JP2751003B2 - New substances, their production methods and their uses - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel substance, a method for producing the same, and a use thereof. The present invention relates to a novel substance into which a substituent is introduced, a method for producing the same, and a separating agent for chromatography containing the novel substance.

[0002]

2. Description of the Related Art Compounds in which a polysaccharide such as cellulose or amylose or a derivative thereof is physically supported on silica gel by a physical method have been used as a separating agent for chromatography, particularly as a separating agent for optical resolution. It is known. However, since this compound has low solvent resistance, there is a drawback that usable eluents are limited when used in liquid chromatography or the like. In addition, the usefulness of the polysaccharide cannot be sufficiently brought out, and the washing solution used for washing the contaminated column is limited, which causes column deterioration.

In order to solve such problems, it has been proposed to use a compound obtained by chemically bonding a polysaccharide derivative to silica gel. However, in this case, since the site to be chemically bonded to the silica gel cannot be selected, the higher order structure of the saccharide itself is affected, and the useful property of the polysaccharide is greatly reduced. In addition, there is a drawback that the quality of the obtained silica gel compound varies greatly due to the unspecified binding site.

Accordingly, the present inventors have studied the use of silica gel as a porous carrier, and a substance having a structure in which a polysaccharide is chemically bonded at the reducing end thereof on the inner and outer surfaces of the pores as a separating agent for chromatography. Already, the present inventors lactonized the reducing end of the oligosaccharide, reacted it with, for example, 3-aminopropyltriethoxysilane, and silane-treated the sugar chain via the amide bond at the 1-position carbon at the reducing end of the sugar moiety. A compound represented by the following formula A, in which Rb and m ₂ have the same meanings as described above, and m ₃ is 2
N is an integer of 0 to 3, and n is preferably 0, but n silane treatment agents may be partially polymerized. )
(Japanese Patent Application No. 4-311042).

[0005]

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Further, the present inventors have developed a technique for polymerizing a sugar chain (Japanese Patent Application No. 4-358797). By utilizing this method, the compound represented by the formula A was successfully used as a primer for enzyme synthesis to extend the sugar moiety to an arbitrary degree of polymerization by enzymatic action. In addition, a polysaccharide derivative of a silane treatment agent obtained by enzymatic synthesis is bonded to silica gel using the silane portion, whereby a compound represented by the following formula B (where Rb has the same meaning as described above) ).

[0007]

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(Wherein, Rb, Z, m ₁ and m ₂ have the same meanings as described above.)

All or a part of the hydroxyl group in the sugar moiety of the compound represented by the formula B thus obtained is substituted with, for example, an isocyanate derivative or the like, and the compound represented by the following general formula (1) is substituted. The compounds of the invention can be obtained.

[0010]

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Alternatively, the reducing end of the oligosaccharide is oxidized to give glucanate, the sugar is extended to an arbitrary degree of polymerization by enzymatic action using this as a primer, and an acid is added to the reaction mixture to form a lactone. . The resulting lactonized polysaccharide is amide-bonded to silica gel surface-treated to have an amino group to obtain a compound represented by the above formula B. The compound of the present invention represented by the above general formula (1) is obtained by substituting all or part of the hydroxyl group of the sugar moiety of the obtained compound represented by the formula B with, for example, an isocyanate derivative or the like. be able to.

The compound represented by the above general formula (1) synthesized by these methods is prepared by applying a silane treatment agent to the inner and outer surfaces of a pore of a porous carrier such as silica gel with carbon at the reducing terminal of the sugar moiety. Through a chemical bond. The present inventors have found that this novel compound can solve the above-mentioned problems, and have completed the present invention.

[0013]

That is, according to the present invention, a polysaccharide or a derivative thereof is chemically bonded to the inside and outside surfaces of pores of a porous carrier at a reducing end of a polysaccharide site.
New substance whose main structure is the structure represented by

[0014]

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(Where R is Ra,

[0016]

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[0017]

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Wherein Ra is a hydrogen atom or a substituent selected from a substituted or unsubstituted alkyl group, a substituted or unsubstituted phenyl group and a substituted or unsubstituted heterocyclic compound; The degree of substitution of R is preferably 30 to 100% from the viewpoint of remarkable improvement. Rb
Denotes a group in which a substituted or unsubstituted methylene group, a substituted or unsubstituted phenylene group, a hetero atom, or the like is covalently bonded. Z is the surface of the porous carrier, a halogen atom, an alkyl group,
Indicates any of an alkoxy group, a phenyl group, a silane treatment agent and a sugar-bonded silane treatment agent. m ₁ indicates the number of monosaccharide units, and indicates an average number of 10 to 500. Also,
m ₂ represents an integer of 1 to 20. ) And chemically bonding the oligosaccharide having a degree of polymerization of 3 or more and 10 or less with a silane treatment agent at the reducing end, and then elongating the resulting oligosaccharide derivative to an average degree of polymerization of about 11 to 500 by enzymatic action. ,
Next, a method for producing a novel substance represented by the above general formula (1), characterized in that the silane moiety present at the sugar terminal is chemically bonded to a porous carrier, the reducing end of an oligosaccharide having a degree of polymerization of 3 to 10 Is obtained by oxidizing the aldehyde group of the above with an oxidizing agent, extending the sugar chain to an average degree of polymerization of about 11 to 500 by enzymatic action, and then adding an acid, wherein the acid is added. An object of the present invention is to provide a method for producing a substance and a chromatographic separating agent containing the novel substance represented by the general formula (1).

The oligosaccharide used for producing the novel compound of the present invention can be a substrate that can be synthesized into a polysaccharide by enzyme synthesis, and may be either a natural oligosaccharide or a synthetic oligosaccharide. Specifically, α-1,4-glucan oligomer (maltooligosaccharide), β-1,4-glucan oligomer (cellooligosaccharide), α-1,6-glucan oligomer (isomaltooligosaccharide), β-1,6 -Glucan oligomer (genthio-oligosaccharide), α-1,3-glucan oligomer (nigero-oligosaccharide), β-1,3-glucan oligomer (laminari oligosaccharide), α-1,2-glucan oligomer, β-1,2-glucan Oligomers (sophorooligosaccharides), β-1,4-chitooligosaccharides, β-1,4
-N-acetylchitooligosaccharide, β-1,4-galactan (lactooligosaccharide), α-1,6-galactan (meligosaccharide), β-2,1-fructan (inulooligosaccharide), β-2,6- Fructan, β-1,4-xylan, β-1,3-xylan, β-1,4-mannan, α
-1,6-mannan and the like can be mentioned as preferable ones. The number average degree of polymerization of these oligosaccharides is 3 or more,
The upper limit is not particularly limited, but is preferably about 3 to 10 from the viewpoint of reactivity in lactonization and reductive amination and handling.

