And R is the same or different acetyl, benzoyl, pivaloyl,
Chloroacetyl group, levulinoyl group, benzyl group, p-
Represents a methoxybenzyl group, an allyl group, a t-butyldimethylsilyl group, a t-butyldiphenylsilyl group, a trimethylsilyl group or a trityl group. ] 3. The method according to claim 1, wherein the deprotection of the protecting group after the condensation reaction is performed by acid decomposition, enzymatic decomposition, or using a metal reagent. 4. The method according to claim 3, wherein the deprotection of the protecting group after the condensation reaction is performed using a metal reagent. 5. A compound of the general formula (1) produced by the method according to claim 1. [Wherein, R ¹ , R ² , R ³ and R ⁴ represent the same or different hydrogen atoms, lower alkyl groups or lower acyl groups, R ⁵ represents a hydrogen atom, lower alkyl groups or lower acyl groups, X represents a monosaccharide residue (however, a hydrogen atom of a hydroxyl group in the sugar residue may be substituted with a lower alkyl group or a lower acyl group), and n is an integer of 0 to 4. A chromanol glycoside represented by the formula: 6. The following general formulas (8), (9), (10),
The chromanol glycoside according to claim 5, which is represented by (11) or (12). Embedded image Embedded image Embedded image Embedded image Embedded image [However, in the formulas (8), (9), (10), (11) and (12), R ¹ , R ² , R ³ and R ⁴ are the same or different hydrogen atoms, lower alkyl groups or lower acyl groups. R ⁵ represents a hydrogen atom, a lower alkyl group or a lower acyl group, n is an integer of 0 to 4, and R is the same or different acetyl group, benzoyl group, pivaloyl group, chloroacetyl group, levulinoyl group , Benzyl group,
represents a p-methoxybenzyl group, an allyl group, a t-butyldimethylsilyl group, a t-butyldiphenylsilyl group, a trimethylsilyl group or a trityl group. ]