Next, as the enzyme used in the present invention, a hydrolase, a glycosyltransferase, a polymerizing enzyme and the like can be used, and more specifically, an enzyme capable of synthesizing an oligosaccharide into a polysaccharide or having such an enzyme. It may be a microorganism.
For example, phosphorylase, dextran sucrose,
Examples include levansucrase, pullulan, and the like, and microorganisms capable of producing these enzymes.

The polysaccharide has an average degree of polymerization of 11 or more, and there is no particular upper limit, but usually 500 or less is preferable.

The porous carrier used in the present invention is a porous inorganic carrier or a porous organic carrier, and specifically, silica gel, diatomaceous earth, porous glass, hydroxyapatite, alumina, titanium oxide, magnesia And porous organic carriers such as polyacrylamide and polyacrylate, and silica gel is particularly preferred. The silica gel has a particle size of 1 to 1000 μm, preferably 2 to 100 μm, and an average pore size of 10 ° to 100 μm.
m, preferably 5 to 500 nm. By using a porous membrane as the porous carrier, a new separation membrane for chromatography can be obtained.

Next, a silane treatment agent having an amino group is used. When the reducing terminal is lactonized or when the reducing terminal is reduced in the presence of a reducing agent for amination, the primary amine is used. Are preferred. The silane treatment agent can be arbitrarily used, such as one commercially available as a silane coupling agent or one synthesized so as to have an amino group.

Further, it has a spacer for bonding the silane treatment agent and the sugar, that is, two or more functional groups of the same or different kind, and one of the functional groups chemically bonds to the reducing end of the sugar and the other functional group. And those chemically bonded to the silane treatment agent may be used. Here, the functional group includes, for example, a vinyl group, an amino group, a hydroxyl group, a carboxyl group, an aldehyde group, an isocyanate group, an isothiocyanate group, a thiol group, a silanol group, an epoxy group, an ether group, an ester group, and an amide group. , Halogen atoms and the like.
The silane treatment may be any treatment capable of binding to these functional groups, and representative silane treatments are shown below. Note that Rb in the general formula (1) represents a group in which a substituted or unsubstituted methylene group, a substituted or unsubstituted phenylene group, a hetero atom, or the like is covalently bonded. Or a part of a spacer in which a silane treatment agent is chemically bonded.

[0025]

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[0026]

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[0027]

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[0028]

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[0029]

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[0030]

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[0031]

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[0032]

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In the above formula, n ₁ is an integer of ₁ to 3, R ¹ is a hydrogen atom or an alkyl chain having about 1 to 20 carbon atoms or a derivative thereof, and R ² is an alkyl chain having about 1 to 20 carbon atoms or a derivative thereof. , X represents at least one substituted or unsubstituted alkoxy group having 1 to 10 carbon atoms, a halogen atom (preferably a chlorine atom), a hydroxyl group, a substituted or unsubstituted phenoxy group, and Y represents a halogen atom.

The substituent (R) introduced into a part or all of the hydroxyl group of the polysaccharide moiety of the compound represented by the above general formula (1) modifies the hydroxyl group, and is used in one molecule of the polysaccharide moiety. It is also possible to introduce two or more different substituents into the hydroxyl group. Ra in the substituent (R) is selected from a substituted or unsubstituted alkyl group, a substituted or unsubstituted phenyl group and a substituted or unsubstituted heterocyclic compound;
Specifically, methyl, ethyl, propyl, butyl, t-butyl, phenyl, methylphenyl, dimethylphenyl, ethylphenyl, diethylphenyl, trimethylsilylphenyl, alkoxyphenyl, dialkoxy Examples include a phenyl group, a halogenated phenyl group, a dihalogenated phenyl group, a phenylazophenyl group, a naphthalene group, an anthracene group, a pyridyl group, and a furyl group.

The introduction of these substituents into the hydroxyl group of the sugar moiety of the compound represented by the formula B can be carried out by a known method.

Hereinafter, a method for producing the novel compound of the present invention represented by the general formula (1) will be described. Production Method 1 Reaction A method for synthesizing compounds represented by the following formulas A and A 'is exemplified.

[0037]

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[0038]

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(In the above formula, Rb, m ₂ , m ₃ and n have the same meanings as described above.)

A method for converting an oligosaccharide into a lactone and amide-bonding it with a silane treatment agent is disclosed in Japanese Patent Application No. 4-31104.
The compound represented by the above formula A can be synthesized with reference to the description in the specification of No. 2. In this case, if water is mixed, polymerization of the silane treatment agents occurs, and therefore it is preferable to perform the treatment under anhydrous conditions. Further, when performing a chemical bond between the oligosaccharide and the silane treatment agent, the saccharide can be produced by chemically bonding with a silane treatment agent having an amino group in the presence of a reducing agent without lactonizing the reducing terminal aldehyde group of the sugar. Is possible (preparation of a compound of formula A ′, wherein n is from 0 to
It is an integer of 3, and n is preferably 0, but n silane treatment agents may be partially polymerized, and m ³ represents an integer of 2 to 9, respectively. ) (Glycoconjugate J. (1986), 3,311-31
9 ELIZABETHKALLIN etc. Reference).

The reducing agent used at this time is Na
Boron compounds such as BH ₄ , NaBH ₃ CN, borane pyridine complex, borane dimethylamine complex, and borane trimethylamine. further,
It is also possible to chemically bond the sugar reducing terminal to the silane treating agent via a spacer. That is, it is possible to form an amide bond or reductive amination at the amino group, which is one of the functional groups of the spacer, and at the sugar reducing terminal, and to chemically bond with the silane treatment agent at the other functional group.

Reaction Next, a method for enzymatically synthesizing the sugar moiety of the compounds represented by the formulas A and A 'obtained above will be exemplified. Formula A obtained using a maltooligosaccharide having four or more sugars
Alternatively, when the sugar chain of the compound represented by the formula A ′ is synthesized by enzyme, phosphorylase can be used as the enzyme. As the phosphorylase used at this time, for example, there is one derived from potato (see Starch Chemistry, Vol. 36, No. 4, p. 257-264 (1989)).

Using a compound represented by the formula A or A ', phosphorylase as an enzyme and potassium glucose monophosphate as a substrate, sterile water or a buffer such as maleic acid (pH 5-8, preferably 6-7,
The pH is adjusted with hydrochloric acid, potassium hydroxide or the like. Also,
When synthesizing a compound having an average degree of polymerization of 30 or more, dimethyl sulfoxide (DMSO) should be added in an amount of 10 to prevent aging.
It is preferable to add 30% (W / W). ) In room temperature to 55
The enzyme synthesis is carried out at a temperature of preferably from 35 to 45 ° C. In addition,
The average degree of polymerization (m ₁ ) can be determined by quantifying the amount of phosphoric acid released from glucose monophosphate or by a GPC method using an approximate value from a standard curve obtained from commercially available amylose (manufactured by Nakano Vinegar). The reaction is stopped by inactivating the enzyme at the degree of polymerization (m ₁ ), the polysaccharide derivative is precipitated in ethanol, washed with ethanol, acetone, ether, hexane, etc., dried under reduced pressure, and subjected to the following formulas C and C ′. Can be obtained.

[0044]

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(In the formula, Rb and m ₁ have the same meanings as described above, and n is an integer of 0 to 3, preferably 3.
Some ethoxy groups may be removed during enzyme synthesis. )

[0046]

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(In the formula, Rb and m ₁ have the same meanings as described above, and n has the same meaning as described above.)

Reaction The bonding of the compounds represented by the formulas C and C 'obtained above to silica gel is exemplified. Equation C,
The compound represented by C ′ is converted to anhydrous DMSO or anhydrous LiCl—
The compound is dissolved in a DMA solution or the like, pyridine is added as a catalyst, and the silane moiety of the compound represented by the formulas C and C ′ is combined with silica gel in the same manner as in a conventionally known silane treatment method to form the following formulas B and B To obtain the compound represented by

[0049]

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[0050]

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(In the formula, Rb, Z, m ₁ and m ₂ have the same meanings as described above.)

Production Method 2 Reaction A polysaccharide derivative having a reducing terminal lactonized
It may be abbreviated as lactonized polysaccharide. 3) shows a production example.
A glucanate salt obtained by oxidizing an oligosaccharide reducing end (see Japanese Patent Application No. 4-311042) is used to extend a sugar chain by enzymatic synthesis, and then lactonize the reducing end by acid treatment. For example, when a maltooligosaccharide having four or more sugars is used as an oligosaccharide, a glucanate salt obtained by oxidizing the reducing end is used as a primer, phosphorylase is used as an enzyme, and potassium glucose monophosphate is used as a substrate. Buffers such as acids (pH 5-8, preferably 6-7, pH
Adjustment is performed with hydrochloric acid, potassium hydroxide or the like. When synthesizing a compound having an average polymerization degree of 30 or more, dimethyl sulfoxide (DMSO) is added in an amount of 10 to 30% to prevent aging.
(W / W) may be added. The enzyme synthesis is carried out at room temperature to 55 ° C, preferably 35 to 45 ° C. Incidentally, the average degree of polymerization (m ₁ ) can be determined by quantifying the amount of phosphoric acid released from glucose monophosphate or an approximate value from a standard curve using commercially available amylose (manufactured by Nakano Vinegar) in the GPC method. The reaction is stopped by deactivating the enzyme at an arbitrary degree of polymerization (m ₁ ). Thereafter, a strong acid such as hydrochloric acid is added to the reaction solution to lower the pH to 0 to 4, preferably 1 to 2, Is lactonized, and the polysaccharide derivative is precipitated in ethanol, washed with ethanol, acetone, ether, hexane, etc., and then dried under reduced pressure, so that amylose having a lactone at the reducing end (hereinafter may be abbreviated as lactonized amylose). .)

Further, after obtaining the lactonized polysaccharide, it is possible to form an amide bond with the amino group of the spacer, or it is possible to lactone the reducing end of the oligosaccharide and bind the spacer, followed by enzyme synthesis. Is also possible.

Reaction The binding between the lactonized polysaccharide and the surface-treated silica gel will be exemplified. Silane treating agent having an amino group on the inner and outer surfaces of the pores of silica gel, for example, 3-
The surface treatment of aminopropyltriethoxysilane can be carried out by a conventionally known method. The lactonized polysaccharide obtained by the above reaction is dissolved in a solvent such as DMSO, and the amino group existing on the surface of the silica gel and 5
An amide bond is formed by reacting at 0 to 70 ° C. Next, the unreacted lactonized polysaccharide is removed by washing with a solvent such as DMSO, acetone and hexane, and dried under reduced pressure to obtain the compound represented by the formula B.

When using a polysaccharide to which a spacer is bound as described in the reaction, a silane-treating agent capable of binding to the spacer or a silane-treating agent derivative synthesized to bind to the spacer is used. It is also possible to apply a surface treatment to silica gel.

Production method 3 The hydroxyl group of the sugar moiety of the compound represented by the formula B or B 'obtained by the above production method 1 or 2 and, for example, 4-methylphenylisocyanate; 3,5-dimethylphenylisocyanate; 3,5-dichlorophenyl Isocyanate: phenyl isocyanate or the like is reacted in an anhydrous DMA / pyridine solution or an anhydrous DMSO / pyridine solution to replace all or a part of the hydroxyl group in the sugar moiety of the compound. This reaction is
It can be performed in a known manner. The amount of the compound to be chemically bonded to the silica gel is not particularly limited, but is usually preferably 5 to 50% by weight. In order to eliminate the influence of the residual silanol group in the obtained compound of the present invention, the performance as a separating agent can be improved by performing an endcapping treatment by a conventionally known method. In the present invention, when showing the structure of each compound, the structure showing the position of the hydroxyl group of the sugar and the like is partially simplified for convenience and generality.

[0057]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples. Synthesis Example 1 (1-1) Compound A ₁ represented by the following formula

[0058]

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(In the formula, n has the same meaning as described above.)

[0060] Using 5 g of maltopentaose,
Compound A ₁ by the method described in JP-A-311042.
Was synthesized. Compound A ₁ is the stretching vibration of bending vibration and C = O of N-H of the secondary amide is observed at 1540 cm ^-1 and 1640 cm ^-1 from the infrared absorption spectrum of FIG. 1, ¹³ C-NMR (solvent : DMSO-d6, compound A ₁ concentration 5% (W / V), the reference substance TMS, 60 ℃, 4
00 MHz), the chemical shift value of the carbon derived from the amide bond is 172 ppm, the carbons at the 4- and 6-positions of the ring-opened sugar are found at 82.7 ppm and 62.8 ppm, respectively. 1
8.1 ppm, assumed main structure of Compound A ₁ from the 3-position of the carbon is upfield shifted and 41.0ppm things and propyl groups found in 57.7ppm is the same as that of the compound A Is done.

(1-2) Compound C ₁ represented by the following formula
Synthesis of

[0062]

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(In the formula, the average degree of polymerization m ₄ calculated by the phosphoric acid determination is 19, and n has the same meaning as described above.)

[0064] dissolved in a compound synthesized above 1-1 A ₁ 1.2 g of glucose 1 phosphoric acid (G1P) and 12 g (adjusted to pH 6) sterilized ultrapure water 300 ml, the crude phosphorylase potato derived therein 180units After the addition, the mixture was allowed to stand at 30 ° C. for about 3 hours. For sugar chain elongation, sample a part of the reaction solution and inactivate the enzyme with trichloroacetic acid.
Tracking was performed by quantifying the amount of free phosphoric acid by the e-Subbarow method.

The reaction solution was inactivated in a hot water bath to deactivate the enzyme, and after filtration, 100% ethanol was added in the same amount as the reaction solution to precipitate the produced sugar. In addition, 50% ethanol,
After washing with 100% ethanol and diethyl ether,
2 hours and dried in vacuo at ° C., to give compound C ₁ of 2.2 g.
Next, the compound was subjected to gel filtration liquid chromatography analysis, and amylose standard reagent (manufactured by Nakano Vinegar Co., Ltd.)
It was confirmed that the average degree of polymerization m ₄ calculated from the standard curve was 31 and the average degree of polymerization m ₄ calculated from the phosphoric acid determination was 19.

¹ H-NMR (solvent: DMSO-d6, compound B ₁ concentration 5% (W / V), reference material TMS, 60
M, 400 MHz) from the silane treatment agent.
m, 1.53 ppm (the methylene proton at the 3-position is masked by a sugar-derived proton) and the methyl proton of the ethoxy group is 1.15 ppm (the methylene proton is masked by a sugar-derived proton). it was suggested the compounds C _1.

(1-3) Synthesis of a compound represented by the following general formula (2)

[0068]

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(Wherein Rc is a hydrogen atom or

[0070]

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The degree of substitution was 41 based on the elemental analysis value.
%, Where m ₄ is considered to be approximately the same as compound C _1, and Z ₁ is a silica gel surface, an ethoxy group and either a silane treatment or a sugar-bonded silane treatment. )

Compound C synthesized in (1-2) of Example 1
The ₁ 1.0 g was dissolved in anhydrous DMSO14ml, preactivated (180 ° C., 2 hours in a vacuum drying) to leave the silica gel (YMC Co., Ltd., average pore size 12 nm, particle size 5 [mu] m) was added in 3g, further pyridine 4ml 90 ° C under nitrogen
In reacted for 12 hours, it was chemically bonded with the silane portion of the silanol groups and the compound C ₁ of the silica gel. The surface-treated silica gel obtained in this manner is applied to a glass filter G4.
Was filtered using, the residue was washed DMSO, tetrahydrofuran, methanol, acetone, hexane, except Compound C _1, etc. unbound, and vacuum dried for two hours at 60 ° C..
For this product, the binding of sugar was confirmed by elemental analysis.
The elemental analysis values were as follows: C: 6.25%, H: 1.53%,
N: 0.06%.

This surface-treated silica gel was treated with anhydrous DMSO8
and 3 ml of anhydrous pyridine, and 3.0 ml of 4-methylphenylisocyanate was added to the reaction solution.
The reaction was carried out at 80 ° C. for 5 hours under nitrogen to modify the hydroxyl group of the sugar moiety chemically bonded to the silica gel surface. After confirming that an excess of isocyanate group remained in the solution by confirming stretching vibration between C and N at 2270 cm ^-1 in the IR spectrum, the surface-treated silica gel in the reaction solution was treated with tetrahydrofuran, methanol, After washing with acetone and hexane to remove impurities,
For 2 hours under vacuum. Then, for this compound I
R spectrum analysis and elemental analysis were performed. The results are shown in FIG. 2 and Table 1.

[0074]

[Table 1]

As a result, 173 was found in the IR spectrum.
It was confirmed that stretching vibration of a carbonyl group (absorption of C 第 O of a secondary carbamate) was observed around 0 cm ⁻¹ , and furthermore, binding to silica gel was confirmed by elemental analysis, and the above-mentioned general formula (2) was obtained. The compound represented by was obtained.

In order to improve the performance of the thus-obtained compound represented by the general formula (2) as a separating agent for chromatography, end capping generally performed was performed using trimethylchlorosilane. The product thus obtained was filtered using a glass filter G4, and the residue was washed with tetrahydrofuran, methanol, acetone, and hexane in the same manner as described above, dried at 60 ° C. for 2 hours under reduced pressure, and separated for chromatography. Agent. The results of evaluating this product as a separating agent for various racemic compounds
It is shown in the table. In Table 2, k ₁ is a holding capacity ratio, which is obtained by the following equation, and α is a separation coefficient, which is obtained by the following equation. Also, k ₂ is k ₁ and a retention capacitance ratio as well,
It is obtained by the following equation.

[0077]

(Equation 1)

[0078]

(Equation 2)

[0079]

(Equation 3)

[0080]

[Table 2]

[0081]

[Table 3]

Example 2 (2-1) Synthesis of compound C ₂ represented by the following formula

[0083]

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(In the formula, the average degree of polymerization m ₄ calculated by the phosphoric acid determination is 79, and n has the same meaning as described above.)

[0085] In the same manner as in Example 1 (1-2), to synthesize a compound C ₂ represented by the above formula. That is, the compound is dissolved A ₁ 0.3 g of glucose 1 phosphoric acid (G1P) 12 g synthesized in Example 1 (1-1) DMSO105ml, sterilized ultrapure water 300 ml (adjusted to pH 6), potato-derived therein After adding 210 units of crude phosphorylase, the mixture was allowed to stand at 30 ° C. for about 4 hours. Sugar chain elongation was monitored by sampling a part of the reaction solution, inactivating the enzyme with trichloroacetic acid, and quantifying the amount of free phosphoric acid by the Fiske-Subbarow method.

The reaction solution was treated in a water bath to deactivate the enzyme, and after filtration, 100% ethanol was added in the same amount as the reaction solution to precipitate the produced sugar. Furthermore, 50% ethanol, 100% ethanol, washed with diethyl ether to give the above compound C ₂ of 2.2g was vacuum dried for two hours at 60 ° C.. Next, the compound was subjected to gel filtration liquid chromatography analysis, and the average degree of polymerization m ₄ calculated from a standard curve using an amylose standard reagent (manufactured by Nakano Vinegar) was 85, and the average degree of polymerization m calculated from the phosphoric acid determination was 85%.
₄ was confirmed to be 79.

(2-2) Synthesis of compound represented by general formula (3) represented by the following formula

[0088]

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(Wherein Rc is a hydrogen atom or

[0090]

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The degree of substitution is 90% based on the calculated weight.
% Or more. In the formula, m ₄ is considered to be almost the same as compound C _2, and Z ₁ has the same meaning as described above. )

Compound C ₂ 1.0 synthesized in the above (2-1)
g was dissolved in 14 ml of anhydrous DMSO and activated (18
3 g of silica gel (average pore size: 100 nm, particle size: 7 μm) (manufactured by Daiso Corporation) dried at 0 ° C. for 2 hours, further added with 4 ml of pyridine, and added with nitrogen at 90 ° C. under nitrogen.
After reacting for 2 hours, silanol group of silica gel and compound C
Chemical bonding was carried out at the silane part of No. ₂ . The surface-treated silica gel thus obtained was filtered using a glass filter G4, and the residue was washed with DMSO, tetrahydrofuran, methanol, acetone and hexane to obtain unbound compound C.
Except for ₂ etc., it was vacuum dried at 60 ° C. for 2 hours.

This surface-treated silica gel was treated with anhydrous DMSO8
and 3 ml of anhydrous pyridine. 1.5 ml of 3,5-dimethylphenylisocyanate was added to the reaction mixture, and the mixture was reacted at 80 ° C. for 5 hours under nitrogen. Was modified. After confirming that an excess isocyanate group remained in the solution and that stretching vibration between C = N was observed at 2270 cm ^-1 in the IR spectrum, the surface-treated silica gel in the reaction solution was treated with tetrahydrofuran, methanol , Acetone and hexane to remove impurities.
Vacuum dried at 2 ° C. for 2 hours. Next, the compound was subjected to IR spectrum analysis and elemental analysis. Table 1 shows the results of elemental analysis.

Further, in the IR spectrum, 1730c
It was confirmed that stretching vibration of the carbonyl group (absorption of C ＝ O of the secondary carbamate) was observed around m ⁻¹ , and further, the bond to silica gel was confirmed from the elemental analysis value, and the above-mentioned general formula (3) was obtained. The compound represented by was obtained.

In order to improve the performance of the obtained compound as a separating agent for chromatography, generally used end-capping was performed using trimethylchlorosilane. The product thus obtained was filtered using a glass filter G4, and the residue was treated with tetrahydrofuran and
After washing with methanol, acetone and hexane,
It was dried under vacuum (reduced pressure) for a time to obtain a separating agent for chromatography. Table 2 shows the results of evaluating this product as a separating agent for various racemic compounds.

Example 3 (3-1) Synthesis of lactonized amylose represented by the following formula

[0097]

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[0098] (wherein, the average degree of polymerization m ₅ calculated by phosphoric acid quantification is 15.) Using maltopentaose 18.9g according to known methods,
This was added to a KOH-methanol solution in an iodine-methanol solution and reacted to give {O-α-D-glucopyranosyl- (1 → 4)} _4- D-glucanate potassium 19.7 g.
(Hereinafter sometimes abbreviated as G5 glucanate potassium). 2.13 g of potassium G5 glucanate and 32 g of glucose monophosphate (G1P) are dissolved in 800 ml of sterilized ultrapure water (adjusted to pH 6), and 240 units of crude potato-derived phosphorylase is added thereto. It was left still. For sugar chain elongation, sample a part of the reaction solution, deactivate the enzyme with trichloroacetic acid,
It was followed by quantifying free phosphoric acid by the bbarow method.

The reaction solution was heated in a water bath to inactivate the enzyme, filtered, and then acidified to pH 1 by adding concentrated hydrochloric acid to convert the glucanate salt to lactone. Thereafter, 100% ethanol was added in the same amount as the amount of the reaction solution to precipitate the produced sugar. In addition, 50% ethanol,
After washing with 100% ethanol and diethyl ether,
Vacuum drying at 2 ° C. for 2 hours gave 1.01 g of the lactonized amylose represented by the above formula. Subjected to gel filtration liquid chromatography analysis of this compound, the standard curve by amylose standard reagent (Nakanosumise), average degree of polymerization m ₅ the calculated was 37. The average degree of polymerization m ₅ calculated from the phosphoric acid determination was 15. Further, from the IR spectrum, it was confirmed that stretching vibration between C = O of the lactone was observed at 1740 cm ^-1 .

(3-2) Synthesis of lactonized amylose represented by the following formula

[0101]

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[0102] (wherein, the average degree of polymerization m ₅ calculated by phosphoric acid quantification is 29.) G5 Group qanat potassium synthesized above (3-1) 0.
53 g and glucose monophosphate (G1P) 16 g 240 m
l of sterilized ultrapure water, dissolved in 105 ml of DMSO, pH6
After adding 400 units of crude potato-derived phosphorylase, the mixture was allowed to stand at 45 ° C. for about 2 hours.
Sugar chain elongation was monitored by sampling a part of the reaction solution, inactivating the enzyme with trichloroacetic acid, and quantifying free phosphoric acid by the Fiske-Subbarow method.

The reaction solution was heated in a water bath to inactivate the enzyme, filtered, and then acidified to pH 1 by adding concentrated hydrochloric acid to convert the glucanate salt to lactone. Thereafter, 100% ethanol was added in the same amount as the amount of the reaction solution to precipitate the produced sugar. In addition, 50% ethanol,
After washing with 100% ethanol and diethyl ether,
Vacuum drying at 2 ° C. for 2 hours gave 3.0 g of the lactonized amylose represented by the above formula. Subjected to gel filtration liquid chromatography analysis of this compound, the standard curve by amylose standard reagent (manufactured by Nakanosumise), average degree of polymerization m ₅ the calculated was 47. The average degree of polymerization m ₅ calculated from the phosphoric acid determination was 29.

(3-3) Synthesis of lactonized amylose represented by the following formula

[0105]

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(In the formula, the average degree of polymerization m ₅ calculated by phosphoric acid quantification is 92.) G5 glucanate potassium synthesized in the above (3-1) was 0.1%.
265 g and glucose monophosphate (G1P) 16 g for 240
Dissolve in 105 ml of sterile ultrapure water, DMSO
After adjusting to 6, the mixture was added with 550 units of crude potato-derived phosphorylase and allowed to stand at 45 ° C. for about 5 hours. For sugar chain elongation, sample a part of the reaction solution,
After inactivating the enzyme with trichloroacetic acid, Fiske-Subbarow
It was followed by quantifying free phosphoric acid by the method.

The reaction solution was heated in a water bath to inactivate the enzyme, filtered, and then acidified to pH 1 by adding concentrated hydrochloric acid to convert the glucanate salt to lactone. Thereafter, 100% ethanol was added in the same amount as the amount of the reaction solution to precipitate the produced sugar. In addition, 50% ethanol, 100
% Ethanol and diethyl ether, and
After vacuum drying for 2 hours, 2.48 g of the lactonized amylose represented by the above formula was obtained. Subjected to gel filtration liquid chromatography analysis of this compound, the standard curve by amylose standard reagent (manufactured by Nakanosumise), average degree of polymerization m ₅ the calculated was 147. The average degree of polymerization m ₅ calculated from the phosphoric acid determination was 92.

(3-4) Synthesis of surface-treated silica gel Part 1 Silica gel (manufactured by Daiso, average pore size 100 nm, particle size 7) previously activated (180 ° C., vacuum drying for 2 hours)
36 μm of anhydrous benzene and 3 ml of anhydrous pyridine were added to 10 g of μm), 2 ml of 3-aminopropyltriethoxysilane was added, and the mixture was reacted at 90 ° C. for 12 hours. The surface-treated silica gel thus obtained was washed with methanol, acetone and hexane, and then dried under vacuum at 60 ° C. for 2 hours.

(3-5) Synthesis of Surface-treated Silica Gel Part 2 Silica gel (manufactured by Daiso, average pore size 100 nm, particle size 7) previously activated (180 ° C., vacuum drying for 2 hours)
To 10 g, 12 ml of anhydrous benzene and 1 ml of anhydrous pyridine were added, and 0.7 ml of 3- (2-aminoethylaminopropyl) trimethoxysilane was added.
The reaction was performed for 2 hours. The surface-treated silica gel thus obtained was washed with methanol, acetone and hexane, and then dried under vacuum at 60 ° C. for 2 hours.

(3-6) Synthesis of surface-treated silica gel Part 3 Silica gel (manufactured by Daiso, average pore size 100 nm, particle size 7) previously activated (180 ° C., vacuum drying for 2 hours)
12 ml of anhydrous benzene and 1 ml of anhydrous pyridine were added to 10 g of μm), 0.7 ml of 3- [2-aminoethylaminoethylamino) propyltrimethoxysilane was added, and the mixture was reacted at 90 ° C. for 12 hours. The surface-treated silica gel thus obtained was washed with methanol, acetone and hexane, and then dried under vacuum at 60 ° C. for 2 hours.

(3-7) Synthesis of Compound Represented by General Formula (4) 3 g of the surface-treated silica gel having an aminopropyl group bonded to the surface obtained in the above (3-4) was added to the above (3-1) 1) of the lactonized amylose obtained in
Then, the solution dissolved in ml was added and amide-bonded at 80 ° C. for 12 hours. The surface-treated silica gel was filtered using a glass filter G4, and the residue was washed with DMSO, tetrahydrofuran, methanol, acetone and hexane to remove unbound lactonized amylose and the like, followed by vacuum drying at 60 ° C. for 2 hours. Next, the sugar-bonded surface-treated silica gel was dispersed in anhydrous DMSO (8 ml) and anhydrous pyridine (3 ml), 3,5-dimethylphenyl isocyanate (1.5 ml) was added to the reaction solution, and the mixture was reacted at 80 ° C. for 5 hours.
Excess isocyanate group remaining in the solution was confirmed by confirming stretching vibration between C = N at 2270 cm ^-1 in the IR spectrum. The sugar-bonded surface-treated silica gel in the reaction solution was washed with tetrahydrofuran, methanol, acetone, and hexane, and dried under vacuum at 60 ° C. for 2 hours. This compound was subjected to IR spectrum analysis and elemental analysis. Table 1 shows the results of elemental analysis.

Also, in the IR spectrum, 1730c
It was confirmed that stretching vibration of a carbonyl group (absorption of C の O of the secondary carbamate) was observed around m ⁻¹ , and furthermore, the bond to silica gel was confirmed from the elemental analysis value, and the following general formula (4) Was obtained as a separating agent for chromatography. Table 2 shows the results of evaluating this product as a separating agent for various racemic compounds.

[0113]

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(Wherein Rc is a hydrogen atom or

[0115]

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The degree of substitution was 90% based on the weight calculation.
That is all. Where m ₄ is the m of the lactonized amylose
_It is considered to be almost the same as _5, and Z ₁ has the same meaning as described above. )

Example 4 Synthesis of a compound represented by the following general formula (5)

[0118]

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(Where Rc is

[0120]

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The degree of substitution was 90% based on the weight calculation.
That is all. Where m ₄ is the m of the lactonized amylose
_It is considered to be almost the same as _5, and Z ₁ has the same meaning as described above. )

The lactonized amylose 1 obtained in Example 3 (3-2) was added to 3 g of the surface-treated silica gel having an aminopropyl group bonded to the surface obtained in Example 3 (3-4).
g dissolved in 8 ml of anhydrous DMSO.
For 12 hours. This surface-treated silica gel is filtered using a glass filter G4, and the residue is treated with DMS.
It was washed with O, tetrahydrofuran, methanol, acetone and hexane to remove unbound lactonized amylose and the like, followed by vacuum drying at 60 ° C for 2 hours. Next, the sugar-bonded surface-treated silica gel was dispersed in anhydrous DMSO (8 ml) and anhydrous pyridine (3 ml), and 3,5-dimethylphenyl isocyanate (1.5 ml) was added to the reaction solution.
After reacting for 2 hours, it was confirmed in the IR spectrum that the excess isocyanate group remained in the solution at 2270 c.
This was confirmed by recognizing stretching vibration between C = N at m ⁻¹ . The sugar-bonded surface-treated silica gel in the reaction solution was washed with tetrahydrofuran, methanol, acetone, and hexane, and dried under vacuum at 60 ° C. for 2 hours. This compound was subjected to IR spectrum analysis and elemental analysis. Table 1 shows the results of elemental analysis.

Further, in the IR spectrum, 1730c
The stretching vibration of the carbonyl group (absorption of CＣO of the secondary carbamic acid ester) was observed around m ⁻¹ , and the bond to silica gel was confirmed from the elemental analysis values. Was obtained as a separating agent for chromatography. Table 2 shows the results of evaluating this product as a separating agent for various racemic compounds.

Example 5 Synthesis of compound represented by the following general formula (6)

[0125]

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(Where Rc is

[0127]

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The degree of substitution was 90% by weight calculation.
That is all. Where m ₄ is the m of the lactonized amylose
_It is considered to be almost the same as _5, and Z ₁ has the same meaning as described above. )

The lactonized amylose 1 obtained in Example 3 (3-3) was added to 3 g of the surface-treated silica gel having an aminopropyl group bonded to the surface obtained in Example 3 (3-4).
g dissolved in 8 ml of anhydrous DMSO.
For 12 hours. This surface-treated silica gel is filtered using a glass filter G4, and the residue is treated with DMS.
It was washed with O, tetrahydrofuran, methanol, acetone and hexane to remove unbound lactonized amylose and the like, followed by vacuum drying at 60 ° C for 2 hours. Next, the sugar-bonded surface-treated silica gel was dispersed in anhydrous DMSO (8 ml) and anhydrous pyridine (3 ml), and 3,5-dimethylphenyl isocyanate (1.5 ml) was added to the reaction solution.
After reacting for 2 hours, it was confirmed in the IR spectrum that the excess isocyanate group remained in the solution at 2270 c.
This was confirmed by recognizing stretching vibration between C = N at m ⁻¹ . The sugar-bonded surface-treated silica gel in the reaction solution was washed with tetrahydrofuran, methanol, acetone, and hexane, and dried under vacuum at 60 ° C. for 2 hours. This compound was subjected to IR spectrum analysis and elemental analysis. Table 1 shows the results of elemental analysis.

Also, in the IR spectrum, 1730c
A stretching vibration of a carbonyl group (absorption of C ＝ O of a secondary carbamate) was observed around m ⁻¹ , and further, a bond to silica gel was confirmed from an elemental analysis value. Was obtained as a separating agent for chromatography. Table 2 shows the results of evaluating this product as a separating agent for various racemic compounds.

Example 6 Synthesis of a compound represented by the following general formula (7)

[0132]

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(Wherein Rc is a hydrogen atom or

[0134]

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The degree of substitution was 90% by weight calculation.
That is all. Where m ₄ is the m of the lactonized amylose
₅ and is considered substantially the same, Z ₂ is one of the silica gel surface, a methoxy group and a silane treatment agent or sugar binding silanizing agent. )

Example 3 3 g of the surface-treated silica gel having amino groups bonded to the surface obtained in (3-5) was added to Example 3
Lactonized amylose 1 synthesized in the same manner as (3-3)
g dissolved in 8 ml of anhydrous DMSO.
For 12 hours to obtain a compound represented by the above general formula (7) in the same manner as in Example 5. Table 1 shows the elemental analysis values of this compound. Table 2 shows the results of evaluating this compound as a separating agent for chromatography and as a separating agent for various racemic compounds.

Example 7 Synthesis of a compound represented by the following general formula (8)

[0138]

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(Wherein Rc is a hydrogen atom or

[0140]

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The degree of substitution was 90% based on the weight calculation.
That is all. Where m ₄ is the m of the lactonized amylose
_It is considered to be almost the same as _5, and Z ₂ has the same meaning as described above. )

Example 3 3 g of surface-treated silica gel having amino groups bonded to the surface obtained in (3-6) was added to Example 3
Lactonized amylose 1 synthesized in the same manner as (3-3)
g was dissolved in 8 ml of dry DMSO.
An amide bond was formed at 12 ° C. for 12 hours to obtain a compound represented by the general formula (8) in the same manner as in Example 5. Table 1 shows the elemental analysis values of this compound. Table 2 shows the results of evaluating this compound as a separating agent for chromatography and as a separating agent for various racemic compounds.

Example 8 Synthesis of a compound represented by the following general formula (9)

[0144]

Embedded image (Where Rc is a hydrogen atom or

[0145]

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The degree of substitution was 90% based on the weight calculation.
That is all. Where m ₄ is the m of the lactonized amylose
₅ and is considered substantially the same, Z ₂ is one of the silica gel surface, a methoxy group and a silane treatment agent or sugar binding silanizing agent. )

(8-1) Synthesis of surface-treated silica gel Silica gel (manufactured by Fuji Silysia Chemical Ltd., having an average pore diameter of 50 nm, previously activated (180 ° C., vacuum drying for 2 hours)
12 ml of anhydrous benzene and ml of anhydrous pyridine were added to 10 g of a particle size of 5 μm), 0.7 ml of 3- (2-aminoethylaminopropyl) triethoxysilane was added, and 90 ° C.
For 12 hours. The surface-treated silica gel thus obtained was washed with methanol, acetone and hexane, and then dried under vacuum at 60 ° C. for 2 hours.

(8-2) 1 g of lactonized amylose synthesized in the same manner as in Example 3 (3-3) was added to 3 g of the surface-treated silica gel having an amino group bonded to the surface obtained in (8-1). A solution dissolved in 8 ml of anhydrous DMSO was added,
The amide bond was performed at 80 ° C. for 12 hours. The surface-treated silica gel was filtered using a glass filter G4, and the residue was washed with DMSO, tetrahydrofuran, methanol, acetone and hexane to remove unbound lactonized amylose and the like, followed by vacuum drying at 60 ° C. for 2 hours. The sugar-bonded silica gel is dispersed in a solution composed of 8 ml of anhydrous DMA-LiCl and 3 ml of pyridine.
After adding 5 g of 3,5-dichlorophenyl isocyanate (prepared from 3,5-dichloroaniline according to a conventional method) dissolved in 5 ml of MA and reacting at 80 ° C. for 12 hours under nitrogen, excess isocyanate groups are contained in the solution. It was confirmed by the presence of C = N stretching vibration at 2270 cm ^-1 in the IR spectrum. This sugar-bonded surface-treated silica gel is treated with
It was washed with acetone and hexane and dried in vacuum at 60 ° C. for 2 hours. This compound was subjected to IR spectrum analysis and elemental analysis. Table 1 shows the results of elemental analysis.

In the IR spectrum, 1730c
A stretching vibration of a carbonyl group (absorption of CＣO of the secondary carbamate) was observed around m ⁻¹ , and further, a bond to silica gel was confirmed from an elemental analysis value. Was obtained as a separating agent for chromatography. Table 3 shows the results of evaluating this as a separating agent for various racemic compounds.

[0150]

[Table 4]

Example 9 Synthesis of a compound represented by the following general formula (10)

[0152]

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(Wherein Rc is a hydrogen atom or

[0154]

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The degree of substitution was 90% by weight calculation.
That is all. Where m ₄ is the m of the lactonized amylose
₅ and is considered substantially the same, Z ₂ is one of the silica gel surface, a methoxy group and a silane treatment agent or sugar binding silanizing agent. )

[0156] Example 3 was added to 3 g of the surface-treated silica gel having amino groups bonded to the surface obtained in Example 8 (8-1).
Lactonized amylose 1 synthesized in the same manner as (3-3)
g was dissolved in 8 ml of dry DMSO.
Amide bonding was performed at 12 ° C. for 12 hours. This surface-treated silica gel is filtered using a glass filter G4, and the residue is DM
It was washed with SO, tetrahydrofuran, methanol, acetone and hexane to remove unbound lactonized amylose and the like, followed by vacuum drying at 60 ° C for 2 hours.

The saccharide-bonded silica gel was treated with anhydrous DMA.
-Dispersed in a liquid composed of 8 ml of LiCl and 3 ml of pyridine, and 2 ml of phenyl isocyanate was added to the reaction solution. The mixture was reacted at 80 ° C. for 12 hours under nitrogen. This was confirmed by confirming stretching vibration between C = N at 2270 cm ^-1 in the IR spectrum. This sugar-bonded surface-treated silica gel is treated with tetrahydrofuran, methanol, acetone,
Washed with hexane and vacuum dried at 60 ° C. for 2 hours. This compound was subjected to IR spectrum analysis and elemental analysis. Table 1 shows the results of elemental analysis.

In the IR spectrum, 1730c
A stretching vibration of a carbonyl group (absorption of CＣO of a secondary carbamate) was observed around m ⁻¹ , and further, a bond to silica gel was confirmed from an elemental analysis value. Was obtained as a separating agent for chromatography. Table 4 shows the results of evaluating this product as a separating agent for various racemic compounds.

[0159]

[Table 5]

Application Examples Preparation of Optical Separation Column and Optical Resolution A novel substance obtained in each of Examples 1 to 9 was packed in a 0.46 × 25 cm stainless steel empty column by a slurry filling method. The filling device was PS-1 manufactured by Kyoto Chromato.
A 0, PS-20 autopacking system was used. Using this column, the optical resolution was evaluated. The high-speed chromatographic system is Waters 510 Pump and
A UV detector or the like was used. As a comparative example, the results of a separating agent prepared by physically coating an amylose tris (3,5-dimethylphenyl carbide) derivative on a surface-treated silica gel were used. The results are shown in Tables 2, 3 and 4 (see CHEMISTRY LETTERS. Pp. 1857-1860, 1987). As is clear from the optical division examples in Table 2, the general formulas (6), (7), and
Any of the compounds represented by (8) is a racemic α
The value was higher than that of the comparative example.

Further, as is apparent from the optical resolution examples in Table 3, the compound represented by the general formula (9) was changed to an eluent containing tetrahydrofuran to give a racemate which could not be separated in the comparative example. The bodies (10) and (11) could be separated. As is clear from the optical division example in Table 4,
For the compound represented by the general formula (10), a racemate which could not be separated in the comparative example became separable. It should be noted that the α value of the racemic body was significantly improved as compared with the comparative example.

Further, in order to examine the solvent resistance of the column for optical resolution prepared using the novel substance of the present invention, the optical resolution was examined after flowing a tetrahydrofuran (THF) solution at a flow rate of 1 ml / min for 2 hours. However, no change was observed, and it was confirmed that the composition had excellent solvent resistance. The analysis was performed using an eluent → hexane: IPA = 90: 10, Δ represents 9
5: 5 is shown. * Indicates an eluent → hexane: tetrahydrofuran = 80: 20. The flow rate is 0.5 ml / mi.
n, at room temperature.

[0163]

The novel substance of the present invention has excellent solvent resistance and is useful as a chromatographic separating agent for resolving optically active substances. Further, according to the present invention, the novel substance can be efficiently produced by a simple method.

[Brief description of the drawings]

1 is an IR spectrum of the compound of formula A _1, obtained in Example 1 (1-1).

FIG. 2 is an IR spectrum of the compound represented by the general formula (2) obtained in Example 1 (1-3).

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Hirofumi Akano 2-46-28 Ariwaki-cho, Handa-shi, Aichi Prefecture

Claims (8)

(57) [Claims] 1. A polysaccharide or a derivative thereof is chemically bonded to inner and outer surfaces of pores of a porous carrier at a reducing end of a polysaccharide site,
A novel substance having a structure represented by the following general formula (1) as a main structure. Embedded image (Wherein R is Ra, Embedded image Wherein Ra is a hydrogen atom or a substituent selected from a substituted or unsubstituted alkyl group, a substituted or unsubstituted phenyl group and a substituted or unsubstituted heterocyclic compound, and Rb is a substituted or unsubstituted A compound in which a methylene group, a substituted or unsubstituted phenylene group, a hetero atom or the like is covalently bonded. Z represents any one of a porous carrier surface, a halogen atom, an alkyl group, an alkoxy group, a phenyl group, a silane treatment agent and a sugar-bonded silane treatment agent. m ₁ indicates the number of monosaccharide units, and on average 10 to 500
Indicates the number of M ₂ represents an integer of 1 to 20. ) 2. An oligosaccharide derivative having an average degree of polymerization of 11 to 500 after being chemically bonded to a silane-treating agent at the reducing end of an oligosaccharide having a degree of polymerization of 3 to 10 by an enzymatic action.
The method for producing a novel substance according to claim 1, wherein the substance is extended to a certain extent and then chemically bonded to a porous carrier at a silane moiety present at the sugar terminal. 3. An aldehyde group at the reducing end of an oligosaccharide having a degree of polymerization of 3 or more and 10 or less is oxidized by an oxidizing agent, and then extended to an average degree of polymerization of a sugar chain of about 11 to 500 by enzymatic action, and then an acid is added. The method for producing a novel substance according to claim 1, wherein 4. The method for producing a novel substance according to claim 1, wherein the enzyme used in claim 2 or 3 is any one of a hydrolase, a glycosyltransferase and a polymerizing enzyme. 5. The method for producing a novel substance according to claim 2, wherein the hydrolase according to claim 4 is phosphorylase. 6. The method for producing a novel substance according to claim 2, wherein dimethyl sulfoxide is added to the reaction solution when the enzyme is synthesized using the enzyme according to claim 5. 7. The method for producing a novel substance according to claim 2 or 3, wherein a substituent is introduced into the sugar moiety hydroxyl group of the novel substance according to claim 1 using an ether bond, an ester bond or a urethane bond. 8. A separating agent for chromatography containing the novel substance according to claim 1.